Expression of indoleamine 2,3-dioxygenase and production of quinolinic acid by human microglia, astrocytes, and neurons

There is good evidence that the kynurenine pathway (KP) and one of its end products, quinolinic acid (QUIN) play a role in the pathogenesis of several major neurological diseases. While QUIN has been shown to be produced in neurotoxic concentrations by macrophages and microglia, the capacity of astrocytes and neurons to produce QUIN is controversial. Using interferon gamma (IFN-gamma)-stimulated primary cultures of human mixed brain cells, we assayed expression of the KP regulatory enzyme indoleamine 2,3-dioxygenase (IDO) and QUIN production by immunocytochemistry. Using IFN-gamma-stimulated purified cultures of neurons, astrocytes, microglia and macrophages, we studied IDO expression by RT-PCR and production of QUIN using mass spectrometry. We found that astrocytes, neurons, and microglia expressed IDO but only microglia were able to produce detectable amounts of QUIN. However, astrocytes and neurons had the ability to catabolize QUIN. This study also provides the first evidence of IDO expression and lack of production of QUIN in culture of primary human neurons.     

Localization of HIV-1 in human brain using polymerase chain reaction/in situ hybridization and immunocytochemistry

Human immunodeficiency virus type 1 (HIV-1) infects the brains of a majority of patients with the acquired immunodeficiency syndrome (AIDS), and has been linked to the development of a progressive dementia termed “HIV-associated dementia.” This disorder results in severe cognitive, behavioral, and motor deficits. Despite this neurological dysfunction, HIV-1 infection of brain cells does not occur significantly in neurons, astrocytes, or oligodendrocytes, but is restricted to brain macrophages and microglia. To identify possible low-level or latent infection of other brain cells, we combined the techniques of the polymerase chain reaction with in situ hybridization for the detection of HIV DNA, and used immunocytochemistry to identify the HIV-expressing cells. In the 21 adult brains studied (15 AIDS and 6 seronegative control brains), we found that polymerase chain reaction/in situ hybridization was both sensitive and specific for identifying HIV-infected cells. In all brains, the majority of infected cells were macrophages and microglia. In several brains, however, a substantial minority of cells harboring HIV DNA were identified as astrocytes. Neurons, oligodendrocytes, and endothelial cells were not infected with HIV, even in cases with HIV-associated dementia. These findings confirm previous data regarding the importance of macrophage/microglial infection, and essentially exclude neuronal infection in pathogenetic models of HIV-associated neurological disease. These data also demonstrate that latent or low-level infection of astrocytes occurs in AIDS, a finding that may be of importance in understanding HIV neuropathogenesis.     

Expression of CD137 and its ligand in human neurons, astrocytes, and microglia: modulation by FGF-2

CD137 (ILA, 4-1BB), a member of the tumor necrosis factor receptor family, and its ligand CD137-L were assayed by RT-PCR and immunocytochemistry in cultured human brain cells. Results demonstrated that both neurons and astrocytes expressed specific RNA for CD137 and its protein, which was found both on the plasma membrane and in the cytoplasm. Surprisingly, microglia, which also expressed CD137 mRNA, showed negative immunostaining. CD137-L-specific RNA was detected only in astrocytes and neurons. When brain cells were treated with fibroblast growth factor-2 (FGF-2), upregulation of CD137 but not of its ligand was observed in neurons and astrocytes. Protein localization was also affected. In microglia, an inhibition of RNA expression was induced by treatment, whereas CD137-L remained negative. Our data are the first demonstration that human brain cells express a protein found thus far in activated immunocompetent cells and epithelia. Moreover, they suggest not only that CD137 and CD137-L might play a role in interaction among human brain cells, but also that FGF-2 might have an immunoregulatory function in brain, modulating interaction of the central nervous system with peripheral immunocompetent cells.     

