Induction of COX-2 enzyme and down-regulation of COX-1 expression by lipopolysaccharide (LPS) control prostaglandin E2 production in astrocytes

Pathological conditions and pro-inflammatory stimuli in the brain induce cyclooxygenase-2 (COX-2), a key enzyme in arachidonic acid metabolism mediating the production of prostanoids that, among other actions, have strong vasoactive properties. Although low basal cerebral COX-2 expression has been reported, COX-2 is strongly induced by pro-inflammatory challenges, whereas COX-1 is constitutively expressed. However, the contribution of these enzymes in prostanoid formation varies depending on the stimuli and cell type. Astrocyte feet surround cerebral microvessels and release molecules that can trigger vascular responses. Here, we investigate the regulation of COX-2 induction and its role in prostanoid generation after a pro-inflammatory challenge with the bacterial lipopolysaccharide (LPS) in astroglia. Intracerebral administration of LPS in rodents induced strong COX-2 expression mainly in astroglia and microglia, whereas COX-1 expression was predominant in microglia and did not increase. In cultured astrocytes, LPS strongly induced COX-2 and microsomal prostaglandin-E2 (PGE2) synthase-1, mediated by the MyD88-dependent NFκB pathway and influenced by mitogen-activated protein kinase pathways. Studies in COX-deficient cells and using COX inhibitors demonstrated that COX-2 mediated the high production of PGE2 and, to a lesser extent, other prostanoids after LPS. In contrast, LPS down-regulated COX-1 in an MyD88-dependent fashion, and COX-1 deficiency increased PGE2 production after LPS. The results show that astrocytes respond to LPS by a COX-2-dependent production of prostanoids, mainly vasoactive PGE2, and suggest that the coordinated down-regulation of COX-1 facilitates PGE2 production after TLR-4 activation. These effects might induce cerebral blood flow responses to brain inflammation

The neurovascular unit as a selective barrier to polymorphonuclear granulocyte (PMN) infiltration into the brain after ischemic injury

The migration of polymorphonuclear granulocytes (PMN) into the brain parenchyma and release of their abundant proteases are considered the main causes of neuronal cell death and reperfusion injury following ischemia. Yet, therapies targeting PMN egress have been largely ineffective. To address this discrepancy we investigated the temporo-spatial localization of PMNs early after transient ischemia in a murine transient middle cerebral artery occlusion (tMCAO) model and human stroke specimens. Using specific markers that distinguish PMN (Ly6G) from monocytes/macrophages (Ly6C) and that define the cellular and basement membrane boundaries of the neurovascular unit (NVU), histology and confocal microscopy revealed that virtually no PMNs entered the infarcted CNS parenchyma. Regardless of tMCAO duration, PMNs were mainly restricted to luminal surfaces or perivascular spaces of cerebral vessels. Vascular PMN accumulation showed no spatial correlation with increased vessel permeability, enhanced expression of endothelial cell adhesion molecules, platelet aggregation or release of neutrophil extracellular traps. Live cell imaging studies confirmed that oxygen and glucose deprivation followed by reoxygenation fail to induce PMN migration across a brain endothelial monolayer under flow conditions in vitro. The absence of PMN infiltration in infarcted brain tissues was corroborated in 25 human stroke specimens collected at early time points after infarction. Our observations identify the NVU rather than the brain parenchyma as the site of PMN action after CNS ischemia and suggest reappraisal of targets for therapies to reduce reperfusion injury after strok

La Via JAK/STAT com a mediadora de respostes a l'estrès oxidatiu, La inflamació i la immunitat innata en astròcits

