Cerebrovascular inflammation contributes to diverse central nervous system (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 ischaemia, trauma and seizures. However, the regulatory and effector mechanisms that underlie neutrophil-mediated neurotoxicity are poorly understood. Here we show that mouse neutrophils are not inherently toxic to neurons but that trans-endothelial migration across IL-1 stimulated brain endothelium triggers neutrophils to acquire a neurotoxic phenotype that causes rapid death of cultured neurons. Neurotoxicity was induced by 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 de-condensed DNA associated with proteases, which are known as neutrophil extracellular traps (NETs). The blockade of histone-DNA complexes attenuated transmigrated neutrophil-induced neuronal death, whilst 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 de-condensed 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, namely the release of proteases and de-condensed DNA triggered by phenotypic transformation during cerebrovascular transmigration. Such mechanisms have important implications for neuroinflammatory disorders, notably in the development of anti-leukocyte therapies.

BackgroundThe innate immune response in the brain is initiated by pathogen-associated molecular patterns (PAMPS) or danger-associated molecular patterns (DAMPS) produced in response to central nervous system (CNS) infection or injury. These molecules activate members of the Toll-like receptor (TLR) family, of which TLR4 is the receptor for bacterial lipopolysaccharide (LPS). Although neurons have been reported to express TLR4, the function of TLR4 activation in neurons remains unknown.MethodsTLR4 mRNA expression in primary mouse glial and neuronal cultures was assessed by RT-PCR. Mouse mixed glial, neuronal or endothelial cell cultures were treated with LPS in the absence or the presence of a TLR4 specific antagonist (VIPER) or a specific JNK inhibitor (SP600125). Expression of inflammatory mediators was assayed by cytometric bead array (CBA) and ELISA. Activation of extracellular-signal regulated kinase 1/2 (ERK1/2), p38, c-Jun-N-terminal kinase (JNK) and c-Jun was assessed by Western blot. The effect of conditioned media of untreated- versus LPS-treated glial or neuronal cultures on endothelial activation was assessed by neutrophil transmigration assay, and immunocytochemistry and ELISA were used to measure expression of intercellular cell adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1).ResultsLPS induces strong release of the chemokines RANTES and CXCL1 (KC), tumor necrosis factor-α (TNFα) and IL-6 in primary mouse neuronal cultures. In contrast, LPS induced release of IL-1α, IL-1β and granulocyte-colony stimulating factor (G-CSF) in mixed glial, but not in neuronal cultures. LPS-induced neuronal KC expression and release were completely blocked by VIPER. In glial cultures, LPS induced activation of ERK1/2, p38 and JNK. In contrast, in neuronal cultures, LPS activated JNK but not ERK1/2 or p38, and the specific JNK inhibitor SP600125 significantly blocked LPS-induced KC expression and release. Finally, conditioned medium of LPS-treated neuronal cultures induced strong expression of ICAM-1 and VCAM-1 on endothelial cells, and induced infiltration of neutrophils across the endothelial monolayer, which was inhibited by VIPER.ConclusionThese data demonstrate for the first time that neurons can play a role as key sensors of infection to initiate CNS inflammation.

The study of the equine immune system and inflammatory responses, by measuring cytokine expression, can provide important insight into disease pathogenesis in the horse. A set of quantitative real time polymerase chain reaction (QPCR) assays for the equine cytokines IL-1α, IL-1β, IL-6, IL-8 and TNF-α were validated using QPCR primers and probes which were generated for the equine IL-1α, IL-1β, IL-6, IL-8, TNF-α and 18S genes. Amplification efficiency, intra-assay and inter-assay variation were determined using 10-fold dilutions of plasmid for each gene. Under these conditions the amplification efficiencies of the primers and probes ranged from 99% to 101%. The mean coefficient of variation (CV) across five sets of plasmid DNA for both intra-assay and inter-assay variation was 0.63% (range 0.2% to 1.8%). Amplification efficiency was also determined using 2-fold dilutions of cDNA and under these conditions amplification efficiency ranged from 83% to 95%. The specificity of amplification was confirmed by DNA sequencing of reaction products. The QPCR assays were also evaluated using three sets of cDNA from equine monocyte derived macrophages (EMDM) stimulated for 1 hour with lipopolysaccharide (LPS). The general trend was the same for all three samples with IL-1α showing the greatest induction and IL-6 the lowest induction. The range of cytokine induction was greater than has previously been reported with values ranging from 12-fold to 30,000-fold. We present a set of QPCR primers and probes that are suitable for quantitation of expression of a set of equine cytokines. The primers and probes have been rigorously analyzed, and we demonstrate that they are specific for the desired genes, have a high amplification efficiency and the assays are highly reproducible.