Purinergic signaling induces cyclooxygenase-1-dependent prostanoid synthesis in microglia: roles in the outcome of excitotoxic brain injury

Cyclooxygenases (COX) are prostanoid synthesizing enzymes constitutively expressed in the brain that contribute to excitotoxic neuronal cell death. While the neurotoxic role of COX-2 is well established and has been linked to prostaglandin E(2) synthesis, the role of COX-1 is not clearly understood. In a model of N-Methyl-D-aspartic acid (NMDA) induced excitotoxicity in the mouse cerebral cortex we found a distinctive temporal profile of COX-1 and COX-2 activation where COX-1, located in microglia, is responsible for the early phase of prostaglandin E(2) synthesis (10 minutes after NMDA), while both COX-1 and COX-2 contribute to the second phase (3-24 hours after NMDA). Microglial COX-1 is strongly activated by ATP but not excitatory neurotransmitters or the Toll-like receptor 4 ligand bacterial lipopolysaccharide. ATP induced microglial COX-1 dependent prostaglandin E(2) synthesis is dependent on P2X7 receptors, extracellular Ca(2+) and cytoplasmic phospholipase A2. NMDA receptor activation induces ATP release from cultured neurons leading to microglial P2X7 receptor activation and COX-1 dependent prostaglandin E(2) synthesis in mixed microglial-neuronal cultures. Pharmacological inhibition of COX-1 has no effect on the cortical lesion produced by NMDA, but counteracts the neuroprotection exerted by inhibition of COX-2 or observed in mice lacking the prostaglandin E(2) receptor type 1. Similarly, the neuroprotection exerted by the prostaglandin E(2) receptor type 2 agonist butaprost is not observed after COX-1 inhibition. P2X7 receptors contribute to NMDA induced prostaglandin E(2) production in vivo and blockage of P2X7 receptors reverses the neuroprotection offered by COX-2 inhibition. These findings suggest that purinergic signaling in microglia triggered by neuronal ATP modulates excitotoxic cortical lesion by regulating COX-1 dependent prostanoid production and unveil a previously unrecognized protective role of microglial COX-1 in excitotoxic brain injury.     

Microglial expression of prostaglandin EP3 receptor in excitotoxic lesions in the rat striatum

Prostaglandins (PG) are produced by the enzymatic activity of cyclooxygenase (COX). PGs and COX have been implicated in the pathophysiology of excitotoxicity and neurodegeneration in the central nervous system (CNS). The PGE2 receptor EP3 is the most abundantly expressed PGE2 receptor subtype in the brain. So far, in the innate rat brain EP3 receptors have been found exclusively in neurons. The aim of this study was to investigate whether EP3 expression in the brain changes under neurodegenerative circumstances such as an acute excitotoxic lesion. Intrastriatal injection of quinolinic acid (QUIN) resulted in a loss of EP3-positive striatal neurons, while simultaneously small glial-shaped EP3-positive cells appeared. Five days after lesioning, 63% of the glial-shaped EP3-positive cells could be identified as ED-1 expressing microglial cells. This percentage increased to 82% after 10 days, suggesting that most of the EP3-positive ED-1-negative cells on day 5 may be microglia which did not yet express ED-1. ED-1-positive microglia also expressed COX-1. These experiments show for the first time that activated microglial cells in excitotoxic lesions express in vivo the PGE2 receptor EP3 and the PGE2 synthesizing enzyme COX-1. Activation of EP3 receptor downregulates cAMP formation and may counteract the upregulation of cAMP formation via EP2 receptors, which has been linked to the anti-inflammatory effects of PGs. This change in EP3-receptor expression in microglia might participate in acute or chronic microglial activation in a variety of brain diseases such as ischemia or Alzheimer's disease (AD). Investigation of the expression of different PGE2 receptor subtypes might promote a better understanding of the pathophysiology of these diseases as well as leading to a modulation of microglial activation by a more specific interference with selective EP receptors than can be achieved by inhibiting global PG synthesis by selective or non-selective COX inhibitors.     

