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.     

CXCR4 is the primary receptor for feline immunodeficiency virus in astrocytes

Feline astrocytes were productively infected with the Crandell feline kidney (CrFK) cell-adapted feline immunodeficiency virus (FIV) Petaluma strain in a primary culture. They expressed mRNA of CXCR4, and the FIV infection was blocked by stromal cell-derived factor 1alpha (SDF-1alpha), SDF-1beta, or the bicyclam AMD3100 in a dose-dependent manner. These observations suggest that, like FIV infection in CrFK cells and lymphocytes, the virus uses CXCR4 as a primary receptor for infecting astrocytes and this can be a possible natural model for AIDS dementia complex.     

Soluble HIV-1 infected macrophage secretory products mediate blockade of long-term potentiation: a mechanism for cognitive dysfunction in HIV-1-associated dementia

It is generally accepted that viral and cellular products from immune competent mononuclear phagocytes (MP) (brain macrophages and microglia) underlie the neuropathogenesis of HIV-1-associated dementia (HAD). What remains unanswered, however, is the composition of and mechanisms for such MP-induced neurological dysfunctions. In attempts to address these issues culture fluids from HIV-1ADA-infected monocyte-derived macrophages (MDMs) (depleted or enriched with progeny virus) were placed onto the CA1 area of rat hippocampal brain slices (the site of mammalian learning and memory) and neuronal long-term potentiation (LTP) assayed. LTP was induced by high frequency stimulation (HFS). Lipopolysaccharide (LPS) served as a surrogate macrophage activator. Synaptic strength was assayed by the initial slope of evoked field excitatory postsynaptic potentials (EPSPs). Synaptic potentiation following HFS was observed in slices incubated with uninfected (control) MDM culture fluids. The magnitude of the LTP response was 150.2 +/- 21.10% compared to basal levels (n=6). Synaptic strength was enhanced in virus-infected (135.7+/-28.9%, n=8) and LPS-activated MDM (123.3+/-5.1%, n=7) but at lower levels than controls. The lowest levels of LTP were in brain slices incubated with virus-infected and LPS-activated MDM fluids at (109.5+/-9.9% n=12). Interestingly, bath application of progeny HIV-1 virions showed minimal LTP effects. Virus-infected, LPS-activated MDM fluids, with progenyvirus, reduced synaptic strength but were not statistically different than replicate culture fluids depleted of virus. In contrast, IL-1beta and quinolinic acid, significantly diminished synaptic strength. These results, taken together, suggest that soluble HIV-1-infected MDM secretory products, but not virus per se, significantly affect LTP. This electrophysiological system, which monitors neuronal function following cell exposure to HIV-1 infected materials could provide a novel testing ground for therapeutics designed to protect brain function in HAD.     

HIV-1 infected and immune competent mononuclear phagocytes induce quantitative alterations in neuronal dendritic arbor: Relevance for HIV-1-associated dementia

Neuronal loss, alterations in dendritic arbor, and decreased synaptic density, in infected brain tissue, are neuropathological signatures of HIV-1-associated dementia (HAD). Brain mononuclear phagocyte (MP) (macrophage and microglia) secretory products can effect neuronal compromise, although the underlying mechanism(s) remain incompletely defined. To these ends, we quantitatively assessed the effects of virus-infected and/or immune activated MP secretory products on multiple aspects of neuronal morphology. Rat cortical and hippocampal neurons were exposed to secretory products from HIV-1-infected and lipopolysaccharide (LPS)-activated human monocyte-derived macrophage (MDM). Our assays for alterations in neuronal dendritic arbor and cell loss included the quantification of neurofilament (NF), neuron-specific enolase (NSE), and MAP-2 by ELISA and cellular morphology. MDM conditioned media (MCM) enhanced neuronal survival. HIV-1 infection or activation by LPS had modest neurotoxic effects. In contrast, the combination of HIV-1 infection and activation of MDM produced significant neurotoxicity. Such MDM products altered dendritic arbor, decreased synaptic density, and increased LDH release. Comparable neurotrophic/toxic responses were observed when neurons were exposed to MCM collected from 12 separate human donors. Similar responses were observed with MCM from human fetal microglia, further supporting the role of HIV-1-infected and immune-activated brain MP in the overall neurotoxic responses. This work provides quantitative measures of neuronal damage by which virus infected and activated MP can elicit neuronal injury in HAD.     

