Cannabinoids' Role in Modulating Central and Peripheral Immunity in Neurodegenerative Diseases.

Cannabinoids (the endocannabinoids, the synthetic cannabinoids, and the phytocannabinoids) are well known for their various pharmacological properties, including neuroprotective and anti-inflammatory features, which are fundamentally important for the treatment of neurodegenerative diseases. The aging of the global population is causing an increase in these diseases that require the development of effective drugs to be even more urgent. Taking into account the unavailability of effective drugs for neurodegenerative diseases, it seems appropriate to consider the role of cannabinoids in the treatment of these diseases. To our knowledge, few reviews are devoted to cannabinoids' impact on modulating central and peripheral immunity in neurodegenerative diseases. The objective of this review is to provide the best possible information about the cannabinoid receptors and immuno-modulation features, peripheral immune modulation by cannabinoids, cannabinoid-based therapies for the treatment of neurological disorders, and the future development prospects of making cannabinoids versatile tools in the pursuit of effective drugs.

Selected cannabis cultivars modulate glial activation: in vitro and in vivo studies.

INTRODUCTION: Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system characterized by neuroinflammation, demyelination and axonal loss. Cannabis, an immunomodulating agent, is known for its ability to treat MS effectively. However, due to variations in the profile of secondary metabolites, especially cannabinoids, among cannabis cultivars, the effectiveness of cannabis treatment can vary, with significant variability in the effects on different biological parameters. For screening available cultivars, cellular in vitro as well as pre-clinical in vivo assays, are required to evaluate the effectiveness of the wide range of chemical variability that exists in cannabis cultivars. This study evaluated comparatively three chemically diverse cannabis cultivars, CN2, CN4 and CN6, containing different ratios of phytocannabinoids, for their neuroinflammatory activity in MS model. MATERIALS AND METHODS: In vitro experiments were performed with lipopolysaccharide (LPS)-activated BV-2 microglia and primary glial cells to evaluate the effect of different cannabis cultivars on nitric oxide (NO) and inflammatory cytokines, as well as inducible nitric oxide synthase (iNOS) protein expression. An in vivo experiment using the experimental autoimmune encephalomyelitis (EAE) MS model was conducted using Myelin oligodendrocyte glycoprotein (MOG) as the activating peptide. The cannabis extracts of the cultivars CN2, CN4, CN6 or vehicle, were intraperitoneally injected with clinical scores given based on observed symptoms over the course of study. At the end of the experiment, the mice were sacrificed, and splenocyte cytokine secretion was measured using ELISA. Lumbar sections from the spinal cord of treated MS mice were evaluated for microglia, astrocytes and CD4+ cells. RESULTS: Extracts of the CN2 cultivar contained tetrahydrocannabinolic acid (THCA) and tetrahydrocannabinol (THC) without cannabidiol (CBD), and a number of monoterpenes. CN4 contained cannabidiolic acid (CBDA) and tetrahydrocannabidiolic acid (THCA), with significant amounts of THC: CBD in a 1:1 ratio, as well as sesquiterpenes and some monoterpenes; and CN6 contained primarily CBDA and THCA, as well as THC and CBD in a 2:1 ratio, with some sesquiterpenes and no monoterpenes. All extracts were not cytotoxic in glial cells up to 50 µg/ml. Dose dependent inhibition of LPS-induced BV2 as well as primary microglial NO secretion confirmed the anti-inflammatory and anti-oxidative activity of the three cannabis cultivars. CN2 but not CN4 reduced both astrocytosis and microglial activation in lumbar sections of EAE mice. In contrast, CN4 but not CN2 significantly decreased the secretion of TNFα and Interferon γ (IFNγ) in primary splenocytes extracted from EAE mice. CONCLUSIONS: While both cannabis cultivars, CN2 and CN4, significantly reduced the severity of the clinical signs throughout the course of the study, they modulated different inflammatory mediators and pathways, probably due to differences in their phytocannabinoid composition. This demonstrates the differential potential of cannabis cultivars differing in chemotype to regulate neuroinflammation and their potential to treat MS.