Temporal expression of osteopontin and CD44 in rat brains with experimental cryolesions

Expression of osteopontin and CD44 in the brain was studied after cryolesioning to understand how osteopontin and its receptor, CD44, are involved in processes in the brains of rats with cryolesions. Western blot analysis showed that osteopontin increased significantly at days 4 and 7 post-injury and declined slightly thereafter in cryolesioned brains in comparison with levels in sham-operated controls. An immunohistochemical study localized osteopontin in activated microglia/macrophages in the core lesions, where the majority of macrophages proliferate. Osteopontin was also detected temporarily in some neurons and a few astrocytes in the lesion periphery on days 4 and 7 post-injury, but the immunoreactivity in macrophages, neurons, and astrocytes disappeared by day 14 post-injury. There was some CD44, a receptor for osteopontin, in the brain cells of sham-operated rats. After injury, intense CD44 immunostaining was seen in the majority of macrophages and in reactive astrocytes, but not in neurons, in the ipsilateral lesions after day 4 post-injury, and this immunoreactivity remained on day 14 post-injury. These findings suggest that activated microglia/macrophages and some neurons are major sources of osteopontin during the early stage of brain damage induced by a cryolesion and that osteopontin interacts with CD44 expressed on astrocytes and activated microglia/macrophages in the damaged cerebral cortex, possibly mediating cell migration after cryolesioning in the rat brain.     

Proteomics analysis of human astrocytes expressing the HIV protein Tat

Astrocyte infection in HIV has been associated with rapid progression of dementia in a subset of HIV/AIDS patients. Astrogliosis and microglial activation are observed in areas of axonal and dendritic damage in HIVD. In HIV-infected astrocytes, the regulatory gene tat is over expressed and mRNA levels for Tat are elevated in brain extracts from individuals with HIV-1 dementia. Tat can be detected in HIV-infected astrocytes in vivo. The HIV-1 protein Tat transactivates viral and cellular gene expression, is actively secreted mainly from astrocytes, microglia and macrophages, into the extracellular environment, and is taken up by neighboring uninfected cells such as neurons. The HIV-1 protein Tat released from astrocytes reportedly produces trimming of neurites, mitochondrial dysfunction and cell death in neurons, while protecting its host, the astrocyte. We utilized proteomics to investigate protein expression changes in human astrocytes intracellularly expressing Tat (SVGA-Tat). By coupling 2D fingerprinting and identification of proteins by mass spectrometry, we identified phosphatase 2A, isocitrate dehydrogenase, nuclear ribonucleoprotein A1, Rho GDP dissociation inhibitor alpha, beta-tubulin, crocalbin like protein/calumenin, and vimentin/alpha-tubulin to have decreased protein expression levels in SVGA-Tat cells compared to the SVGA-pcDNA cells. Heat shock protein 70, heme oxygenase-1, and inducible nitric oxide synthase were found to have increased protein expression in SVGA-Tat cells compared to controls by slotblot technique. These findings are discussed with reference to astrocytes serving as a reservoir for the HIV virus and how Tat promotes survival of the astrocytic host.     

Expression of the endoplasmic reticulum stress response marker, BiP, in the central nervous system of HIV-positive individuals

The prevalence of HIV-associated neurocognitive impairment (NCI), which includes HIV-associated dementia (HAD) and minor cognitive and motor disorder (MCMD), has been increasing. HIV-infected and/or activated macrophages/microglia in the brain initiate the neurodegeneration seen in HIV-associated NCI via soluble neurotoxic mediators, including reactive oxygen species, viral proteins and excitotoxins. Neurotoxic factors released by macrophages/microglia injure neurones directly and alter astrocytic homeostatic functions, which can lead to excitotoxicity and oxidative stress-mediated neuronal injury. Often, cells respond to oxidative stress by initiating the endoplasmic reticulum (ER) stress response. Thus, we hypothesize that ER stress response is activated in HIV-infected cortex. We used immunofluorescence and immunoblotting to assess expression patterns of the ER stress proteins, BiP and ATF6, in HIV-positive cortical autopsy tissue. Additionally, we performed immunofluorescence using cell type-specific markers to examine BiP staining in different cell types, including neurones, astrocytes and macrophages/microglia. We observed a significant increase in BiP expression by both immunoblotting and immunofluorescence in HIV-positive cortex compared with control tissue. Additionally, phenotypic analysis of immunofluorescence showed cell type-specific increases in BiP levels in neurones and astrocytes. Further, ATF-6beta, an ER stress response initiator, is up-regulated in the same patient group, as assessed by immunoblotting. These results suggest that ER stress response is activated in HIV-infected cortex. Moreover, data presented here indicate for the first time that numbers of macrophages/microglia increase in brains of MCMD patients, as has been observed in HAD.     

