No abstract

Laminin , a major glycoprotein component of vessel basement membranes , is recognized by ␤ 1 -and ␤ 3 -integrins expressed on endothelial cells. To determine how endothelial cell integrins might function in multiple sclerosis (MS) lesions , integrin laminin receptors and laminin were analyzed in central nervous system samples from MS patients and controls by immunohistochemistry. In active MS lesions , endothelial cell VLA-6 and ␤ 1 subunits were decreased compared to controls whereas ␣ v subunit and VLA-1 were increased. In chronic inactive lesions ␤ 1 , VLA-6 and ␣ v were the same as controls but VLA-1 remained increased. ␣ 3 subunit was constant in all samples. By immunoelectron microscopy VLA-1 , VLA-6 , ␤ 1 , and laminin were distributed throughout endothelial cells; ␣ v was adjacent to and on luminal surfaces; ␣ v and VLA-1 were on intercellular junctions. These results indicate distinct regulation and functions of these integrins in different lesion stages. In active lesions decreased endothelial cell ␤ 1 /VLA-6 could result in their detachment from laminin thereby facilitating leukocyte transvascular migration and bloodbrain barrier breakdown. Laminins (Ln) are major glycoprotein components of extracellular matrix and vessel basement membranes (BM).1 Peptide sequences of Ln and of other extracellular matrix molecules are recognized by ␣ 1 ␤ 1 (VLA-1), ␣ 2 ␤ 1 (VLA-2), ␣ 3 ␤ 1 (VLA-3), ␣ 6 ␤ 1 (VLA-6), ␣ 6 ␤4, and ␣ v ␤ 3 integrins expressed on the surfaces of many cell types. 2,3Integrin-mediated recognition of extracellular matrix molecules results in intracellular signaling that affects a range of cell behaviors. 4 In endothelial cells these signals affect focal adhesions and cytoskeletal organization, ie, actin fiber assembly. Therefore, integrin-mediated endothelial cell recognition of Ln and other BM molecules may determine cell-to-cell adhesiveness and mediate behaviors such as spreading, retraction, polarization, and migration that are essential for the maintenance and normal functioning of blood vessels. 5-7Inflammatory cytokines such as interleukin-1 (IL-1), tumor necrosis factor-␣ (TNF-␣), interferon-␥ (IFN␥), and transforming growth factor-␤ (TGF␤), growth factors such as fibroblast growth factor, and reactive oxygen intermediates induce changes in the levels and surface distribution of endothelial cell integrins in vitro. 8 -13 In immune reactions these alterations likely affect endothelial cell recognition of BM molecules and result in contraction of the endothelial cells, producing defects or denudation of the vascular lining. Cytokines may also be bound by Ln 14 and influence extracellular matrix turnover. 15,16 Thus, both endothelial cell integrin expression and the BM may undergo numerous modifications over the course of cellular immune reactions. Indeed, in diverse inflammatory conditions alterations of endothelial cell integrin Ln receptor expression have been documented. [17][18][19][20] These studies suggest that endothelial cell integrin expression changes over time in complex patt...

Moraxella bovis, the causative agent of infectious bovine keratoconjunctivitis, exhibits several virulence factors, including pili, haemolysin, leukotoxin, and proteases. The pili are filamentous appendages which mediate bacterial adherence. Prior studies have shown that Q-piliated M. bovis Epp63 are more infectious and more pathogenic than I-piliated and non-piliated isogenic variants, suggesting that Q pili per se, or traits associated with Q-pilin expression, promote the early association of Q-piliated bacteria with bovine corneal tissue. In order to better evaluate the role of Q pili in M. bovis attachment, several M. bovis strains and a recombinant P. aeruginosa strain which elaborates M. bovis Q pili but not P. aeruginosa PAK pili, were evaluated using an in vitro corneal attachment assay. For each strain tested, piliated organisms attached better than non-piliated bacteria. M. bovis Epp63 Q-piliated bacteria adhered better than either the I-piliated or non-piliated isogenic variants. Finally, recombinant P. aeruginosa organisms elaborating M. bovis Q pili adhered better than the parent P. aeruginosa strain which did not produce M. bovis pili. These results indicate that the presence of pili, especially Q pili, enhances the attachment of bacteria to bovine cornea in vitro.

To elucidate mechanisms of endothelial cell (EC) dysfunction in CNS inflammatory responses and beneficial effects of interferon-beta (IFN-gamma) in multiple sclerosis (MS), we analyzed effects of individual and combinations of soluble inflammatory mediators on the intracellular localization of the EC tight junction-associated molecules zonula occludens-1 and -2 (ZO-1 and ZO-2) in human brain ECs. The cytoplasm in the majority of cells in control EC cultures was clear; ZO-1 and ZO-2 were localized peripherally near sites of cell contact and associated with submembranous cytoplasmic filaments. H2O2 induced reversible time- and concentration-dependent translocation of ZO-1 and ZO-2 to a random distribution within EC cytoplasm and retraction of EC borders. For low concentrations, these effects were accompanied by less prominent submembranous filaments but not by evidence of cytotoxicity, increased cell death or altered amounts of ZO-1. Tumor necrosis factor-beta induced similar alterations but interferon-y did not. Co-treatment with either cytokine increased H2O2 effects whereas IFN-beta reversed H2O2-induced effects. In control white matter samples, EC cytoplasm was clear and ZO-1 was located on cell borders. In inflammatory/demyelinating lesions, EC ZO-1 was diffuse, indicating that the alterations induced in vitro mimic those in active MS lesions. These findings suggest that in MS patients, IFN-beta treatment may counteract inflammatory mediator effects on CNS EC tight junction molecules, thereby preserving EC barrier function.