Siglec-9 is a novel leukocyte ligand for vascular adhesion protein-1 and can be used in PET imaging of inflammation and cancer

Abstract

Leukocyte migration to sites of inflammation is regulated by several endothelial adhesion molecules. Vascular adhesion protein-1 (VAP-1) is unique among the homing-associated molecules as it is both an enzyme that oxidizes primary amines and an adhesin. Although granulocytes can bind to endothelium via a VAP-1-dependent manner, the counter-receptor(s) on this leukocyte population is(are) not known. Here we used a phage display approach and identified Siglec-9 as a candidate ligand on granulocytes. The binding between Siglec-9 and VAP-1 was confirmed by in vitro and ex vivo adhesion assays. The interaction sites between VAP-1 and Siglec-9 were identified by molecular modeling and confirmed by further binding assays with mutated proteins. Although the binding takes place in the enzymatic groove of VAP-1, it is only partially dependent on the enzymatic activity of VAP-1. In positron emission tomography, the 68 Gallium-labeled peptide of Siglec-9 specifically detected VAP-1 in vasculature at sites of inflammation and cancer. Thus, the peptide binding to the enzymatic groove of VAP-1 can be used for imaging conditions, such as inflammation and cancer. (Blood. 2011;118(13):3725-3733) IntroductionLeukocyte migration from the blood into the nonlymphoid tissues is a hallmark of inflammation. Several molecules on the endothelial cell surface and their counter-receptors on leukocytes mediate a multistep adhesion cascade featuring tethering, rolling, activation, adhesion, crawling, and transmigration phases. 1,2 Vascular adhesion protein-1 (VAP-1/AOC3) is an endothelial cell molecule that is rapidly translocated from the intracellular storage granules to the endothelial cell surface on inflammation. It contributes to several steps in the extravasation cascade and controls trafficking of lymphocytes, granulocytes, and monocytes to sites of inflammation. VAP-1 has unique features distinct from other conventional adhesion molecules because, besides being an adhesin, it is also an enzyme. It catalyzes oxidative deamination of primary amines and produces hydrogen peroxide, aldehyde, and ammonium. 3 The end products of the enzymatic activity are highly potent inflammatory mediators and can up-regulate other adhesion molecules, such as E-and P-selectin, ICAM-1, and VCAM-1. 4,5 We recently found the first lymphocyte ligand for VAP-1, Siglec-10. 6 It is expressed on B cells, monocytes, and eosinophils but is absent from granulocytes. 7 However, VAP-1 is also involved in granulocyte migration to sites of inflammation. This has been demonstrated in studies with acute inflammation models (peritonitis, lung, and air pouch inflammation) in mouse. In these studies, significant reduction in granulocyte migration to sites of inflammation was obtained with a function blocking anti-VAP-1 antibody and a small molecular inhibitor against VAP-1. [8][9][10] Contribution of VAP-1 both at the rolling and transmigration steps during leukocyte extravasation has been demonstrated, and the enzymatic activity of VAP-1 seems to be important in these proc...

Migration of lymphocytes into hepatic sinusoids

“…VAP-1 binds to Sialic Acid-binding Immunoglobulin-like Lectin (Siglec) molecules; T cell and NK cell adhesion acts via Siglec-10, while granulocyte adhesion to VAP-1 is mediated by Siglec-9 [3,4], as well as the interaction of CD44 with hyaluronan [5]. Integrin-mediated adhesion receptors also play an important role in the localization of lymphocytes to the liver.…”

Multivalent Interactions of Human Primary Amine Oxidase with the V and C22 Domains of Sialic Acid-Binding Immunoglobulin-Like Lectin-9 Regulate Its Binding and Amine Oxidase Activity

“…Siglec-9 is also a leukocyte trafficking molecule and its expression is rapidly up-regulated on the leukocyte surface after inflammation stimuli [6]. Recently, we have identified Siglec-9 and Siglec-10 as counter receptors for human primary amine oxidase (hAOC3; also called vascular adhesion protein-1, VAP-1) on the endothelial cell surface [6, 7].…”

Gallium-labelled peptides for imaging of inflammation

“…2) have been labelled with 68 Ga and evaluated in VAP-1 knockout (KO) and VAP-1 KOTG mice (mouse VAP-1-deficient mice expressing human VAP-1 as a transgene on the endothelium under Tie-1 promoter) [15], and in different experimental models of inflammation, e.g. osteomyelitis, bone healing, sterile skin inflammation, as well as in tumour xenograft models [16][17][18][19][20][21]. This research is being done in cooperation with Prof. Sirpa Jalkanen (Turku, Finland) whose group has a strong background in the characterization of the VAP-1 molecule.…”

“…Using a phage display approach, the groups of Jalkanen and Roivainen recently discovered that Siglec-9 is a granulocyte ligand for VAP-1 and that PET with a 68 Ga-labelled peptide of Siglec-9 specifically detects VAP-1 in vasculature at sites of inflammation and cancer [20]. Although granulocytes can bind to endothelium in a VAP-1-dependent manner, the counter-receptor(s) of this leucocyte population were not previously known.…”