HIV-1-infected and/or immune activated macrophages regulate astrocyte SDF-1 production through IL-1beta

Stromal cell-derived factor 1 alpha (SDF-1alpha) and its receptor CXCR4 play important roles in the pathogenesis of human immunodeficiency virus type one (HIV-1)-associated dementia (HAD) by serving as a HIV-1 co-receptor and affecting cell migration, virus-mediated neurotoxicity, and neurodegeneration. However, the underlying mechanisms regulating SDF-1 production during disease are not completely understood. In this report we investigated the role of HIV-1 infected and immune competent macrophage, the principal target cell and mediator of neuronal injury and death in HAD, in regulating SDF-1 production by astrocytes. Our data demonstrated that astrocytes are the primary cell type expressing SDF-1 in the brain. Immune-activated or HIV-1-infected human monocyte-derived-macrophage (MDM) conditioned media (MCM) induced a substantial increase in SDF-1 production by human astrocytes. This SDF-1 production was directly dependent on MDM IL-1beta following both viral and immune activation. The MCM-induced production of SDF-1 was prevented by IL-1beta receptor antagonist (IL-1Ra) and IL-1beta siRNA treatment of human MDM. These laboratory observations were confirmed in severe combined immunodeficient (SCID) mice with HIV-1 encephalitis (HIVE). In these HIVE mice, reactive astrocytes showed a significant increase in SDF-1 expression, as observed by immunocytochemical staining. Similarly, SDF-1 mRNA levels were increased in the encephalitic region as measured by real time RT-PCR, and correlated with IL-1beta mRNA expression. These observations provide direct evidence that IL-1beta, produced from HIV-1-infected and/or immune competent macrophage, induces production of SDF-1 by astrocytes, and as such contribute to ongoing SDF-1 mediated CNS regulation during HAD.     

Neuropathologic and neuroinflammatory activities of HIV-1-infected human astrocytes in murine brain

The balance between astrocyte and microglia neuroprotection and neurotoxicity defines the tempo of neuronal dysfunction during HIV-1-associated dementia (HAD). Astrocytes maintain brain homeostasis and respond actively to brain damage by providing functional and nutritive neuronal support. In HAD, low-level, continuous infection of astrocytes occurs, but the functional consequences of this infection are poorly understood. To this end, human fetal astrocytes (HFA) and monocyte-derived macrophages (MDM) were infected with HIV-1DJV and HIV-1NL4-3 (neurotropic and lymphotropic strains respectively) and a pseudotyped Vesicular Stomatitis Virus (VSV/HIV-1NL4-3) prior to intracranial injection into the basal ganglia of severe combined immunodeficient mice. Neuropathological and immunohistochemical comparisons for inflammatory and neurotoxic activities were performed amongst the infected cell types at 7 or 14 days. HIV-1-infected MDM induced significant increases in Mac-1, glial fibrillary acidic protein, ionized calcium-binding adapter molecule 1, and proinflammatory cytokine RNA and/or protein expression when compared with HSV/HIV-1- and HIV-1-infected HFA and sham-operated mice. Levels of neuron-specific nuclear protein, microtubule-associated protein 2, and neurofilament antigens were reduced significantly in the brain regions injected with human MDM infected with HIV-1DJV or VSV/HIV-1. We conclude that HIV-1 infection of astrocytes leads to limited neurodegeneration, underscoring the early and active role of macrophage-driven neurotoxicity in disease.     

Interleukin-6 production by astrocytes: Induction by the neurotransmitter norepinephrine