An Emerging Strategy for Neuroinflammation Treatment: Combined Cannabidiol and Angiotensin Receptor Blockers Treatments Effectively Inhibit Glial Nitric Oxide Release.

Cannabidiol (CBD), the major non-psychoactive phytocannabinoid found in cannabis, has anti-neuroinflammatory properties. Despite the increasing use of CBD, little is known about its effect in combination with other substances. Combination therapy has been gaining attention recently, aiming to produce more efficient effects. Angiotensin II activates the angiotensin 1 receptor and regulates neuroinflammation and cognition. Angiotensin receptor 1 blockers (ARBs) were shown to be neuroprotective and prevent cognitive decline. The present study aimed to elucidate the combined role of CBD and ARBs in the modulation of lipopolysaccharide (LPS)-induced glial inflammation. While LPS significantly enhanced nitric oxide synthesis vs. the control, telmisartan and CBD, when administered alone, attenuated this effect by 60% and 36%, respectively. Exposure of LPS-stimulated cells to both compounds resulted in the 95% inhibition of glial nitric oxide release (additive effect). A synergistic inhibitory effect on nitric oxide release was observed when cells were co-treated with losartan (5 µM) and CBD (5 µM) (by 80%) compared to exposure to each compound alone (by 22% and 26%, respectively). Telmisartan and CBD given alone increased TNFα levels by 60% and 40%, respectively. CBD and telmisartan, when given together, attenuated the LPS-induced increase in TNFα levels without statistical significance. LPS-induced IL-17 release was attenuated by CBD with or without telmisartan (by 75%) or telmisartan alone (by 60%). LPS-induced Interferon-γ release was attenuated by 80% when telmisartan was administered in the absence or presence of CBD. Anti-inflammatory effects were recorded when CBD was combined with the known anti-inflammatory agent dimethyl fumarate (DMF)/monomethyl fumarate (MMF). A synergistic inhibitory effect of CBD and MMF on glial release of nitric oxide (by 77%) was observed compared to cells exposed to MMF (by 35%) or CBD (by 12%) alone. Overall, this study highlights the potential of new combinations of CBD (5 µM) with losartan (5 µM) or MMF (1 µM) to synergistically attenuate glial NO synthesis. Additive effects on NO production were observed when telmisartan (5 µM) and CBD (5 µM) were administered together to glial cells.

Therapeutic Potential of Phytocannabinoid Cannabigerol for Multiple Sclerosis: Modulation of Microglial Activation In Vitro and In Vivo.

Multiple sclerosis (MS) is a widespread chronic neuroinflammatory and neurodegenerative disease. Microglia play a crucial role in the pathogenesis of MS via the release of cytokines and reactive oxygen species, e.g., nitric oxide. Research involving the role of phytocannabinoids in neuroinflammation is currently receiving much attention. Cannabigerol is a main phytocannabinoid, which has attracted significant pharmacological interest due to its non-psychotropic nature. In this research, we studied the effects of cannabigerol on microglial inflammation in vitro, followed by an in vivo study. Cannabigerol attenuated the microglial production of nitric oxide in BV2 microglia and primary glial cells; concomitant treatment of the cells with cannabigerol and telmisartan (a neuroprotective angiotensin receptor blocker) decreased nitric oxide production additively. Inducible nitric oxide synthase (iNOS) expression was also reduced by cannabigerol. Moreover, tumor necrosis factor-α (TNF-α), a major cytokine involved in MS, was significantly reduced by cannabigerol in both cell cultures. Next, we studied the effects of cannabigerol in vivo using a mice model of MS, experimental autoimmune encephalomyelitis (EAE). The clinical scores of EAE mice were attenuated upon cannabigerol treatment; additionally, lumbar sections of EAE mice showed enhanced neuronal loss (relative to control mice), which was restored by cannabigerol treatment. Altogether, the set of experiments presented in this work indicates that cannabigerol possesses an appealing therapeutic potential for the treatment of MS.