Interleukin-13 enhances cyclooxygenase-2 expression in activated rat brain microglia: implications for death of activated microglia

Brain inflammation has recently attracted widespread interest because it is a risk factor for the onset and progression of brain diseases. In this study, we report that cyclooxygenase-2 (COX-2) plays a key role in the resolution of brain inflammation by inducing the death of microglia. We previously reported that IL-13, an anti-inflammatory cytokine, induced the death of activated microglia. These results revealed that IL-13 significantly enhanced COX-2 expression and production of PGE(2) and 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) in LPS-treated microglia. Two other anti-inflammatory cytokines, IL-10 and TGF-beta, neither induced microglial death nor enhanced COX-2 expression or PGE(2) or 15d-PGJ(2) production. Therefore, we hypothesized that the effect of IL-13 on COX-2 expression may be linked to death of activated microglia. We found that COX-2 inhibitors (celecoxib and NS398) suppressed the death of microglia induced by a combination of LPS and IL-13 and that exogenous addition of PGE(2) and 15d-PGJ(2) induced microglial death. Agonists of EP2 (butaprost) and peroxisome proliferator-activated receptor gamma (ciglitazone) mimicked the effect of PGE(2) and 15d-PGJ(2), and an EP2 antagonist (AH6809) and a peroxisome proliferator-activated receptor gamma antagonist (GW9662) suppressed microglial death induced by LPS in combination with IL-13. In addition, IL-13 potentiated LPS-induced activation of JNK, and the JNK inhibitor SP600125 suppressed the enhancement of COX-2 expression and attenuated microglial death. Taken together, these results suggest that IL-13 enhanced COX-2 expression in LPS-treated microglia through the enhancement of JNK activation. Furthermore, COX-2 products, PGE(2) and 15d-PGJ(2), caused microglial death, which terminates brain inflammation.     

Anti-inflammatory effects of kinins via microglia in the central nervous system

Kinins are important biologically active peptides that are up-regulated after lesions in both the peripheral and central (CNS) nervous systems. Microglia are immune cells in the CNS and play an important role in the defense of the neuronal parenchyma. In cultured murine microglia, bradykinin (BK) induces mobilization of intracellular Ca2+, microglial migration, and increases the release of nitric oxide and prostaglandin E2. On the other hand, BK attenuates lipopolysaccharide-activated TNF-alpha and IL-1beta release. These results suggest that BK functions as a signal in brain trauma and may have an anti-inflammatory role in the CNS.     

Bradykinin decreases nitric oxide release from microglia via inhibition of cyclic adenosine monophosphate signaling

Bradykinin (BK) is a major potent inflammatory mediator outside the central nervous system. In Alzheimer's disease, BK release and BK receptor expression in brain tissues are upregulated relatively early during the course of the disease. Hence, BK was believed to promote neuroinflammation. However, BK was recently reported to possess anti-inflammatory and neuroprotective roles. Exposure of BV2 microglial cell line to BK lead to a decrease in NO release from unstimulated cells as well as a dose-dependent attenuation, mediated by both B1 and B2 receptors, in lipopolysaccharide (LPS)-induced NO production. In this study we examined whether cyclic adenosine monophosphate (cAMP) signaling is involved in BK-mediated effect in microglial nitric oxide (NO) production. A protein kinase A (PKA) inhibitor mimicked the effects of BK, while cAMP elevating agents antagonized BK-mediated NO decrease. Moreover, BK inhibited the activation of cAMP responsive element binding protein (CREB). In addition, BK protected microglial cells from death triggered by combinations of LPS and each of the cAMP elevating agents. Finally, the addition of Gα_i protein inhibitor abrogated the effects of BK on NO release, and the expression of Gα_i protein in the plasma membrane was induced by BK. These results suggest that BK-mediated reduction in microglial NO production depends on coupling to G_i protein and also involves inhibition of cAMP-PKA-CREB signaling.     