Astrocytes contribute to the immunocompetence of the central nervous system (CNS) via their expression of class II major histocompatibility complex (MHC) antigens and the production of inflammatory cytokines such as interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). Of these cytokines, IL-6 is of particular interest because one of its many immune and inflammatory actions is the promotion of immunoglobulin synthesis, and it is thought that IL-6 expression within the brain exacerbates autoimmune diseases of the CNS, which are marked by local immunoglobulin production. Several stimuli induce astrocyte IL-6 expression, including such inducible endogenous factors as IL-1β and TNF-α. We have investigated the possibility that a constitutively present endogenous factor, the neurotransmitter norepinephrine (NE), can induce astrocyte IL-6 production. We report that NE induces both IL-6 mRNA and protein in primary neonatal rat astrocytes, with optimal induction at 10 μM. IL-6 protein induction by NE is comparable to that seen with IL-1β or TNF-α, and NE synergizes with these cytokines for a ten-fold enhanced effect. In contrast to astrocytes, microglia are relatively unresponsive to NE, IL-1β and TNF-α for IL-6 production. Experiments with the β-adrenergic receptor agonist isoproterenol, and α and β-adrenergic receptor antagonists (propranolol, phentolamine, atenolol, and yohimbine) indicate that β₂ and α₁-adrenergic receptors are involved in NE induction of astrocyte IL-6 expression. These results help to further the understanding of neuron-glial interactions, and the role of astrocytes and adrenergic activity in immune responses within the CNS.     

Interleukin-1 and Tumor Necrosis Factor-α Synergistically Mediate Neurotoxicity: Involvement of Nitric Oxide and of N-Methyl-D-aspartate Receptors

The cytokines interleukin (IL)-1 and tumor necrosis factor (TNF)-α, produced by glial cells within the brain, appear to contribute to the neuropathogenesis of several inflammatory neurodegenerative diseases; however, little is known about the mechanism underlying cytokine-induced neurotoxicity. Using human fetal brain cell cultures composed of neurons and glial cells, we investigated the injurious effects of IL-1 β and TNF-α, cytokines which are known to induce nitric oxide (NO) production by astrocytes. Although neither cytokine alone was toxic, IL-1 β and TNF-α in combination caused marked neuronal injury. Brain cell cultures treated with IL-1 β plus TNF-α generated substantial amounts of NO. Blockade of NO production with a NO synthase inhibitor was accompanied by a marked reduction (about 45%) of neuronal injury, suggesting that NO production by astrocytes plays a role in cytokine-induced neurotoxicity. Addition of N-methly-D-aspartate (NMDA) receptor antagonists to brain cell cultures also blocked IL-1 β plus TNF-α-induced neurotoxicity (by 55%), implicating the involvement of NIMDA receptors in cytokine-induced neurotoxicity. Treatment of brain cell cultures with IL-1 β plus TNF-α was found to inhibit [³H]-glutamate uptake and astrocyte glutamine synthetase activity, two major pathways involved in NMDA receptor-related neurotoxicity. These in vitro findings suggest that agents which suppress NO production or inhibit NMDA receptors may protect against neuronal damage in cytokine-induced neurodegenerative diseases.     

HIV-1-infected and/or immune-activated macrophage-secreted TNF-alpha affects human fetal cortical neural progenitor cell proliferation and differentiation

Neurogenesis, tied to the proliferation, migration and differentiation of neural progenitor cells (NPC) is affected during neurodegenerative diseases, but how neurogenesis is affected during HIV-1 associated dementia (HAD) has not been fully addressed. Here we test the hypothesis that HIV-1-infected and/or immune-activated brain macrophages affect NPC proliferation and differentiation through the regulation of cytokines. We showed that human monocyte-derived macrophages (MDM) conditioned medium (MCM) induces a dose dependent increase in NPC proliferation. Conditioned media from lipopolysaccharide (LPS)-activated MDM (LPS-MCM) or HIV-infected MCM (HIV-MCM) induced a profound increase in NPC proliferation. HIV-infected and LPS-activated MCM (HIV+LPS-MCM) induced the most robust increase in NPC proliferation. Moreover, LPS-MCM and HIV+LPS-MCM decreased beta-III-tubulin and increased GFAP expression, demonstrating an induction of gliogenesis and inhibition of neurogenesis. The increase of NPC proliferation and gliogenesis correlated with increases in production of TNF-alpha by infected/activated MDM. Although both IL-1beta and TNF-alpha induced NPC proliferation and gliogenesis, these effects were only partially abrogated by soluble TNF-alpha receptors R1 and R2 (TNF-R1R2), but not by the IL-1 receptor antagonist (IL-1ra). This indicated that the HIV-1-infected/LPS-activated MCM-mediated effects were, in part, through TNF-alpha. These observations were confirmed in severe combined immunodeficient (SCID) mice with HIV-1 encephalitis (HIVE). In these HIVE mice, NPC injected with HIV-infected MDM showed more astrocyte differentiation and less neuronal differentiation compared to NPC injection alone. These observations demonstrated that HIV-1-infected and immune-activated MDM could affect neurogenesis through induction of NPC proliferation, inhibition of neurogenesis, and activation of gliogenesis.     