Recombinant adeno-associated virus serotype 2 effectively transduces primary rat brain astrocytes and microglia

Recombinant adeno-associated virus-2 (rAAV2) under control of the chicken beta actin promoter/truncated CMV enhancer (CBA) was investigated for its ability to transduce primary cultures of rat brain neurons, microglia and astrocytes. This vector was highly effective in all three cell types in heparin-sensitive manners (astrocytes, microglia and neurons transduced by >98%, 75%, and 95%, respectively). However, astrocytes co-cultured with neurons were not transduced. rAAV2/CBA is an important new method for genetic manipulation of brain cells, though this may be modulated by interactions among cell types.     

Human immunodeficiency virus-associated depression: contributions of immuno-inflammatory, monoaminergic, neurodegenerative, and neurotrophic pathways

In the era of greatly improved pharmacological treatment of HIV infection through highly active antiretroviral therapy (HAART), HIV patients experience reduced viral loads, reduced opportunistic infections, increased CD4+ T cell count, and greater life expectancy. Although life expectancy is increased, patients often develop neurological disturbances that may persist for long periods, seriously jeopardizing quality of life and adherence to the medication protocols of HAART. For these reasons, HIV-associated neurological disorders have gained importance in both clinical and basic investigations of HIV infection. Depression is the most prevalent neuropsychiatric disorder among people living with HIV. In this review, we discuss how HIV can predispose infected individuals to depression by several interrelated mechanisms. These include inducing chronic elevation of cytokines through activation of microglia and astrocytes; decreasing monoaminergic function; inducing neurotoxicity, especially in dopaminergic neurons; and reducing brain-derived neurotrophic factor. These viral pathways interact with psychosocial factors to create the depressive state. HIV depression has a great impact on quality of life and implementation of antiretroviral therapy, and thus, recognition of these modes of action is significant for understanding HIV neuropathology and for selecting modalities for pharmacologic treatment.     

Proinflammatory cytokines and HIV-1 synergistically enhance CXCL10 expression in human astrocytes

HIV encephalitis (HIVE), the pathologic correlate of HIV-associated dementia (HAD) is characterized by astrogliosis, cytokine/chemokine dysregulation, and neuronal degeneration. Increasing evidence suggests that inflammation is actively involved in the pathogenesis of HAD. In fact, the severity of HAD/HIVE correlates more closely with the presence of activated glial cells than with the presence and amount of HIV-infected cells in the brain. Astrocytes, the most numerous cell type within the brain, provide an important reservoir for the generation of inflammatory mediators, including interferon-gamma inducible peptide-10 (CXCL10), a neurotoxin and a chemoattractant, implicated in the pathophysiology of HAD. Additionally, the proinflammatory cytokines, IFN-gamma and TNF-alpha, are also markedly increased in CNS tissues during HIV-1 infection. In this study, we hypothesized that the interplay of host cytokines and HIV-1 could lead to enhanced expression of the toxic chemokine, CXCL10. Our findings demonstrate a synergistic induction of CXCL10 mRNA and protein in human astrocytes exposed to HIV-1 and the proinflammatory cytokines. Signaling molecules, including JAK, STATs, MAPK (via activation of Erk1/2, AKT, and p38), and NF-kappaB were identified as instrumental in the synergistic induction of CXCL10. Understanding the mechanisms involved in HIV-1 and cytokine-mediated up-regulation of CXCL10 could aid in the development of therapeutic modalities for HAD.     

