Lymphocytes leave the blood using a sequential adhesion cascade. Vascular adhesion molecule-1 (VAP-1) is a surface-expressed endothelial glycoprotein, which belongs to a distinct subgroup of monoamine oxidases. We show here that catalytic activity of VAP-1 on primary endothelial cells directly regulates lymphocyte rolling under defined laminar shear. VAP-1 seems to bind to a primary amino group presented on the lymphocyte surface and oxidatively deaminate it in a reaction, which results in the formation of a transient covalent bond between the two cell types. Instead, soluble reaction products (aldehydes and hydrogen peroxide) are not needed for the VAP-1-dependent rolling. Enzymatic regulation of lymphocyte adhesion to endothelium provides a previously unrecognized rapid way of controlling the extravasation process.
Leukocyte migration from the blood to tissues is a prerequisite for normal immune responses. We produced mice deficient in an endothelial cell-surface oxidase (amine oxidase, copper containing-3 [AOC3], also known as vascular adhesion protein-1 [VAP-1]) and found that this enzyme is needed for leukocyte extravasation in vivo. Real-time imaging shows that AOC3 mediates slow rolling, firm adhesion, and transmigration of leukocytes in vessels at inflammatory sites and lymphoid tissues. Absence of AOC3 results in reduced lymphocyte homing into lymphoid organs and in attenuated inflammatory response in peritonitis. These data alter the paradigm of leukocyte extravasation cascade by providing the first physiological proof for the concept that endothelial cell surface enzymes regulate the development of inflammatory reactions in vivo and suggest that this enzyme should be useful as an anti-inflammatory target.
IntroductionWhite blood cells of the polymorphonuclear series (polymorphonuclear leukocytes [PMNs] or granulocytes) are practically absent from healthy tissues. Upon inflammation, however, rapid influx of PMNs from the blood into the affected tissue takes place. PMNs leave the blood mainly via postcapillary venules and they do not recirculate to any significant extent. Lymphocytes, on the other hand, not only exit at the sites of inflammation but also continuously recirculate between the blood and lymphoid tissues. The physiologic recirculation of lymphocytes is supported by specialized high endothelial venules in lymphoid tissues, and this leukocyte type returns back to the blood via the lymphatic vasculature. Monocytes, the third major leukocyte type, normally leave the blood at low levels in various tissues under normal conditions to replenish the tissue macrophage pools, and at high levels during the later phase of inflammation. The extravasation process of all leukocyte classes consists of a series of carefully controlled steps. [1][2][3] First, the leukocyte makes initial tethers with the luminal surface of the blood vessel and starts to roll along the endothelial lining. If it receives appropriate activation signals, it can adhere in a shear-resistant manner to the endothelium. Finally, the leukocyte transmigrates through the vascular wall and continues its odyssey toward the chemotactic inflammation-induced signals within the tissue. Multiple adhesion and signaling molecules act in concert to execute the emigration cascade. One of the endothelial molecules involved in lymphocyte trafficking is vascular adhesion protein-1 (VAP-1). 4 VAP-1 is a cell-surface enzyme belonging to a specific group of amine oxidases (semicarbazide-sensitive amine oxidases [SSAOs] Enzyme Commission 1.4.3.6) that catalyze oxidative deamination of primary amines. 5,6 Here we questioned what the possible mechanistic connection is between the adhesive and enzymatic activity of VAP-1. By using adenoviral constructs encoding native or mutated VAP-1, chemical inhibitors of SSAO activity, and function-blocking anti-VAP-1 monoclonal antibodies (mAbs) in in vitro flow chamber assays we were able to study the role of VAP-1 during PMN rolling, firm adhesion, and transmigration. Interestingly, VAP-1 primarily supported the transmigration step during the PMN extravasation process. Experiments with the SSAO inhibitors and VAP-1 mutants showed that the enzymatic activity of VAP-1 is a prerequisite for the adhesive function of this molecule. Finally, we showed in a rat model of acute inflammation that the new, specific, and potent SSAO inhibitor also blocks the recruitment of PMNs to affected tissue in vivo. These data show that VAP-1 functions in leukocyte adhesion in a step-wise fashion via separate antibody epitope-dependent and oxidation-dependent steps. These experiments are the first to show that an oxidative enzymatic reaction on the luminal surface of endothelial cells regulates PMN emigration in vitro and in vivo. Patients, materials, ...
