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Biomedical subjects

M N Woodroofe

Publications and source records attributed to M N Woodroofe.

At least 19 recordsLinked to original sources
PubMed2006

ADAMTS-1 and -4 are up-regulated following transient middle cerebral artery occlusion in the rat and their expression is modulated by TNF in cultured astrocytes.

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.

ADAM Proteins↗
PubMed2006

Upregulation of ADAM-17 expression in active lesions in multiple sclerosis.

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.

ADAM Proteins↗
PubMed2006

Expression of ADAMTS-1, -4, -5 and TIMP-3 in normal and multiple sclerosis CNS white matter.

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.

ADAM Proteins↗
PubMed2005

Differential expression of ADAMTS-1, -4, -5 and TIMP-3 in rat spinal cord at different stages of acute experimental autoimmune encephalomyelitis.

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.

ADAM Proteins↗
PubMed2003

Cytokine regulation of MCP-1 expression in brain and retinal microvascular endothelial cells.

Chemokines have a pivotal role in the selective mediation and amplification of inflammation. The CNS vascular endothelial cells, which form part of the blood-brain barrier (BBB) and blood-retinal barrier (BRB), are ideally situated to present chemokines to circulating lymphocytes leading to their recruitment. Monocyte-chemoattractant protein-1 (MCP-1), also known as CCL2, a potent chemoattractant of T cells and monocytes, has been implicated in inflammatory and angio-proliferative brain and retinal disease. In this study, MCP-1 expression by CNS endothelial cells was investigated in vitro. Rat brain (GP8/3.9) and retinal (JG2/1) vascular endothelial cell lines expressed MCP-1 constitutively in vitro as assessed by immunocytochemistry and enzyme linked immunosorbant assay (ELISA). Upregulation of secreted MCP-1 was observed following activation with the pro-inflammatory cytokines TNF-alpha, IL-1 beta and IFN-gamma, and was reduced following dexamethasone treatment. Functional chemotactic activity of brain and retinal endothelial cell supernatants was demonstrated in an in vitro chemotaxis assay, which was inhibited by anti-MCP-1 antibodies. These findings suggest that endothelial cell-derived MCP-1 plays a key role in leukocyte recruitment across the blood-brain and blood-retinal barriers in vivo.

Animals↗
PubMed2003

Longitudinal study of chemokine receptor expression on peripheral lymphocytes in multiple sclerosis: CXCR3 upregulation is associated with relapse.

The interaction between chemokines and their receptors leads to selective recruitment of cells to foci of inflammation. Cross-sectional studies have reported significantly different expression of chemokine receptors CXCR3, CCR5 and CCR2 on peripheral blood lymphocytes in multiple sclerosis (MS) compared with controls. Cells expressing these receptors are likely to play a pathogenic role as suggested by studies of experimental autoimmune encephalomyelitis. Also, immunogenetic studies of nonfunctional CCR5 receptors in MS patients, due to 32delta deletion, demonstrated a delay in time to next relapse. The aims of this study were to detect any changes in the serial expression of chemokine receptors CCR2, CCR3, CCR5 and CXCR3 on peripheral blood CD4+ lymphocytes from patients with MS and to correlate the changes with relapses. Upregulation of CXCR3 expression on peripheral blood CD4+ lymphocytes was associated with all relapses and CCR5 expression was significantly affected with all relapses. Clinical recovery, with or without intravenous methylprednisolone treatment, coincided with the return of CXCR3 towards baseline in all but one case. Fluctuation in the expression of CXCR3 and CCR5 was also significantly greater in clinically stable patients with MS compared with controls, which may be due to subclinical disease activity. These findings provide further support for the view that CXCR3 and CCR5 antagonists could have a therapeutic value in MS.

Adult↗
PubMed2002

Expression of chemokines in the CSF and correlation with clinical disease activity in patients with multiple sclerosis.