HIV-1 and IL-1 beta regulate Fas ligand expression in human astrocytes through the NF-kappa B pathway

Reactive astrogliosis is a prominent pathological feature of HIV-1-associated dementia (HAD). We hypothesized that in HAD, astrocytes activated with proinflammatory stimuli such as IL-1beta express Fas ligand (FasL), a death protein. IL-1beta and HIV-1-activated astrocytes expressed FasL mRNA and protein. Luciferase reporter constructs showed that IL-1beta and HIV-1 upregulated FasL promoter activity (p<0.001). The NF-kappaB pathway was involved as shown by inhibition with SN50 and dominant negative IkappaBalpha mutants. Brain extracts from HAD patients had significantly elevated FasL levels compared to HIV-seropositive (p<0.001) and seronegative individuals (p<0.01). We propose that astrocyte expression of FasL may participate in neuronal injury in HAD.     

HIV-1 infected immune competent mononuclear phagocytes influence the pathways to neuronal demise

Secretory products from HIV-1-infected immune-competent mononuclear phagocytes (MP) damage neuronal dendritic arbor (Zheng et al., 2001). The mechanism behind neuronal injury and whether it is species and/or viral strain dependent is not fully understood. To these ends, we investigated whether HIV-1-infected and lipopolysachharide (LPS)-activated MDM elicit neuronal injury in primary human neurons. Neuronal damage was compared to that seen in rat neurons. Utilizing a spectrum of HIV-1 strains to infect human monocyte-derived macrophages (MDM), productive viral replication proved necessary, but not sufficient, for neuronal injury. Neuronal demise was induced by virion-free HIV-1-infected and immune-activated MDM culture supernatants. Maximal alterations in glutamate mediated neuronal signaling, resulted from exposure to secretory products from HIV-1-infected and immune-activated MDM. Apoptosis was the predominant mechanism of cell death induced by HIV-1-infected and LPS-treated MDM. Importantly, neuronal injury and increases in calcium influx mediated by HIV-1-infected and immune-activated MDM culture supernatants was partially blocked by the N-methyl D-aspartate (NMDA) receptor antagonist, MK 801. These data support a primary role for immune-activation in MP neurotoxic activities. The upregulation of NMDA receptor sensitive soluble factors and neuronal apoptosis by HIV-1-infected and immune-activated MDM provide unique insights into links between soluble factors, produced as a consequence of MP immunity, and neuronal demise in HAD.     

Regulation of tissue inhibitor of metalloproteinase-1 by astrocytes: links to HIV-1 dementia

The neuropathogenesis of HIV-1-associated dementia (HAD) revolves around the secretion of toxic molecules from infected and immune-competent mononuclear phagocytes. Astrocyte activation occurs in parallel but limited insights are available for its role in neurotoxicity and cognitive dysfunction. One means in which astrocytes may affect disease is through their production of tissue inhibitors of metalloproteinases (TIMPs). TIMPs are regulators of matrix metalloproteinases, enzymes that affect blood-brain barrier integrity through altering the extracellular matrix. We hypothesized that in response to injury and inflammation in HAD, astrocytes regulate the production of TIMP-1, the inducible type of TIMP that is important in inflammation. To address astrocyte-mediated TIMP-1 regulation in HAD, we evaluated the responses of primary human to IL-1beta and HIV-1. TIMP-1 levels in plasma, CSF, and brain tissue of control, HIV-1 infected patients without cognitive impairment, and HAD patients were also studied. Our data show that an upregulation of TIMP-1 results from astrocytes acutely activated with IL-1beta. In contrast, CSF and brain tissue samples from HAD patients showed reduced TIMP-1 levels compared to seronegative controls. MMP-2 levels in brains showed the opposite. Consistent with this, prolonged activation of astrocytes led to a reduction in TIMP-1 and MMP-2, but a sustained elevation in MMP-1. Our data suggest that in diseased brain tissue, the ability of astrocytes to counteract the destructive effects of MMP through expression of TIMP-1 is diminished by chronic activation. Our studies reveal new opportunities for repair-based therapeutic strategies in HAD.     

