[Show abstract][Hide abstract] ABSTRACT: Multiple sclerosis (MS) is a chronic inflammatory neurodegenerative disease, considered to be autoimmune in origin. Post-translational modification of central nervous system proteins, including glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP), through citrullination of arginine residues, may lead to exposure of neoepitopes, triggering autoimmunity. Here we investigated the expression of citrullinated proteins in active MS lesions, MS normal appearing white matter and control brain white matter. We demonstrate increased citrullinated GFAP and MBP by immunohistochemistry and western blotting in areas of ongoing demyelination, suggesting a pivotal role for deimination of GFAP and MBP in MS pathogenesis MS.
Journal of Neuroimmunology 05/2014; 273:85-95. · 3.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aims of these studies were to identify the cytokine and chemokine expression profile of nucleus pulposus (NP) cells and to determine the relationships between NP cell cytokine and chemokine production and the characteristic tissue changes seen during intervertebral disc (IVD) degeneration.
Real-time q-PCR cDNA Low Density Array (LDA) was used to investigate the expression of 91 cytokine and chemokine associated genes in NP cells from degenerate human IVDs. Further real-time q-PCR was used to investigate 30 selected cytokine and chemokine associated genes in NP cells from non-degenerate and degenerate IVDs and those from IVDs with immune cell infiltrates ('infiltrated'). Immunohistochemistry (IHC) was performed for four selected cytokines and chemokines to confirm and localize protein expression in human NP tissue samples.
LDA identified the expression of numerous cytokine and chemokine associated genes including 15 novel cytokines and chemokines. Further q-PCR gene expression studies identified differential expression patterns in NP cells derived from non-degenerate, degenerate and infiltrated IVDs. IHC confirmed NP cells as a source of IL-16, CCL2, CCL7 and CXCL8 and that protein expression of CCL2, CCL7 and CXCL8 increases concordant with histological degenerative tissue changes.
Our data indicates that NP cells are a source of cytokines and chemokines within the IVD and that these expression patterns are altered in IVD pathology. These findings may be important for the correct assessment of the 'degenerate niche' prior to autologous or allogeneic cell transplantation for biological therapy of the degenerate IVD.
Arthritis research & therapy 12/2013; 15(6):R213. · 4.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The objective of the study was to investigate how inflammatory cytokines, IL-1β and TNF-α control Notch signaling activity in nucleus pulposus (NP) cells. An increase in expression of selective Notch receptors (Notch1, 2), ligand (Jagged 2) and target genes (Hes1, Hey1 and Hey2) was observed in NP cells following cytokine treatment. A concomitant increase in Notch signaling as evidenced by induction in activity of target gene Hes1 and Hey1 promoters and reporter 12xCSL was seen. Moreover, treatment increased activity of a 2 kb Notch2 promoter. Treatment of cells with NF-κB and MAPK inhibitors abolished the inductive effect of cytokines on Notch2 promoter and its expression. Gain and loss-of-function studies confirmed the inductive effect of p65 on Notch2 promoter activity. In contrast, p50 blocked the cytokine induction of promoter activity. Supporting the promoter studies, lentiviral delivery of sh-p65, and sh-IKKβ significantly decreased cytokine dependent change in Notch2 expression. Interestingly, MAPK signaling showed an isoform specific control of Notch2 promoter, p38α/β2/δ, ERK1 and ERK2 contributed to cytokine dependent induction while p38γ played no role. Analysis of human NP tissues showed that Notch 1, 2 and Hey2 expression correlated with each other. Moreover, expression of Notch2 and IL-1β as well as the number of cells immunopositive for Notch2 significantly increased in histologically degenerate discs compared to non-degenerate discs. Taken together these results explain the observed dysregulated expression of Notch genes in degenerative disc disease. Thus controlling IL-1β and TNF-αactivities during disc disease may restore Notch signaling and nucleus pulposus cell function.