Accumulation of Non-Transferrin-Bound Iron by Neurons, Astrocytes, and Microglia

Neurodegenerative conditions such as Alzheimer’s disease, Parkinson’s disease, and hemorrhagic stroke are associated with increased levels of non-transferrin-bound iron (NTBI) in the brain, which can promote Fenton chemistry. While all types of brain cells can take up NTBI, their efficiency of accumulation and capacity to withstand iron-mediated toxicity has not been directly compared. The present study assessed NTBI accumulation in cultures enriched in neurons, astrocytes, or microglia after exposure to ferric ammonium citrate (FAC). Microglia were found to be the most efficient in accumulating iron, followed by astrocytes, and then neurons. Exposure to 100 μM FAC for 24 h increased the specific iron content of cultured neurons, astrocytes, and microglial cells by 30-, 80-, and 100-fold, respectively. All cell types accumulated iron against the concentration gradient, resulting in intracellular iron concentrations that were several orders of magnitude higher than the extracellular iron concentrations. Accumulation of these large amounts of iron did not affect the viability of the cell cultures, indicating a high resistance to iron-mediated toxicity. These findings show that neurons, astrocytes and microglia cultured from neonatal mice all have the capacity to accumulate and safely store large quantities of iron, but that glial cells do this more efficiently than neurons. It is concluded that neurodegenerative conditions involving iron-mediated toxicity may be due to a failure of iron transport or storage mechanisms, rather than to the presence of high levels of NTBI.     

Biolistic expression of the macrophage colony stimulating factor receptor in organotypic cultures induces an inflammatory response

The receptor for macrophage colony-stimulating factor (M-CSFR; c-fms) is expressed at increased levels by microglia in Alzheimer's disease (AD) and in mouse models for AD. Increased expression of M-CSFR on cultured microglia results in a strong proinflammatory response, but the relevance of this cell culture finding to intact brain is unknown. To determine the effects of increased microglial expression of M-CSFR in a complex organotypic environment, we developed a system for biolistic transfection of microglia in hippocampal slice cultures. The promoter for the Mac-1 integrin alpha subunit CD11b is active in cells of myeloid origin. In the brain, CD11b expression is restricted to microglia. Constructs consisting of the promoter for CD11b and a c-fms cDNA or an enhanced green fluorescent protein (EGFP) cDNA were introduced into monotypic cultures of microglia, neurons, and astrocytes. Strong CD11b promoter activity was observed in microglia, whereas little activity was observed in other cell types. Biolistic transfection of organotypic hippocampal cultures with the CD11b/c-fms construct resulted in expression of the c-fms mRNA and protein that was localized to microglia. Furthermore, biolistic overexpression of M-CSFR on microglia resulted in significantly increased production by the hippocampal cultures of the proinflammatory cytokines interleukin (IL)-1alpha macrophage inflammatory protein (MIP-1alpha), and trends toward increased production of IL-6 and M-CSF. These findings demonstrate that microglial overexpression of M-CSFR in an organotypic environment induces an inflammatory response, and suggest that increased microglial expression of M-CSFR could contribute to the inflammatory response observed in AD brain.     

The role of macrophage/microglia and astrocytes in the pathogenesis of three neurologic disorders: HIV-associated dementia,Alzheimer disease,and multiple sclerosis