Microglial Activation Is Modulated by Captopril: in Vitro and in Vivo Studies.

The renin-angiotensin system (RAS) is an important peripheral system involved in homeostasis modulation, with angiotensin II (Ang II) serving as the main effector hormone. The main enzyme involved in Ang II formation is angiotensin-converting enzyme (ACE). ACE inhibitors (ACEIs) such as captopril (Cap) are predominantly used for the management of hypertension. All of the components of the RAS have also been identified in brain. Centrally located hormones such as Ang II can induce glial inflammation. Moreover, in Alzheimer's disease (AD) models, where glial inflammation occurs and is thought to contribute to the propagation of the disease, increased levels of Ang II and ACE have been detected. Interestingly, ACE overexpression in monocytes, migrating to the brain was shown to prevent AD cognitive decline. However, the specific effects of captopril on glial inflammation and AD remain obscure. In the present study, we investigated the effect of captopril, given at a wide concentration range, on inflammatory mediators released by lipopolysaccharide (LPS)-treated glia. In the current study, both primary glial cells and the BV2 microglial cell line were used. Captopril decreased LPS-induced nitric oxide (NO) release from primary mixed glial cells as well as regulating inducible NO synthase (iNOS) expression, NO, tumor necrosis factor-α (TNF-α) and induced interleukin-10 (IL-10) production by BV2 microglia. We further obtained data regarding intranasal effects of captopril on cortical amyloid ß (Aß) and CD11b expression in 5XFAD cortex over three different time periods. Interestingly, we noted decreases in Aß burden in captopril-treated mice over time which was paralleled by increased microglial activation. These results thus shed light on the neuroprotective role of captopril in AD which might be related to modulation of microglial activation.

Candesartan ameliorates brain inflammation associated with Alzheimer's disease.

AIMS: Alzheimer's disease (AD) pathology is associated with brain inflammation involving microglia and astrocytes. The renin-angiotensin system contributes to brain inflammation associated with AD pathology. This study aimed to investigate the role of candesartan, an angiotensin II type 1 receptor blocker, in modulation of glial functions associated with AD. METHODS: Focusing on the role of candesartan in glial inflammation, we evaluated inflammatory mediators' levels, secreted by lipopolysaccharide-induced microglia following candesartan treatment. Also, short-term intranasal candesartan effects on amyloid burden and microglial activation were investigated in 5 familial AD mice. RESULTS: Candesartan showed anti-inflammatory effects and shifted microglial activation toward a more neuroprotective phenotype. Candesartan decreased the lipopolysaccharide-induced nitric oxide synthase and cyclooxygenase-2 expression levels, which was accompanied by an induction of arginase-1 expression levels and enhanced Aß1-42 uptake by microglia. Moreover, intranasally administered candesartan to AD mice model significantly reduced the amyloid burden and microglia activation in the hippocampus. CONCLUSIONS: These results thus shed light on the neuroprotective role of candesartan in the early stage of AD, which might relate to modulation of microglial activation states.

Involvement of the Bradykinin B1 Receptor in Microglial Activation: In Vitro and In Vivo Studies.

