(A) Immunofluorescence for CD45 and -dystroglycan in CNS sections of clodronate-treated mice at days 19 and 21 after MOG immunization and corresponding gelatin in situ zymographies (ZYM) coupled with staining for CD45 or pan-laminin reveal loss of -dystroglycan staining and gelatinase activity at the parenchymal border (arrows) only in brains of clodronate-liposomeCtreated mice at day 21. at sites of leukocyte infiltration. The migration of leukocytes through interstitial extracellular matrices has recently received considerable attention. Sophisticated in vitro assays using fibrous collagen matrices and three-dimensional investigation of leukocyte migration suggest a 1-integrinC and protease-independent mode of leukocyte movement within interstitial matrices (1). Although these studies are physiologically more relevant than studies of random migration on or through immobilized substrates, they do not reflect the complexity of the in vivo situation nor are they relevant to the specialized migration processes required Celastrol to cross basement membranes (BMs). The BM is the first barrier encountered by emigrating leukocytes subsequent to penetration of the vascular endothelial monolayer. Transmigration of this barrier remains difficult to investigate in vitro and the most physiological studies use in vivo inflammatory models (2, 3) or intravital approaches (4). BMs are tight assemblies of specialized extracellular matrix molecules. Together with the endothelial cell monolayer, the BM presents a barrier to the movement of proteins and cells across the blood vessel wall. Our work has shown that blood vessel endothelium has a specialized BM characterized by the presence of two laminin isoforms, laminins 8 and 10 (5). Studies by Karnovsky et al. were the first to demonstrate that central nervous system (CNS) vessels are particularly impermeable to the movement of small molecules and elucidated the ultrastructural basis of this bloodCbrain barrier (BBB) (6). Post-capillary venules in the CNS are ensheathed by a second BM known as the parenchymal BM, produced by the astrocytes and associated leptomeningeal cells (6), which is characterized by presence of laminins 1 and 2 (5). A similar differential expression of cellular receptors for extracellular matrix molecules at the endothelial and parenchymal borders also exists. In particular, dystroglycan is exclusively expressed on the astrocyte endfeet (5, 7, 8). Dystroglycan exists as an extracellular -subunit and a transmembrane -subunit, which are products of the same gene and result from posttranslation processing of the molecule (9). The -dystroglycan subunit is a receptor for several BM components of the parenchymal BM, including laminins 1 and 2, perlecan and agrin (10), as well as the extracellular neuronal component, neurexin (11), and is considered to anchor the astrocyte endfeet to the parenchymal BM. Collectively, the endothelial cell layer, astrocyte endfeet, and their associated BMs constitute the cellular BBB and defects in any one of these components compromises the barrier function of CNS vessels (11, 12). Using a mouse model of experimental autoimmune encephalomyelitis (EAE), we have shown that encephalitogenic T cells interact with the endothelial BM laminins, but not with the parenchymal BM laminins, despite having the cellular receptors capable of mediating such interactions (5). In the course of EAE, leukocytes accumulate in the perivascular space defined by the inner endothelial BM and the outer parenchymal BM, leading to focal leukocyte build up known as perivascular cuffs. Clinical symptoms, however, only become apparent after leukocyte penetration of the parenchymal BM. These results indicate the mechanism of leukocyte transmigration of the inner endothelial cell BM differs from that used to penetrate the parenchymal BM and that the latter is definitely a disease-relevant step. A delay in the onset of EAE symptoms has been observed in several mouse strains, some of which suggest a delay in the penetration of the outer parenchymal border. These include macrophage-depleted mice (13), TNF- KO mice (14), and L-selectin KO mice (15). In the macrophage-depleted mice, leukocyte transendothelial cell migration is not impeded, but rather deficiencies happen at the level of transmigration of the parenchymal BM and. MMPs have been extensively analyzed in multiple sclerosis and EAE, demonstrating activity of MMP-14/MMP-2 (16) and MMP-9 (22), and possibly also of MMP-7 (23) and MMP-8 (24). migration of leukocytes through interstitial extracellular matrices has recently received substantial