Journal of Biological Chemistry 04/2013; · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objective
To investigate tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β) regulation of CCL3 expression in nucleus pulposus (NP) cells and in macrophage migration. Methods
Quantitative reverse transcription–polymerase chain reaction and immunohistochemistry were used to measure CCL3 expression in NP cells. Transfections were used to determine the role of NF-κB, CCAAT/enhancer binding protein (C/EBPβ), and MAPK on cytokine-mediated CCL3 promoter activity. The effect of NP-conditioned medium on macrophage migration was measured using a Transwell system. ResultsAn increase in CCL3 expression and promoter activity was observed in NP cells after TNFα or IL-1β treatment. Treatment of cells with NF-κB and MAPK inhibitors abolished the effect of the cytokines on CCL3 expression. The inductive effect of p65 and C/EBPβ on the CCL3 promoter was confirmed through gain-of-function and loss-of-function studies. Notably, cotransfection with p50 completely blocked cytokine- and p65-dependent induction. In contrast, c-Rel and RelB had little effect on promoter activity. Lentiviral transduction with short hairpin RNA for p65 (shp65) and shIKKβ significantly decreased the TNFα-dependent increase in CCL3 expression. Analysis of degenerated human NP tissue samples showed that CCL3, but not CCL4, expression correlated positively with the grade of tissue degeneration. Importantly, treatment of macrophages with conditioned medium of NP cells treated with TNFα or IL-1β promoted their migration. Pretreatment of macrophages with an antagonist of CCR1, the primary receptor for CCL3 and CCL4, blocked cytokine-mediated migration. Conclusion
Our findings indicate that TNFα and IL-1β modulate the expression of CCL3 in NP cells by controlling the activation of MAPK, NF-κB, and C/EBPβ signaling. The CCL3–CCR1 axis may play an important role in promoting macrophage infiltration in degenerated, herniated discs.
[Show abstract][Hide abstract] ABSTRACT: ADAMTS-13 is the Von Willebrand factor (vWF) cleaving protease, responsible for the cleavage and down-regulation of the pro-thrombotic properties of ultra large VWF multimers. It is expressed predominantly by the hepatic stellate cells of the liver, but is also found to be expressed in other tissues, including brain. Reduced ADAMTS-13 is associated with a variety of thrombotic microangiopathies. Since the cellular origin and regulation of ADAMTS-13 expression in the brain is unknown, we aimed to investigate this in four different central nervous system (CNS)-derived cell lines, SHSY-5Y (human neuroblastoma), U373 (human astroglioma), CHME-3 (human foetal microglia) and hCMEC/D3 (adult human brain endothelial cells). All cell lines expressed ADAMTS-13 mRNA constitutively with neuroblastoma cells showing the highest expression. Interleukin (IL)-1β down-regulated ADAMTS-13 mRNA expression in astroglioma cells and microglial cells whereas TNF and IL-6 treatment showed no significant differences in ADAMTS-13 mRNA expression in any cell line tested. ADAMTS-13 protein expression was reduced in a dose-dependent manner only in astroglioma cells following stimulation by IL-1β. The ability of IL-1β to significantly reduce ADAMTS-13 mRNA expression in human microglia and astroglioma cells suggests a role in the haemostasis of the local microenvironment under inflammatory conditions. This is the first report of ADAMTS-13 expression in cells of the CNS; however, its function remains to be determined.
