Eduardo Molina-Holgado

LPS/IFN-γ cytotoxicity in oligodendroglial cells: Role of nitric oxide and protection by the anti-inflammatory cytokine IL-10

Proinflammatory mediators have been implicated in demyelinating disorders, including multiple sclerosis, whereas it has been proposed that the anti‐inflammatory cytokines interleukin‐ (IL‐) 4 and IL‐10 participate in disease recovery. The present study analysed the effect of interferon‐γ (IFN‐γ) and bacterial endotoxin (lipopolysaccharide, LPS) on proliferation and survival of progenitors and differentiated oligodendrocytes. We also investigated the presence of receptors for IL‐4 and IL‐10 in oligodendroglial cells and explored a possible protective action of IL‐4 and IL‐10 in cultures following LPS/IFN‐γ. Finally, the role of endogenous nitric oxide (NO) on cell viability and the modulatory action of IL‐4 and IL‐10 on inducible nitric oxide synthase (iNOS) expression were also analysed. We report that LPS and/or IFN‐γ reduced proliferation and viability of oligodendroglial cells. Cell death, presumably by apoptosis as evidence by TUNEL and Annexin V binding, was observed following LPS/IFN‐γ, progenitors being more sensitive than differentiated cells. At both developmental stages, LPS/IFN‐γ‐treated cultures expressed iNOS protein and released micromolar concentrations of NO. In progenitors, LPS/IFN‐γ‐mediated cell damage was partially dependent on endogenous NO production, whereas NO was fundamental for cytotoxicity of differentiated oligodendrocytes. Both cell types expressed mRNA for IL‐4 and IL‐10 receptors and expression of IL‐10 receptors at the protein level was also demonstrated. Treatment with either cytokine inhibited the expression of iNOS resulting from the proinflammatory stimulation. IL‐10 was more effective than IL‐4 in suppressing iNOS expression and, interestingly, IL‐10 conferred protection against oligodendroglial death evoked by LPS/IFN‐γ. Our data raise the question of whether IL‐10 may play a protective role in demyelinating diseases, not only downregulating the function of inflammatory cells but also promoting survival of progenitors and differentiated oligodendrocytes.

Interleukin-1 Regulates Proliferation and Differentiation of Oligodendrocyte Progenitor Cells

Interleukin-1 (IL-1) is a pleiotropic cytokine expressed during normal CNS development and in inflammatory demyelinating diseases, but remarkably little is known about its effect on oligodendroglial cells. In this study we explored the role of IL-1beta in oligodendrocyte progenitors and differentiated oligodendrocytes. The effects of IL-1beta were compared to those of IL-1 receptor antagonist, the specific inhibitor of IL-1 activity, since progenitors and differentiated oligodendrocytes produce IL-1beta and express IL-1 receptors. Unlike other proinflammatory cytokines (TNFalpha and IFNgamma), IL-1beta was not toxic for oligodendrocyte lineage cells. However, this cytokine inhibited proliferation of oligodendrocyte progenitors in the presence of growth factors (PDGF plus bFGF). This was evidenced by a significant decrease in both cells incorporating bromodeoxyuridine (45%) and total cell numbers (57%) after 6 days of treatment. Interestingly, IL-1beta blocked proliferation at the late progenitor/prooligodendrocyte (O4+) stage but did not affect proliferation of early progenitors (A2B5+). Inhibition of proliferation paralleled with promotion of differentiation, as revealed by the increased percentage of R-mab+ cells (6.7-fold). Moreover, when oligodendrocyte progenitors were allowed to differentiate in the absence of growth factors, treatment with IL-1beta promoted maturation to the MBP+ stage (4.2-fold) and survival of differentiating oligodendrocytes (2.1-fold). Regarding intracellular signaling, IL-1beta activated the p38 mitogen-activated protein kinase (MAPK) but not the p42/p44 MAPK and, when combined with growth factors, intensified p38 activation but inhibited the growth-factor-induced p42/p44 activation. IL-1beta also induced a time-dependent inhibition of PFGF-Ralpha gene expression. These results support a role for IL-1beta in promoting mitotic arrest and differentiation of oligodendrocyte progenitors as well as maturation and survival of differentiating oligodendrocytes.