Macrophage/microglia (M phi) are the principal immune cells in the central nervous system (CNS) concomitant with inflammatory brain disease and play a significant role in the host defense against invading microorganisms. Astrocytes, as a significant component of the blood-brain barrier, behave as one of the immune effector cells in the CNS as well. However, both cell types may play a dual role, amplifying the effects of inflammation and mediating cellular damage as well as protecting the CNS. Interactions of the immune system, M phi, and astrocytes result in altered production of neurotoxins and neurotrophins by these cells. These effects alter the neuronal structure and function during pathogenesis of HIV-1-associated dementia (HAD), Alzheimer disease (AD), and multiple sclerosis (MS). HAD primarily involves subcortical gray matter, and both HAD and MS affect sub-cortical white matter. AD is a cortical disease. The process of M phi and astrocytes activation leading to neurotoxicity share similarities among the three diseases. Human Immunodeficiency Virus (HIV)-1-infected M phi are involved in the pathogenesis of HAD and produce toxic molecules including cytokines, chemokines, and nitric oxide (NO). In AD, M phis produce these molecules and are activated by beta-amyloid proteins and related oligopeptides. Demyelination in MS involves M phi that become lipid laden, spurred by several possible antigens. In these three diseases, cytokine/chemokine communications between M phi and astrocytes occur and are involved in the balance of protective and destructive actions by these cells. This review describes the role of M phi and astrocytes in the pathogenesis of these three progressive neurological diseases, examining both beneficent and deleterious effects in each disease.     

Growth hormone prevents human immunodeficiency virus-induced neuronal p53 expression

Growth hormone (GH) is neuroprotective, presumably through its actions on GH receptor-mediated pathways. Here, we examined the effects of GH using in vitro and in vivo assays of human immunodeficiency virus (HIV)-induced neuronal injury. Neuronal cultures were in assays of neurotoxicity induced by supernatants from HIV-1 tat-transfected monocytoid cells (Tat supernatant). GH treatment reduced neuronal death compared with untreated cultures (p < 0.001), which was blocked by a GH receptor antagonist, B2036. Tat supernatant-induced p53 expression in neurons was also reduced by GH treatment. Expression of both p53 and GH receptor were increased in brain tissue from HIV-infected persons compared with controls (p < 0.05). Mice receiving intrastriatal implants of Tat supernatant and treated with GH showed less neurobehavioral abnormalities together with reduced neuroinflammation and neuronal injury compared with untreated animals (p < 0.01). Three acquired immunodeficiency syndrome-defined patients with neurocognitive impairment were serially evaluated during daily GH treatment showing a sustained improvement in neuropsychological performance (p < 0.01). GH prevents neuronal death through its actions on neurons involving a p53-mediated pathway and also improved in vivo neurological function, indicating that GH may have a role in the treatment of HIV-induced neurodegeneration.     

Molecular and cellular mechanisms of neuronal cell death in HIV dementia

The deaths of neurons, astrocytes and endothelial cells have been described in patients with HIV (human immunodeficiency virus) dementia. HIV-1 does not infect neurons; instead, neurotoxic substances shed by infected glia and macrophages can induce a form of programmed cell death called apoptosis in neurons. These neurotoxins include the HIV-1 proteins Tat and gp120, as well as pro-inflammatory cytokines, chemokines, excitotoxins and proteases. In this article we review the evidence for apoptosis of various cell types within the brain of HIV-infected patients, and describe in vitro and in vivo experimental studies that have elucidated the mechanisms by which HIV causes apoptosis of brain cells.     

EVALUATION OF INTRACEREBRAL LESIONS IN PATIENTS WITH ACQUIRED IMMUNODEFICIENCY SYNDROME. NEUROPATHOLOGICAL FINDINGS AND EXPERIMENTAL DATA