Modulation of network-driven, GABA-mediated giant depolarizing potentials by SDF-1alpha in the developing hippocampus

Chemokine stromal cell-derived factor-1 (SDF-1, or CXCL12) plays an important role in brain development and functioning. Whole-cell patch clamp recordings were conducted on CA3 neurons in hippocampal slices prepared from neonatal rats between postnatal days 2 and 6 to study the modulatory effects of SDF-1alpha on network-driven, gamma-aminobutyric-acid-mediated giant depolarizing potentials (GDPs), a hallmark of the developing hippocampus. We found that SDF-1alpha, the only natural ligand for chemokine CXC motif receptor 4 (CXCR4), decreased GDP firing without significant effects on neuronal passive membrane properties in neonatal hippocampal neurons. The SDF-1alpha-mediated decrease in GDP firing was blocked by T140, a CXCR4 receptor antagonist, suggesting that SDF-1alpha modulates GDP firing via CXCR4. We also showed that endogenous SDF-1 exerts a tonic inhibitory action on GDPs in the developing hippocampus. As SDF-1/CXCR4 are highly expressed in the developing brain and GDPs are involved in activity-dependent synapse formation and functioning, the inhibitory action of SDF-1alpha on GDPs may reflect a potential mechanism for chemokine regulation of neural development in early neonatal life.     

High-level expression of functional chemokine receptor CXCR4 on human neural precursor cells

Neural precursor cells (NPCs) are self-renewing, multipotent progenitors that give rise to neurons, astrocytes and oligodendrocytes in the central nervous system (CNS). Fetal NPCs have attracted attention for their potential use in studying normal CNS development. Several studies of rodent neural progenitors have suggested that chemokines and their receptors are involved in directing NPC migration during CNS development. In this study, we established a consistent system to culture human NPCs and examined the expression of chemokine receptors on these cells. NPCs were found to express the markers nestin and CD133 and to differentiate into neurons, astrocytes and oligodendrocytes at the clonal level. Flow cytometry and RNase protection assay (RPA) indicated that NPCs express high levels of CXCR4 and low levels of several other chemokine receptors. When examined using a chemotaxis assay, NPCs were able to respond to CXCL12/SDF-1alpha, a ligand of CXCR4. Treatment with anti-CXCR4 antibody or HIV-1 gp120 abolished the migratory response of NPCs towards CXCL12/SDF-1alpha. These findings suggest that CXCR4 may play a significant role in directing NPC migration during CNS development.     

HIV-1, chemokines and neurogenesis

HIV-1 infection of the brain results in a large number of behavioural defecits accompanied by diverse neuropathological signs. However,it is not clear how the virus produces these effects or exactly how the neuropathology and behavioural defecits are related. In this article we discuss the possibility that HIV-1 infection may negatively impact the process of neurogenesis in the adult brain and that this may contribute to HIV-1 related effects on the nervous system. We have previously demonstrated that the development of the dentate gyrus during embryogenesis requires signaling by the chemokine SDF-1 via its receptor CXCR4. We demonstrated that neural progenitor cells that give rise to dentate granule neurons express CXCR4 and other chemokine receptors and migrate into the nascent dentate gyrus along SDF-1 gradients. Animals deficient in CXCR4 receptors exhibit a malformed dentate gyrus in which the migration of neural progenitors is stalled. In the adult, neurogenesis continues in the dentate gyrus. Adult neural progenitor cells existing in the subgranlar zone, that produce granule neurons, express CXCR4 and other chemokine receptors, and granule neurons express SDF-1 suggesting that SDF-1/CXCR4 signaling is also important in adult neurogenesis. Because the cellular receptors for HIV-1 include chemokine receptors such as CXCR4 and CCR5 it is possible that the virus may interfere with SDF-1/CXCR4 signaling in the brain including disruption of the formation of new granule neurons in the adult brain.     