Induction of COX-2 and PGE2 biosynthesis by IL-1β is mediated by PKC and mitogen-activated protein kinases in murine astrocytes

Interleukin‐1 (IL‐1) is an important mediator of immunoinflammatory responses in the brain. In the present study, we examined whether prostaglandin E2 (PGE2) production after IL‐1β stimulation is dependent upon activation of protein kinases in astroglial cells. Astrocyte cultures stimulated with IL‐1β or the phorbol ester, PMA significantly increased PGE2 secretion. The stimulatory action of IL‐1β on PGE2 production was totally abolished by NS‐398, a specific inhibitor of cyclo‐oxygenase‐2 activity, as well as by the protein synthesis inhibitor cycloheximide, and the glucocorticoid dexamethasone. Furthermore, IL‐1β induced the expression of COX‐2 mRNA. This occurred early at 2 h, with a maximum at 4 h and declined at 12 h. IL‐1 β treatment also induced the expression of COX‐2 protein as determined by immunoblot analysis. In that case the expression of the protein remained high at least up to 12 h. Treatment of cells with protein kinase C inhibitors (H‐7, bisindolylmaleimide and calphostin C) inhibited IL‐1β stimulation of PGE2. In addition, PKC‐depleted astrocyte cultures by overnight treatment with PMA no longer responded to PMA or IL‐1. The ablation of the effects of PMA and IL‐1β on PGE2 production, likely results from down‐regulation of phorbol ester sensitive‐PKC isoenzymes. Immunoblot analysis demonstrated the translocation of the conventional isoform cPKC‐α from cytosol to membrane following treatment with IL‐1β. In addition, IL‐1β treatment led to activation of extracellular signal‐regulated kinase (ERK1/2) and p38 subgroups of MAP kinases in astroglial cells. Interestingly, the inhibition of ERK kinase with PD 98059, as well as the inhibition of p38 MAPK with SB 203580, prevented IL‐1β‐induced PGE2 release. ERK1/2 activation by IL‐1β was sensitive to inhibition by the PKC inhibitor bisindolylmaleimide suggesting that ERK phosphorylation is a downstream signal of PKC activation. These results suggest key roles for PKC as well as for ERK1/2 and p38 MAP kinase cascades in the biosynthesis of PGE2, likely by regulating the induction of cyclo‐oxygenase‐2, in IL‐1β‐stimulated astroglial cells.

Role of CB1 and CB2 receptors in the inhibitory effects of cannabinoids on lipopolysaccharide-induced nitric oxide release in astrocyte cultures.

The purpose of this study was to investigate the role of the central cannabinoid receptor (CB1) in mediating the actions of the endogenous cannabinoid agonist anandamide and the synthetic cannabinoid CP‐55940. Activation of primary mouse astrocyte cultures by exposure to bacterial lipopolysaccharide (LPS) caused a marked (approximately tenfold) increase in nitric oxide (NO) release. Coincubation with the cannabinoid agonists anandamide or CP‐55940 markedly inhibited release of NO (–12% to –55%). This effect was abolished by SR‐141716A (1 μM), a CB1 receptor antagonist. SR‐141716A alone also significantly increased NO release in response to LPS, suggesting that endogenous cannabinoids modify inflammatory responses. In contrast, coincubation with the CB2 receptor antagonist SR‐144528 (1 μM) abolished the inhibitory effects of the endogenous cannabinoid anandamide on LPS‐induced NO release, although this may reflect nonspecific effects of this ligand or cannabinoid actions through atypical receptors of anandamide. We also showed that endogenous or synthetic cannabinoids inhibit LPS‐induced inducible NO synthase expression (mRNA and protein) in astrocyte cultures. These results indicate that CB1 receptors may promote antiinflammatory responses in astrocytes.