The importance of brain inflammation to Alzheimer's disease (AD) pathogenesis has been accepted of late, with it currently being held that brain inflammation aggravates AD pathology. One important aspect of brain inflammation is the recruitment and activation of microglia, a process termed microgliosis. Kinins and bradykinin (BK), in particular, are major pro-inflammatory mediators in the periphery, although all of the factors comprising the kinin system have also been described in the brain. Moreover, it was shown that the amyloid ß (Aß) peptide (a component of AD plaques) enhances kinin secretion and activates BK receptors that can, in turn, stimulate Aß production. Still, the role of bradykinin in modulating brain inflammation and AD is not completely understood. In this study, we aimed to investigate the roles of the bradykinin B1 receptor (B1R) and bradykinin B2 receptor (B2R) in regulating microglial secretion of pro-inflammatory factors in vitro. Furthermore, the effects of intranasal administration of specific B1R and B2R antagonists on Aß burden and microglial accumulation in the brains of transgenic AD mice were studied. The data obtained show that neither R-715 (a B1R antagonist) nor HOE 140 (a B2R antagonist) altered microglial cell viability. However, R-715, but not HOE 140, markedly increased lipopolysaccharide-induced nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α) release, as well as inducible nitric oxide synthase expression in BV2 microglial cells. Neither antagonist altered NO nor TNF-α production in non-stimulated cells. We also showed that intranasal administration of R-715 but not HOE 140 to 8-week-old 5X familial AD mice enhanced amyloid burden and microglia/macrophage accumulation in the cortex. To conclude, we provide evidence supporting a role of B1R in brain inflammation and in the regulation of amyloid deposition in AD mice, possibly with microglial/macrophage involvement. Further studies are required to test whether modulation of this receptor can serve as a novel therapeutic strategy for AD.

Intranasal telmisartan ameliorates brain pathology in five familial Alzheimer's disease mice.

The renin-angiotensin system (RAS) is a major circulative system engaged in homeostasis modulation. Angiotensin II (Ang II) serves as its main effector hormone upon binding to its primary receptor, Ang II receptor type 1 (AT1R). It is well established that an intrinsic independent brain RAS exists. Abnormal AT1R activation both in the periphery and in the brain probably contributes to the development of Alzheimer's disease (AD) pathology that is characterized, among others, by brain inflammation. Moreover, treatment with drugs that block AT1R (AT1R blockers, ARBs) ameliorates most of the clinical risk factors leading to AD. Previously we showed that short period of intranasal treatment with telmisartan (a brain penetrating ARB) reduced brain inflammation and ameliorated amyloid burden (a component of Alzheimer's plaques) in AD transgenic mouse model. In the present study, we aimed to examine the long-term effect of intranasally administrated telmisartan on brain inflammation features including microglial activation, astrogliosis, neuronal loss and hippocampus-dependent cognition in five-familial AD mouse model (5XFAD). Five month of intranasal treatment with telmisartan significantly reduced amyloid burden in the cortex and hippocampus of 5XFAD mice as compared with the vehicle-treated 5XFAD group. Similar effects were also observed for CD11b staining, which is a marker for microglial accumulation. Telmisartan also significantly reduced astrogliosis and neuronal loss in the cortex of 5XFAD mice compared with the vehicle-treated group. Improved spatial acquisition of the 5XFAD mice following long-term intranasal administration of telmisartan was also observed. Taken together, our data suggest a significant role for AT1R blockage in mediating neuronal loss and cognitive behavior, possibly through regulation of amyloid burden and glial inflammation.

Angiotensin Converting Enzyme Inhibitors Ameliorate Brain Inflammation Associated with Microglial Activation: Possible Implications for Alzheimer's Disease.

Angiotensin converting enzyme (ACE) converts Angiotensin I to a potent vasoconstrictor angiotensin II (ANG II). ACE inhibitors (ACEIs) are widely used for the management of hypertension. All components of the renin-angiotensin system (RAS) have also been identified in the brain. In addition to cytokines, neuromodulators such as ANG II can induce neuroinflammation. Moreover, in Alzheimer's disease (AD) models, where neuroinflammation occurs and is thought to contribute to the propagation of the disease, increased levels of ANG II and ACE have been detected. However, the specific effect of ACEIs on neuroinflammation and AD remains obscure. The present study suggests that captopril and perindopril, centrally active ACEIs, may serve as modulators for microglial activation associated with AD. Our in vitro study investigated the effect of both ACEIs on nitric oxide (NO), tumor necrosis factor- α (TNF-α) release and inducible NO synthase (iNOS) expression in lipopolysaccharide (LPS)-induced BV2 microglia. Exposure of BV2 microglia to ACEIs significantly attenuated the LPS-induced NO and TNF-α release. In vivo, short term intranasal administration of perindopril or captopril to 5 Familial AD (5XFAD) mice significantly reduced amyloid burden and CD11b expression (a microglial marker) or only CD11b expression respectively, in the cortex of 5XFAD. Long-term intranasal administration of captopril to mice reduced amyloid burden with no effect on CD11b expression. We provide evidence that intranasal delivery of ACEI may serve as an efficient alternative for their systemic administration, as it results in the attenuation of microglial accumulation and even the reduction of Amyloid ß (Aß) plaques.

