Elisabetta Polazzi

Inducible nitric oxide synthase expression in activated rat microglial cultures is downregulated by exogenous prostaglandin E2 and by cyclooxygenase inhibitors

Prostaglandins and nitric oxide (NO) are among the numerous substances released by activated microglial cells, the brain resident macrophages, and they mediate several important microglial functions. We have previously shown that cyclooxygenase‐2 (COX‐2) and inducible NO synthase (iNOS), the two key enzymes in prostaglandin and NO synthesis, respectively, are rapidly co‐induced in rat neonatal microglial cultures activated by bacterial endotoxin (lipopolysaccharide [LPS]) and that COX‐2 expression appears to be under the negative control of endogenous as well as exogenous NO. In this study we show that exogenous prostaglandin E2 (PGE2), which is known to increase cyclic adenosine monophosphate (cAMP) levels in microglial cells, downregulates LPS‐induced iNOS expression in a dose‐dependent manner. The involvement of cAMP in the PGE2‐dependent inhibition of iNOS is supported by several pieces of evidence. First, iNOS expression was also inhibited by agents such as isoproterenol and forskolin, which cause an elevation of cAMP levels, and by dibutyryl cAMP (dbcAMP), a cAMP stable analogue. Second, the inhibitory effect of PGE2 was mimicked by 11‐deoxy‐16,16‐dm PGE2, a selective agonist at the PGE2 receptor subtype EP2, coupled to the activation of adenylyl cyclase, but not by sulprostone, a potent agonist at receptor subtypes EP3 and EP1, associated with an inhibition of adenylyl cyclase activity and intracellular Ca2+ elevation, respectively. Third, the inhibitory effect of PGE2 on NO synthesis was blocked by SQ 22,536, a specific inhibitor of adenylyl cyclase. Interestingly, the abrogation of endogenous prostanoid production by several COX inhibitors caused a reduction of iNOS expression, suggesting a positive modulatory effect of endogenous prostanoids of iNOS expression, as opposed to the inhibitory effect of exogenous PGE2.

Interferon-γ and nitric oxide down-regulate lipopolysaccharide-induced prostanoid production in cultured rat microglial cells by inhibiting cyclooxygenase-2 expression

Abstract: We have used purified microglial cultures obtained from neonatal rat brains to study some aspects of the regulation of prostanoid production in these cells. We found that nitric oxide, which is released into the culture medium along with prostanoids when the cells are exposed to lipopolysaccharide (1–100 ng/ml), can down‐regulate prostanoid biosynthesis. Indeed, the abrogation of endogenous nitric oxide production, obtained by depleting the medium of the precursor l‐arginine or by blocking the activity of nitric oxide synthase by the specific inhibitor NG‐monomethyl‐l‐arginine, led to a remarkable increase in lipopolysaccharide‐induced prostanoid release. Moreover, the nitric oxide‐generating compound 3‐morpholinosydnonimine caused a substantial reduction of prostanoid formation, in the absence of endogenous nitric oxide, suggesting that both endogenous and exogenous nitric oxide may inhibit the induced prostanoid production. We also found that interferon‐γ potentiated the effect of lipopolysaccharide on nitrite accumulation as previously described by others and depressed the lipopolysaccharide‐evoked production of prostaglandin E2, prostaglandin D2, and thromboxane. It is interesting that the inhibitory effect of interferon‐γ on prostanoid production did not appear to depend on the potentiation of NO release, as it was present also when the endogenous synthesis of nitric oxide was abrogated. Additional experiments showed that interferon‐γ and nitric oxide act mainly by down‐regulating the lipopolysaccharide‐induced enzymatic activity and expression (analyzed by western blot) of cyclooxygenase‐2. Our data indicate that microglial prostanoid biosynthesis induced by proinflammatory stimuli, such as lipopolysaccharide, is tightly regulated by nitric oxide. Interferon‐γ appears to affect the balance between these local mediators by favoring nitric oxide production and inhibiting the prostanoid cascade and may thus contribute to the modulation of inflammation, local immune reactivity, and neuronal damage.

