Microglial cell upregulation of HIV-1 expression in the chronically infected promonocytic cell line U1: the role of tumor necrosis factor-alpha.

Culture supernatants from lipopolysaccharide (LPS)-treated murine microglial cells were found to markedly induce the expression of human immunodeficiency virus (HIV)-1 in the chronically infected human promonocytic cell line U1 as detected by measurements of HIV-1 p24 antigen release into U1 culture supernatants. Antibody to tumor necrosis factor (TNF)-alpha had an inhibitory effect on the induction of virus by microglial cell supernatants. Also, treatment of microglia with pentoxifylline, an inhibitor of TNF-alpha production, resulted in suppressed amounts of TNF in the supernatants of LPS-treated microglia and in a reduced stimulatory capacity of these supernatants on HIV-1 expression in U1 cells. These findings support the concept that TNF-alpha production by glial cells plays a pathogenetic role in HIV-1-associated brain disease by promoting the expression of the virus in infected cells.     

Morphine amplifies HIV-1 expression in chronically infected promonocytes cocultured with human brain cells

Previous studies have shown that morphine promotes the replication of human immunodeficiency virus (HIV)-1 in peripheral blood mononuclear cell cocultures. In the present study, we tested the hypothesis that morphine would amplify HIV-1 expression in the chronically infected promonocytic clone U1 when cocultured with lipopolysaccharide-stimulated human fetal brain cells. Marked upregulation of HIV-1 expression was observed in these cocultures (quantified by measurement of HIV-1 p24 antigen levels in supernatants), and treatment of brain cells with morphine resulted in a bell-shaped dose-dependent enhancement of viral expression. The mechanism of morphine's amplifying effect appears to be opioid receptor-mediated and to involve enhanced production of tumor necrosis factor-α by microglial cells.     

Human microglial cell isolation from adult autopsy brain: Brain pH,regional variation,and infection with human immunodeficiency virus type 1

Microglia are the main source of productive infection by human immunodeficiency virus type 1 (HIV-1) in the central nervous system (CNS). Infection of microglia is difficult to study because nonhuman microglia are not infected by HIV-1, adult human microglia from surgically removed brain tissues are scarce, and fetal human microglial cells differ from adult cells in potentially important ways. Adult autopsies are a potential source of brain tissue for HIV-1 research, but the technique of isolating and infecting cells postmortem is not completely standardized. The authors determined optimal conditions for isolating and infecting adult microglial cells using 18 adult autopsy brain specimens from HIV-1-infected and noninfected decedents. The yield of mixed glial cells overall was on average 0.5 x 10(6) cells per gram of wet tissue. There was no correlation between the number of microglia isolated and the postmortem interval (PMI), HIV seropositivity, age, or gender. Brain pH accounted for about 41% of yield variability; a pH of less than 6.0 generally was not compatible with adequate cell recovery. The highest microglial cell yields were derived from anterior brain sectors (frontal lobe and temporal lobe) versus occipital lobe and cerebellum. A PMI of up to 25.5 h produced excellent cell yields in frontal lobe samples with high brain pH. HIV-1 infection of frontal lobe microglia was 100% successful using both CXCR4- and CCR5-tropic strains of HIV-1. With proper selection of cases and brain region, autopsy brain specimens are a dependable source of viable microglial cells to study CNS HIV-1 infection.     

HIV-1-infected and/or immune-activated macrophages regulate astrocyte CXCL8 production through IL-1beta and TNF-alpha: involvement of mitogen-activated protein kinases and protein kinase R