Multifunctional effects of bradykinin on glial cells in relation to potential anti-inflammatory effects

Kinins have been reported to be produced and act at the site of injury and inflammation. Despite many reports that they are likely to initiate a particular cascade of inflammatory events, bradykinin (BK) has anti-inflammatory effects in the brain mediated by glial cells. In the present review, we have attempted to describe the complex responses and immediate reaction of glial cells to BK. Glial cells express BK receptors and induce Ca(2+)-dependent signal cascades. Among them, production of prostaglandin E(2) (PGE(2)), via B(1) receptors in primary cultured microglia, has a negative feedback effect on lipopolysaccharide (LPS)-induced release of tumor necrosis factor-alpha (TNF-alpha) via increasing intracellular cyclic adenosine monophosphate (cAMP). In addition, BK up-regulates the production of neurotrophic factors such as nerve growth factor (NGF) via B(2) receptors in astrocytes. These results suggest that BK may have anti-inflammatory and neuroprotective effects in the brain through multiple functions on glial cells. These observations may help to understand the paradox on the role of kinins in the central nervous system and may be useful for therapeutic strategy.     

PGE1 and PGE2 modify platelet function through different prostanoid receptors

There is evidence that the overall effects of prostaglandin E(2) (PGE(2)) on human platelet function are the consequence of a balance between promotory effects of PGE(2) acting at the EP3 receptor and inhibitory effects acting at the EP4 receptor, with no role for the IP receptor. Another prostaglandin that has been reported to affect platelet function is prostaglandin E(1) (PGE(1)), however the receptors that mediate its actions on platelet function have not been fully defined. Here we have used measurements of platelet aggregation and P-selectin expression induced by the thromboxane A(2) mimetic U46619 to compare the effects of PGE(1) and PGE(2) on platelet function. Their effects on vasodilator-stimulated phosphoprotein (VASP) phosphorylation, as a marker of cAMP, were also determined. We also investigated the ability of the selective prostanoid receptor antagonists CAY10441 (IP antagonist), DG-041 (EP3 antagonist) and ONO-AE3-208 (EP4 antagonist) to modify the effects of the prostaglandins on platelet function. The results obtained confirm that PGE(2) interacts with EP3 and EP4 receptors, but not IP receptors. In contrast PGE(1) interacts with EP3 and IP receptors, but not EP4 receptors. In both cases the overall effects on platelet function reflect the balance between promotory and inhibitory effects at receptors that have opposite effects on adenylate cyclase.     

Prostaglandin E2 EP1 receptors: downstream effectors of COX-2 neurotoxicity

Cyclooxygenase-2 (COX-2), a rate-limiting enzyme for prostanoid synthesis, has been implicated in the neurotoxicity resulting from hypoxia-ischemia, and its inhibition has therapeutic potential for ischemic stroke. However, COX-2 inhibitors increase the risk of cardiovascular complications. We therefore sought to identify the downstream effectors of COX-2 neurotoxicity, and found that prostaglandin E(2) EP1 receptors are essential for the neurotoxicity mediated by COX-2-derived prostaglandin E(2). EP1 receptors disrupt Ca(2+) homeostasis by impairing Na(+)-Ca(2+) exchange, a key mechanism by which neurons cope with excess Ca(2+) accumulation after an excitotoxic insult. Thus, EP1 receptors contribute to neurotoxicity by augmenting the Ca(2+) dysregulation underlying excitotoxic neuronal death. Pharmacological inhibition or gene inactivation of EP1 receptors ameliorates brain injury induced by excitotoxicity, oxygen glucose deprivation and middle cerebral artery (MCA) occlusion. An EP1 receptor inhibitor reduces brain injury when administered 6 hours after MCA occlusion, suggesting that EP1 receptor inhibition may be a viable therapeutic option in ischemic stroke.     

