RESUMEN
Granulocyte extravasation from the blood into tissues is a prerequisite for a proper inflammatory response. It is regulated by a multistep adhesion cascade consisting of successive contacts between leukocyte surface receptors and their endothelial ligands on vessels. Vascular adhesion protein 1 (VAP-1) is an endothelial surface glycoprotein with two functions. It is an enzyme (monoamine oxidase) and an adhesion molecule for lymphocytes. Its function in binding of granulocytes or in leukocyte trafficking into sites of inflammation in vivo has remained unknown. Here we show that treatment of rabbits with anti-VAP-1 monoclonal antibodies abrogates approximately 70% of granulocyte extravasation into a site of an experimental inflammation. Using intravital microscopy, VAP-1 blockade is shown to increase the velocity of the rolling granulocytes and the frequency of their jerky skippings during the rolling. In addition, the number of firmly bound leukocytes decreased by 44% when VAP-1 was rendered nonfunctional. Our results suggest that VAP-1 functions as a molecular brake early in the adhesion cascade and consequently decreases the firm adherence; it may also directly influence the transmigration step. These data elucidate a new interplayer in the granulocyte extravasation process and provide a novel physiological function for a member of the monoamine oxidase family.
Asunto(s)
Amina Oxidasa (conteniendo Cobre)/fisiología , Moléculas de Adhesión Celular/fisiología , Endotelio Vascular/fisiología , Granulocitos/fisiología , Amina Oxidasa (conteniendo Cobre)/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Adhesión Celular , Moléculas de Adhesión Celular/inmunología , Comunicación Celular , Movimiento Celular , Granulocitos/efectos de los fármacos , Hemodinámica , Inflamación , Ratones , Microscopía por Video , Monoaminooxidasa/metabolismo , Peritonitis/fisiopatología , Peritonitis/prevención & control , Conejos , Sialoglicoproteínas/fisiologíaRESUMEN
Lymphocyte binding to vascular endothelium is a prerequisite for the movement of immune cells from the blood into lymphoid tissues and into sites of inflammation. Human vascular adhesion protein-1 (VAP-1) is an endothelial glycoprotein involved in this interaction. It also displays an enzymatic (monoamine oxidase) activity. Here we examined how recombinant human VAP-1 mediates lymphocyte binding using rotatory and flow chamber binding assays. VAP-1 cDNA transfected into an endothelial cell line, which does not bind lymphocytes, renders the cell line capable of binding lymphocytes in a shear-dependent manner. VAP-1 transfectants bound lymphocytes 5 times better than monocytes with a preference for T killer cells, and no specific granulocyte adherence was detectable. The binding is partially inhibited by anti-VAP-1 monoclonal antibodies or by blocking lymphocyte L-selectin and CD18 integrins, but not by inhibition of several other homing-associated molecules. In contrast, CD44 ligation on lymphocytes markedly upregulates their VAP-1-dependent adhesion, suggesting that the VAP-1 counterreceptor can be activated via CD44. The transfectant model also allowed us to perform detailed structure-function analyses of VAP-1. We show that the exposed integrin-binding motif RGD or the enzymatic activity is not indispensable for VAP-1-dependent adhesion. Together, these data show that VAP-1 can reconstitute the lymphocyte-endothelial adhesion cascade under shear and propose a critical role for VAP-1 in lymphocyte emigration from the blood.
Asunto(s)
Amina Oxidasa (conteniendo Cobre)/fisiología , Moléculas de Adhesión Celular/fisiología , Endotelio Vascular/fisiología , Linfocitos/fisiología , Amina Oxidasa (conteniendo Cobre)/genética , Amina Oxidasa (conteniendo Cobre)/inmunología , Secuencia de Aminoácidos/genética , Secuencia de Aminoácidos/fisiología , Anticuerpos Monoclonales/farmacología , Adhesión Celular/fisiología , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/inmunología , Endotelio Vascular/citología , Humanos , Receptores de Hialuranos/fisiología , Selectina L/fisiología , Antígeno-1 Asociado a Función de Linfocito/fisiología , Subgrupos Linfocitarios/fisiología , Estrés Mecánico , TransfecciónRESUMEN
Human vascular adhesion protein-1 (VAP-1) is a dual-function molecule with adhesive and enzymatic properties. In addition to synthesis in endothelial cells, where it mediates lymphocyte binding, VAP-1 is expressed in smooth muscle cells. Here we studied the expression, biochemical structure, and function of VAP-1 in muscle cells and compared it to those in endothelial cells. VAP-1 is expressed on the plasma membrane of all types of smooth muscle cells, but it is completely absent from cardiac and skeletal muscle cells. In tumors, VAP-1 is retained on all leiomyoma cells, whereas it is lost in half of leiomyosarcoma samples. In smooth muscle VAP-1 predominantly exists as a approximately 165-kd homodimeric glycoprotein, but a trimeric (approximately 250 kd) form of VAP-1 is also found. It contains N-linked oligosaccharide side chains and abundant sialic acid decorations. In comparison, in endothelial cells dimeric VAP-1 is larger, no trimeric forms are found, and VAP-1 does not have N-glycanase-sensitive oligosaccharides. Unlike endothelial VAP-1, VAP-1 localized on smooth muscle cells does not support binding of lymphocytes. Instead, it deaminates exogenous and endogenous primary amines. In conclusion, VAP-1 in smooth muscle cells is structurally and functionally distinct from VAP-1 present on endothelial cells.
