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MMP-2和MMP-9在白细胞跨基底膜（BM）迁移中的作用机制。越来越多的数据表明，体外研究表明的基质金属蛋白酶对ECM的一般消化在体内不会发生。相反，先进的质谱技术的出现揭示了MMPs在调节生长因子、趋化因子和细胞受体活性中的更微妙的作用。本文以小鼠实验性自身免疫性脑脊髓炎（EAE）为实验模型，探讨MMP-2和MMP-9在T淋巴细胞跨BM迁移中的作用。先前使用该模型的研究已经精确地定义了T淋巴细胞迁移穿过CNS毛细血管后小静脉的步骤，揭示了除了内皮单层及其BM之外，还存在第二屏障，实质BM和星形胶质细胞端足，其渗透与局灶性MMP-2/ MMP-9活性相关。只有在穿透实质边界时，疾病症状才变得明显，表明这是一个关键的疾病限制步骤。我们的工作表明，在双敲除小鼠（DKO）中MMP-2和MMP-9的消融导致对EAE的抗性和白细胞2向CNS迁移的缺乏。然而，DKO小鼠不是免疫缺陷的，并且当使用骨髓嵌合小鼠仅从白细胞中消除MMP-2和MMP- 9时，EAE确实发生，尽管具有迟发性和较轻的症状。这表明EAE中的MMPs有两个来源，白细胞和CNS驻留来源，后者足以穿透实质边界。我们以前的研究确定巨噬细胞作为EAE中明胶酶的一个来源，并确定了实质边界的底物（肌营养不良蛋白聚糖）。最近的数据表明CNS驻留衍生的MMP-2和MMP-9参与产生吸引白细胞进入脑实质所必需的趋化梯度。我们提出了一个新的范例，其中白细胞和CNS-居民来源的MMP-2/MMP-9有不同的功能，在T细胞transmigration的BM，并建议在这里定义这些功能，使用小鼠EAE模型，并专注于建立趋化梯度。

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.