Indomethacin decreases EP2 prostanoid receptor expression in colon cancer cells

Nonsteroidal anti-inflammatory drugs (NSAIDs) can decrease the risk of colorectal cancer; however, it has not been established if this effect is solely through their ability to inhibit cyclooxygenase (COX). In this study the effects of indomethacin, a potent NSAID and nonselective COX inhibitor, was examined in LS174T human colon cancer cells. These cells were found to express EP2 prostanoid receptors, but not the EP1, EP3 or EP4 subtypes. Pretreatment of LS174T cells with indomethacin produced a complete inhibition of prostaglandin E(2) (PGE(2)) stimulated cyclic AMP (cAMP) formation in a dose dependent manner with an IC(50) of 21 microM. Interestingly, the inhibition of PGE(2)-stimulated cAMP formation by indomethacin was accompanied by a decrease in EP2 mRNA expression and by a decrease in the whole cell specific binding of [(3)H]PGE(2). Thus, treatment of LS174T cells with indomethacin causes a down regulation of EP2 prostanoid receptors expression that may be independent of COX inhibition.     

Prostaglandin E2 induced caspase-dependent apoptosis possibly through activation of EP2 receptors in cultured hippocampal neurons

Cyclooxygenase-2 (COX-2) induction and prostaglandin E2 elevation have been reported to occur after cerebral ischemic insult. To evaluate whether the cyclooxygenase-2 reaction product prostaglandin E2 is directly related to induction of apoptosis in neuronal cells, the effect of prostaglandin E2 on cell viability was examined in hippocampal cells. Prostaglandin E2 (5-25 microM) induced apoptosis in a dose-dependent manner 48 h after addition to the cells, which was characterized by cell shrinkage, nuclear condensation or fragmentation and attenuated by a protein synthesis inhibitor, cycloheximide. Neither 17-phenyl trinor-prostaglandin E2 (an EP1 agonist) nor sulprostone (an EP3 agonist) induced cell death, whereas butaprost (an EP2 agonist) induced apoptosis. Prostaglandin E2 increased the intracellular concentration of cAMP, and the selective EP2 agonist butaprost also induced apoptosis accompanied by increasing cAMP levels in hippocampal cells. Moreover, a cell permeable cAMP analog, dibutyryl cAMP also induced apoptosis in hippocampal cells. These findings suggest that prostaglandin E2-induced apoptosis was mediated through a mechanism involving the cAMP-dependent pathway. In addition, prostaglandin E2 activated caspase-3 activity in a dose-dependent manner and a caspase-3 inhibitor prevented the prostaglandin E2-induced apoptosis. We showed in this report that prostaglandin E2 directly induced apoptosis in hippocampal neurons. Moreover, it is likely that the direct effects of prostaglandin E2 on hippocampal neurons were mediated by activation of EP2 receptors followed by elevation of the intracellular cAMP levels.     

Protein kinase C activation reduces microglial cyclic AMP response to prostaglandin E2 by interfering with EP2 receptors

We studied the modulation by protein kinase C (PKC) of the cyclic AMP (cAMP) accumulation induced by prostaglandin (PG) E2 in rat neonatal microglial cultures. Short pretreatment of microglia with phorbol 12-myristate 13-acetate (PMA) or 4beta-phorbol 12,13-didecanoate, which activate PKC, but not with the inactive 4alpha-phorbol 12,13-didecanoate, substantially reduced cAMP accumulation induced by 1 microM PGE2. The action of PMA was dose and time dependent, and the maximal inhibition (approximately 85%) was obtained after 10-min preincubation with 100 nM PMA. The inhibitory effect of PMA was mimicked by diacylglycerol and was prevented by the PKC inhibitor calphostin C. As PMA did not affect isoproterenol- or forskolin-stimulated cAMP accumulation, we investigated whether activation of PKC decreased cAMP production by acting directly at PGE2 EP receptors. Neither sulprostone (10(-9)-10(-5) M), a potent agonist at EP3 receptors (coupled to adenylyl cyclase inhibition), nor 17-phenyl-PGE2 (10(-6)-10(-5) M), an agonist of EP1 receptors, modified cAMP accumulation induced by forskolin. On the contrary, 11-deoxy-16,16-dimethyl PGE2, which does not discriminate between EP2 and EP4 receptors, both coupled to the activation of adenylyl cyclase, and butaprost, a selective EP2 agonist, induced a dose-dependent elevation of cAMP that was largely reduced by PMA pretreatment, as in the case of PGE2. These results indicated EP2 receptors as a possible target of PKC and suggest that PKC-activating agents present in the pathological brain may prevent the cAMP-mediated microglia-deactivating function of PGE2.     

