Open in a separate window Figure 6 Effect of different anti-uPAR mAbs on leukocytic cell adhesion to endothelium

Open in a separate window Figure 6 Effect of different anti-uPAR mAbs on leukocytic cell adhesion to endothelium. indicate that 2 integrinCmediated leukocyteCendothelial cell interactions and recruitment to inflamed areas require the presence of uPAR and define a new phenotype for uPAR-deficient mice. Moreover, uPAR ligation differentially modulates leukocyte adhesion to endothelium and provides novel targets for therapeutic strategies in inflammation-related vascular pathologies. Keywords: leukocyte, endothelial cells, urokinase receptor, 2 integrin, inflammation Leukocyte activation and adhesion to the endothelium and the subsequent transendothelial migration are pivotal steps in the recruitment of cells to inflamed tissue. This highly coordinated multistep process requires tight regulation (1, 2). This includes the induction of genes coding for adhesion molecules and the modification of ligand-binding affinities of adhesion receptors on leukocytes as well as their change in avidity due to adhesion receptor clustering of leukocytes. Uncontrolled activation of leukocytes or endothelial cells leads to pathological chronic inflammation causing atherosclerosis, rheumatoid arthritis, and other disease states. The 2 2 integrin family of adhesion receptors consists of the four members, LFA-1 (L2, CD11a/CD18),1 Mac-1 (M2, CD11b/CD18, CR3), p150,95 (X2, CD11c/ CD18), and D2 (CD11d/CD18) (3, 4). In acute inflammation, LFA-1 and Mac-1 are the predominant 2 integrins mediating leukocyte adhesion to vascular endothelium. Mac-1 is constitutively expressed on neutrophils and monocytes, whereas LFA-1 is predominantly expressed on lymphocytes, but recent data underline its important contribution in neutrophil recruitment (5, 6). Leukocyte activation via cytokines, chemoattractants, or PMA induces both conformational changes in 2 integrins necessary for enhanced ligand recognition and translocation of Mac-1 to the cell surface (2, 7). Likewise, integrin activation is achieved extracellularly in vitro by the divalent cation Mn2+ (8). After activation, Mac-1 and LFA-1 firmly bind to intercellular adhesion molecule (ICAM)-1 (CD54) expressed on vascular endothelial (1) and smooth muscle cells (9). 2 integrins have been reported to form complexes with other plasma membrane proteins such as CD63 (10), the immunoglobulin receptor FcRIIIB (CD16b) (11) and the urokinase receptor (uPAR, CD87) (12, 13) suggesting possible functional interaction. uPAR consists of three homologous domains and is anchored to the plasma membrane by a glycolipid moiety that is susceptible to dissociation by phosphatidyl-inositolC specific phospholipase C (piPLC) (14). Defactinib Intact uPAR binds the protease uPA (urinary-type plasminogen activator) as well as the adhesive protein vitronectin with high affinity (15), and thereby plays a critical role in pericellular proteolysis and modulation of cellular contacts in adhesion and migration (16, 17). Although uPAR lacks its own transmembrane and cytoplasmic domain, uPA binding has been reported to transduce signals to the cell interior in leukocytes resulting in calcium mobilization (18), Defactinib protein kinase phosphorylation (12, 19C21), and other cellular effects (18, 21, 22). For some of these functions, it has been suggested that uPAR uses related transmembrane integrins as signal transduction devices (23). In fact, uPAR has been localized together with different integrins in focal adhesion areas (24), and increased uPAR expression Mouse monoclonal to CEA. CEA is synthesised during development in the fetal gut, and is reexpressed in increased amounts in intestinal carcinomas and several other tumors. Antibodies to CEA are useful in identifying the origin of various metastatic adenocarcinomas and in distinguishing pulmonary adenocarcinomas ,60 to 70% are CEA+) from pleural mesotheliomas ,rarely or weakly CEA+). and localization of the receptor to the leading edge of migrating monocytes appears to be essential for locomotion or invasiveness of cells independent of uPA activity (20, 25). It has been shown in vitro that uPAR crucially influences integrin function (26): the presence of uPAR inhibited 1 integrinC mediated cell binding to fibronectin, whereas uPAR favored 2 integrinCdependent monocyte adhesion to fibrinogen, one of the adhesive ligands of Mac-1. Moreover, using uPA antisense or ligation with uPA inhibited Mac-1 binding to and degradation of fibrinogen (27, 28). Based on these studies, uPAR has been proposed to form a functional unit with integrins on the cell surface. These relationships prompted us to investigate the contribution of uPAR in 2 integrinCmediated cell-to-cell interactions in vivo using uPAR knockout mice, as well as in vitro, to elute the mechanism of receptor cross-talk. Evidence is provided that uPAR is required for 2 integrinC dependent leukocyte adhesion and recruitment. Materials and Methods Materials Manganese chloride was obtained from (Munich, Germany) and PMA from (Paisley, Scotland). piPLC was from Oxford Glyco-Systems (Abingdon, UK). Intact recombinant soluble uPAR as well as the chymotrypsin-cleaved truncated form lacking Defactinib domain 1 were produced as previously described (29, 30) and were provided by Defactinib Dr. Niels Behrendt (Finsen Laboratory, Copenhagen, Denmark). uPA (Medac, Hamburg, Germany) was inactivated by diisopropyl-fluorophosphate (Serva, Heidelberg, Germany) as previously described (31). Antibodies The following mouse antiChuman uPAR mAbs were used in vitro. mAb no. 3936 (IgG2a-type), provided by Dr. Richard Hart (American Diagnostica, Greenwich, CT), is known to block uPA binding by recognizing an epitope of uPAR that has not been clearly identified yet (32). (Fab)2 fragments were generated using digestion by immobilized pepsin followed by protein ACSepharose affinity chromatography (< 0.05 was regarded as significant..