Matrix metalloproteinase-12 is expressed in phagocytotic macrophages in active multiple sclerosis lesions

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Abstract

Matrix metalloproteinases (MMPs) are proteases involved in extracellular matrix (ECM) remodeling, leukocyte infiltration into lesions and myelin degradation in the central nervous system (CNS) disease multiple sclerosis (MS). We have investigated whether MMP-12 (macrophage metalloelastase) is expressed in MS lesions at various stages. In control patient tissue and (p)reactive MS lesions, only occasional microglial and astrocyte staining was detected. In contrast, in active demyelinating lesions, phagocytic macrophages were MMP-12 positive. A lower proportion of phagocytes was positive for MMP-12 in chronic active demyelinating lesions and inactive lesions. This suggests a role for MMP-12 during demyelination in MS.

Introduction

Matrix metalloproteinases (MMPs) are proteases involved in the remodeling of the extracellular matrix. More than 20 different members of this family have been identified so far, and not only can they can degrade a broad range of components of the extracellular matrix (ECM), such as collagens, laminins and fibronectins, but also cytokines and growth factors Chandler et al., 1997, DeClerck, 2000. Over the past few years, evidence has emerged for an involvement of MMPs in central nervous system (CNS) diseases such as multiple sclerosis (MS), human immunodeficiency virus associated dementia (HIVD), stroke and Alzheimer's disease (reviewed in Hartung and Kieseier, 2000, Yong et al., 2001).

MMPs may contribute to CNS disease in several ways. MMPs degrade numerous components of the basal lamina of cerebral vessels and are capable of causing blood–brain barrier leakage in animal models Anthony et al., 1998, Rosenberg et al., 1992. MMPs also aid in the migration of leukocytes across cerebral vessels and within the parenchyma Leppert et al., 1995, Lou et al., 1999, Stuve et al., 1996. Furthermore, MMPs contribute to neuronal cell death and axonal damage Vos et al., 2000, Newman et al., 2001. MMPs may also play a role in demyelinating diseases such as MS, as there is in vitro evidence that several MMPs, including MMP-12 (macrophage metalloelastase), degrade myelin basic protein, one of the major proteins in the myelin sheets of oligodendrocytes (Chandler et al., 1996).

MMP-12 resembles other members of the MMP family in that it contains a pro-peptide region, a catalytic domain and C-terminal hemopexin like domain. Shapiro et al. (1993) showed that MMP-12 is expressed in human alveolar macrophages, but since then MMP-12 production has also been shown in other cell types and organs, e.g. in brain tumors (Kachra et al., 1999) and placenta (Belaaouaj et al., 1995). MMP-12 is secreted as a proform, but the MMP-12 protein (approximately 52 kDa) can undergo rapid NH2 and COOH terminal processing to a fully mature and active form of approximately 22 kDa. The active form has been reported to cleave a wide variety of substrates including elastin, collagen type IV, laminin, myelin basic protein (MBP), and to release TNF from a pro-TNF fusion peptide in vitro Chandler et al., 1996, Gronski et al., 1997. MMP-12 has been implicated in various diseases, including smoke-induced lung emphysema, in which it seems to play a pivotal role in the degradation of the basal lamina and infiltration of macrophages that lead to emphysema (Hautamaki et al., 1997).

It is unknown whether MMP-12 is involved in MS pathology. However, MMP-12 is found in neuroinflammatory lesions in the widely used animal model for MS, experimental allergic encephalomyelitis (EAE), in particular in foamy macrophages in demyelinating lesions Anthony et al., 1998, Pagenstecher et al., 1998. Involvement of other MMP members in MS has been demonstrated, for examples MMP-9 levels in CSF of MS patients are higher than in control patients Gijbels et al., 1992, Leppert et al., 1998, Waubant et al., 1999. Further MMP-2, -3, -7 and -9 have been localized to cells in MS lesions in immunohistochemical studies, most notably to phagocytic macrophages Anthony et al., 1997, Cossins et al., 1997, Cuzner et al., 1996, Maeda and Sobel, 1996.

In the present study, we examined whether MMP-12 is expressed in MS lesions. By using immunohistochemistry, we studied MMP-12 expression in a range of different MS lesions stages, from the (p)reactive stage to chronic inactive lesions De Groot et al., 2001, Van der Valk and De Groot, 2000. Our results indicate that, whereas in earlier and later MS lesion stages MMP-12 immunoreactivity is limited, in active demyelinating MS lesions foamy macrophages are MMP-12 positive, which suggests a role for MMP-12 during leukocyte migration into MS lesions or during demyelination.

Section snippets

Materials and methods

Human brain tissue was obtained at autopsy (with relatively short post-mortem intervals; see Table 1) from 22 MS cases and 7 cases without a history of brain disease. The autopsies were performed under the management of the Netherlands Brain Bank (NBB), Amsterdam (coordinator Dr. R. Ravid). The NBB protocol was approved by the Ethics committee of the VU Medical Center in Amsterdam. All patients and their next of kin had given written consent for autopsy, and for use of their brain and spinal

MMP-12 immunohistochemistry: positive controls

As a positive control for MMP-12 staining, normal placenta tissue was used. Fetal endothelial cells in the villi stained strongly for MMP-12, both when using the MMP-12 CAT (Fig. 1) and HPD antibody (not shown). Smooth muscle cells surrounding the endothelial cells are moderately positive. Trophoblasts and mesenchymal cells also showed immunoreactivity. The few macrophages within the placenta appeared mostly negative. The intervillous space was negative for both antibodies.

We also investigated

Discussion

This study has been the first to demonstrate the presence of MMP-12 protein in MS lesions, in particular in foamy macrophages in actively demyelinating lesions. Other CNS cells, such as astrocytes and microglia, were only occasionally positive for the MMP-12 antibodies. This is in accordance with findings of MMP-12 staining in several animal models for neuroinflammatory disease, including EAE (Pagenstecher et al., 1998) and experimental autoimmune neuritis (Hughes et al., 1998). These and other

Acknowledgments

We thank Dr. W. Kamphorst from the Department of Pathology, VU Medical Center, Amsterdam, for the neuropathological assessment of the brain tissue. We also thank the Netherlands Brain Bank team for providing the material for this study. We also thank Dr. C. Polman (department of Neurology, VU Medical Center, Amsterdam) for determining the clinical MS subtype diagnosis of the patients in this study. This study was supported by the Dutch foundation “Stichting Vrienden van MS Research,” grant

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