Induction of nitric oxide synthase activity in human astrocytes by interleukin-1β and interferon-γ

Nitric oxide (NO) is a short-lived, diffusible molecule that has a variety of biological activities including vasorelaxation, neurotransmission, and cytotoxicity. In the central nervous system, a constitutive form of nitric oxide synthase (NOS) has been localized in a subset of neurons and in endothelial cells. In addition, both constitutive and LPS-inducible NOS has been demonstrated in rat astrocytes and microglia in vitro. In this report, we present evidence for the production of NO, as measured by the production of nitrite, in highly enriched human fetal astrocyte cultures stimulated with IL-1β. The production of nitrite paralleled the induction of NADPH diaphorase enzyme activity in the perikarya of the majority of stimulated astrocytes. The IL-1β-induced nitrite production by astrocytes was markedly enhanced when cells were co-stimulated with IFN-γ or TNF-α (IFN-γ > TNF-α); LPS had no effect used as a single agent or in combination with other cytokines. N^GMMA and N^G-nitro-arginine, competitive inhibitors of NOS, diminished the accumulation of nitrite, but calmodulin antagonists (trifluoperazine, W-5 and W-7) had little or no inhibitory effect. Human fetal microglia, in contrast to astrocytes, failed to secrete significant amounts of nitrite in response to various stimuli. The results demonstrate the presence of an inducible form of NOS in human fetal astrocytes; human microglia, in turn, may control astrocyte NO production by providing IL-1β as an activating signal.     

Distinct Expressions of Three Cytokines by IL-1-Stimulated Astrocytes in Vitro and in AIDS Brain

Interleukin-1 (IL-1) is elevated in brain tissue of individuals who died with acquired immunodeficiency syndrome (AIDS) and other diseases where this cytokine likely stimulates reactive astrocytosis. IL-1 stimulates, among others, production of interleukin-6 (IL-6), granulocyte macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-α (TNF-α) in cultured astrocytes and astrocytoma cell lines. These and other cytokines may contribute to the neuropathogenesis after infection by human immunodeficiency virus type-1 (HIV-1). For example, concentration of TNF-α is increased in brain tissue of individuals who died with AIDS and correlates with the severity of AIDS Dementia Complex (ADC). TNF-α and IL-6 have been immunocytochemically detected in brain tissue but they have not been localized to astrocytes. We, therefore, examined the expression of IL-6, GM-CSF, and TNF-α in human primary astrocytes and astrocytoma cell lines U251 and 253 exposed to IL-1 in serum-free medium. In addition, we immunocytochemically assayed GM-CSF expression by astrocytes in brain tissue (n = 8). The three cytokines were differentially induced in cultured astrocytes by IL-1. The astrocytoma cell lines recapitulated cytokine-specific patterns of expression in astrocytes. The patterns were characterized by amounts produced, compartmentalization (intra- and/or extracellular), time courses, and optimal doses of IL-1 for induction. GM-SCF-like immunoreactivity was detected in some but not all, GFAP⁺ cells. GM-CSF⁺/GFAP⁺ cells were detected in only three of seven cases containing GM-CSF immunoreactivity. Thus, a discrepancy may exist between human astrocytic cytokine expression in vitro and in tissue. Novel methods therefore may need to be developed to recapitulate in vitro the heterogeneity of astrocytic cytokine expression in AIDS and other brain tissue.     

Intracellular CXCR4 signaling, neuronal apoptosis and neuropathogenic mechanisms of HIV-1-associated dementia.