Up-regulation of cyclooxygenase-2 expression in cultured microglia by prostaglandin E2, cyclic AMP and non-steroidal anti-inflammatory drugs.

Cyclooxygenase‐2, the inducible isoform of cyclooxygenase, is highly expressed in microglial cells activated by bacterial lipopolysaccharide and is a major regulatory factor in the synthesis of prostanoids, such as prostaglandins, prostacyclin and thromboxanes. Since prostanoids are potent modulators of inflammation, immune responses and neurotoxicity, the regulation of their synthesis may be crucial for balancing microglial neuroprotective and neurotoxic activities. The present study shows that expression of cyclooxygenase–2 and prostanoid production in cultured rat microglia activated by lipopolysaccharide is up‐regulated by cyclic AMP (CAMP), as indicated by experiments performed in the presence of adenylyl cyclase activators, cAMP analogues and protein kinase A‐specific inhibitors. Exogenous prostaglandin E2 (PGE2), which elevates the cAMP level in microglial cells, also increased the lipopolysaccharide‐induced expression of cyclooxygenase–2 and production of thromboxane in a dose– and time‐dependent manner. The observations that the lipopolysaccharide‐induced prostanoid production was specifically increased by 11‐deoxy‐16,16‐dm PGE2, a selective agonist at the PGE2 receptor EP2 coupled to the activation of adenylyl cyclase, and that the enhancing effect of PGE2 was partially prevented by specific inhibitors of adenylyl cyclase and protein kinase A, suggest that the up‐regulation of cyclooxygenase–2 expression by PGE2 is mediated by CAMP, through a putative microglial EP2 receptor. Unexpectedly, non‐steroidal anti‐inflammatory drugs such as indomethacin and 6–methoxy naphthalene acetic acidic, which inhibit cyclooxygenase enzymatic activity and abrogate prostanoid synthesis, caused a moderate but consistent up‐regulation of cyclooxygenase–2 expression. In conclusion, while the strong up‐regulation of cyclooxygenase–2 expression by exogenous PGE2 appears to be mediated by EP2 receptors and CAMP, the limited down‐regulation caused by anti‐inflammatory drug treatments may be either due to arachidonic acid metabolites other than PGE2, or to PGE2 itself, acting through a distinct CAMP‐independent signalling pathway.

Down‐regulation of microglial cyclo‐oxygenase‐2 and inducible nitric oxide synthase expression by lipocortin 1

Activated microglial cells are believed to play an active role in most brain pathologies, during which they can contribute to host defence and repair but also to the establishment of tissue damage. These actions are largely mediated by microglial secretory products, among which are prostaglandins (PGs) and nitric oxide (NO). The anti‐inflammatory protein, lipocortin 1 (LC1) was reported to have neuroprotective action and to be induced by glucocorticoids in several brain structures, with a preferential expression in microglia. In this paper we tested whether the neuroprotective effect of LC1 could be explained by an inhibitory effect on microglial activation. We have previously shown that bacterial endotoxin (LPS) strongly stimulates PGE2 and NO production in rat primary microglial cultures, by inducing the expression of the key enzymes cyclo‐oxygenase‐2 (COX‐2) and inducible nitric oxide synthase (iNOS), respectively. Dexamethasone (DEX, 1–100u2003nM) and LC1‐derived N‐terminus peptide (peptide Ac2‐26, 1–100u2003μgu2003ml−1) dose‐dependently inhibited the production of both PGE2 and NO from LPS‐stimulated microglia. The inhibitory effects of DEX on NO and of the peptide on NO and PGE2 synthesis were partially abrogated by a specific antiserum, raised against the N‐terminus of human LC1. The peptide Ac2‐26 did not affect arachidonic acid release from control and LPS‐stimulated microglial cultures. Western blot experiments showed that the LPS‐induced expression of COX‐2 and iNOS was effectively down‐regulated by DEX (100u2003nM) and peptide Ac2‐26 (100u2003μgu2003ml−1). In conclusion, our findings support the hypothesis that LC1 may foster neuroprotection by limiting microglial activation, through autocrine and paracrine mechanisms.