Monocyte infiltration is an important pathogenic event in human immunodeficiency virus type one (HIV-1) associated dementia (HAD). CXCL8 (Interleukin 8, IL-8), a CXC chemokine that elicits chemotaxis of neutrophils, has recently been found to recruit monocytes or synergistically enhance CCL2-mediated monocyte migration. In this report, we demonstrate CXCL8 levels in the cerebrospinal fluid of HAD patients are higher than HIV-1 seropositive patients without neurological impairment. The underlying mechanisms regulating CXCL8 production during disease are not completely understood. We investigated the role of HIV-1-infected and immune-competent macrophages, the principal target cell and mediator of neuronal injury in HAD, in regulating astrocyte CXCL8 production. Immune-activated and HIV-1-infected human monocyte-derived-macrophages (MDM) conditioned media (MCM) induced production of CXCL8 by human astrocytes. This CXCL8 production was dependent on MDM IL-1β and TNF-α production following viral and immune activation. CXCL8 production was reduced by inhibitors for mitogen-activated protein kinases (MAPKs), including p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinases (ERK1/2). Moreover, prolonged IL-1β or TNF-α treatment activated double-stranded RNA-activated protein kinase (PKR). Inhibition of PKR prevented elevated CXCL8 production in astrocytes. We conclude that IL-1β and TNF-α, produced from HIV-1-infected and immune-competent macrophages, are critical in astrocyte CXCL8 production. Multiple protein kinases, including p38, JNK, ERK1/2, and PKR, participate in the inflammatory response of astrocytes. These observations will help to identify effective therapeutic strategies to reduce high-levels of CXCL8-mediated CNS inflammation during HAD.     

Generation and characterization of mouse microglial cell lines

A murine cell line (MMGT1) has been established after transfection of primary microglial cell cultures with a v-myc-containing plasmid. This cell line was comparable with primary microglial cells with respect to morphology, presence of acetylated low density lipoprotein receptor, non-specific estrase, CD63, major histocompatibility complex antigens and CD11, and binding for Ricinus communis agglutinin. Primary microglia as well as MMGT1 cells were negative for glial fibrillary acidic protein. Different MMGT1 strains were obtained after subcloning, two of which resembled histocytes (F4/80 and BM-8). These cell strains, MMGT12 and 16, were able to opsonize latex beads, and could be induced by endotoxins (LPS) to secrete TNF-α, IL-1, IL-6, TGF-β, and EGF. The other subclones had intermediate (MCA519, ER-MP20) or mixed macrophage characteristics and did not react to endotoxin by an increase in TNF-α, IL-1, and TGF-β. Our newly established murine microglia lines may prove to be useful models to study inflammation and repair in the brain.     

Modulation by tumor necrosis factor-α of human astroglial cell production of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF)

Phagocyte survival and function are enhanced by GM-CSF and G-CSF. The production of both CSFs can be induced in mesenchymal cells by tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1). We have recently demonstrated that IL-1α and β induced the production of GM-CSF and G-CSF by two human astroglial cell lines. In the present study, we examined the effects TNF-α on the production of GM-CSF and G-CSF by U87MG, a human astroglial cell line that constitutively expresses GM-CSF and G-CSF, and U373MG, a second human astroglial cell line does not produce CSF. We demonstrate that U87MG can be induced to increase its production of GM-CSF and G-CSF by exposure to TNF-α while U373MG is induced to produce GM-CSF but not G-CSF. These responses, measured by accumulation of elevated levels of CSF protein and mRNA, are rapid and sensitive. The implications of these findings to the immunopathogenesis of central nervous system infections are discussed.     

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.     

Induction of human immunodeficiency virus type 1 replication in human glial cells after proinflammatory cytokines stimulation: Effect of IFNγ, IL1β, and TNFα on differentiation and chemokine production in glial cells

