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Overexpression of the inducible cyclooxygenase (COX-2) and inducible NO synthase (iNOS) in activated brain macrophages (microglia) and astrocytes appears central to many neuroinflammatory conditions. 15-Deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) is a ligand for the peroxisome proliferator-activated receptor (PPAR)gamma. It has been proposed as an inhibitor of microglial activation, based on the study of iNOS down-regulation in rodent microglia. Because iNOS induction after cytokine activation remains controversial in human microglia, we examined the effect of 15d-PGJ(2) and other PPAR agonists on human microglia and astrocytes, using COX-2 induction as an index of activation. We found that PPAR alpha ligands (clofibrate and WY14643) enhanced IL-1 beta-induced COX-2 expression in human astrocytes and microglia, while inhibiting IL-1 beta plus IFN-gamma induction of iNOS in astrocytes. This is the first description of an inhibition of iNOS uncoupled from that of COX-2. 15d-PGJ(2) suppressed COX-2 induction in human astrocytes. It prevented NF-kappa B binding to the COX-2 promoter through a new pathway that is the repression of NF-kappa Bp50 induction by IL-1 beta. In contrast, 15d-PGJ(2) increased c-Jun and c-Fos DNA-binding activity in astrocytes, which may result in the activation of other inflammatory pathways. In human microglia, no effect of 15d-PGJ(2) on COX-2 and NF-kappa Bp65/p50 induction was observed. However, the entry of 15d-PGJ(2) occurred in microglia because STAT-1 and c-Jun expression was modulated. Our data suggest the existence of novel pathways mediated by 15d-PGJ(2) in human astrocytes. They also demonstrate that, unlike astrocytes and peripheral macrophages or rodent brain macrophages, human microglia are not subject to the anti-inflammatory effect of 15d-PGJ(2) in terms of COX-2 inhibition.

Bacterial DNA and synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs stimulate cells of the immune system to secrete a variety of cytokines and chemokines. This function can be carried out by microglia and astrocytes in the CNS. To evaluate the effect of CpG ODN on microglia and astrocytes, purified cells were isolated and cultured in vitro. CpG ODN rapidly up-regulated their production of IL-1beta, IL-6, IL-12, TNFalpha, MIP-1alpha and/or MIP-1beta. In vivo, systemically administered CpG ODN up-regulated the expression of mRNA encoding cytokines and chemokines in normal mouse brain. These findings suggest that CpG ODN can directly activate immune cells of the CNS.

Microglial cells and astrocytes isolated from human embryonic proencephalon were compared to monocyte-derived macrophages (MDM) for their ability to replicate human cytomegalovirus (HCMV) in vitro. A specific cytopathic effect was observed in microglial cells and astrocytes, but not in MDM. A high percentage of glial cells but a low percentage of MDM expressed immediate-early and late viral antigens. The ability of HCMV-infected microglial cells and astrocytes to release viral particles in their supernatants was significantly higher than that of infected MDM. Human microglial cells and astrocytes at an early stage of development are highly susceptible to HCMV infection.

The understanding of immune surveillance and inflammation regulation in cerebral tissue is essential in the therapy of neuroimmunological disorders. We demonstrate here that primary human glial cells were able to produce alpha- and beta-chemokines (IL-8 > growth related protein alpha (GROalpha) >> RANTES > microphage inflammatory protein (MIP)-1alpha and MIP-1beta) in parallel to PGs (PGE2 and PGF2alpha) after proinflammatory cytokine stimulation: TNF-alpha + IL-1beta induced all except RANTES, which was induced by TNF-alpha + IFN-gamma. Purified cultures of astrocytes and microglia were also induced by the same combination of cytokines, to produce all these mediators except MIP-1alpha and MIP-1beta, which were produced predominantly by astrocytes. The inhibition of PG production by indomethacin led to a 37-60% increase in RANTES, MIP-1alpha, and MIP-1beta but not in GROalpha and IL-8 secretion. In contrast, inhibition of IL-8 and GRO activities using neutralizing Abs resulted in a specific 6-fold increase in PGE2 but not in PGF2alpha production by stimulated microglial cells and astrocytes, whereas Abs to beta-chemokines had no effect. Thus, the production of PGs in human glial cells down-regulates their beta-chemokine secretion, whereas alpha-chemokine production in these cells controls PG secretion level. These data suggest that under inflammatory conditions, the intraparenchymal production of PGs could control chemotactic gradient of beta-chemokines for an appropriate effector cell recruitment or activation. Conversely, the elevated intracerebral alpha-chemokine levels could reduce PG secretion, preventing the exacerbation of inflammation and neurotoxicity.