CB2 cannabinoid receptors promote mouse neural stem cell proliferation

Neurospheres are clonal cellular aggregates of neural stem/precursor cells that grow in culture as free‐floating clusters. Activation of CB1 cannabinoid receptors, which are expressed by these cells, promotes proliferation. In the present study we investigated the expression of CB2 cannabinoid receptors and the effect of exogenous cannabinoids on neural stem/precursor cell proliferation. Neurospheres containing nestin‐positive and sn‐1 diacylglycerol lipase α‐positive cells expressed both CB1 and CB2 receptors, which were maintained through several passages. Application of the non‐selective cannabinoid agonist (HU‐210, 0.5 µm) stimulated bromodeoxyuridine incorporation and neurosphere formation. This action involved both CB1 and CB2 receptors as neurosphere formation was stimulated by either selective CB1 [arachidonyl‐2’chloroethylamide/(all Z)‐N‐(2‐cycloethyl)‐5,8,11,14‐eicosatetraenamide (ACEA), 200 nm and 1 µm] or CB2 (JWH‐056, 0.5 µm) agonists. In addition, CB1 or CB2 antagonists (1 µm SR‐141716A and SR‐144528, respectively) blocked basal proliferation, suggesting that endogenous cannabinoids are implicated in neurosphere proliferation. In addition, cannabinoid agonist‐stimulated proliferation was reduced by the Akt translocation inhibitor BML‐257 (12.5 µm), suggesting a role for phosphoinositide‐3 kinase signalling. Together, our results suggest that cannabinoids stimulate proliferation of neural stem/precursor cells acting on both CB1 and CB2 cannabinoid receptors through a phosphoinositide‐3 kinase/Akt pathway.

Neurobehavioral and Immunological Consequences of Prenatal Immune Activation in Rats. Influence of Antipsychotics

Increasing evidence suggests that pre- or perinatal events that influence the immune system contribute to the development of behavioral or neuropsychiatric disorders. For instance, exposure of pregnant rats to the bacterial endotoxin lipopolysaccharide (LPS) disrupts sensorimotor information processing, as assessed by the prepulse inhibition test (PPI), and also the immune function in adult offspring, which might be of particular relevance as regards schizophrenia. However, the consequences of maternal LPS exposure during pregnancy on synaptic functioning in adult offspring and, more importantly, the therapeutic opportunity to re-establish PPI and immune function have still to be demonstrated. In this work, we analyzed the consequences of prenatal LPS exposure on dopaminergic neurotransmission and presynaptic markers in adult brain areas related to PPI circuitry. In addition, we tested whether oral treatment with the typical antipsychotic drug haloperidol (HAL) could reinstate PPI performances and cytokine serum levels in six-month-old male rats with prenatal LPS exposure. Both sensory information processing deficits and immune anomalies induced by prenatal exposure to LPS were accompanied by changes in dopaminergic neurotransmission and synaptophysin expression. It is important to note that PPI disruption and serum increases in cytokines induced by prenatal LPS exposure were both reversed by HAL. Taken together, these results demonstrate the critical influence of prenatal immune events on the functioning of adult nervous and immune systems, in association with the putative role of the immune system in the development of behavior relevant to schizophrenia.

The endogenous cannabinoid anandamide potentiates interleukin-6 production by astrocytes infected with Theiler's murine encephalomyelitis virus by a receptor-mediated pathway

Theilers murine encephalomyelitis virus (TMEV) infection of a susceptible strain of mice results in virus persistence in the brain and chronic primary immune‐mediated demyelination, which resembles multiple sclerosis. Recent attention has focused on the anti‐inflammatory and immunosuppressive properties of interleukin‐6, a pleiotropic cytokine involved in the regulation of immunological responses, acute phase protein production and hematopoiesis. Anandamide (arachidonoyl ethanolamine) is a natural brain constituent that binds a specific brain cannabinoid receptor. In this study we investigated whether anandamide can modify interleukin‐6 production by primary cultures of murine brain cortical astrocytes infected with TMEV. Astrocytes from susceptible (SJL/J) and resistant (BALB/c) strains of mice infected with TMEV (105 PFU/well) increased IL‐6 release over a period of 24 h. Anandamide caused an enhancement of the release of IL‐6 by TMEV‐infected astrocytes in a concentration‐dependent manner (1–25 μM). Treatment of TMEV‐infected astrocytes with 10 μM arachidonyl trifluoromethyl ketone, a potent inhibitor of the amidase that degrades anandamide, was found to potentiate the effects of anandamide on IL‐6 release. A novel and selective cannabinoid receptor antagonist, SR 141617A, blocked the enhancing effects of anandamide on IL‐6 release by TMEV‐infected astrocytes, suggesting a cannabinoid receptor‐mediated pathway. The physiological implications of these results are unknown, but may be related to the hypothesis of the protective effects of cannabinoids on neurological disorders like multiple sclerosis.

