Potential roles of cytokines and chemokines in human intervertebral disc degeneration: interleukin-1 is a master regulator of catabolic processes.

OBJECTIVE: These studies investigated cytokine and chemokine receptor profiles in nucleus pulposus (NP) cells, and the effects of receptor stimulation on mRNA levels of extracellular matrix (ECM) components, degrading enzymes and cytokine and chemokine expression. METHOD: Immunohistochemistry (IHC) was performed to localise expression of CD4, CCR1, CXCR1 and CXCR2 in human NP tissue samples. Effects of cytokine and chemokine stimulation was performed to investigate effects related to ECM remodelling and modulation of cytokine and chemokine mRNA expression. RESULTS: IHC identified CD4, CCR1, CXCR1 and CXCR2 expression by NP cells. Differential expression profiles were observed for CD4 and CXCR2 in tissue samples from degenerate and infiltrated IVDs. In vitro stimulations of primary human NP cultures with IL-16, CCL2, CCL3, CCL7 or CXCL8 did not identify any modulatory effects on parameters associated with ECM remodelling or expression of other cytokines and chemokines. Conversely, IL-1 was seen to modulate ECM remodelling and expression of all other cytokines and chemokines investigated. CONCLUSION: This study demonstrates for the first time that NP cells express a number of cytokine and chemokine receptors and thus could respond in an autocrine or paracrine manner to cytokines and chemokines produced by NP cells, particularly during tissue degeneration. However, this study failed to demonstrate regulatory effects on ECM genes and degradative enzymes or other cytokines and chemokines for any target investigated, with the exception of IL-1. This suggests that IL-1 is a master regulator within the IVD and may exert regulatory potential over a plethora of other cytokines and chemokines.

Brevican and phosphacan expression and localization following transient middle cerebral artery occlusion in the rat.

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.

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.

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.

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.

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.

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.

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.

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.

Migration responses of human monocytic cell lines to alpha- and beta-chemokines.

The beta-chemokines monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 1 alpha (MIP-1alpha), MIP-1beta and regulated on activation, normal T cells, expressed and secreted (RANTES) induced the in vitro migration of the monocytic cell line MonoMac-6. MCP-1 exhibits the most potent chemotactic effect on this cell line while MIP-1alpha, RANTES and to a lesser extent MIP-1beta were more moderate inducers of cell migration. MonoMac-6 migration in response to chemokines was shown to be a chemotactic and not a chemokinetic response, which was inhibited by pertussis and cholera toxins suggesting a role for G proteins in chemokine receptor-mediated signalling in these cells; chemotaxis of MonoMac-6 cells in response to MCP-1 was abrogated by the addition of anti-MCP-1 antibody. The response of MonoMac-6 cells to the alpha-chemokines IL-8, IP-10, growth-related peptide (Gro) alpha and MIP-2beta was substantially weaker than to the beta-chemokines. MCP-1 caused an alteration in cellular morphology by increasing ruffling at the cell membrane and the number of cells exhibiting extended pseudopodia. The chemotactic response of MonoMac-6 cells to beta-chemokines was compared with less well-differentiated myelomonocytic cell lines. THP-1 showed a similar, but weaker response to the beta-chemokines while both HL60 and U937 failed to respond to any member of this subfamily when tested under the same conditions. These results suggest that the differentiation status of cells of monocytic lineage may affect their response to beta-chemokines.