The mechanism(s) by which HIV-1 affects neural injury in HIV-1-associated dementia (HAD) remains unknown. To ascertain the role that cellular and viral macrophage products play in HAD neurotoxicity, we explored one potential route for neuronal demise, CXCR4. CXCR4, expressed on lymphocytes and neurons, is both a part of neural development and a co-receptor for HIV-1. Its ligand, stromal cell-derived factor-1alpha (SDF-1alpha), affects neuronal viability. GTP binding protein (G-protein) linked signaling after neuronal exposure to SDF-1alpha, virus-infected monocyte-derived macrophage (MDM) secretory products, and virus was determined. In both human and rat neurons, CXCR4 was expressed at high levels. SDF-1alpha/beta was detected predominantly in astrocytes and at low levels in MDM. SDF-1beta/beta was expressed in HAD brain tissue and upregulated in astrocytes exposed to virus infected and/or immune activated MDM conditioned media (fluids). HIV-1-infected MDM secretions, virus and SDF-1beta induced a G inhibitory (Gi) protein-linked decrease in cyclic AMP (cAMP) and increase inositol 1,4, 5-trisphosphate (IP3) and intracellular calcium. Such effects were partially blocked by antibodies to CXCR4 or removal of virus from MDM fluids. Changes in G-protein-coupled signaling correlated, but were not directly linked, to increased neuronal synaptic transmission, Caspase 3 activation and apoptosis. These data, taken together, suggest that CXCR4-mediated signal transduction may be a potential mechanism for neuronal dysfunction during HAD.     

Role of mitogen-activated protein kinases in inducible nitric oxide synthase and TNFalpha expression in human fetal astrocytes

Astrocytes are important sources of proinflammatory mediators such as iNOS and TNFalpha in the diseased central nervous system. In previous studies, we showed that the cytokine IL-1 plays a critical role in the activation of human astrocytes to express TNFalpha and the inducible form of nitric oxide synthase (iNOS). In the present study, we have addressed the role of the MAP-kinase pathway in the signaling events leading to the induction of these genes. Treatment with SB203580, a specific inhibitor of p38 mitogen-activated protein kinases (MAPK), potently inhibited IL-1-mediated induction of iNOS and TNFalpha in cultures of human fetal astrocytes. In contrast, PD98059, an upstream inhibitor of the extracellular regulated kinase (ERK)1/2 pathway, had little or no effect. Interestingly, SB203580 reduced the mRNA expression for iNOS, TNFalpha, and IL-6, indicating inhibition prior to translation. Transfection of astrocytes with a dominant-negative Jun-NH(2)-terminal kinase (JNK) construct also reduced iNOS expression. Western blot analysis showed phosphorylated p38 and JNK in IL-1-activated astrocytes, and phosphorylated ERK in both resting and activated cells. Electrophoretic mobility shift assay (EMSA) showed that IL-1 induced NF-kappaB and AP-1 DNA complex formation in astrocytes, and that SB203580 inhibited AP-1 complex formation. Taken together, these results demonstrate the differential roles played by the three MAP kinases in human astrocyte inflammatory gene activation and point to a crucial function of p38 and JNK MAP kinases in IL-1-mediated astrocyte activation.     

Cytokine regulation of astrocyte function: in-vitro studies using cells from the human brain

Participation of astrocytes in central nervous system pathophysiology is likely to involve cytokines, both as stimulators and mediators of astrocyte function. We have used highly enriched human astrocyte cultures as an experimental tool to investigate the influence of cytokines on adhesion molecule expression and synthesis of mediators that are probably important in immune and inflammatory reactions involving the nervous system and in cerebral tissue repair. The response of astrocytes to interferon-γ mainly resulted in increased expression of major histocompatibility complex antigens and co-stimulatory molecules (intercellular adhesion molecule-1, LFA-1α) which mediate astrocyte-T-cell interactions. Another co-stimulatory molecule, B7, was neither expressed nor inducible by IFN-γ and other cytokines. TNF-α and IL-1β were more efficient in stimulating synthesis of immunoregulatory and proinflammatory cytokines (IL-6, IL-8 and colony-stimulating factors), cytokine antagonists (TNF-α soluble receptors), or cytokines with a possible neuroprotective role (leukemia inhibitory factor); they also increased expression of some co-stimulatory molecules (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1). Transforming growth factor-β₁ was a strong inducer of leukemia inhibitory factor, but did not affect either major histocompatibility complex/co-stimulatory molecule expression or cytokine synthesis. Thus, different cytokines activate distinct functional programs in astrocytes, which may play a specific role in different brain diseases or at different stages of the same disease. It was additionally observed that the response of human astrocytes to cytokines (in particular the inducible synthesis of certain cytokines) varied greatly depending on the presence or absence of neurons in the culture system. This finding suggests that neuronal-glial interactions may be implicated in determining the activation threshold of astrocytes to inflammatory cytokines.     

