Chun C. Chao

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.

The opioid–cytokine connection

Opioids (exogenous opiates and endogenous opioid peptides) have a diversity of effects on the immune system. Although numerous studies have shown that opioid-induced immunosuppression can be mediated indirectly via the central nervous system (CNS) or through direct interactions with immunocytes, the precise cellular mechanisms underlying the immunomodulatory effects of opioids are largely unknown. In recent years, investigations from several laboratories have indicated that opioids can operate as cytokines, the principal communication signals of the immune system. All of the major properties of cytokines are shared by opioids, i.e., production by immune cells with paracrine, autocrine, and endocrine sites of action, functional redundancy, pleiotropy and effects that are both dose- and time-dependent. Studies of the effects of opioids on peripheral blood mononuclear cells (PBMC) or brain cells cocultured with HIV-infected cells suggest that some of the immunoregulatory actions of opioids are mediated by ultrahigh affinity receptors on PBMC and glial cells. Because the CNS is populated predominantly by astroglia and microglia which have properties of immune cells, it is possible that certain of the CNS effects of opioids involve cytokine-like interactions with glial cells. Although there is mounting evidence supporting the concept that opioids are members of the cytokine family, the relative contribution of the opioids to immunoregulation remains unclear. The importance of opiate addiction in the AIDS epidemic means that gaining a better understanding of the mechanisms of opioid-induced immunomodulation is of more than academic interest.

Altered cytokine release in peripheral blood mononuclear cell cultures from patients with the chronic fatigue syndrome

Chronic fatigue syndrome (CFS) is an idiopathic illness associated with a variety of immunologic abnormalities. To investigate potential pathogenetic mechanisms, we evaluated serum levels and peripheral blood mononuclear cell (PBMC) production of selected cytokines and immunoglobulins. Serum bioactive transforming growth factor beta (TGF-beta) levels were higher (P less than 0.01) in patients with CFS (290 +/- 46 pg/mL) than in control subjects (104 +/- 18 pg/mL), but levels of other cytokines tested were not different. Lipopolysaccharide-stimulated release of interleukin 1 beta (IL-1 beta), IL-6, and tumor necrosis factor-alpha was increased (P less than 0.05) in PBMC cultures from patients with CFS versus control subjects; enhanced (P less than 0.01) IL-6 release to phytohemagglutinin was also observed. In contrast, TGF-beta release in response to lipopolysaccharide was depressed (P less than 0.01) in PBMC cultures derived from patients with CFS. No differences in IL-2 and IL-4 or immunoglobulin production were observed. The enhanced release of inflammatory cytokines by stimulated PBMC from patients with CFS suggests that these cells are primed for an increased response to immune stimuli. These data also suggest an association between abnormal regulation of TGF-beta production in vivo and in vitro with the immunologic consequence of CFS.

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.

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.

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.

Stress and Pathogenesis of Infectious Disease

Abstract Despite inherent difficulties in defining and measuring stress, a scientific framework has been provided in recent years for understanding how disruptive life experiences might be translated into altered susceptibility to infectious diseases. Studies of the effects of stress on pathogenesis of infectious disease are highly relevant to assessment of the biological importance of the immune impairments that have been associated with stress. With a few notable exceptions, investigations of viral infections in humans and in animal models support the hypothesis that stress promotes the pathogenesis of such infections. Similar conclusions can be drawn from studies of bacterial infections in humans and animals and from a small number of studies of parasitic infections in rodent models. While many of these studies have substantial limitations, the data nonetheless suggest that stress is a potential cofactor in the pathogenesis of infectious disease. Given recent unprecedented advances in the neurosciences, in immunology, and in the field of microbial pathogenesis, the relationship between stress and infection should be a fruitful topic for interdisciplinary research.

A case-control study to assess possible triggers and cofactors in chronic fatigue syndrome**

PURPOSE To assess possible triggers and cofactors for chronic fatigue syndrome (CFS) and to compare levels of selected cytokines between cases and an appropriately matched control group. PATIENTS AND METHODS We conducted a case-control study of 47 cases of CFS obtained through a regional CFS research program maintained at a tertiary care medical center. One age-, gender-, and neighborhood-matched control was identified for each case through systematic community telephone sampling. Standardized questionnaires were administered to cases and controls. Sera were assayed for transforming growth factor-beta (TGF-beta), interleukin-1 beta, interleukin-6, tumor necrosis factor-alpha, and antibody to Borrelia burgdorferi and Babesia microti. RESULTS Cases were more likely to have exercised regularly before illness onset than controls (67% versus 40%; matched odds ratio (MOR) = 3.4; 95% CI = 1.2 to 11.8; P = 0.02). Female cases were more likely to be nulliparous prior to onset of CFS than controls (51% versus 31%; MOR = 8.0; 95% CI = 1.03 to 170; P = 0.05). History of other major factors, including silicone-gel breast implants (one female case and one female control), pre-morbid history of depression (15% of cases, 11% of controls) and history of allergies (66% of cases, 51% of controls) were similar for cases and controls. However, cases were more likely to have a diagnosis of depression subsequent to their diagnosis of CFS compared to a similar time frame for controls (MOR = undefined; 95% CI lower bound = 2.5; P < 0.001). Positive antibody titers to B burgdorferi (one case and one control) and B microti (zero cases and two controls) were also similar. CONCLUSIONS Further investigation into the role of prior routine exercise as a cofactor for CFS is warranted. This study supports the concurrence of CFS and depression, although pre-morbid history of depression was similar for both groups.