Luisa Minghetti

Microglia as effector cells in brain damage and repair: focus on prostanoids and nitric oxide

Microglial cells are believed to play an active role in brain inflammatory, immune and degenerative processes. Depending on the magnitude of microglial reaction, on the type of stimulus and on the concurrence of other local factors, microglia can contribute to host defence and repair, or to the establishment and maintenance of brain damage. Many of the effects of microglial cells can be ascribed to the numerous substances that these cells can synthesize and release in response to a variety of stimuli (cytokines, pro-inflammatory substances, neurotransmitters, toxins, etc.). The present article deals with two classes of compounds that activated microglial cells can produce in large amounts: prostanoids (that derive from arachidonic acid through the cyclooxygenase pathway), and nitric oxide (that is synthesized from arginine by nitric oxide synthase). Prostanoids and nitric oxide have a number of common targets, on which they may exert similar or opposite actions, and have a crucial role in the regulation of inflammation, immune responses and cell viability. Their synthesis can massively increase when the inducible isoforms of cyclooxygenase and nitric oxide synthase are expressed. The metabolic pathways of prostanoids and nitric oxide are finely tuned by the respective end-products, by cyclic AMP and by a number of exogenous factors, such as cytokines, glucocorticoids, lipocortin-1 and others. Some of these factors (e.g. transforming growth factor-beta 1, interleukin-10, lipocortin-1) may be secreted by microglial cells themselves, and act in an autocrine-paracrine way. In view of the neuroprotective role attributed to some prostaglandins and to the cytotoxicity of excessive levels of nitric oxide or its derivatives, the balance between prostanoid and nitric oxide levels may be crucial for orienting microglial reactions towards neuroprotection or neurotoxicity.

Role of inflammation in neurodegenerative diseases

Purpose of reviewInflammation is a self-defensive reaction aimed at eliminating or neutralizing injurious stimuli, and restoring tissue integrity. In neurodegenerative diseases inflammation occurs as a local response driven by microglia, in the absence of leucocyte infiltration. Like peripheral inflammation, neuroinflammation may become a harmful process, and it is now widely accepted that it may contribute to the pathogenesis of many central nervous system disorders, including chronic neurodegenerative diseases. This review addresses some of the most recent advances in our understanding of neuroinflammation. Recent findingsThe presence of activated microglia surrounding amyloid plaques and increased levels of complement elements, cytokines, chemokines and free radicals support the existence of a self-propagating toxic cycle and provide a rationale for anti-inflammatory approaches to prevent or delay neurodegeneration. Nonetheless, recent studies have provided evidence that chronic stimulation leads microglia to acquire an anti-inflammatory phenotype, characterized by activated morphology and induction of neuroprotective and immunoregulatory molecules. The causes and consequences of this atypical phenotype have just begun to be unravelled. SummaryAlthough significant advances have been made in our knowledge of degenerative diseases, there remains controversy regarding whether neuroinflammation and microglial activation are beneficial or detrimental. Strategies aimed at both preventing and boosting microglial activation are presently under investigation, and these studies might reveal new potentially effective treatments for these neurological disorders.

Role of the peroxisome proliferator-activated receptor-γ (PPAR-γ) and its natural ligand 15-deoxy-Δ12,14-prostaglandin J2 in the regulation of microglial functions

The peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) is a member of a large group of nuclear receptors controlling the proliferation of peroxisomes that is involved in the downregulation of macrophage functions. Here, we report that PPAR‐γ was constitutively expressed in rat primary microglial cultures and that such expression was downregulated during microglial activation by endotoxin (LPS). The presence of the PPAR‐γ natural ligand 15‐deoxy‐Δ12,14‐prostaglandin J2 (15d‐PGJ2) counteracted the repression of PPAR‐γ expression caused by LPS. In microglial cultures stimulated by LPS, interferon‐γ (IFN‐γ) or by their combination, 15d‐PGJ2 reduced the production of nitric oxide (NO) and the expression of inducible NO synthase (iNOS). The inhibitory effect was dose‐dependent and did not involve an elevation of cyclic AMP, a second messenger known to inhibit NOS expression in microglia. In addition, 15d‐PGJ2 down‐regulated other microglial functions, such as tumour necrosis factor‐α (TNF‐α) synthesis and major histocompatibility complex class II (MHC class II) expression. The effects of 15d‐PGJ2 occurred, at least in part, through the repression of two important transcription factors, the signal transducer and activator of transcription 1 and the nuclear factor κB, known to mediate IFN‐γ and LPS cell signalling. Our observations suggest that 15d‐PGJ2, the synthesis of which is likely to occur within the brain, could play an important role in preventing brain damage associated with excessive microglial activation.