HIV-1 Envelope Glycoprotein 120 Regulates Brain IL-1β System and TNF-α mRNAs In Vivo

Human immunodeficiency virus type I (HIV-1)-derived envelope glycoprotein 120 (gp120) is proposed to play an important role in HIV-1 neuropathology. Gp120 may act through mediators including proinflammatory cytokines. Here, we investigated the regulation of the IL-1β system [IL-1β, IL-1 receptor type I (IL-1RI), IL-1 receptor antagonist (IL-1Ra)], TNF-α and TGF-α mRNAs in the rat central nervous system (CNS) in response to the constant intracerebroventricular (ICV) microinfusion of HIV-1 gp120 for 72 h and 144 h. The results show that gp120: (1) increased IL-1β and IL-1Ra mRNAs levels in the same samples from the cerebellum, hypothalamus and midbrain, with the largest increase in the hypothalamus; (2) induced profiles of IL-1β mRNA and IL-1Ra mRNA that were highly intercorrelated; (3) increased the hypothalamic TNF-α mRNA levels; and (4) did not affect the IL-1RI mRNA and TGF-α mRNA levels in any brain region. A dysregulation in the IL-1β/IL-1Ra CNS balance and a mutual induction and synergistic activity of IL-1β and TNF-α could result in a deleterious amplification cycle of cellular activation and cytotoxicity with implications to HIV-1-associated encephalitis, encephalopathy, and neurological manifestations.     

Cytokine induction of heat shock protein expression in human oligodendrocytes: An interleukin-1-mediated mechanism

In this study, we examined the role of cytokines, known to be in elevated levels in multiple sclerosis (MS) plaques, in regulating oligodendrocyte (ODC) expression of heat shock protein (hsp) in human brain-derived glial cell cultures. Using dual-stain immunohistochemistry, we initially compared the ability of a mixture of cytokines (IL-1α, IL-1β, IL-2, IL-6, IL-8, TNF-α, TNF-β, IFN-β and IFN-γ) with that of physical stimuli such as heat shock and peroxide, to increase cellular expression of the mainly inducible hsp72 species in mixed glial cell cultures (containing ODC, astrocytes and microglia). Similar to heat shock and peroxide, the cytokine mixture induced hsp72 expression only in ODC (70 ± 5% vs. a baseline of 3 ± 1% positive cells). When used individually, however, only IL-1α (79 ± 3%), IFN-γ (70 ± 2%) and TNF-α (65 ± 5%) induced ODC hsp72 expression in mixed glial cell cultures. In purified ODC preparations, only IL-1α induced hsp72 expression (84 ± 4%). An IL-1 receptor antagonist (IL-1ra), abrogated hsp72 induction by IL-1α (16 ± 3%) as well as that due to IFN-γ (14 ± 1%) and TNF-α (13 ± 2%) in mixed glial cell cultures. Furthermore, ODC express IL-1 receptors, detected by confocal laser scanning microscopy. Our data indicate that cytokines mediate hsp induction in ODC possibly via a final common pathway involving IL-1 binding to its receptor on ODC. Such interaction could enhance any putative ODC-immune interactions which are dependent on hsp molecule recognition.     

Inhibition of astrocyte TNFα expression by extracellular potassium

TNFα and IL-6 are cytokines of great interest, given the numerous biological activities and the documented expression in several central nervous system (CNS) pathologies. In this report, we have examined cultures of IL-1- or IL-1/IFNγ-activated human fetal astrocytes as a model to study mechanisms of cytokine regulation in the inflamed CNS. Since one of the major functions of astrocytes is spatial buffering of K⁺ ions, we examined the effect of high extracellular KCl on astrocyte cytokine expression by ribonuclease protection assay and ELISA. Results demonstrate that astrocyte TNFα production was potently inhibited by K⁺ with 44 and 89% inhibition at 25 and 55 mM K+, respectively. In contrast, astrocyte IL-6 inhibition required higher concentrations of K+ (≥75 mM). These results demonstrate a novel role for astrocyte potassium channel activity in modulation of glial cytokine production.