Journal of Molecular Neuroscience 07/2011; 46(2):343-51. · 2.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Clarification of the pathophysiological mechanisms of intra-arterial thromboembolism may lead to novel treatments for cerebrovascular disease. There is increasing evidence for the role of von Willebrand factor (VWF) cleaving protease (ADAMTS-13) in modulating the thrombotic cascade in high flow arterial settings. VWF multimers are rich in ultra large forms (ULVWF) which can rapidly bind its primary platelet receptor resulting in spontaneous aggregation of platelets. Under normal conditions, these ULVWF are regulated by rapid proteolysis converting them to smaller, less active forms. The protease responsible for cleavage of ULVWF is ADAMTS-13. We measured the ADAMTS-13 antigen and ADAMTS-13 activity levels in the plasma of consecutive patients with transient ischaemic attack (TIA), compared with nonstroke controls, in a hospital based TIA clinic. This was compared with VWF levels. Samples were analysed in the acute phase and at 3 months postevent. In our pilot study patients with partial anterior circulation type TIAs had significantly reduced ADAMTS-13 activity compared to controls (p=0.0394). No significant differences were seen between high- and low-risk ABCD2 scored patients. We did not observe any significant changes in VWF levels in our study sample. Our pilot study suggests a potential role for altered ADAMTS-13 activity in the pathophysiology of TIA.
Journal of neurology, neurosurgery, and psychiatry 11/2010; 81(11):e69. · 4.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The ADAMTS enzymes (a disintegrin and metalloproteinase with thrombospondin type 1-like motifs) have important roles in central nervous system (CNS) physiology and pathology. This current study aimed to analyse the expression of ADAMTS-9 following transient middle cerebral artery occlusion (tMCAo) in the rat, a model of focal cerebral ischaemia. Using real-time RT-PCR, ADAMTS-9 mRNA was demonstrated to be significantly up-regulated in tMCAo brain tissue compared to sham-operated at 24h post-ischaemia. The mature form of the ADAMTS-9 protein was only detected by Western blotting in brains subjected to tMCAo at 24h. In situ hybridisation demonstrated that ADAMTS-9 mRNA was expressed by neurones in tMCAo tissue. This study indicates that ADAMTS-9 expression is modulated in response to cerebral ischaemia in vivo and further research will resolve whether it plays a role in the subsequent degenerative or repair processes.
[Show abstract][Hide abstract] ABSTRACT: The ECM (extracellular matrix) is a complex molecular framework that provides physical support to cells and tissues, while also providing signals for cell growth, migration, differentiation and survival. The ECM of the CNS (central nervous system) is unusual in that it is rich in CSPGs (chondroitin sulfate proteoglycans), hyaluronan and tenascins. The CSPGs are widely expressed throughout the developing and adult CNS and have a role in guiding or limiting neurite outgrowth and cell migration. Alterations in the synthesis or breakdown of the ECM may contribute to disease processes. Here, we examine changes in the brain-specific CSPGs, brevican and phosphacan, following transient middle cerebral artery occlusion, a model of stroke in the rat. We have investigated their expression at various time points as well as their spatial relationship with ADAMTS-4 (a disintegrin and metalloprotease with thrombospondin motifs 4). The co-localization of ADAMTS or its activity may indicate a functional role for this matrix-protease pair in degeneration/regeneration processes that occur in stroke.
Biochemical Society Transactions 09/2007; 35(Pt 4):692-4. · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) -1, -4 and -5 proteases have been identified in the CNS at the mRNA level. These glutamyl endopeptidases, inhibited by tissue inhibitor of metalloproteinases (TIMP)-3, are key enzymes in the degradation of the aggregating chondroitin sulphate proteoglycans (CSPGs), and may therefore play a role in CNS extracellular matrix (ECM) changes in multiple sclerosis (MS). We have investigated ADAMTS and TIMP-3 expression in normal and MS CNS white matter by real-time RT-PCR, western blotting and immunohistochemistry. We report for the first time the presence of ADAMTS-1, -4 and -5 in normal and MS white matter. Levels of ADAMTS-1 and -5 mRNA were decreased in MS compared to normal tissue, with no significant change in ADAMTS-4 mRNA levels. Protein levels of ADAMTS-4 were significantly higher in MS tissue compared to normal tissue. Immunohistochemical studies demonstrated that ADAMTS-4 was associated predominantly with astrocytes with increased expression within MS lesions. TIMP-3 mRNA was significantly decreased in MS compared to controls. These studies suggest a role for ADAMTS-4 in the pathogenesis of MS. Further studies on the activity of ADAMTS-4 will enable a better understanding of its role in the turnover of the ECM of white matter in MS.