OBJECTIVE: To define the chemokine profile in the CSF of patients with multiple sclerosis (MS) and compare it with three control groups; patients with benign headache (headache), non-inflammatory neurological diseases (NIND), and other inflammatory neurological diseases (IND). In addition, the correlations of CSF chemokine concentrations with chemokine receptor expression on CSF CD4(+) T cells and with clinical disease activity were assessed. METHODS: Forty three patients with MS, 24 with IND, 44 with NIND, and 12 with benign headache undergoing diagnostic or therapeutic lumbar puncture were included. Supernatant fluid from CSF was analysed for four beta (CCL2, CCL3, CCL4, CCL5) and two alpha (CXCL9, CXCL10)chemokines by enzyme linked immunosorbent assay (ELISA). Chemokine receptors CCR3, CCR5, and CXCR3 on CD4(+) T cells from eight patients with MS were analysed using directly conjugated fluorescent labelled monoclonal antibodies and flow cytometry. RESULTS: CXCL10, formerly interferon-gamma inducible protein-10 (IP-10), was significantly increased and CCL2, formerly monocyte chemoattractant protein-1 (MCP-1), was significantly reduced in the CSF of patients with MS and IND compared with those with benign headache and NIND. Concentrations of CXCL10 were significantly greater in patients with relapsing-remitting compared with secondary progressive MS and correlated significantly with CXCR3 expression on CSF CD4(+) T cells from patients with MS. Concentrations of CXCL10 decreased and CCL2 concentrations increased as time from the last relapse increased in patients with MS. CONCLUSION: Increased CXCL10 and decreased CCL2 concentrations in the CSF are associated with relapses in MS. Although serial values from individual patients were not available, this study suggests that CXCL10 and CCL2 may return towards baseline concentrations after a relapse. Correlation of CXCL10 with CD4(+) T cell expression of CXCR3 was consistent with its chemoattractant role for activated lymphocytes. Thus CXCL10 neutralising agents and CXCR3 receptor antagonists may be therapeutic targets in MS.

Adult↗
PubMed2002

Expression of chemokines in cerebrospinal fluid and serum of patients with chronic inflammatory demyelinating polyneuropathy.

Chemokines are likely to contribute to the pathogenesis of chronic inflammatory demyelinating polyneuropathy (CIDP), as evidenced by data from experimental autoimmune neuritis. The alpha and beta chemokines in the cerebrospinal fluid (CSF) and serum from patients with CIDP were analysed using an enzyme linked immunosorbent assay. CXCL9, CXCL10, and CCL3 were raised in the CSF in CIDP compared with controls and non-demyelinating neuropathies (p < 0.001). Although the CSF levels of CCL2 were significantly higher than the serum levels for all groups, the difference between groups was not significant. CXCL9, CXCL10, and CCL3 may contribute to the pathogenesis of CIDP by recruiting inflammatory T cells and monocytes to spinal nerve roots, while CCL2 is likely to play a physiological role.

Adult↗
PubMed2001

Immunoregulation of microglial functional properties.

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.

Animals↗
PubMed2001

Extracellular nucleotides differentially regulate interleukin-1beta signaling in primary human astrocytes: implications for inflammatory gene expression.