Regulation of neuronal P53 activity by CXCR 4

Abnormal activation of CXCR 4 during inflammatory/infectious states may lead to neuronal dysfunction or damage. The major goal of this study was to determine the coupling of CXCR 4 to p53-dependent survival pathways in primary neurons. Neurons were stimulated with the HIV envelope protein gp120(IIIB) or the endogenous CXCR 4 agonist, SDF-1 alpha. We found that gp120 stimulates p53 activity and induces expression of the p53 pro-apoptotic target Apaf-1 in cultured neurons. Inhibition of CXCR 4 by AMD 3100 abrogates the effect of gp120 on both p53 and Apaf-1. Moreover, gp120 neurotoxicity is markedly reduced by the p53-inhibitor, pifithrin-alpha. The viral protein also regulates p53 phosphorylation and expression of other p53-responsive genes, such as MDM 2 and p21. Conversely, SDF-1 alpha, which can promote neuronal survival, increases p53 acetylation and p21 expression in neurons. Thus, the stimulation of different p53 targets could be instrumental in determining the outcome of CXCR 4 activation on neuronal survival in neuro-inflammatory disorders.     

Complex effects of stromal cell-derived factor-1 alpha on melanin-concentrating hormone neuron excitability

Stromal cell-derived factor 1alpha (SDF-1alpha), a chemoattractant for leucocytes and neurons, and its receptor, CXCR4 are expressed in subsets of neurons of specific brain areas. In rat lateral hypothalamic area (LHA) we show, using immunocytochemistry, that CXCR4 is localized within melanin-concentrating hormone (MCH)-expressing neurons, mainly involved in feeding behaviour regulation. We investigated whether SDF-1alpha may control MCH neuronal activity. Patch-clamp recordings in rat LHA slices revealed multiple effects of SDF-1alpha on the membrane potential of MCH neurons, indirect through glutamate/GABA release and direct through GIRK current activation. Moreover, SDF-1alpha at 0.1-1 nM decreased peak and discharge frequency of action potential evoked by current pulses. These effects were further confirmed in voltage-clamp experiments, SDF-1alpha depressing both potassium and sodium currents. At 10 nM, however, SDF-1alpha increased peak and discharge frequency of action potential evoked by current pulses. Using a specific CXCR4 antagonist, we demonstrated that only the depressing effect on AP discharge was mediated through CXCR4 while the opposite effect was indirect. Together, our studies reveal for the first time a direct effect of SDF-1alpha on voltage-dependent membrane currents of neurons in brain slices and suggest that this chemokine may regulate MCH neuron activity.     

Role of activated astrocytes in neuronal damage: Potential links to HIV-1-associated dementia

HIV-1-associated dementia (HAD) is an important complication of HIV-1 infection. Reactive astrogliosis is a key pathological feature in HAD brains and in other central nervous system (CNS) diseases. Activated astroglia may play a critical role in CNS inflammatory diseases such as HAD. In order to test the hypothesis that activated astrocytes cause neuronal injury, we stimulated primary human fetal astrocytes with HAD-relevant pro-inflammatory cytokine IL-1beta. IL-1beta-activated astrocytes induced apoptosis and significant changes in metabolic activity in primary human neurons. An FITC-conjugated pan-caspase inhibitor peptide FITC-VAD-FMK was used for confirming caspase activation in neurons. IL-1beta activation enhanced the expression of death protein FasL in astrocytes, suggesting that FasL is one of the potential factors responsible for neurotoxicity observed in HAD and other CNS diseases involving glial inflammation. Our data presented here add to the developing picture of role of activated glia in HAD pathogenesis.     

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.     

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.     