[Show abstract][Hide abstract] ABSTRACT: ADAM-17, a disintegrin and metalloproteinase, is the major proteinase responsible for the cleavage of membrane-bound tumour necrosis factor (TNF) as well as being an active sheddase of other cytokines, cytokine receptors, growth factors and adhesion molecules. TNF is a major proinflammatory cytokine that has been identified as having a pathogenic role in inflammatory diseases within the CNS including multiple sclerosis (MS). Here we report the cellular origin and distribution of ADAM-17 expression within clinically and neuropathologically confirmed MS and normal control white matter, assessed by immunohistochemistry, western blotting and PCR. ADAM-17 expression was associated with the blood vessel endothelium, activated macrophages/microglia and parenchymal astrocytes in MS white matter. Increased levels of ADAM-17 immunoreactivity were displayed in active lesions with evidence of recent myelin breakdown. Further studies into the functional role of ADAM-17 in the pathogenesis of MS and other inflammatory conditions are required.
[Show abstract][Hide abstract] ABSTRACT: ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) enzymes are a recently described group of metalloproteinases. The substrates degraded by ADAMTS-1, -4 and -5 suggest that they play a role in turnover of extracellular matrix in the central nervous system (CNS). ADAMTS-1 is also known to exhibit anti-angiogenic activity. Their main endogenous inhibitor is tissue inhibitor of metalloproteinases (TIMP)-3. The present study was designed to investigate ADAMTS-1, -4 and -5 and TIMP-3 expression after experimental cerebral ischaemia and to examine whether cytokines known to be up-regulated in stroke could alter their expression by astrocytes in vitro. Focal cerebral ischaemia was induced by transient middle cerebral artery occlusion in the rat using the filament method. Our results demonstrate a significant increase in expression of ADAMTS-1 and -4 in the occluded hemisphere but no significant change in TIMP-3. This was accompanied by an increase in mRNA levels for interleukin (IL)-1beta, IL-1 receptor antagonist (IL-1ra) and tumour necrosis factor (TNF). ADAMTS-4 mRNA and protein were up-regulated by TNF in primary human astrocyte cultures. The increased ADAMTS-1 and -4 in experimental stroke, together with no change in TIMP-3, may promote ECM breakdown after stroke, enabling infiltration of inflammatory cells and contributing to brain injury. In vitro studies suggest that the in vivo modulation of ADAMTS-1 and -4 may be controlled in part by TNF.
Brain Research 06/2006; 1088(1):19-30. · 2.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Experimental autoimmune encephalomyelitis (EAE) is an animal model of inflammatory demyelination, a pathological event common to multiple sclerosis (MS). During CNS inflammation there are alterations in the extracellular matrix (ECM). A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS)-1, -4 and -5 are proteases present in the CNS, which are able to cleave the aggregating chondroitin sulphate proteoglycans, aggrecan, phosphacan, neurocan and brevican. It is therefore important to investigate changes in their expression in different stages of EAE induction. We have investigated expression of ADAMTS-1, -4, -5 and tissue inhibitor of metalloproteinase (TIMP)-3, by real-time RT-PCR. We have also examined protein expression of ADAMTS-1, -4 and -5 by western blotting and immunocytochemistry in spinal cord from animals at different stages of disease progression. Our study demonstrated a decrease in ADAMTS-4 mRNA and protein expression. TIMP-3 was decreased at the mRNA level although protein levels were increased in diseased animals compared to controls. Our study identifies changes in ADAMTS expression during the course of CNS inflammation which may contribute to ECM degradation and disease progression.
