John M. Clements

Matrix metalloproteinases, tumor necrosis factor and multiple sclerosis: an overview

The matrix metalloproteinases (MMPs) are a family of at least 14 zinc-dependent enzymes which are known to degrade the protein components of extracellular matrix. In addition, MMPs and related enzymes can also process a number of cell surface cytokines, receptors, and other soluble proteins. In particular we have shown that the release of the pro-inflammatory cytokine, tumor necrosis factor-alpha, from its membrane-bound precursor is an MMP-dependent process. MMPs are expressed by the inflammatory cells which are associated with CNS lesions in animal models of multiple sclerosis (MS) and in tissue from patients with the disease. MMP expression will contribute to the tissue destruction and inflammation in MS. Drugs which inhibit MMP activity are effective in animal models of MS and may prove to be useful therapies in the clinic.

Matrix metalloproteinase expression during experimental autoimmune encephalomyelitis and effects of a combined matrix metalloproteinase and tumour necrosis factor-α inhibitor

Matrix metalloproteinases (MMPs) are a large family of Zn2+ endopeptidases that are expressed in inflammatory conditions and are capable of degrading connective tissue macromolecules. MMP-like enzymes are also involved in the processing of a variety of cell surface molecules including the pro-inflammatory cytokine TNF-alpha. MMPs and TNF-alpha have both been implicated in the pathology associated with neuro-inflammatory diseases (NIDs), particularly multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). We have shown that BB-1101, a broad spectrum hydroxamic acid-based combined inhibitor of MMP activity and TNF processing, reduces the clinical signs and weight loss in an acute EAE model in Lewis rats. However, little is known about which MMPs are involved in the neuroinflammatory process. In order to determine the optimum inhibitory profile for an MMP inhibitor in the treatment of NID, we investigated the profile of MMP expression and activity during EAE. The development of disease symptoms was associated with a 3-fold increase in MMP activity in the cerebrospinal fluid (CSF), which could be inhibited by treatment with BB-1101, and an increase in 92 kDa gelatinase activity detected by gelatin substrate zymography. Quantitative PCR analysis of normal and EAE spinal cord revealed the expression of at least seven MMPs. Of these, matrilysin showed the most significant change, being elevated over 500 fold with onset of clinical symptoms and peaking at maximum disease severity. Of the other six MMPs detected, 92 kDa gelatinase showed a modest 5 fold increase which peaked at the onset of clinical signs and then declined during the most severe phase of the disease. Matrilysin was localised by immunohistochemistry to the invading macrophages within the inflammatory lesions of the spinal cord. Matrilysins potent broad spectrum proteolytic activity and its localisation to inflammatory lesions in the CNS suggest this enzyme could be particularly involved in the pathological processes associated with neuro-inflammatory disease.

Matrix metalloproteinase expression in an experimentally-induced DTH model of multiple sclerosis in the rat CNS

In an experimentally-induced DTH model of MS, we examined mRNA and protein expression of a range of MMPs and of TNFalpha to establish the contribution that individual MMPs might make to the pathogenesis. In control rat brain, mRNA for all of the MMPs examined was detectable. However, by immunohistochemistry, only MMP-2 could be detected. In the DTH lesions, significant increases in the level of mRNA expression were observed for MMP-7, MMP-8, MMP-12, and TNFalpha. Where expression of MMP mRNA was increased, there was a corresponding increase in protein expression detected by immunohistochemistry. To determine whether the upregulated MMPs could invoke destructive events in the CNS, highly purified activated MMP-7, MMP-8, and MMP-9 were stereotaxically injected into the brain parenchyma. All provoked recruitment of leukocytes and BBB breakdown. In addition, MMPs 7 and 9 induced loss of myelin staining. In conclusion, specific MMPs are upregulated in DTH lesions; for the most part, measurement of mRNA was a predictor of increased protein expression. From our injections of MMPs, it is clear that the upregulated MMPs in the DTH lesions could participate in the disruption of the BBB, leukocyte recruitment, and tissue damage.

Enhanced expression of MMP-7 and MMP-9 in demyelinating multiple sclerosis lesions

The pathology of multiple sclerosis (MS) is characterised by breakdown of the blood-brain barrier accompanied by infiltration of macrophages and T cells into the central nervous system (CNS). Myelin is degraded and engulfed by the macrophages, producing lesions of demyelination. Some or all of these mechanisms might involve proteinases, and here we have studied the cellular localisation and distribution of two matrix metalloproteinases (MMPs), MMP-7 (matrilysin) and MMP-9 (92-kDa gelatinase), in the normal human CNS and active demyelinating MS lesions. Cryostat sections of CNS samples were immunostained with antisera to MMP-7 and MMP-9. In addition, non-radioactive in situ hybridisation (ISH) was performed using a digoxygenin-labelled riboprobe to detect the expression of MMP-7. MMP-7 immunoreactivity was weakly detected in microglial-like cells in normal brain tissue sections, and was very strong in parenchymal macrophages in active demyelinating MS lesions. This pattern of expression was confirmed using ISH. MMP-7 immunoreactivity was not detected in macrophages in spleen or tonsil, indicating that it is specifically induced in infiltrating macrophages in active demyelinating MS lesions. MMP-9 immunoreactivity was detected in a few small blood vessels in normal brain tissue sections, whereas many blood vessels stained positive in CNS tissue sections of active demyelinating MS lesions. The up-regulation of MMPs in MS may contribute to the pathology of the disease.