In this paper we present the results of post-mortem examinations of the central nervous system in 61 male patients who died with Acquired Immunodeficiency Syndrome (AIDS); it includes 23 patients with reported neurological abnormalities at the time of presentation. The analysis revealed central nervous system (CNS) neoplasms (lymphoma, Kaposi's sarcoma) and a variety of inflammatory lesions (bacterial, fungal, protozoal and viral) in 32 cases. A total of 11 patients without opportunistic infections showed significant brain abnormalities characterized by microglial nodules and/or multinucleated giant cells, changes which are probably related to infection by human immunodeficiency virus (HIV). In addition, we describes results from a series of experiments designed to define the target cell population of HIV in the brain. The expression of CD4 complex--putative receptor for HIV--was investigated using short-term cultured brain cells taken from embryonic brain anlage and from different regions of fetal brain; glioma cells were also used. Cells derived from normal embryonic and fetal brain, as well as glioma cells, were examined with respect to their susceptibility to HIV. CD4 antigen expression could be demonstrated only on glioma cells of the permanent glioma line 85HG-59 comprised of cells with properties characteristic of astrocytes. Nevertheless, normal embryonic and fetal brain cells as well as glioma cells could be infected by HIV as documented by immunocytochemical methods and southern blot analysis. HIV infected brain cells showed reduced growth rate and altered growth pattern. This study emphasizes the diversity of HIV conditioned CNS impairments, suggesting that genomic variability of HIV may result in varying cell type preference of the virus. The experimental data indicate that CD4 expression in brain cells is probably not 'conditio sine qua non' for HIV susceptibility. The alterations of HIV-infected brain cells demonstrated provide further evidence for a direct involvement of HIV in the pathogenesis of AIDS-related neurological syndromes.     

Regulation of the cell adhesion molecule CD44 after nerve transection and direct trauma to the mouse brain

CD44 is a cell surface glycoprotein involved in cell adhesion during neurite outgrowth, leukocyte homing, and tumor metastasis. In the current study, we examined the regulation of this molecule 4 days after neural trauma in different forms of central and peripheral injury. Transection of the hypoglossal, vagus, or sciatic nerve led to the appearance of CD44-immunoreactivity (CD44-IR) on the surface of the affected motoneurons, their dendrites, and their axons. Fimbria fornix transection led to CD44-IR on a subpopulation of cholinergic neurons in the ipsi- and contralateral medial septum and diagonal band of Broca and colocalized with galanin-IR. Central projections of axotomized sensory neurons to the spinal cord (substantia gelatinosa, Clarke's column) also showed an increase in CD44-IR, which was abolished by spinal root transection. Nonneuronal CD44-IR was mainly restricted to sites of direct injury. In the crushed sciatic nerve, CD44-IR was found on the demyelinating Schwann cells and on infiltrating monocytes and granulocytes. Direct parasagittal transection of the cerebral cortex led to CD44-IR on resident astrocytes and on leukocytes entering the injured forebrain tissue. CD44-IR also increased on reactive retinal astrocytes and microglia after the optic nerve crush. Additional time points in the retina and hypoglossal nucleus (days 1, 2, and 14) and cerebral cortex (day 2) injury models also showed the same cell type pattern for the CD44-IR. Finally, polymerase chain reaction analysis confirmed the posttraumatic expression of CD44 mRNA and detected only the standard haematopoietic CD44 splice isoform both in direct and indirect brain injury models. Overall, the current study shows the widespread, graded appearance of CD44-IR on neurons and on nonneuronal cells, depending on the form of neural injury. Here, the ability of CD44 to bind to a variety of extracellular matrix and cell adhesion proteins and its common presence in different forms of brain pathology could suggest an important role for this cell surface glycoprotein in the neuronal, glial, and leukocyte response to trauma and in the repair of the damaged nervous system.     

Inducible nitric oxide synthase expression is selectively induced in astrocytes isolated from adult human brain

Inducible nitric oxide synthase (iNOS) expression has been shown to be differentially regulated among different cell types and species. In cultures of primary human fetal glial cells, we have shown that astrocytes rather than microglia express iNOS. In the present study, we extended these findings to primary cultures of astrocytes and microglia derived from adult human brains. Mixed cultures of adult brain tissue were stimulated with IL-1β and IFNγ, a combination known to induce iNOS maximally in human fetal cells, and the expression of iNOS was determined by immunocytochemistry. Cell types were determined by morphology as well as immunocytochemistry for GFAP (astrocytes) and CD68 (microglia). The results showed that in cultures of adult human glia, iNOS was expressed following stimulation with cytokines, and the expression was restricted to astrocytes. Astrocyte iNOS immunoreactivity was detected both in the cytosol and in a discrete paranuclear region, a pattern noted in human fetal astrocytes. These results demonstrate that the ability to express iNOS is common to both fetal and adult human astrocytes.