Peter Thornton

Inflammation and brain injury: Acute cerebral ischaemia, peripheral and central inflammation

Inflammation is a classical host defence response to infection and injury that has many beneficial effects. However, inappropriate (in time, place and magnitude) inflammation is increasingly implicated in diverse disease states, now including cancer, diabetes, obesity, atherosclerosis, heart disease and, most relevant here, CNS disease. A growing literature shows strong correlations between inflammatory status and the risk of cerebral ischaemia (CI, most commonly stroke), as well as with outcome from an ischaemic event. Intervention studies to demonstrate a causal link between inflammation and CI (or its consequences) are limited but are beginning to emerge, while experimental studies of CI have provided direct evidence that key inflammatory mediators (cytokines, chemokines and inflammatory cells) contribute directly to ischaemic brain injury. However, it remains to be determined what the relative importance of systemic (largely peripheral) versus CNS inflammation is in CI. Animal models in which CI is driven by a CNS intervention may not accurately reflect the clinical condition; stroke being typically induced by atherosclerosis or cardiac dysfunction, and hence current experimental paradigms may underestimate the contribution of peripheral inflammation. Experimental studies have already identified a number of potential anti-inflammatory therapeutic interventions that may limit ischaemic brain damage, some of which have been tested in early clinical trials with potentially promising results. However, a greater understanding of the contribution of inflammation to CI is still required, and this review highlights some of the key mechanism that may offer future therapeutic targets.

Interleukin-1-induced neurotoxicity is mediated by glia and requires caspase activation and free radical release.

Interleukin (IL)‐1 expression is induced rapidly in response to diverse CNS insults and is a key mediator of experimentally induced neuronal injury. However, the mechanisms of IL‐1‐induced neurotoxicity are unknown. The aim of the present study was to examine the toxic effects of IL‐1 on rat cortical cell cultures. Treatment with IL‐1β did not affect the viability of pure cortical neurones. However, IL‐1 treatment of cocultures of neurones with glia or purified astrocytes induced caspase activation resulting in neuronal death. Neuronal cell death induced by IL‐1 was prevented by pre‐treatment with the IL‐1 receptor antagonist, the broad spectrum caspase inhibitor Boc‐Asp‐(OMe)‐CH2F or the antioxidant α‐tocopherol. The NMDA receptor antagonist dizolcipine (MK‐801) attenuated cell death induced by low doses of IL‐1β but the α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid receptor antagonist 2,3‐dihydroxy‐6‐nitro‐7‐sulfamoyl‐benzo(F)quinoxaline (NBQX) had no effect. Inhibition of inducible nitric oxide synthase with N(ω)‐nitro‐l‐arginine methyl ester had no effect on neuronal cell death induced by IL‐1β. Thus, IL‐1 activates the IL‐1 type 1 receptor in astrocytes to induce caspase‐dependent neuronal death, which is dependent on the release of free radicals and may contribute to neuronal cell death in CNS diseases.

Platelet interleukin-1alpha drives cerebrovascular inflammation.

White blood cell infiltration across an activated brain endothelium contributes to neurologic disease, including cerebral ischemia and multiple sclerosis. Identifying mechanisms of cerebrovascular activation is therefore critical to our understanding of brain disease. Platelet accumulation in microvessels of ischemic mouse brain was associated with endothelial activation in vivo. Mouse platelets expressed interleukin-1alpha (IL-1alpha), but not IL-1beta, induced endothelial cell adhesion molecule expression (ICAM-1 and VCAM-1), and enhanced the release of CXC chemokine CXCL1 when incubated with primary cultures of brain endothelial cells from wild-type or IL-1alpha/beta-deficient mice. A neutralizing antibody to IL-1alpha (but not IL-1beta) or application of IL-1 receptor antagonist inhibited platelet-induced endothelial activation by more than 90%. Platelets from IL-1alpha/beta-deficient mice did not induce expression of adhesion molecules in cerebrovascular endothelial cells and did not promote CXCL1 release in vitro. Conditioned medium from activated platelets induced an IL-1alpha-dependent activation of mouse brain endothelial cells and supported the transendothelial migration of neutrophils in vitro. Thus, we have identified platelets as a key source of IL-1alpha and propose that platelet activation of brain endothelium via IL-1alpha is a critical step for the entry of white blood cells, major contributors to inflammation-mediated injury in the brain.

Neutrophil Cerebrovascular Transmigration Triggers Rapid Neurotoxicity through Release of Proteases Associated with Decondensed DNA

Cerebrovascular inflammation contributes to diverse CNS disorders through mechanisms that are incompletely understood. The recruitment of neutrophils to the brain can contribute to neurotoxicity, particularly during acute brain injuries, such as cerebral ischemia, trauma, and seizures. However, the regulatory and effector mechanisms that underlie neutrophil-mediated neurotoxicity are poorly understood. In this study, we show that mouse neutrophils are not inherently toxic to neurons but that transendothelial migration across IL-1–stimulated brain endothelium triggers neutrophils to acquire a neurotoxic phenotype that causes the rapid death of cultured neurons. Neurotoxicity was induced by the addition of transmigrated neutrophils or conditioned medium, taken from transmigrated neutrophils, to neurons and was partially mediated by excitotoxic mechanisms and soluble proteins. Transmigrated neutrophils also released decondensed DNA associated with proteases, which are known as neutrophil extracellular traps. The blockade of histone–DNA complexes attenuated transmigrated neutrophil-induced neuronal death, whereas the inhibition of key neutrophil proteases in the presence of transmigrated neutrophils rescued neuronal viability. We also show that neutrophil recruitment in the brain is IL-1 dependent, and release of proteases and decondensed DNA from recruited neutrophils in the brain occurs in several in vivo experimental models of neuroinflammation. These data reveal new regulatory and effector mechanisms of neutrophil-mediated neurotoxicity (i.e., the release of proteases and decondensed DNA triggered by phenotypic transformation during cerebrovascular transmigration). Such mechanisms have important implications for neuroinflammatory disorders, notably in the development of antileukocyte therapies.