Activation of α7 nicotinic acetylcholine receptor by nicotine selectively up-regulates cyclooxygenase-2 and prostaglandin E2 in rat microglial cultures

BackgroundNicotinic acetylcholine (Ach) receptors are ligand-gated pentameric ion channels whose main function is to transmit signals for the neurotransmitter Ach in peripheral and central nervous system. However, the α7 nicotinic receptor has been recently found in several non-neuronal cells and described as an important regulator of cellular function. Nicotine and ACh have been recently reported to inhibit tumor necrosis factor-α (TNF-α) production in human macrophages as well as in mouse microglial cultures. In the present study, we investigated whether the stimulation of α7 nicotinic receptor by the specific agonist nicotine could affect the functional state of activated microglia by promoting and/or inhibiting the release of other important pro-inflammatory and lipid mediator such as prostaglandin E2.MethodsExpression of α7 nicotinic receptor in rat microglial cell was examined by RT-PCR, immunofluorescence staining and Western blot. The functional effects of α7 receptor activation were analyzed in resting or lipopolysaccharide (LPS) stimulated microglial cells pre-treated with nicotine. Culture media were assayed for the levels of tumor necrosis factor, interleukin-1β, nitric oxide, interleukin-10 and prostaglandin E2. Total RNA was assayed by RT-PCR for the expression of COX-2 mRNA.ResultsRat microglial cells express α7 nicotinic receptor, and its activation by nicotine dose-dependently reduces the LPS-induced release of TNF-α, but has little or no effect on nitric oxide, interleukin-10 and interleukin-1β. By contrast, nicotine enhances the expression of cyclooxygenase-2 and the synthesis of one of its major products, prostaglandin E2.ConclusionsSince prostaglandin E2 modulates several macrophage and lymphocyte functions, which are instrumental for inflammatory resolution, our study further supports the existence of a brain cholinergic anti-inflammatory pathway mediated by α7 nicotinic receptor that could be potentially exploited for novel treatments of several neuropathologies in which local inflammation, sustained by activated microglia, plays a crucial role.

Induction of prostanoid biosynthesis by bacterial lipopolysaccharide and isoproterenol in rat microglial cultures.

Abstract: We have used purified microglial cultures obtained from neonatal rat cerebral cortex to investigate the ability of microglia to release prostanoids after exposure to bacterial lipopolysaccharide, a classic macrophage activator. Release of prostaglandin E2, prostaglandin D2, and thromboxane A2 was low in basal conditions and increased in a dose‐ and time‐dependent way upon lipopolysaccharide treatment (1–100 ng/ml), by a mechanism requiring de novo protein synthesis. When compared with astrocytes, microglial cells appeared to respond more effectively to lipopolysaccharide, being able to release prostanoids after exposure to a 100‐fold lower concentration of lipopolysaccharide. In addition to prostanoids, we also measured the release of leukotriene B4; although lipopolysaccharide failed to stimulate leukotriene B4 release by microglial cells, it doubled the basal production in astrocytes. Lipopolysaccharide enhanced the release of preloaded [3H]arachidonic acid from microglial membrane phospholipids by a mechanism inhibited by the protein synthesis inhibitor cycloheximide, which suggests that the increased availability of arachidonic acid contributed to the enhanced prostanoid production. Lipopolysaccharide, however, also stimulated prostanoid synthesis by inducing cyclooxygenase activity, as shown by determining the activity of newly synthesized enzyme after inactivating the endogenous enzyme with aspirin and by assessing the level of the inducible form of cyclooxygenase by western blot analysis. Among the mechanisms potentially involved in the regulation of microglial prostanoid production, we studied the effect of β‐adrenergic receptor activation. The β‐agonist isoproterenol was inactive by itself but doubled the effect of lipopolysaccharide. The drug appeared to act mainly through the inducible cyclooxygenase; because it did not stimulate arachidonic acid release, it enhanced the lipopolysaccharide‐evoked prostanoid production observed after aspirin pretreatment and induced de novo synthesis of cyclooxygenase detectable by western blot analysis. We suggest that during cerebral inflammatory processes microglia can contribute to the establishment of high prostanoid levels, which can be further elevated by β‐adrenergic activation.

In vitro neuronal and glial differentiation from embryonic or adult neural precursor cells are differently affected by chronic or acute activation of microglia.