IntroductionLymphocyte recirculation is essential for the immune defense. Lymphocytes exit from the blood through the vascular endothelium in small postcapillary veins. They then migrate in the tissue in search of foreign antigens. Lymphocytes are collected from the peripheral tissues back to systemic circulation via the lymphatic vasculature. In the periphery, lymphocytes penetrate into blindended lymphatic vessels present in virtually all organs and are then transported to lymph nodes and other organized lymphatic tissues via the afferent lymphatics. From the lymph nodes, lymphocytes continue their travel via the efferent lymphatic vessels back to the major veins. 1,2 The multistep adhesion cascade controlling lymphocyte extravasation from the blood consists of relatively well-understood phases of specific interactions between the leukocyte and the vascular endothelium that take place under laminar shear stress. 3 First, the blood-borne lymphocyte tethers and starts to roll on the endothelium. If it receives appropriate activation stimuli, it can firmly adhere to the endothelium and finally transmigrate through the vascular wall into the tissue. The adhesion and activation molecules involved in the early parts of the cascade have been studied for a long time (reviewed in Lowe 4 ; Kunkel and Butcher 5 ; and Shimizu et al 6 ), but the equally important molecules mediating the transmigration step started to attract attention only later. 7,8 In contrast to the vascular side, the mechanisms governing leukocyte traffic in and out from lymphatic vessels remain practically unknown. During the last few years, several molecular markers have been defined that allow the identification of these irregular and thin-walled vessels. [9][10][11][12][13][14] In terms of cell migration, however, only the expression of a chemokine receptor CCR7 has been hypothesized to contribute to the chemotactic migration of dendritic cells into the afferent lymph, 15 and sphingosine-1-phosphate receptors may control lymphocyte exit from the lymph node into the efferent lymphatics. 16,17 Mannose receptor 18 and common lymphatic endothelial and vascular endothelial receptor-1 (CLEVER-1) 19 are, to our knowledge, the only molecules directly shown to mediate lymphocyte adhesion to lymphatic vessels.CLEVER-1 is a large glycoprotein expressed in lymphatic vessels and on high endothelial venules (HEVs). 19 In frozen section binding assays, anti-CLEVER-1 monoclonal antibodies (mAbs) inhibit lymphocyte adhesion to HEVs and lymphatic vessels. CLEVER-1 is translated from a large approximately 7800-nucleotide mRNA. It contains 7 fasciclin domains, a proteoglycan link protein-like sequence, 22 epidermal growth factor (EGF)-like repeats, and 2 RGD motifs. 19 The same molecule has been independently characterized as stabilin-1 and FEEL-1. Stabilin-1 was found to be a fasciclin-like hyaluronan receptor homolog, but no function has been alluded to it. 20 FEEL-1, on the other hand, was reported to be a scavenging receptor that binds bacteria and endocytoses adv...
Leukocyte migration from the blood into tissues is pivotal in immune homeostasis and in inflammation. During the multistep extravasation cascade, endothelial selectins (P-and E-selectin) and vascular adhesion protein-1 (VAP-1), a cell-surfaceexpressed oxidase, are important in tethering and rolling. Here, we studied the signaling functions of the catalytic activity of VAP-1. Using human endothelial cells transfected with wild-type VAP-1 and an enzymatically inactive VAP-1 point mutant, we show that transcription and translation of E-and P-selectins are induced through the enzymatic activity of VAP-1. Moreover, use of VAP-1-deficient animals and VAP-1-deficient animals carrying the human VAP-1 as a transgene show a VAP-enzyme activity-dependent induction of P-selectin in vivo. Up-regulation of P-selectin was found both in high endothelial venules in lymphoid tissues and in flat-walled vessels in noninflamed tissues. VAP-1 activity in vivo led to in- IntroductionCoordinated function of the multistep leukocyte extravasation cascade is a prerequisite for leukocyte emigration from the blood into the tissue. Many adhesion and signaling molecules have well-established roles in this process. 1,2 On endothelial cells, selectins (P-selectin [CD62P] and E-selectin [CD62E]) mediate tethering of bloodborne cells to vascular endothelium, and the subsequent rolling along the endothelial lining in a sheardependent manner. 3 The rolling cells can be exposed to activating stimuli, such as chemokines, which can trigger firm, integrindependent adhesion of the leukocytes in the vessel. Finally, the leukocytes diapedese through the vessel wall using adhesion molecules from immunoglobulin and other superfamilies as well as local protease activity.In addition to these well-established interplayers, other molecules are involved in leukocyte trafficking. Among these, enzymes expressed on the cell surface that have their catalytic domains outside the plasma membrane (ectoenzymes) have emerging roles in leukocyte migration. 4 Vascular adhesion protein-1 (VAP-1, also known as amine oxidase copper containing-3 [AOC3]) is an ectoenzyme that belongs to the specific subgroup of oxidases known as semicarbazide-sensitive amine oxidases (SSAOs). 5,6 It catalyzes a reaction in which a primary amine is oxidatively deaminated into an aldehyde, and then hydrogen peroxide and ammonium are released. 7,8 VAP-1/SSAO is a bifunctional molecule that can support leukocyte adhesion under shear conditions via enzymeactivity-dependent and enzyme-activity-independent ways. 4 Monoclonal anti-VAP-1 antibodies that do not block its oxidase activity effectively block lymphocyte and granulocyte binding to endothelial cells in vitro and in vivo. Small-molecule SSAO enzyme inhibitors, on the other hand, are equally effective in perturbing leukocyte-endothelial contacts in vitro and in vivo. [9][10][11][12][13][14] The ability of the oxidase reaction to regulate the expression and/or function of other molecules involved in the emigration process is largely unknown.We h...