[spa] En la isquemia cerebral se desarrolla un proceso inflamatorio que contribuye a incrementar la muerte neural. La presencia de células necróticas promueve la activación de la respuesta inmune innata que contribuye a iniciar el proceso inflamatorio. Las citoquinas actúan de mediadoras de la respuesta inmunitaria e inflamatoria. Durante la reoxigenación después de la isquemia, se generan especies reactivas de oxígeno (ERO) que aumentan el daño por reperfusión. En respuesta a la isquemia cerebral, se activan proteínas de la via de señalización JAK/STAT. Esta vía participa en la respuesta celular a los ERO y a las citoquinas. De modo que la inflamación, la respuesta inmune innata y el estrés oxidativo son los procesos derivados de la isquemia/reperfusión que potencialmente pueden activar la vía JAK/STAT. La vía JAK/STAT utiliza un mecanismo en el cual los factores de transcripción STAT que se hayan en estado latente en el citoplasma son fosforilados en tirosina por las quinasas de la familia Janus, dando lugar a la dimerización y translocación al núcleo de las STATs. Las proteínas STAT modulan la expresión de genes relacionados con la diferenciación, la inflamación y la supervivencia. El objetivo de esta tesis es el estudio de la activación de la vía JAK/STAT en respuesta al estrés oxidativo, la inflamación y la inmunidad innata en astrocitios. La vía Jak2/Stat1 se activa en respuesta a citoquinas proinflamatorias, como IFN-gamma y interleuquina-6, y peróxido de hidrógeno en astrocitos, y promueve la muerte celular a las 24 horas. El tratamiento con el inhibidor de Jak2, AG490, previene de la muerte demostrando la implicación de la vía Jak2/Stat1 en la muerte. La activación de la vía Jak2/Stat1 es específica de peróxidos, ya que tratamientos con otros agentes proxidantes, tales como el sulfato de hierro (donador de iones hidroxilo), el nitroprusiato (donador de óxido nítrico) o el paraquato (donador de aniones superóxido) inducen la generación de EROs pero no activan a Stat1. De manera que el peróxido de hidrógeno induce específicamente la activación de la vía Jak2/Stat1. Paralelamente, los tratamientos con peróxido de hidrógeno y los compuestos antioxidantes trolox y propilgallato, inhiben la generación de EROs pero no detienen la activación de Stat1, mientras que el tratamiento de peróxido de hidrógeno con el antioxidante N-acetil-cisteína no reduce la producción de EROs y si inhibe la activación de Stat1. Estos resultados demuestran que la activación de Stat1 y la generación de EROs son efectos independientes. En el siguiente trabajo se demuestra que los siRNAs (por small interfering RNAs) inducen efectos inespecíficos en determinados tipos celulares de manera dependiente de secuencia e independiente del efecto silenciador. En cultivo glial mixto, el tratamiento con siRNAs aumenta la expresión de Stat1 y de otras moléculas proinflamatorias como iNOS, la citoquina IL-6 y las quimiocinas IP-10 e IP-9. La adición de un grupo O-metilo en la cadena sense del siRNA no impide el silenciamiento de la proteína para la cual es específico, y si inhibe el incremento de la expresión de los marcadores inflamatorios que se han estudiado. La activación de la inmunidad innata se observa en cultivo de glía mixta y en cultivo puro de astrocitos, pero no tiene en cultivo de microglía pura ni en la línea celular no glial 3T3, demostrando que los astrocitos son particularmente sensibles a desarrollar respuestas inmunes innatas frente al tratamiento con siRNAs. En este trabajo se demuestra que los siRNA activan la respuesta inmune innata en astrocitos activando el receptor TLR3, puesto que el tratamiento con siRNA, del mismo modo que el tratamiento con el agonista de TLR3, poly di dC, induce un aumento de la expresión de este receptor. En esta tesis, se pone de manifiesto el papel de la vía JAK/STAT en la modulación del estrés oxidativo, la inflamación y la inmunidad innata

La Via JAK/STAT com a mediadora de respostes a l'estrès oxidatiu, la inflamació i la immunitat innata en astròcits

Memòria presentada per optar al grau de Doctor per la Universitat de Barcelona.-- Tesis elaborada en el Institut d'Investigacions Biomèdiques de Barcelona, Consejo Superior de Investigaciones Científicas, Institut d'Investigacions Biomèdiques August Pi i Sunyer.Peer Reviewe

β2 integrin-mediated crawling on endothelial ICAM-1 and ICAM-2 is a prerequisite for transcellular neutrophil diapedesis across the inflamed blood-brain barrier

