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Specific inhibition of the entry of encephalitogenic T lymphocytes into the central nervous system in multiple sclerosis would provide a means of inhibiting disease without compromising innate immune responses. We show here that targeting lymphocyte interactions with endothelial basement membrane laminins provides such a possibility. In mouse experimental autoimmune encephalomyelitis, T lymphocyte extravasation correlates with sites expressing laminin [alpha]4 and small amounts of laminin [alpha]5. In mice lacking laminin [alpha]4, laminin [alpha]5 is ubiquitously expressed along the vascular tree, resulting in marked and selective reduction of T lymphocyte infiltration into the brain and reduced disease susceptibility and severity. Vessel phenotype and immune response were not affected in these mice. Rather, laminin [alpha]5 directly inhibited integrin [alpha].sub.6][beta].sub.1] migration of T lymphocytes through laminin [alpha]4. The data indicate that T lymphocytes use mechanisms distinct from other immune cells to penetrate the endothelial basement membrane barrier, permitting specific targeting of this immune cell population., During leukocyte extravasation into inflamed tissues, cells quickly traverse the endothelial cell monolayer where they subsequently face the endothelial basement membrane. Previous data has shown that although Leukocyte transmigration of [...]

I. Overview of Endothelial Cell Development, Differentiation, and Maturation II. Expression of Endothelial Cell-Specific Extracellular Matrix Molecules During Endothelial Cell Development/maturation A. Basement membranes in general B. Laminins III. Differential [...]

Specific inhibition of the entry of encephalitogenic T lymphocytes into the central nervous system in multiple sclerosis would provide a means of inhibiting disease without compromising innate immune responses. We show here that targeting lymphocyte interactions with endothelial basement membrane laminins provides such a possibility. In mouse experimental autoimmune encephalomyelitis, T lymphocyte extravasation correlates with sites expressing laminin α4 and small amounts of laminin α5. In mice lacking laminin α4, laminin α5 is ubiquitously expressed along the vascular tree, resulting in marked and selective reduction of T lymphocyte infiltration into the brain and reduced disease susceptibility and severity. Vessel phenotype and immune response were not affected in these mice. Rather, laminin α5 directly inhibited integrin α6β1–mediated migration of T lymphocytes through laminin α4. The data indicate that T lymphocytes use mechanisms distinct from other immune cells to penetrate the endothelial basement membrane barrier, permitting specific targeting of this immune cell population. [ABSTRACT FROM AUTHOR]

The endothelial cell monolayer of cerebral vessels and its basement membrane (BM) are ensheathed by the astrocyte endfeet, the leptomeningeal cells, and their associated parenchymal BM, all of which contribute to establishment of the blood-brain barrier (BBB). As a consequence of this unique structure, leukocyte penetration of cerebral vessels is a multi-step event. In mouse experimental autoimmune encephalomyelitis (EAE), a widely used central nervous system inflammatory model, Ieukocytes first penetrate the endothelial cell monolayer and underlying BM using integrin β1-mediated processes, but mechanisms used to penetrate the second barrier defined by the parenchymal BM and gila limitans remain uninvestigated. We show here that macrophage-derived gelatinase (matrix metalloproteinasc [MMP]-2 and MMP-9) activity is crucial for leukocyte penetration of the parenchymal BM. Dystroglycan, a transmembrane receptor that anchors astrocyte endfeet to the parenchymal BM via high affinity interactions with laminins 1 and 2, perlecan and agrin, is identified as a specific substrate of MMP-2 and MMP-9. Ablation of both MMP-2 and MMP-9 in double knockout mice confers resistance to EAE by inhibiting dystroglycan cleavage and preventing leukocyte infiltration. This is the first description of selective in situ proteolytic damage of a BBB-specific molecule at sites of leukocyte infiltration. [ABSTRACT FROM AUTHOR]

Investigates the expression and function of laminins in the embryonic and mature vasculature. Formation of simple endothelial tubes via processes of vasculogenesis and angiogenesis; Recruitment of mural cells and differentiation into different vessel types; Details on the final and optimal pattern of the vascular network for an organ.