CXC chemokines generate age-related increases in neutrophil-mediated brain inflammation and blood-brain barrier breakdown.

Children are at greater risk than adults of permanent brain damage and mortality following head injury or infection [1-5]. Rodent models have demonstrated a window of susceptibility in young animals during which the brain parenchyma is at greater risk of acute neutrophil-mediated breakdown of the blood-brain barrier [6-7]. The exact mechanism of this age-related susceptibility to brain inflammation has yet to be defined, but animal models have revealed that the potent pro-inflammatory cytokine interleukin-1beta (IL-1beta) initiates an intense acute neutrophil-mediated inflammatory response in the brains of young rats and mice that is not seen in adults [6]. Here, we demonstrate the rapid induction of CXC chemokines (which contain a Cys-X-Cys motif), in particular the cytokine-induced neutrophil chemoattractant CINC-1, following the intracerebral administration of IL-1beta. The CXC chemokines produced a more intense neutrophil response in young rats than in adults. The IL-1beta-induced blood-brain barrier breakdown in young rats could be attenuated by an anti-CINC-1 neutralising antibody. These results show that the immature central nervous system (CNS) is dramatically more susceptible to the chemotactic effects of CXC chemokines. Blocking the CXC chemokine activity associated with brain inflammation inhibits neutrophil-mediated blood-brain barrier damage and represents a significant therapeutic possibility.

Quantitation of matrix metalloproteinases in cultured rat astrocytes using the polymerase chain reaction with a multi‐competitor cDNA standard

Matrix metalloproteinases (MMPs) are a family of Zn2+ endopeptidases that are expressed in many inflammatory conditions and that contribute to connective tissue breakdown and the release of the pro‐inflammatory cytokine tumour necrosis factor‐α (TNF‐α). There is emerging evidence that MMPs have a role in inflammatory disorders of the central nervous system (CNS) such as multiple sclerosis. However, little is known about the expression of MMPs by inflamed tissue within the CNS or by the glia, neurones, and leucocytes which participate in the inflammatory response. To address this issue we have developed a polymerase chain reaction (PCR)‐based method for the quantitation of rat MMP mRNA levels, which we have applied to astrocyte cultures with and without inflammatory stimulation. The technique relies on a competition reaction in which a synthetic standard cDNA is co‐amplified with the target cDNA in the same PCR reaction. Standard multi‐competitor cDNAs, containing priming sites for nine MMPs, and two housekeeping genes were constructed. We have shown that MMP activity is increased over three‐fold in neonatal rat astrocyte cultures following stimulation with lipopolysaccharide (LPS). At the mRNA level, MT‐MMP‐1, 72 kDa gelatinase, and stromelysin‐3 were constitutively expressed and unaffected by LPS treatment, whereas 92 kDa gelatinase, and stromelysin‐1 were strongly induced (1,000‐fold). Stromelysin‐2, rat collagenase, and macrophage metalloelastase were modestly upregulated by LPS treatment. Matrilysin was not expressed. This technique is suitable for quantifying MMP expression in the cells which contribute to inflammation in the CNS and could also be applied directly to tissue samples from animal models of disease.

Reduction of excitotoxicity and associated leukocyte recruitment by a broad-spectrum matrix metalloproteinase inhibitor.

An important step in the cascade leading to neuronal cell death is degradation of laminin and other components of the brain extracellular matrix by microglia‐derived proteases. Excitotoxic cell death of murine hippocampal neurones inu2003vivo can be prevented by inhibitors of tissue plasminogen activator (tPA) or by inhibitors of plasmin. Plasmin is a potent activator of the matrix metalloproteinases (MMPs), which are made by resident and recruited leukocytes following CNS injury. In this study, we show, using Taqman RT‐PCR, that MMP mRNAs, but not other calcium‐dependent proteases such as calpain mRNAs, are acutely up‐regulated after an excitotoxic challenge inu2003vivo. α2‐antiplasmin or BB‐3103, a broad‐spectrum inhibitor of the MMPs, co‐injected with kainic acid into the striatum, inhibits excitotoxic cell death in the rat striatum, and reduces both the number of recruited macrophages and the size of the lesion. We also show that leukocyte populations differentially express MMPs, which may account, in part, for the expression profile we observe in the challenged brain. Our results show that inhibition of the MMPs in the rat will prevent kainic acid‐induced cell death in the brain. These studies suggest that MMP inhibitors have therapeutic potential for use in stroke, and support the increasing evidence that microglial activation may contribute to neuronal cell death.

Different expression patterns of matrix metalloproteinases in inflammatory demyelinating diseases of the central and peripheral nervous system

101 A Comparative Study of MMP and TIMP-2 Production in Rat and Human Foetal CNSVaseular Endothdial Cell Lines K.A. Harkness,Royal Hallamshire Hospital, U.K., M.N. Woodroofe, Sheffield Hallam University, U.K., J.D. Sussman, G.A.B, Davies-Jones, RoyaIHallcu~sh~Hosp~tal, UX. 104 Interferon-b Induces Release of Soluble Adhesion Molecules from Human Cerebral Endothelial Cells B. Kallmann, V. Hum m el, K. Toyka, P, Rieckmann, Umver~ity of Wuerzburg, Germany