Although evidence for human immunodeficiency virus 1 (HIV-1) presence in the central nervous system (CNS) of infected patients is well established, the intensity of viral replication within the brain is not usually known. In vitro, human embryonic microglial cells internalized HIV-1 through a CD4-dependent pathway but were not permissive to viral replication. We observed that HIV replication was induced when CNS cell cultures were stimulated for 14 days by a combination of proinflammatory cytokines including IFNγ, IL1β, and TNFα. After long-term cytokine stimulation, morphologically differentiated glial cells appeared, in which HIV-1 tat antigen was detected after infection. Thus, variations in the stage of maturation/activation of CNS cells under inflammatory conditions probably play a major role in facilitating massive production of HIV-1. We then studied the effect of prolonged cytokine stimulation on the secretion of inflammatory mediators by glial cells. An early increased secretion of prostaglandin F2α and chemokines (RANTES>>MIP-1α>>MIP-1β) was observed, due to both microglia and astrocytes. In contrast to persistent PGF2α production, an extinction of RANTES and MIP-1β but not of MIP-1α secretion occurred during the 14 days of stimulation and was inversely correlated with the ability of glial cells to replicate HIV-1. The study of the secretory factors produced in response to a persistent inflammation could provide a better understanding of the modulation of HIV replication in glial cells. GLIA 23:304–315, 1998. © 1998 Wiley-Liss, Inc.     

HTLV-1 Infection of a Human Monocytic Cell Line and of Primary Cultures of Human Monocyte-Derived Macrophages

This report concerns the pathogenesis of the myelopathy associated with HTLV-1 infection and here we present the results of investigating the effect of HTLV-1 on cultures of human monocytes and of monocyte-derived macrophages. The monocytic cell line U937 was successfully infected by cocultivation with lethally irradiated cells carrying HTLV-1, as well as by inoculation of concentrated cell-free virus preparations. On the basis of immunoperoxidase staining, we determined that 0.3 to 0.5% of the cell population infected by cocultivation, and 0.5 to 0.7% of that treated with the cell-free virus expressed HTLV-1 antigen. U937 cells were also transfected with HTLV-1 proviral DNA. Two days after transfection, specific staining for HTLV-1 p24 antigen was evident in 4 to 5% of the cell population. Primary cultures of monocyte-derived human mocrophages could also be infected with the concentrated cell-free virus preparations and 0.8 to 1.0% of the cells expressed the viral antigen as determined by the alkaline phosphatase-anti-alkaline phosphatase immunocytochemical proceduce. The presence of TNF ß in the medium supernatant of HTLV-1-carrying cells was documented by a bioassay in which L929 cells were used. A cytotoxic effect of cell-free supernatants from virus-carrying cells could be demonstrated in the case of one CNS-derived continuous cell line. No TNF activity was detected in supernatants of U937 cells or of macrophages experimentally infected with HTLV-1.     

M-CSF production by HIV-1-infected monocytes and its intrathecal synthesis implications for neurological HIV-1-related disease

Macrophage-colony stimulating factor (M-CSF) is detectable in the cerebrospinal fluid (SF) of HIV-1-infected patients, and may be produced intrathecally by both reactive astrocytes and cells of the monocyte/macrophage (MO) lineage, microglial cells included. Since MO constitute the target cells for HIV-1 in the central nervous system (CNS), the culture conditions that induce M-CSF production by HIV-1-infected MO were studied. MO cultures infected with supernatants (SN) of HIV-1-infected peripheral blood lymphocyte (PBL) cultures produced only trace or undetectable amounts of M-CSF. Co-cultures of MO with normal PBL released high amounts of M-CSF, suggesting that viable cell-to-cell interactions are required to induce cytokine production by MO and/or PBL. M-CSF production was markedly increased in the MO co-cultured with HIV-1-infected PBL, thus implying that HIV-1 induces increased cytokine synthesis/release by MO and/or PBL only when cell membrane-associated messages are operating. Intracerebrally synthesized M-CSF by HIV-1-infected MO may play a role in promoting viral replication/spread within the CNS, and inducing brain damage by stimulating microglia proliferation, and neurotoxic factor release by these cells.     

