Neutrophils mediate blood-spinal cord barrier disruption in demyelinating neuroinflammatory diseases.

Disruption of the blood-brain and blood-spinal cord barriers (BBB and BSCB, respectively) and immune cell infiltration are early pathophysiological hallmarks of multiple sclerosis (MS), its animal model experimental autoimmune encephalomyelitis (EAE), and neuromyelitis optica (NMO). However, their contribution to disease initiation and development remains unclear. In this study, we induced EAE in lys-eGFP-ki mice and performed single, nonterminal intravital imaging to investigate BSCB permeability simultaneously with the kinetics of GFP(+) myeloid cell infiltration. We observed a loss in BSCB integrity within a day of disease onset, which paralleled the infiltration of GFP(+) cells into the CNS and lasted for ∼4 d. Neutrophils accounted for a significant proportion of the circulating and CNS-infiltrating myeloid cells during the preclinical phase of EAE, and their depletion delayed the onset and reduced the severity of EAE while maintaining BSCB integrity. We also show that neutrophils collected from the blood or bone marrow of EAE mice transmigrate more efficiently than do neutrophils of naive animals in a BBB cell culture model. Moreover, using intravital videomicroscopy, we demonstrate that the IL-1R type 1 governs the firm adhesion of neutrophils to the inflamed spinal cord vasculature. Finally, immunostaining of postmortem CNS material obtained from an acutely ill multiple sclerosis patient and two neuromyelitis optica patients revealed instances of infiltrated neutrophils associated with regions of BBB or BSCB leakage. Taken together, our data provide evidence that neutrophils are involved in the initial events that take place during EAE and that they are intimately linked with the status of the BBB/BSCB.

ß2 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 ß2 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, ß2 integrin-mediated neutrophil crawling on endothelial ICAM-1 and ICAM-2 is a prerequisite for transcellular neutrophil diapedesis across the inflamed BBB.

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 stroke.

HIV-1 Nef interferes with T-lymphocyte circulation through confined environments in vivo.

HIV-1 negative factor (Nef) elevates virus replication and contributes to immune evasion in vivo. As one of its established in vitro activities, Nef interferes with T-lymphocyte chemotaxis by reducing host cell actin dynamics. To explore Nef's influence on in vivo recirculation of T lymphocytes, we assessed lymph-node homing of Nef-expressing primary murine lymphocytes and found a drastic impairment in homing to peripheral lymph nodes. Intravital imaging and 3D immunofluorescence reconstruction of lymph nodes revealed that Nef potently impaired T-lymphocyte extravasation through high endothelial venules and reduced subsequent parenchymal motility. Ex vivo analyses of transendothelial migration revealed that Nef disrupted T-lymphocyte polarization and interfered with diapedesis and migration in the narrow subendothelial space. Consistently, Nef specifically affected T-lymphocyte motility modes used in dense environments that pose high physical barriers to migration. Mechanistically, inhibition of lymph node homing, subendothelial migration and cell polarization, but not diapedesis, depended on Nef's ability to inhibit host cell actin remodeling. Nef-mediated interference with in vivo recirculation of T lymphocytes may compromise T-cell help and thus represents an important mechanism for its function as a HIV pathogenicity factor.

Brain-derived antigens in lymphoid tissue of patients with acute stroke.

In experimental animals, the presence of brain-derived constituents in cervical lymph nodes has been associated with the activation of local lymphocytes poised to minimize the inflammatory response after acute brain injury. In this study, we assessed whether this immune crosstalk also existed in stroke patients. We studied the clinical course, neuroimaging, and immunoreactivity to neuronal derived Ags (microtubule-associated protein-2 and N-methyl d-aspartate receptor subunit NR-2A), and myelin-derived Ags (myelin basic protein and myelin oligodendrocyte glycoprotein) in palatine tonsils and cervical lymph nodes of 28 acute stroke patients and 17 individuals free of neurologic disease. Stroke patients showed greater immunoreactivity to all brain Ags assessed compared with controls, predominantly in T cell zones. Most brain immunoreactive cells were CD68(+) macrophages expressing MHC class II receptors. Increased reactivity to neuronal-derived Ags was correlated with smaller infarctions and better long-term outcome, whereas greater reactivity to myelin basic protein was correlated with stroke severity on admission, larger infarctions, and worse outcome at follow-up. Patients also had more CD69(+) T cells than controls, indicative of T cell activation. Overall, the study showed in patients with acute stroke the presence of myelin and neuronal Ags associated with lymph node macrophages located near activated T cells. Whether the outcome of acute stroke is influenced by Ag-specific activation of immune responses mediated by CD69 lymphocytes deserves further investigation.

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-E(2) (PGE(2)) 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 PGE(2) 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 PGE(2) production after LPS. The results show that astrocytes respond to LPS by a COX-2-dependent production of prostanoids, mainly vasoactive PGE(2), and suggest that the coordinated down-regulation of COX-1 facilitates PGE(2) production after TLR-4 activation. These effects might induce cerebral blood flow responses to brain inflammation.

Astrocyte TLR4 activation induces a proinflammatory environment through the interplay between MyD88-dependent NFκB signaling, MAPK, and Jak1/Stat1 pathways.