Telmisartan Modulates Glial Activation: In Vitro and In Vivo Studies.

The circulating renin-angiotensin system (RAS), including the biologically active angiotensin II, is a fundamental regulatory mechanism of blood pressure conserved through evolution. Angiotensin II components of the RAS have also been identified in the brain. In addition to pro-inflammatory cytokines, neuromodulators, such as angiotensin II can induce (through angiotensin type 1 receptor (AT1R)) some of the inflammatory actions of brain glial cells and influence brain inflammation. Moreover, in Alzheimer's disease (AD) models, where neuroinflammation occurs, increased levels of cortical AT1Rs have been shown. Still, the precise role of RAS in neuroinflammation is not completely clear. The overall aim of the present study was to elucidate the role of RAS in the modulation of glial functions and AD pathology. To reach this goal, the specific aims of the present study were a. to investigate the long term effect of telmisartan (AT1R blocker) on tumor necrosis factor-α (TNF-α), interleukin 1-ß (IL1-ß) and nitric oxide (NO) release from glial cells. b. to examine the effect of intranasally administered telmisartan on amyloid burden and microglial activation in 5X familial AD (5XFAD) mice. Telmisartan effects in vivo were compared to those of perindopril (angiotensin converting enzyme inhibitor). Long-term-exposure of BV2 microglia to telmisartan significantly decreased lipopolysaccharide (LPS) -induced NO, inducible NO synthase, TNF-α and IL1-ß synthesis. The effect of Telmisartan on NO production in BV2 cells was confirmed also in primary neonatal rat glial cells. Intranasal administration of telmisartan (1 mg/kg/day) for up to two months significantly reduced amyloid burden and CD11b expression (a marker for microglia) both in the cortex and hipoccampus of 5XFAD. Based on the current view of RAS and our data, showing reduced amyloid burden and glial activation in the brains of 5XFAD transgenic mice, one may envision potential intervention with the progression of glial activation and AD by using AT1R blockers.

Differential effect of intranasally administrated kinin B1 and B2 receptor antagonists in Alzheimer's disease mice.

An Increasing body of evidence supports a critical role of brain inflammation in the pathogenesis of Alzheimer's disease. A principal aspect of the brain immune response to inflammation is the activation of microglia. It has been shown that the kinin system is activated during brain inflammation and previously we demonstrated that bradykinin B1 receptor agonist reduced microglial activation in vitro. The aim of the present study was to investigate the effects of bradykinin B1 or B2 receptor antagonists on microglial release of pro-inflammatory factors in BV2 microglia. In vivo, we focused on the effects of intranasally given kinin antagonists on amyloid burden and microglia/macrophage marker expression in brains of 5X familial Alzheimer's disease mice. The present data show that pharmacological antagonism of B1 receptor (R-715) but not B2 receptor (HOE-140) markedly increased nitric oxide and tumor necrosis factor alpha release from BV2 microglial cells. We also showed that intranasal treatment with R-715 but not HOE-140 of Alzheimer's mice enhanced amyloid beta burden and microglia/macrophages activation. Taken together, our data reveal a possible role for the bradykinin B1 receptor in neuroinflammation and in the control of Abeta accumulation in transgenic mice, possibly through regulation of glial cell responses.