Asunto(s)
Amina Oxidasa (conteniendo Cobre)/metabolismo , Moléculas de Adhesión Celular/metabolismo , Músculo Liso/metabolismo , Músculo Liso/patología , Amina Oxidasa (conteniendo Cobre)/análisis , Amina Oxidasa (conteniendo Cobre)/fisiología , Anticuerpos Monoclonales , Moléculas de Adhesión Celular/análisis , Moléculas de Adhesión Celular/fisiología , Línea Celular , Transformación Celular Neoplásica , Regulación hacia Abajo , Endotelio/metabolismo , Glicósido Hidrolasas/metabolismo , Humanos , Immunoblotting , Leiomiosarcoma/metabolismo , Linfocitos/metabolismo , Microscopía Inmunoelectrónica , Monoaminooxidasa/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Sialoglicoproteínas , Células Tumorales CultivadasRESUMEN
Reactive arthritis is usually a self-limiting polyarthritis which develops after certain gastrointestinal or urogenital infections. Microbial antigens found in the inflamed joints are thought to play a key role in the development of this disease. It is not known how antigens of the pathogenic organisms migrate from the mucosal tissues into the joints. The data presented here show that mononuclear phagocytes which mediate the dissemination of several intracellular pathogens acquire an enhanced capacity to bind to nonstimulated vascular endothelial cells after phagocytosis of Yersinia enterocolitica O:3, one of the causative organisms of reactive arthritis. The increased binding to previously nonstimulated endothelial cells was mediated by P-selectin, whose translocation to the endothelial cell surface was induced by monocytes with intracellular Yersinia bacteria. These results suggest that mononuclear phagocytes may be responsible for the dissemination of bacterial antigens and the initiation of the joint inflammation in reactive arthritis.
Asunto(s)
Endotelio Vascular/microbiología , Monocitos/fisiología , Selectina-P/metabolismo , Yersinia enterocolitica/fisiología , Antígenos CD18/metabolismo , Adhesión Celular , Células Cultivadas , Endotelio Vascular/citología , Antígeno HLA-B27 , Humanos , Integrina alfa4beta1 , Integrinas/metabolismo , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Monocitos/microbiología , Selectina-P/fisiología , Receptores de Fibronectina/metabolismo , Receptores Mensajeros de Linfocitos/metabolismo , SerotipificaciónRESUMEN
Interactions between lymphocyte surface receptors and their ligands on vascular endothelial cells regulate the exit of lymphocytes from the circulation. Distinct subsets of mononuclear cells bind to high endothelial venules (HEVs) in different lymphoid organs to a different extent, but the molecular mechanisms behind this selectivity have remained poorly characterized. Here we show that vascular adhesion protein-1 (VAP-1) mediates subtype-specific binding of CD8-positive T cells and natural killer cells to human endothelium. VAP-1-dependent, oligosaccharide-dependent peripheral lymph node (PLN) HEV adhesion under shear was independent of L-selectin, P-selectin glycoprotein ligand 1, and alpha4 integrins, the known lymphocyte receptors involved in the initial recognition of endothelial cells. PLN HEV adhesion was also critically dependent on peripheral lymph node vascular addressins (PNAds), but lymphocyte L-selectin was absolutely required for PNAd binding. Most lymphocytes relied on both PNAd and VAP-1 in HEV binding. The overlapping function of L-selectin ligands and VAP-1 in PLN introduces a new control point into the lymphocyte extravasation process. Finally, intravital microscopy revealed that VAP-1 is involved in initial interactions between human lymphocytes and endothelial cells in inflamed rabbit mesenterial venules in vivo. In conclusion, VAP-1 is a novel contact-initiating ligand that discriminates between different subpopulations of mononuclear cells and is an appealing target for selective modulation of adhesion of CD8- and CD16-positive effector cells.