Wen S. Sheng

Role of microglia in central nervous system infections.

SUMMARY The nature of microglia fascinated many prominent researchers in the 19th and early 20th centuries, and in a classic treatise in 1932, Pio del Rio-Hortega formulated a number of concepts regarding the function of these resident macrophages of the brain parenchyma that remain relevant to this day. However, a renaissance of interest in microglia occurred toward the end of the 20th century, fueled by the recognition of their role in neuropathogenesis of infectious agents, such as human immunodeficiency virus type 1, and by what appears to be their participation in other neurodegenerative and neuroinflammatory disorders. During the same period, insights into the physiological and pathological properties of microglia were gained from in vivo and in vitro studies of neurotropic viruses, bacteria, fungi, parasites, and prions, which are reviewed in this article. New concepts that have emerged from these studies include the importance of cytokines and chemokines produced by activated microglia in neurodegenerative and neuroprotective processes and the elegant but astonishingly complex interactions between microglia, astrocytes, lymphocytes, and neurons that underlie these processes. It is proposed that an enhanced understanding of microglia will yield improved therapies of central nervous system infections, since such therapies are, by and large, sorely needed.

Cytokine effects on glutamate uptake by human astrocytes.

Glutamate uptake by astrocytes has been postulated to play a neuroprotective role during brain inflammation. Using primary human fetal astrocyte cultures, we investigated the influence of selected cytokines on glutamate uptake activity. Interleukin (IL)-1β and tumor necrosis factor-α dose-dependently inhibited astrocyte glutamate uptake, whereas interferon (IFN)-γ alone stimulated this activity. The nitric oxide synthase inhibitor, NG-monomethyl-L-arginine, blocked IL-1β-mediated inhibition of glutamate uptake, suggesting involvement of nitric oxide in the effect of IL-1β. IL-1 receptor antagonist protein totally reversed the inhibitory effect of cytokines, suggesting a critical role of IL-1β. The anti-inflammatory cytokine IFN-β blocked cytokine (IL-1β plus IFN-γ)-induced inhibition of glutamate uptake with a corresponding reduction in nitric oxide generation. Taken together, these findings suggest that proinflammatory cytokines inhibit astrocyte glutamate uptake by a mechanism involving nitric oxide, and that IFN-β may exert a therapeutically beneficial effect by blocking cytokine-induced nitric oxide production in inflammatory diseases of the brain.

Cytokine-stimulated astrocytes damage human neurons via a nitric oxide mechanism

Astrocytes have been reported to play a neuropathogenic role within the brain, although little is known about the mechanism underlying astrocyte‐mediated neuronal injury. We investigated the hypothesis that cytokine‐stimulated astrocytes adversely affect neuronal cell survival via generation of the free radical nitric oxide (NO). Primary human astrocytes produced substantial amounts of NO in response to interleukin (IL)‐1α or IL‐1β, which was blocked by the NO synthase inhibitor NG‐monomethyl‐L‐arginine (NMMA). IL‐1β‐induced NO production was markedly potentiated by interferon (IFN)‐γ. IL‐1 receptor agonist protein (IRAP) totally blocked NO generation by cytokine‐stimulated astrocytes. Using reverse transcription‐polymerase chain reaction and sequencing analyses of the astrocyte NO synthase gene, we found a single band encoding for a 615 bp product that was identical to the corresponding sequence reported for human hepatocytes. Treatment of human fetal brain cell cultures with IL‐1β plus IFN‐γ resulted in marked neuronal loss, as assessed by microscopic analysis and measurement of lactate dehydrogenase release. This cytokine‐induced neuronal damage was blocked by simultaneous treatment of the brain cell cultures with NMMA or IRAP, suggesting a critical role of IL‐1. These findings indicate that cytokine‐stimulated astrocytes are neurotoxic via a NO‐mediated mechanism and point to potential new therapies for neurodegenerative disorders that involve cytokines and reactive astrocytes.

Morphine induces apoptosis of human microglia and neurons

Apoptosis plays a critical role in normal brain development and in a number of neurodegenerative diseases. Recently, opiates have been shown to promote apoptotic death of cells of the immune and nervous systems. In this study, we investigated the effect of morphine on apoptosis of primary human fetal microglial cell, astrocyte and neuronal cell cultures. Exposure of microglia and neurons to 10(-6) M morphine potently induced apoptosis of these brain cells (approximately fourfold increase above untreated control cells). In contrast to microglia and neurons, astrocytes were completely resistant to morphine-induced apoptosis. Concentration-response and time-course studies indicated that neurons were more sensitive than microglia to morphines effect on apoptosis. Naloxone blocked morphine-induced apoptosis suggesting involvement of an opiate receptor mechanism. Potent inhibition (>70%) of apoptosis by an inhibitor of caspase-3 as well as co-localization of active caspase-3 and DNA fragmentation in microglia or neurons treated with morphine indicated that caspase-3 is involved in the execution phase of morphine-induced apoptosis. The results of these in vitro studies have implications regarding the potential effect of opiates on fetal brain development and on the course of certain neurodegenerative diseases.