Differential regulation of astrocyte prostaglandin response by kinins: possible role for mitogen activated protein kinases.

The role of kinins, well known as peripheral inflammatory mediators, in the modulation of brain inflammation is not completely understood. The present data show that bradykinin, a B2 receptor agonist, enhanced both basal and lipopolysaccharide (LPS)-induced cyclooxygenase-2 mRNA and protein levels and prostaglandin E2 synthesis in primary rat astrocytes. By contrast, Lys-des-Arg(9)-bradykinin, which is a bradykinin breakdown product and a selective kinin B1 receptor agonist, attenuated both basal and LPS-induced astrocyte cyclooxygenase-2 mRNA levels and prostaglandin E2 production. Pre-treating the cells with p42/p44 MAPK but not with JNK or p38 inhibitors completely abrogated PGE2 synthesis in cells stimulated with LPS in the presence of bradykinin or bradykinin B1 receptor agonist. Bradykinin, but not the bradykinin B1 receptor agonist, augmented p42/p44 MAPK phosphorylation. The phosphorylation of JNK and p38 was not altered upon exposure to Bradykinin or the bradykinin B1 receptor agonist. These results suggest that the dual delayed effect of kinins on PGE2 synthesis may be due to differential regulation of COX-2 and signaling molecules such as p42/p44 MAPKs. Thus, kinins may exert opposing actions on brain inflammation and neurodegenerative diseases.

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 Gαi protein inhibitor abrogated the effects of BK on NO release, and the expression of Gαi protein in the plasma membrane was induced by BK. These results suggest that BK-mediated reduction in microglial NO production depends on coupling to Gi protein and also involves inhibition of cAMP-PKA-CREB signaling.

Distinct modulation of microglial amyloid ß phagocytosis and migration by neuropeptides (i).

Microglial activation plays an integral role in the development and course of neurodegeneration. Although neuropeptides such as bradykinin (BK), somatostatin (SST), and endothelin (ET) are known to be important mediators of inflammation in the periphery, evidence of a similar function in brain is scarce. Using immunocytochemistry, we demonstrate the expression of receptors for BK (B1, B2 subtypes), ET (ETA, ETB subtypes) and SST (SST 2, 3, 4 subtypes) in primary microglia and microglial cell lines. Exposure of BV2 and N9, as well as primary microglial cells to BK or SST increased Aß uptake in a concentration-dependent manner, whereas endothelin decreased Aß uptake. This was caused by increased phagocytosis of Aß since the rate of intracellular Aß degradation remained unaffected. All neuropeptides increased chemotactic activity of microglia. In addition, BK reduced Aß-induced expression of proinflammatory genes including iNOS and COX-2. ET decreased the Aß-induced expression of monocyte chemoattractant protein 1 and interleukin-6. These results suggest that neuropeptides play an important role in chemotaxis and Aß clearance and modulate the brain's response to neuroinflammatory processes.

Glial response to lipopolysaccharide: possible role of endothelins.

Glial inflammation plays a major role in the development of neurodegenerative diseases. Although endothelins (ETs) are known as modulators of inflammation in the periphery, little is known about their possible role in brain inflammation. Previously, we demonstrated that all three endothelins (ET-1, ET-2 and ET-3) enhanced unstimulated synthesis of the glial pro-inflammatory mediators, prostaglandin E2 (PGE2) and nitric oxide (NO). In the present study, glial cells were stimulated in an in vitro model of inflammation by incubation with the bacterial endotoxin lipopolysaccharide (LPS). Indeed, the present study shows that ETs regulate basal and LPS-induced glial inflammation in an opposite fashion. Here we demonstrate that ETs significantly inhibited the LPS-induced glial synthesis of PGE2 and NO, and each of the selective antagonists for ETA and ETB receptors (BQ123 and BQ788 respectively), significantly inhibited the ETs effects in LPS-treated cells. Similar results were observed when expression of key enzymes namely, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in PG and NO synthesis respectively, was measured. ET-1 significantly enhanced the expression of both COX-2 and iNOS. Whereas, it inhibited the LPS-induced expression of both enzymes. These observations suggest a novel neuro-immune feedback pathway through which inflammatory mediators' synthesis is initially enhanced by ETs and are eventually blocked by the same neuropeptide when excessive production of inflammatory mediators occurs following an inflammatory insult.