Opposite regulation of prostaglandin E2 synthesis by transforming growth factor-β1 and interleukin 10 in activated microglial cultures

We have recently shown that prostaglandin E2 (PGE2) synthesis in activated microglia is tightly regulated by several substances (NO, neurotransmitters, pro-inflammatory cytokines), that might originate from intrinsic brain cells or from hematogenous cells infiltrating the brain in the course of inflammatory diseases. In view of the important immunoregulatory and neuroprotective functions recently attributed to PGE2, in the present study we extended our analysis of factors regulating PGE2 synthesis in rat microglial cultures to two anti-inflammatory and immunosuppressive cytokines, transforming growth factor beta1 (TGF-beta1) and interleukin 10 (IL-10), which share with PGE2 the ability to strongly deactivate peripheral macrophages and microglial cells. Moreover, we looked at the effect of the two cytokines on nitric oxide (NO) synthesis, another important microglial effector, whose synthesis is linked to that of PGE2 by complex feed-back mechanisms. We found that while both cytokines inhibited LPS-induced NO release, they had distinct and opposite regulatory activities on PGE2 production. In fact, while TGF-beta1 enhanced LPS-induced PGE2 synthesis, IL-10 showed an inhibitory effect. The two cytokines acted mainly by regulating the LPS-induced expression of the rate limiting enzymes of the two metabolic pathways, cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS). Moreover, TGF-beta1 counteracted the effect of the pro-inflammatory cytokine interferon-gamma, which in the same cultures has been shown to downregulate PGE2 and to upregulate NO synthesis. Although the present in vitro observations cannot be directly extrapolated to the in vivo situation, they may provide a novel clue for understanding the specific role of TGF-beta1 and IL-10 in several neurological diseases such as multiple sclerosis, in which their cerebral level was found to be elevated.

Human immunodeficiency virus type 1 Tat protein stimulates inducible nitric oxide synthase expression and nitric oxide production in microglial cultures.

In order to establish whether the neurotoxicity of the human immunodeficiency virus type 1 (HIV-1) regulatory protein Tat could be related to the production of potentially toxic substances by microglial cells, we examined the ability of recombinant HIV-1 Tat protein to stimulate the release of NO in purified rat microglial cultures. We found that the exposure of microglia to Tat led to a dose dependent expression of the inducible isoform of nitric oxide (iNOS) and NO production. The effect was remarkably enhanced by pretreatment or cotreatment with the proinflammatory cytokine interferon-gamma (IFN-gamma), but not with bacterial lipopolysaccharide (LPS). The high concentrations of Tat required (>100 ng/ml) suggested the viral protein induced transactivation of the iNOS gene, rather than acting through a receptor-mediated mechanism, that generally requires lower concentrations. Indeed, the induction of the iNOS gene by Tat was largely prevented by a specific inhibitor of the nuclear factor-kB (NF-kB), a transcription factor known to be involved in the induction of iNOS by LPS. The activation of NF-kB could largely account for the ability of Tat to induce iNOS expression and to act in synergism with IFN-gamma, which utilizes a different transduction system. On the other hand, the convergence of Tat and LPS on the same target (NF-kB) could explain the lack of synergism between these substances. We propose that the induction of iNOS in microglial cells and the consequent release of high and sustained levels of NO during HIV-1 cerebral infection may be an important step in the cascade of pathological events triggered by Tat. Furthermore, the NO-dependent damage may be exacerbated by the presence of IFN-gamma, which is likely to occur in pathological conditions characterized by glial activation and inflammatory cell infiltration.