Synthetic cannabinoid WIN55,212-2 inhibits generation of inflammatory mediators by IL-1β-stimulated human astrocytes

Activated glial cells have been implicated in the neuropathogenesis of many infectious and inflammatory diseases of the brain. A number of inflammatory mediators have been proposed to play a role in glial cell‐related brain damage; e.g., free radicals such as nitric oxide (NO), cytokines, and chemokines. Our laboratory has been interested in the effect of psychoactive drugs and their derivatives on the production of these mediators. Cannabinoids have been shown to possess immunomodulatory as well as psychoactive properties. We previously have shown that interleukin (IL)‐1β‐stimulated human astrocytes, but not microglia, produce NO. In this study, we investigated the effects of the synthetic cannabinoid WIN55,212‐2 on the production of several key inflammatory mediators by human fetal astrocytes activated by IL‐1β. Expression of the cannabinoid receptors CB1 and CB2 was detected on human astrocytes. WIN55,212‐2 (10−5 M) potently inhibited inducible NO synthase (iNOS) and corresponding NO production by IL‐1β‐stimulated astrocytes. The CB1 and CB2 receptor‐specific antagonists SR141716A and SR144528, respectively, partially blocked this suppressive effect. In addition, treatment of astrocytes with WIN55,212‐2 downregulated in a concentration‐dependent manner IL‐1β‐induced tumor necrosis factor (TNF)‐α release. Treatment with WIN55,212‐2 also inhibited production of the chemokines CXCL10, CCL2 and CCL5 by IL‐1β‐activated astrocytes. These findings indicate that WIN55,212‐2 inhibits the production of inflammatory mediators by IL‐1β‐stimulated human astrocytes and suggest that comparable agents may have therapeutic potential for the management of brain inflammation.

Robust expression of TNF-α, IL-1β, RANTES, and IP-10 by human microglial cells during nonproductive infection with herpes simplex virus

Cytokine (TNF-α/β, IL-1β, IL-6, IL-18, IL-10, and IFN-α/β/γ) and chemokine (IL-8, IP-10, MCP-1, MIP-1α/β, and RANTES) production during herpes simplex virus (HSV) 1 infection of human brain cells was examined. Primary astrocytes as well as neurons were found to support HSV replication, but neither of these fully permissive cell types produced cytokines or chemokines in response to HSV. In contrast, microglia did not support extensive viral replication; however, ICP4 was detected by immunochemical staining, demonstrating these cells were infected. Late viral protein (nucleocapsid antigen) was detected in <10% of infected microglial cells. Microglia responded to nonpermissive viral infection by producing considerable amounts of TNF-α, IL-1β, IP-10, and RANTES, together with smaller amounts of IL-6, IL-8, and MlP-1α as detected by RPA and ELISA. Surprisingly, no interferons (α,β, or γ) were detected in response to viral infection. Pretreatment of fully permissive astrocytes with TNF-α prior to infection with HSV was found to dramatically inhibit replication, resulting in a 14-fold reduction of viral titer. In contrast, pretreatment of astrocytes with IL-1β had little effect on viral replication. When added to neuronal cultures, exogenous TNF-α or IL-1β did not suppress subsequent HSV replication. Exogenously added IP-10 inhibited HSV replication in neurons (with a 32-fold reduction in viral titer), however, similar IP-10 treatment did not affect viral replication in astrocytes. These results suggest that IP-10 possesses direct antiviral activity in neurons and support a role for microglia in both antiviral defense of the brain as well as amplification of immune responses during neuroinflammation.

Inhibition of microglial cell RANTES production by IL-10 and TGF-beta.

Using human fetal microglial cell cultures, we found that the gram‐negative bacterial cell wall component lipopolysaccharide (LPS) stimulated RANTES (regulated upon activation of normal T cell expressed and secreted) production through the protein kinase C signaling pathway and that activation of transcription nuclear factor (NF)‐κB was required for this effect. Similarly, the proinflammatory cytokines interleukin (IL)‐1β and tumor necrosis factor‐α dose‐dependently stimulated microglial cell RANTES production via NF‐κB activation. Anti‐inflammatory cytokines, IL‐10, and transforming growth factor (TGF)‐β sequentially inhibited LPS‐ and cytokine‐induced microglial cell NF‐κB activation, RANTES mRNA expression, and protein release. Proinflammatory cytokines but not LPS also stimulated RANTES production by human astrocytes. These findings demonstrate that human microglia synthesize RANTES in response to proinflammatory stimuli, and that the anti‐inflammatory cytokines IL‐10 and TGF‐β down‐regulate the production of this β‐chemokine. These results may have important therapeutic implications for inflammatory diseases of the brain. J. Leukoc. Biol. 65: 815–821; 1999.