Endothelial cells express a 400-kDa or 240-kDa laminin α chain, depending on their tissue of origin or physiological state [1, 2]. Using differential display and subsequent screening of a mouse endothelial cell cDNA library we here identify the gene coding for the 240-kDa laminin chain as the laminin α4 gene. The complete mouse laminin α4 cDNA sequence is reported and compared with other laminin α chains. In situ hybridization of embryonic and new born mouse tissues revealed expression of laminin α4 mRNA in a subset of endothelium, in particular aortic endothelium, endocardium and endothelium of blood vessels in the skin and in the brain. Strong laminin α4 expression by aortic endothelia was confirmed by data obtained from cultured bovine aortic endothelial cells (BAEC). Isolation of laminin from BAEC conditioned medium revealed a Y-shaped molecule in rotary shadowing. Subsequent sequencing of BAEC laminin resulted in laminin α4, β1 and γ1 amino acid sequences, confirming that laminin α4 is one of the major laminin α chains expressed by aortic endothelium not only in the mouse. In addition, strong laminin α4 mRNA expression occurred in peripheral nerves, cardiac muscle, fat, the dermis of the skin and lung stroma of mouse tissues. The data demonstrate a cytokine and progesterone-regulated differential expression of laminin α4 mRNA in mouse endothelium, suggesting a distinct functional role for this laminin chain in endothelium. [ABSTRACT FROM AUTHOR]

Matrix metalloproteinases (MMPs) have been implicated in the cleavage of several proinflammatory chemokines, thereby mod-ulating their function and having an impact on the inflammatory process. However, in vivo evidence of such a role remains limited. In this study, we use IL-1β-induced peritonitis as a model for an acute immune response, which is initiated by neutrophil influx followed by macrophage infiltration within a few hours of IL-1β injection into the peritoneal cavity. Using single and double knockout mice for MMP-2 and MMP-9, we show that MMP-2 and MMP-9 act synergistically mainly at the initial step of neutrophil recruitment into the peritoneal cavity. The use of bone marrow chimeric mice revealed the cellular sources of MMP-2 and MMP-9 to be distinct, with resident cells being the source of the former and infiltrating leukocytes the source of the latter. We show that the omentum is the main site of neutrophil entry into the peritoneal cavity, where MMP-2 and MMP-9 act synergis-tically to potentiate the action of CXCL5 (ENA-78/ LIX), thereby, promoting neutrophil migration into the peritoneal cavity. To our knowledge, this is the first in vivo demonstration of MMP-2 and MMP-9 processing of a chemokine that has been directly correlated with an enhanced chemoattracting function. [ABSTRACT FROM AUTHOR]

A simplification of the laminin nomenclature is presented. Laminins are multidomain heterotrimers composed of alpha, beta and gamma chains. Previously, laminin trimers were numbered with Arabic numerals in the order discovered, that is laminins-1 to -5. We introduce a new identification system for a trimer using three Arabic numerals, based on the alpha, beta and gamma chain numbers. For example, the laminin with the chain composition alpha5beta1gamma1 is termed laminin-511, and not laminin-10. The current practice is also to mix two overlapping domain and module nomenclatures. Instead of the older Roman numeral nomenclature and mixed nomenclature, all modules are now called domains. Some domains are renamed or renumbered. Laminin epidermal growth factor-like (LE) domains are renumbered starting at the N-termini, to be consistent with general protein nomenclature. Domain IVb of alpha chains is named laminin 4a (L4a), domain IVa of alpha chains is named L4b, domain IV of gamma chains is named L4, and domain IV of beta chains is named laminin four (LF). The two coiled-coil domains I and II are now considered one laminin coiled-coil domain (LCC). The interruption in the coiled-coil of beta chains is named laminin beta-knob (Lbeta) domain. The chain origin of a domain is specified by the chain nomenclature, such as alpha1L4a. The abbreviation LM is suggested for laminin. Otherwise, the nomenclature remains unaltered.

Using gene array technology, we recently observed for the first time an up-regulation of laminin alpha4 chain in human gliomas. The data were validated by semiquantitative reverse transcription-PCR for RNA expression and immunohistochemistry for protein expression. Moreover, increase of the alpha4 chain-containing laminin-8 correlated with poor prognosis for patients with brain gliomas. Therefore, we hypothesized that inhibition of laminin-8 expression by a new generation of highly specific and stable antisense oligonucleotides (Morpholino) against chains of laminin-8 could slow or stop the spread of glioma and its recurrence and thus might be a promising approach for glioma therapy. We next sought to establish an in vitro model to test the feasibility of this approach and to optimize conditions for Morpholino treatment. To develop a model, we used human glioblastoma multiforme cell lines M059K and U-87MG cocultured with normal human brain microvascular endothelial cells (HBMVEC). Using Western blot analysis and immunohistochemistry, we confirmed that antisense treatment effectively blocked laminin-8 protein synthesis. Antisense oligonucleotides against both alpha4 and beta1 chains of laminin-8 were able to block significantly the invasion of cocultures through Matrigel. On average, the invasion was blocked by 62% in cocultures of U-87MG with HBMVEC and by 53% in cocultures of M059K with HBMVEC. The results show that laminin-8 may contribute to glioma progression and recurrence not only as part of the neovascularization process but also by directly increasing the invasive potential of tumor cells.