The cytokine interleukin-1beta (IL-1beta) is a potent activator of human astrocytes, inducing or modulating expression of multiple proinflammatory genes via activation of the transcription factors nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1). In this study, we examined whether IL-1beta signaling is regulated in these cells by extracellular nucleotides that are released at high concentrations under inflammatory conditions and act as ligands for members of the P2 receptor family. Using reporter constructs and electromobility shift assays, we found that cotreatment of astrocyte cultures with ATP (1-100 microm) significantly potentiated IL-1beta-mediated activation of NF-kappaB and AP-1 and that ATP alone activated AP-1. These effects were blocked by the P2 receptor antagonists XAMR 0721, periodate-oxidized ATP, and suramin. A role for ATP in modulating IL-1beta-mediated inflammatory gene expression was supported further by the observation that ATP potentiated the IL-1beta-induced expression of IL-8 mRNA and protein but strongly downregulated IP-10 expression. Reverse transcription-PCR and cloning demonstrated expression of the ATP-responsive P2 receptor subtypes P2Y(1), P2Y(2), and P2X(7), as well as the ATP-insensitive receptor P2Y(4). ADP, a selective agonist for P2Y(1), produced results similar to or greater than those obtained using ATP, whereas 2'-3'-O-(4-benzoyl-benzoyl)-ATP, a selective agonist for P2X(7), was less effective than ATP. In contrast, UTP, a selective agonist for P2Y(2) and P2Y(4), was ineffective. These studies indicate that different P2 receptor subtypes play distinct roles in the modulation of IL-1beta-mediated signal transduction in human astrocytes, and that signaling via P2 receptors may fine-tune the transcription of genes involved in inflammatory responses in the human CNS.

Adenosine Triphosphate↗
PubMed2001

Astrocyte and endothelial cell expression of ADAM 17 (TACE) in adult human CNS.

ADAM 17, also known as TACE, is an important sheddase for a number of proteins, including tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-alpha (TGF-alpha), L-selectin, p75, and p55 TNF receptors, and interleukin-1 receptor II (IL-1RII). The presence of ADAM 17 mRNA in adult mouse and rat CNS was recently reported (Karkkainen et al. Mol Cell Neurosci 15:547-560, 2000). However, the cellular origin of ADAM 17 remains unknown. In this study, we have used an anti-ADAM 17 antibody in an immunohistochemical study of its distribution in human adult CNS tissue. Cells with astrocytic and endothelial morphology were ADAM 17-positive. This finding was further confirmed using double immunofluorescence with antibodies against GFAP and von Willebrand factor, which label astrocytes and endothelial cells, respectively. This study demonstrates that ADAM 17 is expressed by astrocytes and endothelial cells in normal brain tissue and may have a role in normal brain function.

ADAM Proteins↗
PubMed2000

Expression of the beta-chemokine receptors CCR2, CCR3 and CCR5 in multiple sclerosis central nervous system tissue.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) characterised by perivascular inflammatory cell infiltrates and plaques of demyelination. Chemokines have been shown to play an important role in the activation and directional migration of cells to sites of CNS inflammation. The action of chemokines requires the expression of their complementary chemokine receptors by their target cells. We have examined the expression of the beta-chemokine receptors CCR2, CCR3 and CCR5 in post-mortem MS CNS tissue using single- and double-labelling immunocytochemistry techniques. Low levels of CCR2, CCR3 and CCR5 were expressed by microglial cells throughout control CNS tissue. In chronic active MS lesions CCR2, CCR3 and CCR5 were associated with foamy macrophages and activated microglia. CCR2 and CCR5 were also present on large numbers of infiltrating lymphocytes. A smaller number of CCR3-positive lymphocytes were present, but we also noted CCR3 and CCR5 on astrocytes in five of the 14 cases of MS investigated, particularly associated with processes around vessels and at the glia limitans. Ligands for CCR2 and CCR3 include MCP-1 and MCP-3 which were co-localised around vessels with the infiltrating leukocytes, but were also present in unaffected areas of cortex. The elevated expression of CCR2, CCR3 and CCR5 in the CNS in MS suggests these beta-chemokine receptors and their ligands play a role in the pathogenesis of MS.

Adult↗
PubMed2000

Expression of the interferon-gamma-inducible chemokines IP-10 and Mig and their receptor, CXCR3, in multiple sclerosis lesions.