Tumor Necrosis Factor-Alpha Production by Human Fetal Microglial Cells: Regulation by Other Cytokines

Tumor necrosis factor (TNF)-alpha has been postulated to play an important physiologic as well as pathologic role within the developing brain. In the present study, we found that human fetal microglial cells released abundant amounts of TNF-alpha upon stimulation with lipopolysaccharide (LPS). Treatment of microglial cell cultures with antibodies specific to interleukin (IL)-6, IL-10, and transforming growth factor (TGF)-beta augmented LPS-stimulated release of TNF-alpha. Each of these cytokines dose-dependently suppressed TNF-alpha release. Also, TNF-alpha mRNA expression was inhibited by each of these cytokines. By way of contrast, treatment of microglial cell cultures with IL-alpha or IL-1 beta alone or in the presence of LPS, resulted in increased release of TNF-alpha, and IL-1 stimulated the expression of TNF-alpha mRNA. These findings suggest that these cytokines are likely to modify the beneficial and harmful effects of TNF-alpha within the developing brain.

Differential responses of human brain cells to West Nile virus infection

In recent years, West Nile virus (WNV) has emerged as a major cause of encephalitis in the United States. However, the neuropathogenesis of this flavivirus is poorly understood. In the present study, the authors used primary human brain cell cultures to investigate two neuropathogenic features: viral replication and induction of cytokines. Although neurons and astrocytes were found to support productive WNV infection, viral growth was poorly permissive in microglial cells. Compared to neuronal cultures that sustained viral growth for at least 2 weeks, replication peaked in astrocytes by 72 h post infection. In response to viral infection, astrocytes produced chemokines (CXCL10 and CCL5), but none of the cytokines (tumor necrosis factor [TNF]-α, interleukin [IL]-1β, IL-6, interferon α or γ) tested could be detected. Although microglial cells failed to support viral replication, WNV induced production of the proinflammatory cytokines IL-6 and TNF-α. Microglial cells also released robust amounts of the chemokines CXCL10 and CCL2, as well as lower levels of CCL5, in response to WNV infection. WNV-induced chemokine and cytokine production by microglia was coupled with activation of mitogen-activated protein kinase (MAPK) intracellular signaling pathways. Inhibition of p38 MAPK decreased chemokine production in response to WNV. Taken together, these findings suggest that microglial cell responses may influence the neuropathogenesis of WNV infection.

TNF‐α‐induced chemokine production and apoptosis in human neural precursor cells

Recent studies have shown that proinflammatory cytokines damage rodent neural precursor cells (NPCs), a source of self‐renewing, multipotent cells that play an important role in the developing as well as adult brain. In this study, the effects of tumor necrosis factor α (TNF‐α) on cytokine and chemokine production by human NPCs (>98% nestin‐ and >90% A2B5‐positive), obtained from 6‐ to 8‐week‐old fetal brain specimens, were evaluated. NPCs stimulated with this proinflammatory cytokine were found to produce abundant amounts of the chemokines monocyte chemoattractant protein 1 (MCP‐1)/CC chemokine ligand 2 (CCL2) and interferon‐inducible protein 10 (IP‐10)/CXC chemokine ligand 10 (CXCL10) in a time‐ and concentration‐dependent manner. TNF‐α treatment also induced NPC apoptosis. Receptors for TNF [TNFRI (p55) and TNFRII (p75)] mRNA were constitutively expressed on NPCs. However, only TNFRI was involved in TNF‐α‐induced chemokine production and apoptosis by NPCs, as anti‐TNFRI but not anti‐TNFRII antibodies blocked the stimulatory effect. TNF‐α treatment induced p38 mitogen‐activated protein kinase (MAPK) phosphorylation in NPCs, and SB202190, an inhibitor of p38 MAPK, blocked TNF‐α‐induced chemokine production. Thus, this study demonstrated that NPCs constitutively express receptors for TNF‐α, which when activated, trigger via a p38 MAPK signaling pathway production of two chemokines, MCP‐1/CCL2 and IP‐10/CXCL10, which are involved in infectious and inflammatory diseases of the brain.