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 IFNgamma, IL1beta, and TNFalpha. 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 F2alpha and chemokines (RANTES>>MIP-1alpha>>MIP-1beta) was observed, due to both microglia and astrocytes. In contrast to persistent PGF2alpha production, an extinction of RANTES and MIP-1beta but not of MIP-1alpha 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.

Human cytomegalovirus (HCMV) infection can result in neurological symptoms. In vitro replication of the HCMV was studied in primary cultures of microglial cells from the central nervous systems (CNS) of human embryos. The microglial cells were infected with various amounts of either the AD169 laboratory HCMV strain or a clinical HCMV isolate. A specific cytopathic effect occurred within 24 h and persisted for two months. Immunocytochemical tests for immediate early and late viral antigens done one and three days after the infection demonstrated that 60% to 80% of the microglial cells were infected and that 3% to 8% were the site of viral DNA replication. Kinetic studies showed accumulation of viral particles in the supernatant during the first two weeks after the infection. Prestimulation of the cells by PMA 24 h before the infection was associated with increased release of viral particles and with an increased percentage of cells expressing late viral antigens. The microglial cells of the human embryonic CNS are fully permissive targets for the HCMV. The in vitro HCMV model used in this study may prove useful for investigating the pathophysiology of HCMV encephalitis, in particular after mother-to-fetus transmission of the virus.

Secretion of the eicosanoids, nitric oxide (NO.) and superoxide anion (O2.-) was evaluated in human embryonic astrocytes and microglia. An inducible form of cyclo-oxygenase (COX 2) was demonstrated in astrocytes and microglia after IL-1 beta plus IFN-gamma stimulation; since 1) large amounts of PGF2 alpha were released; 2) PGF2 alpha secretion required protein synthesis and was blocked by indomethacin; and 3) the response was delayed and persistent. Using the same inducers, astrocytes, but not microglial cells, produced NO. and had an inducible form of nitric oxide synthase. Conversely, microglial cells were induced by IL-1 beta and IFN-gamma to generate superoxide anions (O2.-) through an NADPH oxidase-dependent pathway. We then investigated interactions between these different pathways of synthesis by inhibition experiments. The cytokine-induced production of PGF2 alpha in astrocytes was not affected by exposure to N-omega-monomethyl-L-arginine, which inhibits NO. production, whereas it was reduced by 40% in microglia. Since microglia did not secrete any detectable NO. in their supernatant, intracellular production of NO. could occur in these cells that positively regulated PGF2 alpha production. Exposure to indomethacin, which prevented PGF2 alpha production in both astrocytes and microglia, resulted in a 64% increase in cytokine-induced NO. production by astrocytes and a 70% inhibition of O2.- generation by stimulated microglia. Finally, superoxide dismutase depletion of O2.- in astrocytes and microglia had no effect on PGF2 alpha production in these cells. These results demonstrate that there are important interactions between the pathways of synthesis of inflammatory mediators in glial cells that could unveil additional regulatory mechanisms.

This study focused on the activation/differentiation of human microglial cells and astrocytes, which is a prerequisite for HIV1 replication. In vitro, IFN gamma induced a differentiation-like morphological change in embryonic microglia and astrocytes, in both primary and in purified culture. This effect was enhanced by TNF alpha which in itself had no effect. IFN gamma did not increase the low level of endogenous TNF alpha, which is not secreted by embryonic cells, but stimulated the expression of TNF alpha R1, although to a relatively low extent. IFN gamma facilitated TNF alpha response through an increase in TNF alpha R1 expression, but probably also through interaction with different intracellular signal transduction pathways.

Four continuous cell lines of human microglial cells were obtained by transfection of enriched cultures of human embryonic brain-derived macrophages with a plasmid encoding for the large T antigen of SV40. The transformed cells had the macrophagic characteristics of adherence and intra-cytoplasmic non-specific esterase activity. They could phagocytize zymosan particles but the phagocytic activity remained low. They expressed several macrophagic antigens but not the monocytic markers CD14, CD4, CD68/Ki-M6 and CD11c. The cells could be activated to express class II major histocompatibility complex antigens after interferon-gamma activation. Finally, interleukin-6 was produced spontaneously by the cells and this production was further increased after interleukin-1 alpha stimulation.