attention. Sophisticated in vitro assays using fibrous collagen matrices and three-dimensional investigation of leukocyte migration suggest a 1-integrinC and protease-independent mode of leukocyte movement within interstitial matrices (1). Although these studies are physiologically more relevant than studies of random migration on or through immobilized substrates, they do not reflect the difficulty of the in vivo scenario nor are they relevant to the specialized migration processes required to mix basement membranes (BMs). The BM is the 1st barrier experienced by emigrating leukocytes subsequent to penetration of the vascular endothelial monolayer. Transmigration of this barrier remains difficult to investigate in vitro and the most physiological studies use in vivo inflammatory models (2, 3) or intravital methods (4). BMs are limited assemblies of specialized extracellular matrix molecules. Together with the endothelial cell monolayer, Rabbit Polyclonal to FCGR2A the BM presents a barrier to the movement of proteins Celastrol and cells across the blood vessel wall. Our work has shown that blood vessel endothelium has a specialised BM characterized by the presence of two laminin isoforms, laminins 8 and 10 (5). Studies by Karnovsky et al. were the first to demonstrate that central nervous system (CNS) vessels are particularly impermeable to the movement of small molecules and elucidated the ultrastructural basis of this bloodCbrain barrier (BBB) (6). Post-capillary venules in the CNS are ensheathed by a second BM known as the parenchymal BM, produced by the astrocytes and connected leptomeningeal cells (6), which is definitely characterized by presence of laminins 1 and 2 (5). A similar differential manifestation of Celastrol cellular receptors for extracellular matrix molecules in the endothelial and parenchymal borders also is present. In particular, dystroglycan is definitely specifically expressed within the astrocyte endfeet (5, 7, 8). Dystroglycan is present as an extracellular -subunit and a transmembrane -subunit, which are products of the same gene and result from posttranslation control of the molecule (9). The -dystroglycan subunit is definitely a receptor for a number of BM components of the parenchymal BM, including laminins 1 and 2, perlecan and agrin (10), as well as the extracellular neuronal component, neurexin (11), and is considered to anchor the astrocyte endfeet to Celastrol the parenchymal BM. Collectively, the endothelial cell coating, astrocyte endfeet, and their connected BMs constitute the cellular BBB and problems in any one of these parts compromises the barrier function of CNS vessels (11, 12). Using a mouse model of experimental autoimmune encephalomyelitis (EAE), we have demonstrated that encephalitogenic T cells interact with the endothelial BM laminins, but not with the parenchymal BM laminins, despite having the cellular receptors capable of mediating such relationships (5). In the course of EAE, leukocytes accumulate in the perivascular space defined from the inner endothelial BM and the outer parenchymal BM, leading to focal leukocyte build up known as perivascular cuffs. Clinical symptoms, however, only become apparent after leukocyte penetration of the parenchymal BM. These results indicate the mechanism of leukocyte transmigration of the inner endothelial cell BM differs from that used to penetrate the parenchymal BM and that the latter is definitely a disease-relevant step. A delay in the onset of EAE symptoms has been observed in several mouse strains, some of which suggest a delay in the penetration of the outer parenchymal border. These include macrophage-depleted mice (13), TNF- KO mice (14), and L-selectin KO mice (15). In the macrophage-depleted mice, leukocyte transendothelial cell migration is not impeded, but rather deficiencies happen at the level of transmigration of the parenchymal BM and the glia limitans, supporting the concept of a double barrier migration process (13). Passive transfer of encephalitogenic T cells in macrophage-depleted mice results in T cell build up in the perivascular cuff, suggesting that macrophages have a primary part associated with penetration of the parenchymal BM and infiltration of the CNS parenchyma (13). The initial transmigration of the endothelial monolayer requires expression of the adhesion molecule 4 integrin by leukocytes (3). Integrin 41 binds to vascular cell adhesion molecule-1 within the endothelial surface in inflamed vessels and induces matrix metalloproteinase-2 (MMP-2) manifestation in encephalitogenic T cells, which has been proposed to.