Protein synthesis dependent effects of kinins on astrocyte prostaglandin synthesis.

It has been shown that kinins and their receptors are over expressed in the brain under pathophysiological conditions such as inflammation. However, little is known about the possible role of kinins, and especially bradykinin in brain inflammation. Although kinins are thought to have immediate effects, peptides may also exert longer and protein synthesis dependent actions. To evaluate this possibility, we assessed the regulation of prostaglandin E(2) synthesis after 15h bradykinin or Lys-des-Arg(9)-bradykinin (B(1) receptor agonist) treatment in rat neonatal astrocytes. Bradykinin, dose dependently stimulated basal and lipopolysaccharide-induced prostaglandin E(2) production, whereas exposure of astrocytes to the B(1) receptor agonist decreased both basal and lipopolysaccharide-induced prostaglandin E(2) release in a dose-dependent manner. These kinin effects on PGE(2) synthesis were completely abrogated by actinomycin-D and cycloheximide, suggesting de novo synthesis of proteins. Bradykinin also increased cyclooxygenase-2 protein levels about 2-fold, while the B(1) receptor agonist decreased cyclooxygenase-2 protein expression. There was no change in cyclooxygenase-1 protein levels after treatment with either of the kinins. Our data suggest a delayed feedback regulatory mechanism of kinins on astrocyte inflammation, whereby astrocyte prostaglandin synthesis is initially enhanced by bradykinin (B(2)) and eventually blocked by kinin breakdown product, acting on B(1) receptors. At least part of this presumed feedback loop could be mediated by de novo protein synthesis of cyclooxygenase-2.

Inhibitory effect of carnosine and N-acetyl carnosine on LPS-induced microglial oxidative stress and inflammation.

Chronic inflammation and oxidative stress have been implicated in the pathogenesis of neurodegenerative diseases. A growing body of research focuses on the role of microglia, the primary immune cells in the brain, in modulating brain inflammation and oxidative stress. One of the most abundant antioxidants in the brain, particularly in glia, is the dipeptide carnosine, beta-alanyl-L-histidine. Carnosine is believed to be involved in cellular defense such as free radical detoxification and inhibition of protein cross-linking. The more stable N-acetyl derivative of carnosine has also been identified in the brain. The aim of the present study was to examine the role of carnosine and N-acetyl carnosine in the regulation of lipopolysaccharide (LPS)-induced microglial inflammation and oxidative damage. In this study, BV2 microglial cells were stimulated with bacterial LPS, a potent inflammatory stimulus. The data shows that both carnosine and N-acetyl carnosine significantly attenuated the LPS-induced nitric oxide synthesis and the expression of inducible nitric oxide synthase by 60% and 70%, respectively. By competitive spectrophotometric measurement and electrospray mass spectrometry analysis, we demonstrated a direct interaction of N-acetyl carnosine with nitric oxide. LPS-induced TNFalpha secretion and carbonyl formation were also significantly attenuated by both compounds. N-acetyl carnosine was more potent than carnosine in inhibiting the release of the inflammatory and oxidative stress mediators. These observations suggest the presence of a novel regulatory pathway through which carnosine and N-acetyl carnosine inhibit the synthesis of microglial inflammatory and oxidative stress mediators, and thus may prove to play a role in brain inflammation.

Lipopolysaccharide differently affects prostaglandin E2 levels in fetal and maternal compartments of perfused human term placenta.