In acute neuroinflammatory states such as meningitis, neutrophils cross the blood-brain barrier (BBB) and contribute to pathological alterations of cerebral function. The mechanisms that govern neutrophil migration across the BBB are ill defined. Using live-cell imaging, we show that LPS-stimulated BBB endothelium supports neutrophil arrest, crawling, and diapedesis under physiological flow in vitro. Investigating the interactions of neutrophils from wild-type, CD11a(-/-), CD11b(-/-), and CD18(null) mice with wild-type, junctional adhesion molecule-A(-/-), ICAM-1(null), ICAM-2(-/-), or ICAM-1(null)/ICAM-2(-/-) primary mouse brain microvascular endothelial cells, we demonstrate that neutrophil arrest, polarization, and crawling required G-protein-coupled receptor-dependent activation of β2 integrins and binding to endothelial ICAM-1. LFA-1 was the prevailing ligand for endothelial ICAM-1 in mediating neutrophil shear resistant arrest, whereas Mac-1 was dominant over LFA-1 in mediating neutrophil polarization on the BBB in vitro. Neutrophil crawling was mediated by endothelial ICAM-1 and ICAM-2 and neutrophil LFA-1 and Mac-1. In the absence of crawling, few neutrophils maintained adhesive interactions with the BBB endothelium by remaining either stationary on endothelial junctions or displaying transient adhesive interactions characterized by a fast displacement on the endothelium along the direction of flow. Diapedesis of stationary neutrophils was unchanged by the lack of endothelial ICAM-1 and ICAM-2 and occurred exclusively via the paracellular pathway. Crawling neutrophils, although preferentially crossing the BBB through the endothelial junctions, could additionally breach the BBB via the transcellular route. Thus, β2 integrin-mediated neutrophil crawling on endothelial ICAM-1 and ICAM-2 is a prerequisite for transcellular neutrophil diapedesis across the inflamed BBB

AG490 prevents cell death after exposure of rat astrocytes to hydrogen peroxide or proinflammatory cytokines: Involvement of the Jak2/STAT pathway

Janus kinases/STAT pathway mediates cellular responses to certain oxidative stress stimuli and cytokines. Here we examine the activation of Stat1 and Stat3 in rat astrocyte cultures and its involvement in cell death. H 2O2, interferon (INF)-γ and interleukin (IL)-6 but not IL-10 caused cell death. Stat1 was phosphorylated on tyrosine (Tyr)-701 after exposure to H2O2, INF-γ or IL-6 but not IL-10. Tyr-705 pStat3 was observed after H2O2, IL-6 and IL-10. Also, H2O2 induced serine (Ser)-727 phosphorylation of Stat1 but not Stat3. The degree of Tyr-701 pStat1 by the different treatments positively correlated with the corresponding reduction of cell viability. AG490, a Jak2 inhibitor, prevented Tyr-701 but not Ser-727, Stat1 phosphorylation. Also, AG490 inhibited Tyr-705 Stat3 phosphorylation induced by H 2O2 and IL-6 but did not prevent that induced by IL-10. Furthermore, AG490 conferred strong protection against cell death induced by INF-γ, IL-6 and H2O2. These results suggest that Jak2/Stat1 activation mediates cell death induced by proinflammatory cytokines and peroxides. However, we found evidence suggesting that AG490 reduces oxidative stress induced by H2O2, which further shows that H2O2 and/or derived reactive oxygen species directly activate Jak2/Stat1, but masks the actual involvement of this pathway in H 2O2-induced cell death.This work was supported by a grant from the Comisión Interministerial de Ciencia y Tecnologia (CICYT; SAF2002-01963). RG has a fellowship from the IDIBAPS and VP is ascribed to the ‘Ramón y Cajal’ programmePeer Reviewe

Activation of Matrix Metalloproteinase-3 and Agrin Cleavage in Cerebral Ischemia/Reperfusion

Matrix metalloproteinase-3 (MMP-3) degrades components of the extracellular matrix and may participate in the pathogenesis of stroke. Here we examine the expression, activation, and cellular location of MMP-3 and the cleavage of agrin, an MMP-3 substrate, following transient middle cerebral artery occlusion in the rat. MMP-3 was activated by ischemia/reperfusion, which was revealed by the appearance of a cleaved form and increased degradation of a substrate. MMP-3 was observed in ischemic neurons, oligodendrocytes, microvasculature, and reactive microglia/macrophages. In cell cultures, MMP-3 expression was observed in neurons and, to a lesser extent, in mature oligodendrocytes, but not in oligodendrocyte progenitors, astrocytes, or microglia. Casein zymography revealed MMP-3 in cultured neurons. Agrin was expressed in cultured neurons and cultured astrocytes. In brain tissue, agrin was detected in neurons, and following ischemia it was also detected in reactive astrocytes. Addition of MMP-3 to protein extracts from control brain caused neuronal agrin degradation. Following ischemia/reperfusion, agrin disappeared from the tissue membrane fraction and a cleaved agrin fragment was found in tissue protein extracts. The present results show MMP-3 activation and neuronal transmembrane agrin cleavage after ischemia/reperfusion. In addition, the finding that MMP-3 cleaves brain agrin strongly suggests that ischemia-induced MMP-3 activation causes agrin cleavage.Peer Reviewe