Exogenous IL-7 induces Fas-mediated human neuronal apoptosis: potential effects during human immunodeficiency virus type 1 infection

The use of exogenous cytokines is part of translational immune-antiretroviral approaches to induce immune reconstitution and possibly eliminate the persistence of human immunodeficiency virus type 1 (HIV-1) in virally suppressed infected individuals on highly active antiretroviral therapy (HAART). Recently, our laboratories demonstrated that interleukin-7 (IL-7) has significant efficiency in stimulating HIV-1 replication from proviral latency in CD4+ T lymphocytes of infected patients. The authors now investigated the possible role of IL-7 in HIV-1-associated dementia (HAD). The authors demonstrated that the IL-7 receptor is expressed on both human neurons (i.e., differentiated NT2 cells) and human astrocytes, with relatively higher mRNA levels in neurons. The translational protein levels of IL-7 receptor alpha were not proportional to those of the mRNA levels in these central nervous system (CNS)-based cell types. Exogenous IL-7 was observed to only slightly down-regulate IL-7 receptor alpha expression on both neurons and astrocytes, as assayed by Western blotting. Instead of promoting survival, surprisingly, exogenous IL-7 induced neuronal apoptosis, as detected by TUNEL assays. Furthermore, IL-7 augmented neuronal apoptosis induced by HIV-1 gp120. Human apoptosis genomic microarray analyses of IL-7-treated human neurons showed up-regulated expression of proapoptotic genes: protein kinases, caspase-10, FAST kinase, tumor necrosis factor (TNF) receptor, and BCL2-antagonist of cell death. These data suggest that IL-7 leads to neuronal apoptosis by a molecular mechanism(s) that occurs via Fas-mediated activation-induced cell death. These studies may therefore not only be key in evaluating the potential use of IL-7 in vivo as a therapeutic modality, but also suggest that IL-7, which is increased endogenously in HIV-1-infected individuals late in disease, may be involved in the neuronal apoptosis demonstrated during HAD.     

Regional and cellular localization of the CXCl12/SDF-1 chemokine receptor CXCR7 in the developing and adult rat brain

The chemokine stromal cell-derived factor-1 (SDF-1) regulates neuronal development via the chemokine receptor CXCR4. In the adult brain the SDF-1/CXCR4 system was implicated in neurogenesis, neuromodulation, brain inflammation, tumor growth, and HIV encephalopathy. Until the recent identification of RDC1/CXCR7 as the second SDF-1 receptor, CXCR4 was considered to be the only receptor for SDF-1. Here we provide the first map of CXCR7 mRNA expression in the embryonic and adult rat brain. At embryonic stages, CXCR7 and CXCR4 were codistributed in the germinative zone of the ganglionic eminences, caudate putamen, and along the routes of GABAergic precursors migrating toward the cortex. In the cortex, CXCR7 was identified in GABAergic precursors and in some reelin-expressing Cajal-Retzius cells. Unlike CXCR4, CXCR7 was abundant in neurons forming the cortical plate and sparse in the developing dentate gyrus and cerebellar external germinal layer. In the adult brain, CXCR7 was expressed by blood vessels, pyramidal cells in CA3, and mature dentate gyrus granule cells, which is reminiscent of the SDF-1 pattern. CXCR7 and CXCR4 overlapped in the wall of the four ventricles. Further neuronal structures expressing CXCR7 comprised the olfactory bulb, accumbens shell, supraoptic and ventromedial hypothalamic nuclei, medial thalamus, and brain stem motor nuclei. Also, GLAST-expressing astrocytes showed signals for CXCR7. Thus, CXCR4 and CXCR7 may cooperate or act independently in SDF-1-dependent neuronal development. In mature neurons and blood vessels CXCR7 appears to be the preponderant SDF-1-receptor.     

Regulation of cell cycle proteins by chemokine receptors: A novel pathway in human immunodeficiency virus neuropathogenesis?