Proton pump inhibitors exert anti-inflammatory effects and decrease human microglial and monocytic THP-1 cell neurotoxicity

To explore whether proton pump inhibitors (PPIs) possess anti-inflammatory effects on microglia, we investigated the effect of lansoprazole (LPZ) and omeprazole (OPZ) on the toxic action towards SH-SY5Y neuroblastoma cells of supernatants from human microglia and THP-1 cells stimulated by lipopolysaccharide combined with interferon-γ. In addition, we studied the effect of LPZ and OPZ on the THP-1 cell production of the pro-inflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-6 using enzyme-linked immunosorbent assays. We found that both PPIs had a protective effect on the toxicity of supernatants and that there was a synergism of this effect with S-ibuprofen (IBP), a typical non-steroidal anti-inflammatory drug (NSAID). A similar protective effect of LPZ was observed with supernatants from stimulated human microglia. We also found that both PPIs significantly reduced the TNF-α secretion from stimulated THP-1 cells in a concentration dependent manner and that there was a trend towards such reduction of IL-6. These results indicate that PPIs possess anti-inflammatory effects and can decrease human microglial and monocytic neurotoxicity. They suggest that PPIs combined with NSAIDs may be effective in the treatment of a broad spectrum of neurodegenerative diseases associated with activated microglia.     

GM-CSF and M-CSF modulate beta-chemokine and HIV-1 expression in microglia

Significant numbers of patients with acquired immunodeficiency syndrome (AIDS) develop CNS infection primarily in macrophages and microglial cells. Therefore, the regulation of human immunodeficiency virus type 1 (HIV-1) infection and activation of the brain mononuclear phagocytes subsequent to infection are important areas of investigation. In the current report, we studied the role of granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage-CSF (M-CSF) in the expression of antiviral beta-chemokines and HIV-1 p24 in cultures of primary human fetal microglia. We found that stimulation with GM-CSF or M-CSF induced macrophage inflammatory proteins (MIP-1alpha and MIP-1beta) and augmented RANTES expression, after HIV-1 infection of microglia. This was not due to the effect of GM-CSF on viral expression because GM-CSF was neither necessary nor stimulatory for viral infection, nor did GM-CSF enhance the expression of env-pseudotyped reporter viruses. Blocking GM-CSF-induced microglial proliferation by nocodazole had no effect on beta-chemokine or p24 expression. The functional significance of the GM-CSF-induced beta-chemokines was suggested by the finding that, in the presence of GM-CSF, exogenous beta-chemokines lost their anti-HIV-1 effects. We further show that although HIV-1-infected microglia produced M-CSF, they failed to produce GM-CSF. In vivo, GM-CSF expression was localized to activated astrocytes and some inflammatory cells in HIV-1 encephalitis, suggesting paracrine activation of microglia through GM-CSF. Our results demonstrate a complex interplay between CSFs, chemokines, and virus in microglial cells and may have bearing on the interpretation of data derived in vivo and in vitro.     

An alpha5beta1 integrin inhibitor attenuates glioma growth

Integrins are heterodimeric transmembrane proteins, which mediate cell–cell and cell–extracellular matrix (ECM) interaction. We show, that an inhibitor of alpha5 beta1 integrin (α5β1), JSM6427, attenuated glioma growth and decreased the density of microglia at the tumor border. 21 days after glioma cell injection into an experimental mouse model, the tumor volume was significantly smaller after treating animals for 14 days with JSM6427 as compared to controls. We could demonstrate the expression of integrin α5β1 on both microglia and glioma cells using flow cytometry. In a slice culture we could compare glioma growth in the presence and absence of microglia. Slices injected with glioma cells were treated with the integrin inhibitor JSM6427 and showed a significant reduction in tumor size as compared to control. Depleting microglial cells from the slice culture by treatment with clodronate liposomes abrogated the effect of JSM6427 on glioma invasion indicating that the presence of microglia is required. We show further, that microglial migration, and proliferation was attenuated dose-dependently by JSM6427.     

