Shuxian Hu

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.

Cutting Edge: TLR2-Mediated Proinflammatory Cytokine and Chemokine Production by Microglial Cells in Response to Herpes Simplex Virus

Recent studies indicate that TLRs are critical in generating innate immune responses during infection with HSV-1. In this study, we investigated the role of TLR2 signaling in regulating the production of neuroimmune mediators by examining cytokine and chemokine expression using primary microglial cells obtained from TLR2−/− as well as wild-type mice. Data presented here demonstrate that TLR2 signaling is required for the production of proinflammatory cytokines and chemokines: TNF-α, IL-1β, IL-6, IL-12, CCL7, CCL8, CCL9, CXCL1, CXCL2, CXCL4, and CXCL5. CXCL9 and CXCL10 were also induced by HSV, but their production was not dependent upon TLR2 signaling. Because TLR2−/− mice display significantly reduced mortality and diminished neuroinflammation in response to brain infection with HSV, the TLR2-dependent cytokines identified here might function as key players influencing viral neuropathogenesis.

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.

Cytokine-mediated neuronal apoptosis

Cytokines have been reported to induce neuronal injury via the free radical nitric oxide (NO); however, the precise mechanism underlying cytokine-mediated neurotoxicity is unclear. We investigated the hypothesis that cytokine-mediated neurotoxicity in primary cultures of human fetal neurons occurs via an apoptotic mechanism triggered by NO. Treatment of mixed neuronal/glial cell cultures with interferon (IFN)-gamma plus interleukin (IL)-1 beta for 13 days induced a high output of NO accompanied by marked neuronal loss. The NO synthase inhibitor N-monomethyl-L-arginine (NMMA) significantly attenuated cytokine-induced neuronal loss, confirming the involvement of NO. Cytokine-mediated neuronal injury was accompanied by morphologic changes and a DNA fragmentation pattern consistent with apoptosis. Treatment of neuronal cell cultures with NMMA protected against cytokine-mediated apoptotic death. These findings, using primary human neuronal cell cultures, support the hypothesis that cytokine-mediated neurotoxicity involving NO proceeds via an apoptotic mechanism. These findings could lead to the development of new therapies for neurodegenerative diseases involving glia, cytokines, and NO.

Modulation of human microglial cell superoxide production by cytokines.

Reactive oxygen intermediates (e.g., superoxide [O2 ‐]) generated by microglia may play a role in host defense and injury within the central nervous system. We investigated the effect of cytokines on human microglial cell O2 ‐ production on stimulation with phorbol myristate acetate. Priming of microglial cell cultures with interferon‐γ or tumor necrosis factor‐α resulted in a dose‐ and time‐dependent enhancement of (O2 ‐ production. The priming effects of these cytokines were mediated through a protein kinase C signal transduction pathway. In contrast, astrocytes did not generate detectable O2 ‐ on phorbol myristate acetate stimulation. Treatment of microglia with transforming growth factor‐β, interleukin‐4, or interleukin‐10 suppressed in a dose‐dependent manner the priming effects of tumor necrosis factor‐α and interferon‐γ. The results of this study have implications for understanding the mechanisms by which cytokines and microglia contribute to processes of host defense and neurodegeneiration via generation of reactive oxygen intermediates. J. Leukoc. Biol. 58: 65–70; 1995.

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.

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 morphines amplifying effect appears to be opioid receptor-mediated and to involve enhanced production of tumor necrosis factor-alpha by microglial cells.