The endocannabinoid system is modulated in response to spinal cord injury in rats

Endocannabinoids are lipid mediators with protective effects in many diseases of the nervous system. We have studied the modulation of the endocannabinoid system after a spinal cord contusion in rats. In early stages, lesion induced increases of anandamide and palmitoylethanolamide (PEA) levels, an upregulation of the synthesizing enzyme NAPE-phospholipase D and a downregulation of the degradative enzyme FAAH. In delayed stages, lesion induced increases in 2-arachidonoylglycerol and a strong upregulation of the synthesizing enzyme DAGL-alpha, that is expressed by neurons, astrocytes and immune infiltrates. The degradative enzyme MAGL was also moderately increased but only 7 days after the lesion. We have studied the cellular targets for the newly formed endocannabinoids using RT-PCR and immunohistochemistry against CB(1) and CB(2) receptors. We observed that CB(1) was constitutively expressed by neurons and oligodendrocytes and induced in reactive astrocytes. CB(2) receptor was strongly upregulated after lesion, and mostly expressed by immune infiltrates and astrocytes. The endocannabinoid system may represent an interesting target for new therapeutical approaches to spinal cord injury.

Activation of the endocannabinoid system as therapeutic approach in a murine model of multiple sclerosis

Theilers murine encephalomyelitis virus‐induced demyelinating disease (TMEV‐IDD) is a well‐characterized murine model of human multiple sclerosis (MS) that closely resembles the chronic and progressive clinical form of the disease. Recent studies have described the involvement of the cannabinoid system in the progression of the disease and the benefits associated with the administration of cannabinoid agonists. With the objective to study whether “indirect” agonists, that is, compounds able to reinforce the physiological endocannabinoid transmission and, therefore, devoid of the psychotropic effects of “direct” agonists, could be suitable agents for the amelioration of MS neurological deficits, we administered the potent and selective anandamide uptake inhibitor UCM707 to TMEV‐infected mice. Our results indicate that treatment during established disease significantly improves the motor function of the diseased mice. At the histological level, UCM707 is able to reduce microglial activation, diminish major histocompatibility complex class II antigen expression, and decrease cellular infiltrates in the spinal cord. Additionally, in microglial cells, UCM707 decreases the production of the proinflammatory cytokines tumor necrosis factor (TNF)‐α, interleukin (IL)‐1β, and IL‐6; reduces nitric oxide levels and inducible nitric oxide synthase expression; and is able to potentiate the action of a subeffective dose of the endocannabinoid anandamide. Overall, these results suggest that agents able to activate the endocannabinoid system could constitute a new series of drugs for the treatment of MS.

Effect of anandamide uptake inhibition in the production of nitric oxide and in the release of cytokines in astrocyte cultures

Astrocytes play a key role regulating aspects of inflammation in the central nervous system (CNS). Several enzymes, such as the inducible nitric oxide synthase (iNOS) or the cyclooxygenase‐2 (COX‐2), along with different inflammatory mediators such as the free radical nitric oxide (NO) or proinflammatory cytokines, have been proposed to be involved in the cell damage associated with neuroinflammation. Recent studies suggest that the endogenous cannabinoid system (ECS) may be involved in the regulation of neuroinflammation. Cannabinoid agonists decrease neurotoxicity and release of proinflammatory factors from activated glial cells and anandamide itself is able to promote antiinflammatory responses in astrocytes via CB1 cannabinoid receptors. The present study is aimed at studying whether UCM707, a potent and selective anandamide uptake inhibitor, is able to inhibit the production of proinflammatory mediators by LPS‐stimulated astrocytes. Our findings indicate that UCM707 is able to reduce NO release, iNOS expression, and the production of the proinflammatory cytokines tumoral necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) in a significant manner, while producing a slight increase in IL‐6 levels. These effects can be reproduced by administration of the synthetic agonist HU210 and partially or totally blocked by administration of CB1 or CB2 selective antagonists, further supporting the involvement of the ECS. These results confirm the ability of UCM707 to reinforce the beneficial effects induced by anandamide and make it an attractive candidate for the management of those pathologies with neuroinflammation as one of their hallmarks.