Matrix metalloproteinase-9 and urokinase plasminogen activator mediate interleukin-1-induced neurotoxicity.

Matrix metalloproteinases (MMPs) are endopeptidases known to mediate acute neuronal injury, but it is unclear whether these proteases are induced by the primary insult or by inflammation associated with injury. We have reported recently that interleukin-1 (IL-1) induces neurotoxicity by an astrocyte-dependent mechanism. The aim of the present study was to test the hypothesis that MMPs mediate IL-1 neurotoxicity in rat, glial-neuronal cocultures. IL-1beta induced the release of astrocytic MMP-9 in cocultures, whilst an antagonist of MMP-9 inhibited IL-1beta-induced neuronal death. Urokinase plasminogen activator (uPA) was constitutively expressed on neuronal membrane fractions, and amiloride (an antagonist of uPA) or plasminogen activator inhibitor (PAI)-1 significantly reduced IL-1beta-induced neurotoxicity. Thus, neuronal uPA contributes to IL-1 neurotoxicity, and may be responsible for activating MMP-9 released from IL-1-primed astrocytes. In summary, IL-1-induced neurotoxicity is dependent on extracellular protease activity, and these mechanisms may contribute to neuronal cell death in CNS diseases.

Interleukin-1 drives cerebrovascular inflammation via MAP kinase-independent pathways.

Cerebrovascular inflammation is triggered by diverse central nervous system (CNS) insults and contributes to disease pathogenesis. The pro-inflammatory cytokine interleukin (IL)-1 is central to this cerebrovascular inflammatory response and understanding the underlying signalling mechanisms of IL-1 actions in brain endothelium may provide therapeutic targets for disease intervention. For the first time, we compare the contributions of p38, JNK and ERK mitogen-activated protein (MAP) kinase and NF-kB pathways to IL-1-induced brain endothelial activation. In cultures of primary mouse brain endothelium and the rat brain endothelial GPNT cell line, interleukin-1β (IL-1β induced a rapid (within 5 minutes) and transient activation of p38 and JNK (but not ERK) MAP kinases. IL-1β also induced nuclear recruitment of nuclear factor (NF)-kB p65. IL-1β-induced brain endothelial expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 was insensitive to MAP kinase inhibitors. IL-1β-induced brain endothelial expression of ICAM-1 and VCAM-1 was inhibited (80-88 %) by the proteasome inhibitor MG132 or the antioxidant caffeic acid phenethyl ester (CAPE), effects suggested to be NF-kB-dependent. IL-1β-induced brain endothelial CXCL1 expression was partially inhibited by JNK MAP kinase or MG132 (62 or 56 %, respectively). However, CXCL1 secretion from brain endothelium was reduced (65 %) only by MG132, and not MAP kinase inhibitors. Similarly, IL-1β-induced neutrophil transendothelial migration was reduced (77-89 %) by MG132, but not MAP kinase inhibitors. In summary, we show that several key components of IL-1β-induced brain endothelial activation (CAM, CXCL1 expression or release and neutrophil transmigration) are largely independent of MAP kinase activity but are reduced by proteasome inhibition, possibly reflecting a requirement for NF-kB activity. Similar mechanisms may contribute to cerebrovascular inflammation in response to CNS injury.

Psychoneuroimmunology of Stroke

Stroke is the major cause of disability in the Western world and is the third greatest cause of death, but there are no widely effective treatments to prevent the devastating effects of stroke. Extensive and growing evidence implicates inflammatory and immune processes in the occurrence of stroke and particularly in the subsequent injury. Several inflammatory mediators have been identified in the pathogenesis of stroke including specific cytokines, adhesion molecules, matrix metalloproteinases, and eicosanoids. An early clinical trial suggests that inhibiting interleukin-1 may be of benefit in the treatment of acute stroke.

48 Endovascular inflammation may occur via tissue-specific mechanisms

Invading neutrophils are important contributors to brain injury in the context of ischaemia. In vitro evidence suggests a key role for platelet-derived interleukin-1α in mediating neutrophil migration across the vascular endothelium. The aim of this study was to determine the role of platelets and IL-1 using in vivo models of vascular inflammation. Two murine models were used. Bacterial endotoxin, lipopolysaccharide (LPS), was injected intraperitoneally into C57BL/6 mice and lavage of the peritoneal cavity was performed 6 h later. LPS was stereotactically injected into the striatum, and the brain fixed and removed 24 h later. Flow cytometry was used to assess neutrophil levels in suspension, while immunohistochemistry was used to assess neutrophil numbers in tissue. To determine the role of platelets, platelet depletion was induced by injection of anti-CD41 antibody 24 h prior to LPS. To determine the role of IL-1, IL-1α/β double knockout mice were used. In the peritoneum, platelet depletion abolished neutrophil migration, indicating a key role for platelets in this process. A robust inflammatory response was seen in serum cytokines after LPS injection and platelet depletion selectively abrogated the increase in serum IL-1α. IL-1α/β double knockout mice revealed a significant difference in LPS-induced neutrophil migration between knockout animals and controls in the encephalitis model, with no difference in the peritonitis model. In an in vivo peritonitis model, neutrophil migration appears to be dependent on platelets, yet independent of IL-1. In an encephalitis model, neutrophil migration appears dependent on IL-1. This may suggest differential migration mechanisms in different vascular beds.