The recruitment of leucocytes to sites of inflammation is an important feature of multiple sclerosis (MS) pathology. Chemokines are involved in the activation and specific directional migration of monocytes and T-lymphocytes to sites of inflammation. Using immunocytochemistry, the expression of the alpha-chemokines, interferon (IFN)-gamma-inducible protein-10 (IP-10) and monokine induced by IFN-gamma (Mig), and their receptor CXCR3 have been examined in post-mortem central nervous system (CNS) tissue from MS cases at different stages of lesion development. In actively demyelinating lesions both IP-10 and Mig protein were predominantly expressed by macrophages within the plaque and by reactive astrocytes in the surrounding parenchyma. CXCR3 was expressed by T cells and by astrocytes within the plaque. Interferon-gamma may stimulate glial cells to express IP-10 and Mig, which continue the local inflammatory response by selectively recruiting activated T-lymphocytes into the CNS.

Adult↗
PubMed2000

Dexamethasone regulation of matrix metalloproteinase expression in CNS vascular endothelium.

Matrix metalloproteinases (MMPs) have been implicated in the early breakdown of the blood-brain barrier in neuroinflammatory disease. Although expression of these enzymes by resident glial cells and recruited immune cells has been described, altered expression of MMPs by the CNS vascular endothelial cells may also contribute to barrier disruption. In the present study, the in vitro expression of MMP-2 and -9 as well as tissue inhibitor of metalloproteinase (TIMP)-2 by rat CNS microvascular endothelial cells has been determined and compared with that by endothelial cell lines derived from rat aorta and high endothelial venules. Primary cultures of rat brain microvascular endothelial cells as well as the rat brain (GP8/3.9) and rat retinal endothelial (JG2/1) cell lines constitutively expressed MMP-2, -9 and TIMP-2. In vitro activation of CNS endothelium with the pro-inflammatory cytokines, tumour necrosis factor-alpha and interleukin-1beta, resulted in selective upregulation of MMP-9 activity, whereas no significant changes were seen in MMP-2 or TIMP-2 levels at 24 h. The addition of dexamethasone partially inhibited the cytokine-induced upregulation of MMP-9. Treatment of GP8/3.9 brain endothelial cells with active MMP-9 caused subtle but distinct alterations in the expression of the junctional protein, ZO-1. Quantitative differences found between CNS and non-CNS endothelial cells in the expression of both MMP-2 and -9, and in the expression of TIMP-2 demonstrate that CNS vascular endothelium is functionally distinct from non-CNS endothelium. These results suggest that cytokine-induced upregulation of MMP-9 expression by the CNS vascular endothelium may play a role in the pathogenesis of blood-brain and blood-retinal barrier breakdown in vivo.

Animals↗
PubMed1999

Chemokines induce migration and changes in actin polymerization in adult rat brain microglia and a human fetal microglial cell line in vitro.

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.

Actins↗
PubMed1999

Chemokine modulation of matrix metalloproteinase and TIMP production in adult rat brain microglia and a human microglial cell line in vitro.

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.

Animals↗
PubMed1998

Expression of monocyte chemoattractant protein-1 and other beta-chemokines by resident glia and inflammatory cells in multiple sclerosis lesions.

Beta-chemokines induce the directional migration of monocytes and T lymphocytes and are thus associated with chronic inflammation. Using immunocytochemistry and in situ hybridisation (ISH) techniques, we have examined the expression of the beta-chemokines monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and RANTES (regulated upon activation, normal T cell expressed and secreted) in post-mortem human brain from multiple sclerosis (MS) cases, at different stages of lesion development. In actively demyelinating MS plaques RANTES expression was restricted to the blood vessel endothelium, perivascular cells and surrounding astrocytes, suggesting a role in the recruitment of inflammatory cells from the circulation. MCP-1 was expressed by astrocytes and macrophages within acute MS lesions, but was restricted to reactive astrocytes in the parenchyma surrounding the lesion. MIP-1alpha was expressed by astrocytes and macrophages within the plaque, while MIP-1beta was expressed by macrophages and microglia within the lesion, and by microglia in surrounding white matter. Glial cells may be stimulated to produce chemokines and continue the local inflammatory response by forming chemotactic gradients to attract T cells and mononuclear phagocytes from the circulation and surrounding tissue.

Adult↗