Matrix metalloproteinase expression during experimental autoimmune encephalomyelitis and effects of a combined matrix metalloproteinase and tumour necrosis factor-alpha 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. Matrilysin's 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.

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.

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-alpha (TNF-alpha). 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.