Tumor necrosis factor α-308 alleles in multiple sclerosis and optic neuritis

Tumor necrosis factor-α (TNF-α), a proinflammatory cytokine, is believed to play an important role in multiple sclerosis (MS) pathogenesis. A bi-allelic polymorphism in the TNF-α promoter region (TNFα-308), has been reported to influence levels of TNF-α production. In the present study, we investigated the TNFα-308 polymorphism in 93 patients with MS, 17 patients with optic neuritis (ON) and 95 healthy individuals using an allele-specific PCR technique. Allelic genotype was compared with TNF-α mRNA expression levels and HLA class II phenotypes. No significant difference regarding the TNFα-308 polymorphism was observed between MS patients and controls. Specifically, the less common allele, TNF2, which is associated with higher expression levels of TNF-α, was somewhat less frequent among MS patients. In fact, analysis of 19 patients homozygous for the MS associated HLA-DR-DQ haplotype HLA-Dw2 showed that this haplotype does not carry the TNF2 allele. In addition, in 47 patients, the TNF-α alleles did not correlate with expression levels measured as numbers of TNF-α expressing cells. Thus, we found no evidence for an important role of TNFα-308 polymorphism for genetic susceptibility to MS.     

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.     

Induction of immunoreactive interleukin-1β and tumor necrosis factor-α in the brains of rabies virus infected rats

Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNFα) are important cytokines in the development of brain inflammation during pathological process. During rabies virus infection, the level of these proinflammatory cytokines are enhanced in the brain. In the present study we determined the cellular localization of these two cytokines by immunocytochemistry in brains of rats infected with rabies virus, at different time-intervals of the disease (day 1, 3, 4, 5 and at final stage day 6 post-infection (p.i.)). Cellular identification of IL-1β (irIL-1β) and TNFα (irTNFα) immunopositive cells was studied using a polyclonal antibody against these cytokines and against glial fibrillary acidic protein (GFAP) to detect astrocytes and GSA-I-B4 isolectin to detect microglial cells and/or infiltrating macrophages. In brains of control and early infected rats, irIL-1β was only detected in fibers located in the hypothalamus, supraoptic and tractus optic nuclei and infundibular nucleus. From day 4 onwards until day 6 p.i., enhanced irIL-1β was found and identified either in activated ameboid and/or infiltrated macrophages (amygdala, thalamus, internal capsula, subtantia nigra, septal nuclei and around blood vessels), or in activated ramified cells (hypothalamus and periventricular nucleus, piriformis and cingulate cortex, hippocampus). IrTNFα was observed in the brains of rats at a final stage of disease (day 5 and 6 p.i.): in the hypothalamus, the amygdala, the internal capsula, the thalamus, the septal nuclei, the hippocampus, the habenular nuclei and around the blood vessels. Ir-TNFα was detected in round cells identified as ameboid microglia and/or infiltrated macrophages. A marked activation of microglial and astroglial cells was observed mainly in the hypothalamus, the thalamus and hippocampus and around the blood vessels, at day 4 p.i. and later, revealing a high central inflammatory reaction in brains of rabies virus infected rats. These results showed that IL-1β and TNFα are produced in the brain both by local microglial cells and infiltrating macrophages during rabies infection. Thus, these cytokines may play an important role in coordinating the dramatic inflammatory response associated with the rabies-encephalopathy as well as in the neural modification and alteration of brain functions.     

Expression of proinflammatory cytokines and its relationship with virus infection in the brain of macaques inoculated with macrophage‐tropic simian immunodeficiency virus