The contribution of microglia to the modulation of neurogenesis under pathological conditions is unclear. Both pro‐ and anti‐neurogenic effects have been reported, likely reflecting the complexity of microglial activation process. We previously demonstrated that prolonged (72 hr) in vitro exposure to lipopolysaccharide (LPS) endows microglia with a potentially neuroprotective phenotype, here referred as to “chronic”. In the present study we further characterized the chronic phenotype and investigated whether it might differently regulate the properties of embryonic and adult neural precursor cells (NPC) with respect to the “acute” phenotype acquired following a single (24 hr) LPS stimulation. We show that the LPS‐dependent induction of the proinflammatory cytokines interleukin (IL)‐1α, IL‐1β, IL‐6, and tumor necrosis factor (TNF)‐α was strongly reduced after chronic stimulation of microglia, as compared with acute stimulation. Conversely, the synthesis of the anti‐inflammatory cytokine IL‐10 and the immunomodulatory prostaglandin E2 (PGE2) was still elevated or further increased, after chronic LPS exposure, as revealed by real time PCR and ELISA techniques. Acutely activated microglia, or their conditioned medium, reduced NPC survival, prevented neuronal differentiation and strongly increased glial differentiation, likely through the release of proinflammatory cytokines, whereas chronically activated microglia were permissive to neuronal differentiation and cell survival, and still supported glial differentiation. Our data suggest that, in a chronically altered environment, persistently activated microglia can display protective functions that favor rather than hinder brain repair processes.

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.

PPAR-γ Agonists as Regulators of Microglial Activation and Brain Inflammation

The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) belongs to a large group of nuclear receptors controlling reproduction, metabolism, development and immune response. Upon activation by specific agonists, these receptors form dimers and translocate to the nucleus, where they act as agonist-dependent transcription factors and regulate gene expression by binding to specific promoter regions of target genes. The observation that PPAR-gamma is involved in the regulation of macrophage differentiation and activation in the peripheral organs has prompted the investigation of the functional role of PPAR-gamma in microglial cells, the main macrophage population of the CNS. The present review summarizes the several lines of evidence supporting that PPAR-gamma natural and synthetic agonists may control brain inflammation by inhibiting several functions associated to microglial activation, such as the expression of surface antigens and the synthesis of nitric oxide, prostaglandins, inflammatory cytokines and chemokines. Moreover, one of the major natural PPAR-gamma agonist, 15d-prostaglandin J(2) may contribute to the safe elimination of activated microglia by inducing apoptosis. Synthetic PPAR-gamma agonists do not entirely reproduce the range of 15d-prostaglandin J(2) effects, suggesting that PPAR-gamma independent mechanisms are also involved in the action of this prostaglandin. In addition to microglia, PPAR-gamma agonists affect functions and survival of other neural cells, including astrocytes, oligodendrocytes and neurons. Although most of the evidence comes from in vitro observations, an increasing number of studies in animal models further supports the potential therapeutic use of PPAR-gamma agonists in human brain diseases including multiple sclerosis, Parkinsons disease and Alzheimers disease.

Microglial activation in chronic neurodegenerative diseases: roles of apoptotic neurons and chronic stimulation.

In chronic neurodegenerative diseases, microglial activation is an early sign that often precedes neuronal death. Increasing evidence indicates that in these chronic pathologies activated microglia sustain a local inflammatory response. Nonetheless, the potential detrimental or protective roles of such reaction remain to date not fully understood, mainly because of the lack of direct evidence of the functional properties acquired by microglia in the course of chronic diseases. Purified microglial cultures have been extensively used to investigate microglial functions associated with activation, but they are often criticized for some experimental constrains, including the abrupt addition of activators, the limited time of stimulation, and the absence of interactions with neurons or other elements of brain parenchyma. To limit these confounding factors, we developed in vitro models in which microglial cells were repeatedly challenged with lipopolysaccharide or co-cultured with healthy, apoptotic, or necrotic neuronal cells. We found that chronic stimulation and interaction with phosphatidylserine-expressing apoptotic cells induced microglial cells to release immunoregulatory and neuroprotective agents (prostaglandin E(2), transforming growth factor-beta, and nerve growth factor), whereas the synthesis of pro-inflammatory molecules (tumor necrosis factor-alpha and nitric oxide) was inhibited. These findings suggest that signals that are relevant to chronic diseases lead to a progressive down-regulation of pro-inflammatory microglial functions and may help in understanding the atypical microglial activation that begins to be recognized in some chronic neuropathologies.

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.