Alessia Nicolini

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, 1–100 nM) and LC1‐derived N‐terminus peptide (peptide Ac2‐26, 1–100 μg ml−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 (100 nM) and peptide Ac2‐26 (100 μg ml−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.

Prolonged exposure of microglia to lipopolysaccharide modifies the intracellular signaling pathways and selectively promotes prostaglandin E2 synthesis

During inflammatory or degenerative processes microglial cells are likely to be exposed to activating agents that persist in brain parenchyma for prolonged periods. As our knowledge on microglial activation is largely based on in vitro studies in which microglial cultures are activated by a single administration of pro‐inflammatory stimuli, we investigated the effects of repeated endotoxin (LPS) challenges on microglial functional state. Primary rat microglial cultures were subjected to one, two or three consecutive LPS‐stimulation and the production of tumor necrosis factor‐α (TNF‐α), nitric oxide (NO), prostaglandin E2 (PGE2) and 15‐deoxy‐Δ12,14‐PGJ2 (15d‐PGJ2) measured. The ability of microglial cells to produce NO, TNF‐α and 15d‐PGJ2 upon the first LPS challenge rapidly declined after the second and the third stimulations, whereas PGE2 synthesis remained constantly elevated. Accordingly, the expression of inducible NO synthase decreased whereas cyclooxygenase‐2 and microsomal PGE synthase remained up‐regulated. The signaling pathways evoked by single or multiple LPS‐stimulation were also profoundly different, when considering the activation of the transcription factors nuclear factor‐kappa B and CREB, and of the p38 MAPK. Our observations suggest that prolonged exposure to LPS, and likely other activating agents, induces in microglia a functional state clearly distinct from that triggered by acute stimulation. The progressive down‐regulation of pro‐inflammatory molecules and the sustained release of PGE2 could have important implications for the resolution of brain inflammation.

Paracetamol effectively reduces prostaglandin E2 synthesis in brain macrophages by inhibiting enzymatic activity of cyclooxygenase but not phospholipase and prostaglandin E synthase

Epidemiological studies indicate that nonsteroidal anti‐inflammatory drugs (NSAIDs) are neuroprotective, although the mechanisms underlying their beneficial effect remain largely unknown. Given their well‐known adverse effects, which of the NSAIDs is the best for neurodegenerative disease management remains a matter of debate. Paracetamol is a widely used analgesic/antipyretic drug with low peripheral adverse effects, possibly related to its weak activity as inhibitor of peripheral cyclooxygenase (COX), the main target of NSAIDs. As microglia play an important role in CNS inflammation and pathogenesis of neurodegenerative diseases, we investigate the effect of paracetamol on rat microglial cultures. Although less potent than other NSAIDs, (indomethacin ≃ NS‐398 > flurbiprofen ≃ piroxicam > paracetamol ≃ acetylsalicylic acid), paracetamol completely inhibited the synthesis of prostaglandin E2 (PGE2) in lipopolysaccharide‐stimulated microglia, when used at concentrations comparable to therapeutic doses. The drug did not affect the expression of the enzymes involved in PGE2 synthesis, i.e., COX‐1, COX‐2, and microsomal PGE synthase, or the release of the precursor arachidonic acid (AA). Paracetamol inhibited the conversion of exogenous AA, but not PGH2, into PGE2 indicating that the target of the drug is COX activity. Consistently, paracetamol inhibited with similar IC50 the synthesis of PGF2α and thromboxane B2, two other COX metabolites. Finally, none of the NSAIDs affected the productions of nitric oxide and tumor necrosis factorα, two inflammatory mediators released by activated microglia. As paracetamol was reported to inhibit PG synthesis in peripheral macrophages with an IC50 at least three orders of magnitude higher than in microglia, we suggest that this drug represents a good tool for treating brain inflammation without compromising peripheral PG synthesis.

Effects of phosphatidylserine on p38 mitogen activated protein kinase, cyclic AMP responding element binding protein and nuclear factor‐κB activation in resting and activated microglial cells

In the last few years, the interaction between phosphatidylserine (PS), a phospholipid that becomes permanently exposed on the external cell surface in the early phases of apoptosis, and its specific receptor (PtdSerR) has emerged as a crucial event for the engulfing of apoptotic cells and for preventing the acquisition of pro‐inflammatory functions by peripheral macrophages. Recently, we demonstrated that PtdSerR is expressed in microglial cultures purified from neonatal rat brain, and that PS‐liposomes, used to mimic apoptotic cells, strongly reduce the lipopolysaccharide (LPS)‐induced release of inflammatory mediators. Here, we show that in resting microglia, PS‐liposomes induce cyclic AMP responding element binding protein (CREB) phosphorylation but do not activate nuclear factor‐κB (NF‐κB) and p38 mitogen‐activated protein kinase (p38), in line with the non‐inflammatory consequences of the recognition and removal of apoptotic cells by macrophages. In LPS‐activated microglia, PS‐liposomes did not affect NF‐κB activation but inhibited the phosphorylation of p38 and delayed that of CREB. To our knowledge, this is the first biochemical evidence of the molecular signaling evoked by PS/PtdSerR interaction possibly related to repression of pro‐inflammatory activities in microglial cells.

Human immunodeficiency virus type‐1 Tat protein induces nuclear factor (NF)‐κB activation and oxidative stress in microglial cultures by independent mechanisms

We have extended our previous findings and shown that human immunodeficiency virus Tat protein, in addition to nitric oxide (NO), stimulated rat microglial cultures to release pro‐inflammatory cytokine interleukin‐1β and tumour necrosis factor‐α in a nuclear factor (NF)‐κB‐dependent manner. At the same time, Tat stimulated the accumulation of free radicals, as indicated by the increased levels of isoprostane 8‐epi‐prostaglandin F2α (8‐epi‐PGF2α), a reliable marker of lipid peroxidation and oxidative stress, by a mechanism unrelated to NF‐κB activation. The presence of free radical scavengers abrogated Tat‐induced 8‐epi‐PGF2α accumulation without affecting NO and cytokine production. Consistently, Tat‐induced IκBα degradation – an index of NF‐κB activation – was not affected by free radical scavengers, but was prevented by an NF‐κB‐specific inhibitor. Our observations indicate that NF‐κB plays a key role in Tat‐dependent microglial activation, and that oxidative stress and NF‐κB activation induced by Tat occur by independent mechanisms.

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.