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.

目的是研究明胶酶，即基质金属蛋白酶-2和基质金属蛋白酶-9，在白细胞跨基底膜迁移中的作用机制。越来越多的数据表明，体外研究表明MMPs对ECM的一般消化不会发生在体内。相反，复杂的基于质谱学的技术的出现揭示了MMPs在调节生长因子、趋化因子和细胞受体的活性方面的更微妙的作用。本研究以小鼠实验性自身免疫性脑脊髓炎(EAE)为实验模型，研究了基质金属蛋白酶-2和基质金属蛋白酶-9在T淋巴细胞跨骨髓间充质干细胞迁移中的作用。以前使用该模型的研究精确地定义了T淋巴细胞跨CNS毛细血管后小静脉迁移的步骤，揭示了除了内皮单分子层及其BM之外，还存在第二个屏障，即实质BM和星形胶质细胞终末足，其穿透与局部的MMP2/MMP9活性有关。只有当穿透实质边界时，疾病症状才会变得明显，这表明这是关键的疾病限制步骤。我们的工作表明，在双基因敲除小鼠(DKO)中消融MMP2和MMP9导致了对EAE的抵抗，并且没有白细胞2向中枢神经系统迁移。然而，DKO小鼠并不存在免疫缺陷，使用骨髓嵌合小鼠，当只从白细胞中去除基质金属蛋白酶-2和基质金属蛋白酶-9时，确实会发生EAE，尽管发病较晚，症状较轻。这表明EAE中有两种来源的MMPs，一种是白细胞，另一种是中枢神经系统驻留的来源，后者足以穿透实质边界。我们以前的研究发现，巨噬细胞是EAE中明胶酶的来源之一，并在实质边界发现了一种底物(营养不良多糖)。最近的数据表明，中枢神经系统派生的基质金属蛋白酶-2和基质金属蛋白酶-9参与了趋化梯度的产生，这是吸引白细胞进入脑实质所必需的。我们提出了一种新的范式，即白细胞和中枢神经系统驻留的基质金属蛋白酶-2/基质金属蛋白酶-9来源在骨髓基质细胞T细胞移行中具有不同的功能，并建议使用小鼠EAE模型来定义这些功能，重点是建立趋化梯度。

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.