Potentiation of PGE2-mediated cAMP production during neuronal differentiation of human neuroblastoma SK-N-BE(2)C cells

The prostaglandin-evoked cAMP production was studied in human neuroblastoma SK-N-BE(2)C cells during neuronal differentiation induced by all-trans retinoic acid. The incubation with 5 microM all-trans retinoic acid for 4-6 days promoted neurite outgrowth of cells. After differentiation, prostaglandin E(2) (PGE(2))-induced cAMP production was dramatically increased, whereas forskolin- and AlF-induced cAMP productions were not changed. The increase reached maximum after 4-days of incubation with all-trans retinoic acid. The differentiation caused an increase in the maximal response and a decrease in the half-maximal effective concentration of the PGE(2)-induced cAMP production. In addition, the binding of [(3)H]PGE(2) to membrane receptors was enhanced in differentiated cells. However, the order of potency of the various prostaglandins (PGE(1) = PGE(2) > PGD(2) = PGF(2alpha) = PGI(2)) in cAMP production did not change during the differentiation, suggesting that mainly E-prostanoid (EP) receptors were involved. Butaprost, an EP(2) receptor specific agonist, increased the cAMP level in a concentration dependent manner and had a similar potentiating effect on cAMP production as PGE(2) upon differentiation. Northern blot analysis using the human cDNA probes shows that the EP(2) mRNA level was about seven times higher in differentiated cells, while the dopamine beta-hydroxylase (DBH) mRNA completely disappeared. Our results, thus, suggest that elevated gene expression of the prostanoid EP(2) receptor results in an increase in the PGE(2)-evoked cAMP production in SK-N-BE(2)C cells during neuronal differentiation.     

Mucosal prostanoid receptors and synthesis in familial adenomatous polyposis

Chronic ingestion of non-steroidal anti-inflammatory medication is reported to delay or, in part, reverse development of polyps in the colon, but the mechanism for this effect is unknown. Using mRNA and immunoglobulin probes, specific for prostanoid receptors and for prostaglandin endoperoxide synthase (COX 1 and 2), we sought to define, by in situ and in vitro techniques, changes in PGE2 receptors and synthesis in cell populations of precancerous familial adenomatous polyposis (FAP) colonic mucosa. In FAP, expression of prostanoid receptors EP3 and EP4 among colonic lamina propria mononuclear and lateral crypt epithelial cells was robust, with 53.9+/-5.3% of mononuclear cells staining EP4+. When sections of normal colonic mucosa were examined by similar techniques, prostanoid receptor EP4 was expressed on only 21.3+/-1.2% of lamina propria mononuclear cells (including CD4+ T lymphocytes), as well as on surface and lateral crypt epithelium, and this distribution was found at the mRNA level as well. When receptor expression was quantitated by densitometry, immunoreactive EP3 protein on deep basolateral (but not other) FAP crypt epithelium was enhanced 2.8-fold over normal, and the number of prostanoid receptor EP4+ mononuclear cells by 2.5-fold. On the other hand, while COX 1 expression in mononuclear cells was prominent in normal and FAP mucosa, densitometric analysis showed immunoreactive prostaglandin endoperoxide synthase levels were further increased in FAP, due to a greater than fourfold elevation of COX 2 expression among mononuclear cells and epithelia. Our data suggest enhanced cell-specific prostanoid receptor expression and increased prostanoid synthesis in precancerous FAP mucosa.     