In order to test the hypothesis that alteration of cell cycle proteins are involved in the neuronal damage caused by human immunodeficiency virus (HIV), the authors have been studying the effect of chemokines on the CDK/Rb/E2F-1 pathway--which is involved in neuronal apoptosis and differentiation. First, they have asked whether CXCR4, the specific receptor for the chemokine SDF-1 and X4-using gp120s, can regulate Rb and E2F-1 activity in cultures of differentiated rat neurons. Although CCR3 and CCR5 are known to mediate infection of microglia by HIV-1, recent evidence indicate that CXCR4 also play important roles in HIV-induced neuronal injury, and dual-tropic isolates that use CXCR4 to infect macrophages have recently been reported. The authors have focused on two specific brain areas in which CXCR4 is physiologically relevant, i.e., the cerebellum and the hippocampus. So far, the data indicate that changes in the nuclear and cytosolic levels of Rb, which result in the functional loss of this protein, are associated with apoptosis in these neurons, and that SDF-1alpha and gp120IIIB affect this pathway. A summary of the findings are presented.     

Functional CXCR4 receptor development parallels sensitivity to HIV-1 gp120 in cultured rat astroglial cells but not in cultured rat cortical neurons

The alpha chemokine receptor CXCR4 is used as the major coreceptor for the cell entry of T-cell-tropic human immunodeficiency virus-1 (HIV-1) isolates. Activation of this coreceptor by its natural ligand SDF1alpha is associated with an intracellular Ca(2+) increase. Because the HIV-1 glycoprotein 120 (gp120) is shedded from the surface of HIV-1-infected cells and is regarded as an injurious molecule in the pathogenesis of HIV-1-associated encephalopathy (HIVE), we investigated the effects of gp120 on the intracellular Ca(2+) regulation of astrocytes and neurons. After 5 days in vitro (DIV), SDF1alpha (50 nM) elicited a pertussis toxin-sensitive intracellular Ca(2+) increase due to Ca(2+) release from internal stores that was reduced by a blocking monoclonal antibody against the CXCR4 receptor in astrocytes and neurons. Parallel with the development of the SDF1alpha response, cells became sensitive to direct application of gp120 (1.25 microg/ml), which, similarly to SDF1alpha, elicited a transient intracellular Ca(2+) increase. However, short-term incubation with gp120 for 60 to 120 min induced a reduction of glutamate- or ATP-evoked intracellular Ca(2+) responses only in astrocytes and not in neurons, although functional CXCR4 receptors were expressed in both cell types. Therefore, our data strongly suggest that the CXCR4 receptor-mediated intracellular signaling pathway of gp120 differs in astrocytes and neurons.     

Neuroanatomical distribution of CXCR4 in adult rat brain and its localization in cholinergic and dopaminergic neurons

Accumulating evidence supports a role of chemokines and their receptors in brain function. Up to now scarce evidence has been given of the neuroanatomical distribution of chemokine receptors. Although it is widely accepted that chemokine receptors are present on glial cells, especially in pathological conditions, it remains unclear whether they are constitutively present in normal rat brain and whether neurons have the potential to express such chemokine receptors. CXCR4, a G protein-coupled receptor for the chemokine stromal cell-derived factor-1 (SDF-1/CXCL12) was reported to have possible implications in brain development and AIDS-related dementia. By dual immunohistochemistry on brain sections, we clearly demonstrate that CXCR4 is constitutively expressed in adult rat brain, in glial cells (astrocytes, microglia but not oligodendrocytes) as well as in neurons. Neuronal expression of CXCR4 is mainly found in cerebral cortex, caudate putamen, globus pallidus, substantia innominata, supraoptic and paraventricular hypothalamic nuclei, ventromedial thalamic nucleus and substantia nigra. Using confocal microscopy, a differential distribution of CXCR4 in neuronal perikarya and dendrites can be observed according to the brain structure. Furthermore, this work demonstrates for the first time the coexistence of a chemokine receptor with classical neurotransmitters. A localization of CXCR4 is thus observed in neuronal cell bodies expressing choline acetyltransferase-immunoreactivity in the caudate putamen and substantia innominata, as well as in tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta. In conclusion, the constitutive neuronal CXCR4 expression suggests that SDF-1/CXCL12 could be involved in neuronal communication and possibly linked up with cholinergic and dopaminergic neurotransmission and related disorders.