Changes in endothelial permeability are crucial in the pathogenesis of many diseases. Adenosine is one of the endogenous mediators controlling endothelial permeability under normal conditions, and an endothelial cell surface enzyme CD73 is a key regulator of adenosine production. Here we report that IFN-b is a novel inducer of CD73. We found that pretreatment with IFN-b dramatically improved the vascular barrier function in lungs after intestinal ischemia-reperfusion injury in wild-type animals in vivo. IFN-b had absolutely no protective effects in CD73-deficient mice, which suffered from more severe lung damage than wild-type mice, showing that IFN-b functions strictly in a CD73-dependent manner. Most importantly, IFN-b treatment initiated after the ischemic period almost completely inhibited vascular leakage during the reperfusion. IFN-b also induced the expression and activity of CD73 and concurrently decreased vascular permeability in cultured human pulmonary endothelial cells. These data show that induction of CD73 and improvement of vascular barrier are new mechanisms for the anti-inflammatory action of IFN-b. Moreover, IFN-b treatment may be useful in alleviating vascular leakage induced by ischemia-reperfusion injury.
Vascular adhesion protein-1 (VAP-1) is an endothelial adhesion molecule mediating leukocyte interactions with blood vessels during leukocyte extravasation. Molecularly VAP-1 is a cell-surface-expressed ecto-enzyme belonging to the group of semicarbazide-sensitive amine oxidases (SSAO; EC 2.4.6.3), which deaminate primary amines. Here we asked whether peptides displaying a suitable free amine group could be a substrate or inhibitor of SSAO and thus regulate VAP-1-mediated leukocyte adhesion. On the basis of a molecular model of VAP-1, we designed synthetic peptides that fit to the substrate channel of VAP-1. One of these lysine-containing peptides effectively inhibits VAP-1-dependent lymphocyte rolling and firm adhesion to primary endothelial cells under physiologically relevant shear conditions. The same peptide inhibits the SSAO activity of endothelial and recombinant VAP-1 in a selective and long-lasting manner. We also show that all enzymatically active VAP-1 is displayed on the cell surface. Our results suggest that, in addition to soluble amines, specific cell-surface-bound molecules containing free NH 2 groups in a suitable position may modulate the enzymatic activity of SSAO. Moreover, the inhibitory peptide diminishes leukocyte interactions with endothelial cells under conditions of shear, and thus it may be useful to treat inflammatory conditions.
VAP-1, an ecto-enzyme expressed on the surface of endothelial cells, is involved in leukocyte trafficking between the blood and tissues under physiological and pathological conditions. In this study, we used VAP-1-deficient mice to elucidate whether absence of VAP-1 alters the immune system under normal conditions and upon immunization and microbial challenge. We found that VAP-1-deficient mice display age-dependent paucity of lymphocytes, in the Peyer’s patches of the gut. IgA concentration in serum was also found to be lower in VAP-1−/− animals than in wild-type mice. Although there were slightly less CD11a on B and T cells isolated from VAP-1-deficient mice than on those from wild-type mice, there were no differences in the expression of gut-homing-associated adhesion molecules or chemokine receptors. Because anti-VAP-1 therapies are being developed for clinical use to treat inflammation, we determined the effect of VAP-1 deletion on useful immune responses. Oral immunization with OVA showed defective T and B cell responses in VAP-1-deficient mice. Antimicrobial immune responses against Staphylococcus aureus and coxsackie B4 virus were also affected by the absence of VAP-1. Importantly, when the function of VAP-1 was acutely neutralized using small molecule enzyme inhibitors and anti-VAP-1 Abs rather than by gene deletion, no significant impairment in antimicrobial control was detected. In conclusion, VAP-1-deficient mice have mild deviations in the mucosal immune system and therapeutic targeting of VAP-1 does not appear to cause a generalized increase in the risk of infection.
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