There is increasing evidence that astrocytes play important roles in immune regulation in the brain. Astrocytes express toll-like receptors (TLR) and build up responses to innate immune triggers by releasing proinflammatory molecules. We investigate signaling pathways and released molecules after astrocyte TLR4 activation. Purified rodent brain astrocyte cultures were treated with the TLR4 activator bacterial lipopolysaccharide (LPS). Tools used to interfere with this system include small interference RNA, inhibitory drugs, and MyD88 or Stat1 deficient mice. LPS induced early activation of the transcription factor NFκB, through the MyD88 adaptor, and expression of TNF-α, VCAM-1, IL-15, and IL-27. LPS also induced delayed Jak1/Stat1 activation, which was MyD88-independent but was not mediated by IFN-ß. Jak1/Stat1 activation induced the expression of negative cytokine regulator SOCS-1 and CXCL10 chemokine (IP-10). Mitogen-activated protein kinases (MAPK) were also involved in TLR4 signaling in a MyD88-independent fashion. p38 exerted a strong influence on LPS-induced gene expression by regulating the phosphorylation of Stat1 and the transcriptional activity of NFκB, while JNK regulated the Jak1/Stat1 pathway, and ERK1/2 controlled the expression of Egr-1 and influenced MyD88-dependent MMP-9 expression. Interplay between these signals was evidenced by the increased induction of MMP-9 in Stat1-deficient cells challenged with LPS, suggesting that Stat1 negatively regulates the expression of MMP-9 induced by LPS. Therefore, astrocytes are responsive to TLR4 activation by inducing a complex set of cell-dependent molecular reactions mediated by NFκB, MAPK and Jak1/Stat1 signaling pathways. Here we identified cross-talking signals generating a proinflammatory environment that will modulate the response of surrounding cells.

Antioxidant CR-6 protects against reperfusion injury after a transient episode of focal brain ischemia in rats.

Oxidative and nitrosative stress are targets for intervention after ischemia/reperfusion. The aim of this study was to explore the effect of CR-6, a vitamin-E analogue that is antioxidant and scavenger of nitrogen-reactive species. Sprague-Dawley rats had the middle cerebral artery (MCA) occluded either for 90 mins or permanently. Cortical perfusion was continuously monitored by laser-Doppler flowmetry. CR-6 (100 mg/kg) was administered orally either at 2 and 8 h after MCA occlusion, or at 2 h only. Infarct volume, neurological deficit, and signs of reperfusion injury were evaluated. CR-6 was detected in plasma and brain by HPLC. CR-6 reduced glutathione consumption in the ischemic brain and superoxide generation in the isolated MCA. CR-6 decreased infarct volume and attenuated the neurological deficit at 1 and 7 days after ischemia/reperfusion, but not after permanent ischemia. Immediately after reperfusion, cortical blood flow values returned to their baseline (+/-20%) in several animals, whereas others showed hyper-perfusion (>20% of baseline). Reactive hyperemia was associated with adverse events such as increased cortical BBB leakage, edema, protein nitrotyrosination, COX-2 expression, and neutrophil accumulation; and with a poorer outcome, and CR-6 attenuated these effects. In conclusion, oral CR-6 administration after transient ischemia protects the brain from reperfusion injury.

Astrocytes are very sensitive to develop innate immune responses to lipid-carried short interfering RNA.

Short interfering RNA (siRNA) inhibits the synthesis of specific proteins through RNA interference (RNAi). However, siRNA can induce innate immune responses that are mediated by toll-like receptors (TLRs) in cells of the immune system. Here, we sought to evaluate whether siRNA can induce such responses in glial cells. We examined the effects of various siRNA sequences prepared with lipids (oligofectamine). Lipid-siRNA induced variable degrees of silencing-independent nonspecific effects, e.g. increased Stat1 and Cox-2 expression and release of IL-6 and IP-10 in primary astroglia. This was prevented through chemical modification of siRNA by nucleoside 2'-O-methylation, without impairing specific gene silencing. Lipid-siRNA also induced nonspecific responses in purified astroglia, but not in microglia, or 3T3 cells. The highest TLR7 and TLR3 mRNA expression was found in microglia and purified astroglia, respectively. Accordingly, the TLR3 agonist poly(I:C) (PIC) induced higher release of IFN-beta in primary and purified astroglia than in microglia. As siRNA, PIC induced IP-10, Stat1, VCAM-1, and Cox-2 and increased TLR3 mRNA expression. The effects of lipid-siRNA in purified astrocytes were attenuated after silencing TLR3 or TLR7 expression, and by the PKR inhibitor 2-aminopurine. Furthermore, lipid-siRNA induced the expression of RIG-I. In contrast, siRNA devoid of lipids did not enter the astrocytes, did not silence gene expression, and did not induce Stat1 or Cox-2. The results show that, in astroglia, lipid-siRNA induces innate immune responses that are mediated, at least in part, by intracellular mechanism dependent on TLR7, TLR3, and helicases.

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

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.

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(2)O(2), interferon (INF)-gamma and interleukin (IL)-6 but not IL-10 caused cell death. Stat1 was phosphorylated on tyrosine (Tyr)-701 after exposure to H(2)O(2), INF-gamma or IL-6 but not IL-10. Tyr-705 pStat3 was observed after H(2)O(2), IL-6 and IL-10. Also, H(2)O(2) 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(2)O(2) and IL-6 but did not prevent that induced by IL-10. Furthermore, AG490 conferred strong protection against cell death induced by INF-gamma, IL-6 and H(2)O(2). 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 H(2)O(2), which further shows that H(2)O(2) and/or derived reactive oxygen species directly activate Jak2/Stat1, but masks the actual involvement of this pathway in H(2)O(2)-induced cell death.

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.