Prostaglandin E2 synthesis is differentially affected by reactive nitrogen intermediates in cultured rat microglia and RAW 264.7 cells

We studied the effects of nitric oxide (NO) on prostanoid production, cyclooxygenase (COX‐2) expression and [3H]arachidonic acid (AA) release in RAW 264.7 macrophagic cells and rat microglial primary cultures. Inhibition of NO synthesis enhanced microglial prostanoid production without affecting that of RAW 264.7 cells. Both 3‐morpholinosydnonimine (SIN‐1), (which, by releasing NO and superoxide, leads to the formation of peroxynitrite), and S‐nitroso‐N‐acetylpenicillamine (SNAP), (which releases only NO), inhibited microglial prostanoid production, by preventing COX‐2 expression. In contrast, in RAW 264.7 cells, SIN‐1 enhanced both basal and LPS‐stimulated prostanoid production by upregulating COX‐2, while SNAP stimulated basal production and slightly inhibited the LPS‐induced production, as a cumulative result of enhanced AA release and depressed COX‐2 expression. Thus, reactive nitrogen intermediates can influence prostanoid production at distinct levels and in different way in the two cell types, and results obtained with RAW 264.7 cells can not be extrapolated to microglia.

Reorientation of prostanoid production accompanies “activation” of adult microglial cells in culture

Using morphological, immunocytochemical, and functional parameters we have previously shown that highly purified adult rat microglial cells undergo a process of “activation” when cultured in a serum‐containing medium in the absence of added proinflammatory substances or other factors (Slepko and Levi: Glia 16:241–246, 1996). Here we studied the lipopolysaccharide (LPS)‐evoked production of two prostanoids, thromboxane A2 (measured as thromboxane B2) (TXB2) and prostaglandin E2 (PGE2), as a function of microglial “activation.” LPS induced a greater time‐ and dose‐dependent release of TXB2, compared to PGE2, in the less “activated” cells. Further “activation” led to amplified synthesis of PGE2 and not of TXB2, so that the TXB2/PGE2 ratio changed from 2.2 to 0.25 between the 2nd and 4th day in culture. Western blot experiments showed that the LPS‐evoked expression of the inducible form of cyclooxygenase (COX) was markedly higher in cells exhibiting a more “activated” phenotype. The expression of the constitutive isoform of COX was low in all conditions, was slightly greater in more “activated” cells, and was not affected by LPS. Neither progression in microglial “activation” nor LPS treatment enhanced thromboxane synthase activity. We hypothesize that reorientation of prostanoid synthesis toward a major production of PGE2 in the more “activated” cells can be largely attributed to an increased inducibility of cellular COX expression, combined with the inability of thromboxane synthase to cope with the increased availability of the COX product prostaglandin H2 (PGH2), the common precursor of TXA2 and PGE2. In view of the different, and at times opposite, functional activity of TXB2 and PGE2, the described change in prostanoid production pattern may contribute to the role of “activated” microglia in inflammation and host defense. J. Neurosci. Res. 49:292–300, 1997.

POSSIBLE ROLE OF MICROGLIAL PROSTANOIDS AND FREE RADICALS IN NEUROPROTECTION AND NEURODEGENERATION

Microglial cells are important effector cells in brain inflammation and immune response. In normal brain, microglial cells show a down-regulated immunophenotype, adapted to the specialised microenvironment of the central nervous system, but they rapidly respond to a number of different insults and become activated. Microglial activation is a natural and defensive process, addressed mainly to host defence and neuroprotection. However, in some circumstances, microglial activation can be inappropriately triggered or deployed to excess, and eventually contribute to the damage or death of the surrounding cells (Kreutzberg, 1996).

Prostaglandin E2 Downregulates Inducible Nitric Oxide Synthase Expression in Microglia by Increasing cAMP Levels

Microglial cells are ontogenetically related to the cells of the mononuclear phagocyte lineage, unlike all the other cells types in the CNS, and are widely spread throughout the brain. In normal brain, resting microglia show a downregulated phenotype adapted to the specialised microenvironment of the CNS. However, they can be rapidly activated to become important effector cells in most brain pathologies. Depending on the type or intensity of the stimulus and the concurrence of other local factors, they may foster neuroprotective and repair processes as well as contribute to the establishment and/or the amplification of tissue damage1.