Role of the gelatinases, MMP-2 and MMP-9, in cellular transmigration of basement membranes. The project deals with strategic cleavage of novel substrates by the gelatinases that are required for generation of a chemotactic gradient in inflamed tissues.

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The aim is to investigate the mechanisms of action of the gelatinases, MMP-2 and MMP-9, in leukocyte migration across basement membranes (BMs). There is increasing data that the general digestion of ECM by MMPs suggested by in vitro studies does not occur in vivo. Rather, the advent of sophisticated mass spectrometry-based techniques has revealed more subtle roles for the MMPs in the modulation of the activity of growth factors, chemotactic factors, and cellular receptors. We propose here to investigate the role of MMP-2 and MMP-9 in T lymphocyte transmigration across BMs in vivo, using murine experimental autoimmune encephalomyelitis (EAE) as an experimental model. Previous studies using this model have precisely defined the steps involved in T lymphocyte migration across the post-capillary venules of the CNS, revealing that in addition to the endothelial monolayer and its BM, a second barrier exists, the parenchymal BM and astrocyte endfeet, penetration of which is associated with focal MMP-2/ MMP-9 activity. Only upon penetration of the parenchymal border do disease symptoms become apparent, indicating that this is a crucial disease-limiting step. Our work has shown that ablation of MMP-2 and MMP-9 in double knockout mice (DKO) results in resistance to EAE and absence of leukocyte 2 migration to the CNS. However, DKO mice are not immune deficient, and when MMP-2 and MMP- 9 are eliminated from leukocytes only, using bone marrow chimeric mice, EAE does develop, albeit with late onset and milder symptoms. This suggests two sources of MMPs in EAE, a leukocyte and a CNS-resident source, and that the latter is sufficient for penetration of the parenchymal border. Our previous studies identified macrophages as one source of the gelatinases in EAE, and have identified a substrate at the parenchymal border (dystroglycan). Recent data suggest the involvement of the CNS-resident-derived MMP-2 and MMP-9 in generation of a chemotactic gradient essential for attracting leukocytes into the brain parenchyma. We propose a new paradigm where leukocyte and CNS-resident sources of MMP-2/MMP-9 have different functions in T cell transmigration of BMs, and propose here to define these functions using the murine EAE model and with focus on the establishment of chemotactic gradients.