Journal of Autoimmunity 03/2006; 26(1):16-23. · 8.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tumour necrosis factor (TNF) is a major immunomodulatory and proinflammatory cytokine implicated in the pathogenesis of multiple sclerosis (MS) and the animal model experimental autoimmune encephalomyelitis (EAE). ADAM-17 cleaves membrane-bound TNF into its soluble form. The distribution and level of ADAM-17 expression within spinal cords of Lewis rats with EAE was investigated. ADAM-17 was associated with endothelial cells in the naïve and pre-disease spinal cords. In peak disease astrocytic and inflammatory cells expressed ADAM-17. Upregulation of ADAM-17 mRNA expression was coupled with a decrease in mRNA levels of its inhibitor TIMP3 suggesting a role for ADAM-17 in EAE pathogenesis.
Journal of Neuroimmunology 08/2005; 164(1-2):1-9. · 3.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Microglia are the resident tissue macrophages of the central nervous system (CNS) parenchyma and are key players in the initiation of an inflammatory response. Microglia rapidly transform from a resting to an activated morphology in response to a variety of disease states. However, they can also be the target of infections, as in the case of HIV. Many of the effector properties of microglia can be attributed to the array of substances they secrete in response to stimuli such as bacterial lipopolysaccharide, cytokines, and chemokines. The products of activated microglia include: cytokines (pro- and anti-inflammatory), chemokines, nitric oxide, superoxide radicals, and proteases. Furthermore, microglia have the ability to present antigen to T cells, migrate in response to chemotactic stimuli, and phagocytose cell debris. This report focuses on the immunomodulatory functions of microglia, with particular attention to chemokines, and highlights their pivotal role in the CNS.
Microscopy Research and Technique 08/2001; 54(1):10-7. · 1.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Matrix metalloproteinases (MMPs) are a family of zinc-dependent enzymes, capable of degrading proteins found in the extracellular matrix. MMPs 2 and 9 are known to be produced by microglia, the resident macrophages of the central nervous system. The control of the secretion of these proteases and the activation of proenzymes by other proteases such as plasmin, as well as the balance between MMP secretion and the secretion of their natural inhibitors (TIMPs), have an important relevance in the pathogenesis of multiple sclerosis (MS). The in vitro control of MMPs 2 and 9, TIMPs 1 and 2, and urokinase-type plasminogen activator by microglia was examined in response to a panel of chemokines (chemotactic cytokines), using ELISA and zymography techniques. The chemokines MCP1, MIP1beta, RANTES, IL-8, and Fractalkine were all found significantly to increase the secretion of MMPs and TIMPs by a human foetal microglial cell line, CHME3, after 24 h stimulation. The chemokines tested, MCP1, MIP1beta, and Fractalkine, were also shown to increase MMP9 secretion by primary isolated rat brain microglia in vitro. MCP1, MIP1alpha/beta, and RANTES significantly decreased the secretion of uPA into culture supernatants in ELISA experiments. These findings suggest an important potential role for the involvement of chemokines in the breakdown of the blood-brain barrier and also the destruction of myelin basic protein in MS.
[Show abstract][Hide abstract] ABSTRACT: Microglia, the resident macrophages of the central nervous system, are the primary cells to respond to injury in the brain, both in inflammation, e.g., in multiple sclerosis, and trauma. Chemokines are potential mediators of microglial cell recruitment to sites of injury; thus, the ability of microglia to migrate in response to a number of chemokines was assessed. The chemokines monocyte chemoattractant protein 1, macrophage inflammatory protein 1alpha, macrophage inflammatory protein 1beta, RANTES (regulated upon activation normal T cell expressed and secreted), interleukin 8, and IP-10 (interferon gamma inducible protein-10), induce migration and changes in the distribution of f-actin in adult rat microglia and a human microglial cell line, CHME3, in vitro. Both cell types show a significant migration response, above control levels, to all the chemokines tested in a typical dose-dependent manner. These chemokines also induced a reorganization of the actin cytoskeleton of the cells. This study indicates that chemokines play an important role in the recruitment of microglia to areas of central nervous system inflammation.
Journal of Neuroscience Research 02/1999; 55(1):17-23. · 2.97 Impact Factor