EP₃ receptors mediate PGE₂-induced hypothalamic paraventricular nucleus excitation and sympathetic activation

Prostaglandin E(2) (PGE(2)), an important mediator of the inflammatory response, acts centrally to elicit sympathetic excitation. PGE(2) acts on at least four E-class prostanoid (EP) receptors known as EP(1), EP(2), EP(3), and EP(4). Since PGE(2) production within the brain is ubiquitous, the different functions of PGE(2) depend on the expression of these prostanoid receptors in specific brain areas. The type(s) and location(s) of the EP receptors that mediate sympathetic responses to central PGE(2) remain unknown. We examined this question using PGE(2), the relatively selective EP receptor agonists misoprostol and sulprostone, and the available selective antagonists for EP(1), EP(3), and EP(4). In urethane-anesthetized rats, intracerebroventricular (ICV) administration of PGE(2), sulprostone or misoprostol increased renal sympathetic nerve activity, blood pressure, and heart rate. These responses were significantly reduced by ICV pretreatment with the EP(3) receptor antagonist; the EP(1) and EP(4) receptor antagonists had little or no effect. ICV PGE(2) or misoprostol increased the discharge of neurons in the hypothalamic paraventricular nucleus (PVN). ICV misoprostol increased the c-Fos immunoreactivity of PVN neurons, an effect that was substantially reduced by the EP(3) receptor antagonist. Real-time PCR detected EP(3) receptor mRNA in PVN, and immunohistochemical studies revealed sparsely distributed EP(3) receptors localized in GABAergic terminals and on a few PVN neurons. Direct bilateral PVN microinjections of PGE(2) or sulprostone elicited sympathoexcitatory responses that were significantly reduced by the EP(3) receptor antagonist. These data suggest that EP(3) receptors mediate the central excitatory effects of PGE(2) on PVN neurons and sympathetic discharge.     

Interleukin-1beta differentially regulates beta2 adrenoreceptor and prostaglandin E2-mediated cAMP accumulation and chloride efflux from Calu-3 bronchial epithelial cells. Role of receptor changes, adenylyl cyclase, cyclo-oxygenase 2, and protein kinase A

Here we tested the effect of interleukin-1beta, a pro-inflammatory cytokine, on cAMP accumulation and chloride efflux in Calu-3 airway epithelial cells in response to ligands binding to adenylyl cyclase-coupled receptors such as the beta2 adrenoreceptor and EP prostanoid receptors. Interleukin-1beta significantly increased isoprenaline-induced cAMP accumulation by increasing beta2 adrenoreceptor numbers via a protein kinase A-dependent mechanism. In contrast, interleukin-1beta significantly impaired prostaglandin E2-induced cAMP accumulation by induction of cyclo-oxygenase-2, prostaglandin E2 production, and a resulting down-regulation of adenylyl cyclase. The cAMP changes were all mirrored by alterations in chloride efflux assessed using the fluorescent chloride probe N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide with interleukin-1beta increasing chloride efflux in response to isoprenaline and reducing the response to prostaglandin E2. Studies with glibenclamide confirmed that chloride efflux was via the cystic fibrosis transmembrane conductance regulator. Calu-3 expresses EP4 receptors, but not EP2, and receptor expression is reduced by interleukin-1beta. Collectively, these results provide mechanistic insight into how interleukin-1beta can differentially regulate cAMP generation and chloride efflux in response to different adenylyl cyclase-coupled ligands in the same cell. These findings have important implications for diseases involving inflammation and abnormal ion flux such as cystic fibrosis.     

