Interleukin-1beta enhances NMDA receptor-mediated intracellular calcium increase through activation of the Src family of kinases.

Interleukin (IL)-1beta is a proinflammatory cytokine implicated in various pathophysiological conditions of the CNS involving NMDA receptor activation. Circumstantial evidence suggests that IL-1beta and NMDA receptors can functionally interact. Using primary cultures of rat hippocampal neurons, we investigated whether IL-1beta affects NMDA receptor function(s) by studying (1) NMDA receptor-induced [Ca2+]i increase and (2) NMDA-mediated neurotoxicity. IL1beta (0.01-0.1 ng/ml) dose-dependently enhances NMDA-induced [Ca2+]i increases with a maximal effect of approximately 45%. This effect occurred only when neurons were pretreated with IL-1beta, whereas it was absent if IL-1beta and NMDA were applied simultaneously, and it was abolished by IL-1 receptor antagonist (50 ng/ml). Facilitation of NMDA-induced [Ca2+]i increase by IL-1beta was prevented by both lavendustin (LAV) A (500 nm) and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) (1 microm), suggesting an involvement of tyrosine kinases. Increased tyrosine phosphorylation of NMDA receptor subunits 2A and 2B and coimmunoprecipitation of activated Src tyrosine kinase with these subunits was observed after exposure of hippocampal neurons to 0.05 ng/ml IL-1beta. Finally, 0.05 ng/ml IL-1beta increased by approximately 30% neuronal cell death induced by NMDA, and this effect was blocked by both lavendustin A and PP2. These data suggest that IL-1beta increases NMDA receptor function through activation of tyrosine kinases and subsequent NR2A/B subunit phosphorylation. These effects may contribute to glutamate-mediated neurodegeneration.

Reactive oxygen species generated by glia are responsible for neuron death induced by human immunodeficiency virus-glycoprotein 120 in vitro.

Human immunodeficiency virus infection is often followed by neurodegeneration, the cause of motor and cognitive impairment in some patients affected by acquired immunodeficiency. Several in vitro data indicate glycoprotein (gp) 120 as one of the substances responsible for the neurodegenerative event that takes place only if non-neuronal cells (glial cells) are present. Our purpose was to investigate the molecular mechanisms through which glial cells could affect neuron viability after exposure to gp120 protein. We used a sandwich co-culture of primary hippocampal neurons and primary glial cells, where the two cell populations face each other but are separable. Exposure of 1-week-old rat hippocampal neurons in co-culture with glia to 600 pM gp120 protein resulted in the death of 30% of neurons after 6 days of treatment. A significant increase of intracellular calcium ([Ca2+]i), evident 72 h after gp120 exposure (control 45.8+/-7.6 nM, gp120 176.5+/-43.6 nM), preceded neuron death. The gp120 protein affected neither the viability nor the morphology or [Ca2+]i of glial cells. However, a significant amount of reactive oxygen species as well as of interleukin-1beta was produced. Treatment of the co-culture with an antibody against interleukin-1beta prevented neuron increase of [Ca2+]i and cell death but not glial production of reactive oxygen species, whereas prior incubation of glial cells with Trolox, an antioxidant analog of vitamin E, down-regulated interleukin-1beta expression and completely prevented neuron cell death. Our results indicate that reactive oxygen species produced in glial cells by gp120 exposure cause neurodegeneration by inducing the synthesis of interleukin-1beta.

Cloricromene, a semi-synthetic coumarin derivative, inhibits tumor necrosis factor-alpha production at a pre-transcriptional level.

Cloricromene decreases myocardial infarct size after ischemic-reperfusion injury in vivo, and it has been suggested that this is due to inhibition of tumor necrosis factor-alpha (TNF-alpha). The purpose of this work was to characterize the mechanism of cloricromene-induced inhibition of TNF-alpha in rat macrophages. Cloricromene inhibited lipopolysaccharide-induced TNF-alpha release in a dose-dependent manner (IC(50)=5.9 +/- 0.8 microM). This was not due to cytotoxicity, as cloricromene was well tolerated up to 500 microM. Cloricromene inhibited lipopolysaccharide-induced expression of TNF-alpha mRNA, which suggests a pre-transcriptional effect. We then investigated the early signal transduction pathway triggered by lipopolysaccharide. The binding of lipopolysaccharide to its receptor CD14 activates protein kinase C and nuclear factor-kappaB (NF-kappaB). Cloricromene inhibited NF-kappaB activation in a dose-dependent manner, but affected protein kinase C translocation only slightly. We then established that cloricromene inhibited lipopolysaccharide-induced cellular oxidative activity, which is important for NF-kappaB activation. Our results show that cloricromene interferes with the early signal transduction pathway triggered by lipopolysaccharide.