The aim of the present study was to examine the effect of lipopolysaccharide (LPS) on the levels of prostaglandin E(2) (PGE(2)) in the perfusates of the fetal and the maternal compartments of perfused human term placental tissue. Term placentas were perfused for 10h in the absence [control, (n=4)] and presence of LPS [LPS=1 microg/kg perfused placental tissue, (n=4)] in the maternal reservoir. Perfusate samples from the fetal and the maternal circulations were collected every 30 min and examined for PGE(2) levels by radio-immunoassay. PGE(2) levels in the fetal circulation were gradually increased reaching significant peak value of 479+/-159 pg/ml, as compared to PGE(2) levels in the maternal circulation (140+/-146 pg/ml) (p<0.05). After 10 hours of perfusion with control medium, PGE(2) levels in the maternal circulation (347+/-144 pg/ml) were significantly higher as compared to the fetal circulation (150+/-57 pg/ml) (p<0.05). In presence of LPS, PGE(2) levels in the fetal circulation increased reaching a peak value of 1028+/-663 pg/ml after 240 min of perfusion. The levels of PGE(2) in the control group after 240 min of perfusion were significantly lower (156+/-77 pg/ml) (p<0.05). No significant differences were detected in the levels of PGE(2) in the perfusate of the maternal compartment in presence of LPS, as compared to control. Our results suggest that the placenta may play an important role in maintaining high levels of PGE(2) in the fetal circulation and low PGE(2) levels in the maternal circulation during normal pregnancy. Moreover, placental PGE(2) release into the fetal and the maternal circulations may be differently affected in presence of intra-uterine infection/inflammation.

Regulation of glial inflammatory mediators synthesis: possible role of endothelins.

Endothelins are well known as modulators of inflammation in the periphery, but little is known about their possible role in brain inflammation. Stimulation of astrocyte prostaglandin, an inflammatory mediator, synthesis was shown so far only by endothelin 3 (ET-3). By contrast, several studies showed no change or slight decrease of basal nitric oxide synthesis after treatment of astrocytes with endothelin 1 (ET-1) and ET-3. However, a significant increase in astrocytic and microglial nitric oxide synthase (NOS) was observed after exposure to ET-1 and ET-3 in a model of forebrain ischaemia. Here we demonstrate that all three endothelins (ET-1, ET-2, ET-3) significantly enhanced the synthesis of prostaglandin E(2) and nitric oxide in glial cells. Each of the selective antagonists for ETA and ETB receptors (BQ123 and BQ788 respectively), significantly inhibited endothelins-induced production of both nitric oxide and prostaglandin E(2). These results suggest a regulatory mechanism of endothelins, interacting with both endothelin receptors, on glial inflammation. Therefore, inhibition of endothelin receptors may have a therapeutic potential in pathological conditions of the brain, when an uncontrolled inflammatory response is involved.

Inhibitory effect of somatostatin on prostaglandin E2 synthesis by primary neonatal rat glial cells.

Glial inflammation plays an integral role in the development of neurodegenerative disease. Although somatostatin is known to be a local anti-inflammatory factor in the periphery, evidence of a similar function in the brain is scarce. The aim of the present study was to investigate the effect of somatostatin on prostaglandin E(2) synthesis in primary neonatal rat glial cells. The data shows that high concentrations of somatostatin (10(-5)-10(-4)) significantly increased prostaglandin synthesis. By contrast, when used at physiologically relevant concentrations (10(-9)-10(-7) M), somatostatin and somatostatin receptor agonists decreased prostaglandin E(2) synthesis in non-stimulated glial cells as well as in lipopolysaccharide-induced prostaglandin synthesis. The inhibitory effect of somatostatin in lipopolysaccharide-treated cells could be mimicked by protein kinase A inhibitor and was prevented by forskolin. These observations suggest the presence of a novel neuro-immune feedback pathway through which somatostatin inhibits glial prostaglandin synthesis, and thus may prove to play a role in brain inflammation. This action of somatostatin may have a therapeutic potential in pathological conditions of the brain, where an inflammatory response is involved.