Acacetin Attenuates Neuroinflammation via Regulation the Response to LPS Stimuli In Vitro and In Vivo

Under normal conditions in the brain, microglia play roles in homeostasis regulation and defense against injury. However, over-activated microglia secrete proinflammatory and cytotoxic factors that can induce progressive brain disorders, including Alzheimer's disease, Parkinson's disease and ischemia. Therefore, regulation of microglial activation contributes to the suppression of neuronal diseases via neuroinflammatory regulation. In this study, we investigated the effects of acacetin (5,7-dihydroxy-4'-methoxyflavone), which is derived from Robinia pseudoacacia, on neuroinflammation in lipopolysaccharide (LPS)-stimulated BV-2 cells and in animal models of neuroinflammation and ischemia. Acacetin significantly inhibited the release of nitric oxide (NO) and prostaglandin E(2) and the expression of inducible NO synthase and cyclooxygenase-2 in LPS-stimulated BV-2 cells. The compound also reduced proinflammatory cytokines, tumor necrosis factor-α and interleukin-1β, and inhibited the activation of nuclear factor-κB and p38 mitogen-activated protein kinase. In an LPS-induced neuroinflammation mouse model, acacetin significantly suppressed microglial activation. Moreover, acacetin reduced neuronal cell death in an animal model of ischemia. These results suggest that acacetin may act as a potential therapeutic agent for brain diseases involving neuroinflammation.     

Activation of prostaglandin E2-EP4 signaling reduces chemokine production in adipose tissue

Inflammation of adipose tissue induces metabolic derangements associated with obesity. Thus, determining ways to control or inhibit inflammation in adipose tissue is of clinical interest. The present study tested the hypothesis that in mouse adipose tissue, endogenous prostaglandin E₂ (PGE₂) negatively regulates inflammation via activation of prostaglandin E receptor 4 (EP4). PGE₂ (5–500nM) attenuated lipopolysaccharide-induced mRNA and protein expression of chemokines, including interferon-γ-inducible protein 10 and macrophage-inflammatory protein-1α in mouse adipose tissue. A selective EP4 antagonist (L161,982) reversed, and two structurally different selective EP4 agonists [CAY10580 and CAY10598] mimicked these actions of PGE₂. Adipose tissue derived from EP4-deficient mice did not display this response. These findings establish the involvement of EP4 receptors in this anti-inflammatory response. Experiments performed on adipose tissue from high-fat-fed mice demonstrated EP4-dependent attenuation of chemokine production during diet-induced obesity. The anti-inflammatory actions of EP4 became more important on a high-fat diet, in that EP4 activation suppressed a greater variety of chemokines. Furthermore, adipose tissue and systemic inflammation was enhanced in high-fat-fed EP4-deficient mice compared with wild-type littermates, and in high-fat-fed untreated C57BL/6 mice compared with mice treated with EP4 agonist. These findings provide in vivo evidence that PGE₂-EP4 signaling limits inflammation. In conclusion, PGE₂, via activation of EP4 receptors, functions as an endogenous anti-inflammatory mediator in mouse adipose tissue, and targeting EP4 may mitigate adipose tissue inflammation.     

Apoptotic Pathways Mobilized in Microglia and Neurones as a Consequence of Chromogranin A-Induced Microglial Activation

Senile plaques of Alzheimer's brain are characterized by activated microglia and immunoreactivity for the peptide chromogranin A. We have investigated the mechanisms by which chromogranin A activates microglia, producing modulators of neuronal survival. Primary cultures of rat brain-derived microglia display a reactive phenotype within 24 h of exposure to 10 nM chromogranin A, culminating in microglial death via apoptotic mechanisms mediated by interleukin-1beta converting enzyme. The signalling cascade initiated by chromogranin A triggers nitric oxide production followed by enhanced microglial glutamate release, inhibition of which prevents microglial death. The plasma membrane carrier inhibitor aminoadipate and the type II/III metabotropic glutamate receptor antagonist (RS)-alpha-methyl-4-sulphonophenylglycine are equally protective. A significant amount of the released glutamate occurs from bafilomycin-sensitive stores, suggesting a vesicular mode of release. Inhibition of this component of release affords significant microglial protection. Conditioned medium from activated microglia kills cerebellar granule cells by inducing caspase-3-dependent neuronal apoptosis. Brain-derived neurotrophic factor is partially neuroprotective, as are ionotropic glutamate receptor antagonists, and, when combined with boiling of conditioned medium, full protection is achieved; nitric oxide synthase inhibitors are ineffective.