The pathogenesis of acquired immunodeficiency syndrome dementia complex (ADC) is still poorly understood. Many studies suggest that proinflammatory cytokines such as IL‐1β and TNF‐α released by microglia/macrophages or astrocytes play a role in CNS injury. A microscopic finding of a microglial nodule with multinucleated giant cells (MNGCs) is a histopathologic hallmark of ADC and named HIV encephalitis. However, in vivo expression of these cytokines in this microenvironment of HIV encephalitis is not yet clarified. One of the main reasons is complexities of brain pathology in patients who have died from terminal AIDS. In this study, we infected two macaques with macrophage‐tropic Simian immunodeficiency virus SIV239env/MERT and examined expression of TNF‐α and IL‐1β in inflammatory lesions with MNGCs and its relation to virus‐infected cells using immunohistochemistry. One macaque showed typical inflammatory lesions with MNGCs in the frontal white matter. Small microglial nodules were also detected in the basal ganglia and the spinal cord. SIVenv positive cells were detected mainly in inflammatory lesions, and seemed to be microglia/macrophages and MNGCs based on their morphology. Expression of IL‐1β and TNF‐α were detected in the inflammatory lesions with MNGCs, and these positive cells were found to be negative for SIVenv by double‐labeling immunohistochemistry or immunohistochemistry of serial sections. There were a few TNF‐α positive cells and almost no IL‐1β positive cells in the area other than inflammatory lesions. Another macaque showed scattered CD3+ cells and CD68+ cells in the perivascular regions of the white matter. SIVenv and TNF‐α was demonstrated in a few perivascular macrophages. These findings indicate that virus‐infected microglia/macrophages do not always express IL‐1β and TNF‐α, which suggests an indirect role of HIV‐1‐infected cells in cytokine‐mediated pathogenesis of ADC. Our macaque model for human ADC may be useful for better understanding of its pathogenesis.     

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.     

In vitro evidence for a dual role of tumor necrosis factor-α in human immunodeficiency virus type 1 encephalopathy

Microglial cell activation, myelin alteration, and abundant tumor necrosis factor (TNF)-α message have been observed in the brains of some human immunodeficiency virus type 1 (HIV - 1)vinfected and demented patients. We therefore used cultures of purified human microglia and oligodendrocytes derived from adult human brain to examine the role of TNF-α in HIV-1 encephalopathy. Human microglia synthesize TNF-α message and protein in vitro. When these cells were infected with HIV-1 JrFL and maintained in the presence of TNF-α antibodies, soluble TNF-α receptors, or the TNF-α inhibitor pentoxifylline, viral replication was delayed or strongly inhibited. Both human microglia and oligodendrocytes express the two TNF receptors, TNF-Rl, which has been implicated in cytotoxicity, and TNF-R2. While TNF-α may enhance HIV-1 replication in an autocrine manner, it is not toxic for microglia. In contrast, recombinant human TNF-α causes oligodendrocyte death in a dose-dependent manner. In situ detection of DNA fragmentation in some cells indicated that oligodendrocyte death may occur by apoptosis. Addition of live microglia or medium conditioned by these cells also resulted in 30 to 40% oligodendrocyte death, which was largely prevented by TNF-α inhibitors. We propose that TNF-α plays a dual role in HIV-1 encephalopathy, enhancing viral replication by activated microglia and damaging oligodendrocytes. Thus, TNF-α inhibitors may alleviate some ofthe neurological manifestations of acquired immunodeficiency syndrome.     

Expression of inducible nitric oxide synthase, interleukin-1 and caspase-1 in HIV-1 encephalitis

Inflammatory cytokines and enzymes such as IL-1 and inducible nitric oxide synthase (iNOS) may play an important role in the pathogenesis of AIDS dementia, a condition associated with infection of the CNS cells by the HIV-1. In this report, we investigated the expression of iNOS, IL-1, and caspase-1 (interleukin-1 converting enzyme) in HIV-1 encephalitis (HIVE) by immunocytochemistry and analyzed their expression with respect to HIV-1 infection and glial activation. In HIVE, all three molecules were expressed at high levels in areas of HIV-1 infection (microglial nodules with HIV-1 p24 immunoreactivity) and in areas of diffuse white matter gliosis. Expression was cell-type specific, with IL-1 and caspase-1 being expressed in macrophages and microglia, and iNOS in activated astrocytes. Multinucleated giant cells, a hallmark of virally infected cells, showed intense staining for both IL-1 and caspase-1, suggesting induction of these molecules by HIV-1. Double immunocytochemistry demonstrated a regional co-localization of astrocyte iNOS and microglial IL-1 and caspase-1. These results support the notion that autocrine and paracrine interactions between HIV-1 infected macrophages and microglia, activated microglia, and astrocytes lead to expression of proinflammatory and neurotoxic molecules. iNOS and caspase-1 may provide additional therapeutic targets for HIVE.