Chondroitin sulfate inhibits lipopolysaccharide-induced inflammation in rat astrocytes by preventing nuclear factor kappa B activation

et al.Chondroitin sulfate (CS) is a glucosaminoglycan (GAG) currently used for the treatment of osteoarthritis because of its antiinflammatory and antiapoptotic actions. Recent evidence has revealed that those peripheral effects of CS may also have therapeutic interest in diseases of the CNS. Since neuroinflammation has been implicated in different neuronal pathologies, this study was planned to investigate how CS could modulate the inflammatory response in the CNS by using rat astrocyte cultures stimulated with lipopolysaccharide (LPS). We have evaluated different proteins implicated in the nuclear factor kappa B (NFK{green}) and Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways employing RT-PCR, western blot and immunofluorescence techniques. At 10 μM, CS prevented translocation of p65 to the nucleus, reduced tumour necrosis factor alpha (TNF-α) mRNA and mitigated cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) induction by LPS. However, it did not modify LPS-induced IP-10 and SOCS-1 mRNA, proteins that participate in the JAK/STAT pathway. The results of this study indicate that CS can potentially reduce neuroinflammation by inhibition of NFK{green}B. Therefore endogenous GAGs could afford neuroimmunomodulatory actions under neurotoxic conditions. © 2010 IBRO.This work was supported by “Cátedra Bioibérica/UAM de Inflamación Crónica y Citoprotección”(CABICYC) Spain and, in part by the Spanish Ministry of Science and Innovation Ref. SAF2009-12150 to MGL, SAF2008-04515-C02-01 to AMP, the Spanish Ministry of Health (Instituto de Salud Carlos III) RETICS-RD06/0026, Comunidad Autónoma de Madrid SAL2006/0275, Fundación CIEN, IS Carlos III, MICINN, N° PI016/09 and Agencia Lain Entralgo, CN N° NDE07/09, Comunidad de Madrid, Spain to AGG.Peer Reviewe

Exposure of glia to pro-oxidant agents revealed selective Stat1 activation by H2O2 and Jak2-independent antioxidant features of the Jak2 inhibitor AG490

12 paginas, 7 figuras.The JAK/STAT pathway is activated in response to cytokines and growth factors. In addition, oxidative stress can activate this pathway, but the causative pro-oxidant forms are not well identified. We exposed cultures of rat glia to H2O2, FeSO4, nitroprussiate, or paraquat. We assessed oxidative stress by measuring reactive oxygen species (ROS) and oxidated proteins, we determined phosphorylated Stat1 (pStat1), and we evaluated the effect of antioxidants (trolox, propyl gallate, and N-acetylcysteine) and of Jak2 (Janus tyrosine kinases) inhibitors (AG490 and Jak2-Inhibitor-II). Pro-oxidant agents induced ROS and protein oxidation, excluding nitroprussiate that induced protein nitrosylation. H2O2, and to a lesser extent FeSO4, increased the level of pStat1, whereas nitroprussiate and paraquat did not. Trolox and propyl gallate strongly prevented ROS formation but they did not abolish H2O2-induced pStat1. In contrast, NAC did not reduce the level of ROS but it prevented the increase of pStat1 induced by H2O2, evidencing a differential effect on ROS formation and on Stat1 phosphorylation. H2O2 induced pStat1 in mixed glia cultures and, to a lesser extent, in purified astroglia, but not in microglia. Jak2 inhibitors reduced H2O2-induced pStat1, suggesting the involvement of this kinase in the increased phosphorylation of Stat1 by peroxide. Unexpectedly, AG490, but not Jak2- Inhibitor-II, reduced ROS formation, and it abrogated lipid peroxidation in microsomal preparations. Furthermore, AG490 reduced ROS in glial cells that were transfected with siRNA to silence Jak2 expression. These findings reveal previously unrecognized Jak2-independent antioxidant properties of AG490, and show that Jak2-dependent Stat1 activation by peroxide is dissociated from ROS generation.Grant sponsor: CICYT; Grant number: SAF2002-01963; Grant sponsor: FIS; Grant number: FS041104-O; Grant sponsor: European Network of Excellence DiMI; Grant number: LSHB-CT-2005-512146.Peer reviewe