Sirpa Jalkanen

Cell-surface enzymes in control of leukocyte trafficking

Leukocyte trafficking between the blood and the tissues is pivotal for normal immune responses. Cell-adhesion molecules (such as selectins and leukocyte integrins) and chemoattractants (such as chemokines) have well-established roles in supporting leukocyte exit from the blood. Emerging data now show that, for both leukocytes and endothelial cells, enzymatic reactions that are catalysed by cell-surface-expressed enzymes with catalytic domains outside the plasma membrane (known as ectoenzymes) also make crucial contributions to this process. Ectoenzymes can function physically as adhesion receptors and can regulate the recruitment of cells through their catalytic activities. Here, we provide new insights into how ectoenzymes — including nucleotidases, cyclases, ADP-ribosyltransferases, peptidases, proteases and oxidases — guide leukocyte traffic.

Vascular Adhesion Protein-1 Is Involved in Both Acute and Chronic Inflammation in the Mouse

Vascular adhesion protein-1 (VAP-1) is an endothelial molecule that possesses both adhesive and enzymatic properties in vitro. So far, however, elucidation of its in vivo function has suffered from the lack of function-blocking reagents that are suitable for use in animal models. In this work we produced monoclonal antibodies against murine VAP-1 and characterized them using in vitro binding assays. We then examined whether the antibodies could prevent leukocyte migration in in vivo inflammation models, including two acute models (peritonitis induced with proteose peptone and interleukin-1 and air pouch inflammation enhanced by CCL21) and one chronic model (autoimmune diabetes in nonobese diabetic mice). Antibodies 7-88 and 7-106 inhibited migration of granulocytes and monocytes in both acute models of inflammation. Strikingly, antibody 7-88 significantly prevented diabetes in a subset of nonobese diabetic mice. The results show for the first time that in mouse models of inflammation, VAP-1 mediates leukocyte trafficking to sites of inflammation and thus is a potential target for anti-inflammatory therapies.

Origins of Serum Semicarbazide-Sensitive Amine Oxidase

Semicarbazide-sensitive amine oxidases (SSAO) are enzymes that are capable of deaminating primary amines to produce aldehyde, ammonia, and hydrogen peroxide. This activity has been associated with vascular adhesion protein-1 (VAP-1) and is found in the serum, endothelium, adipose, and smooth muscle of mammals. Circulating SSAO activity is increased in congestive heart failure, diabetes, and inflammatory liver diseases. To investigate the origin of circulating SSAO activity, two transgenic mouse models were created with full-length human VAP-1 (hVAP-1) expressed on either endothelial (mTIEhVAP-1) or adipose tissues (aP2hVAP-1), with tie-1 and adipocyte P2 promoters, respectively. Under normal conditions a circulating form of hVAP-1 was found at high levels in the serum of mice with endothelium-specific expression and at low levels in the serum of mice with adipose specific expression. The level of circulating hVAP-1 in the transgenic mice varied with gender, transgene zygosity, diabetes, and fasting. Serum SSAO activity was absent from VAP-1 knockout mice and endothelial cell–specific expression of human VAP-1 restored SSAO activity to the serum of VAP-1 knockout mice. Together, these experiments show that in the mouse VAP-1 is the only source of serum SSAO, that under physiological conditions vascular endothelial cells can be a major source of circulating VAP-1 protein and SSAO, and that serum VAP-1 can originate from both endothelial cells and adipocytes during experimental diabetes. An increased endothelial cell capacity for lymphocyte binding and altered expression of redox-sensitive proteins was also associated with the mTIEhVAP-1 transgene.

Lymphocyte homing to the gut: attraction, adhesion, and commitment.

Summary:u2002 Lymphocytes continuously migrate from the blood into the intestine. Naive lymphocytes leave the blood through high endothelial venules in Peyers patches. During the multistep extravasation cascade, they sequentially roll on, firmly adhere to, and transmigrate through the endothelial layer using multiple adhesion molecules and chemotactic signals. In the organized lymphoid tissues of the gut, lymphocytes can become activated, if they meet their cognate antigens transported to Peyers patches through the gut epithelium. During activation and proliferation, the lymphocytes become imprinted by the local dendritic cells, so that after returning to systemic circulation via the efferent lymphatic vasculature, they preferentially home to lamina propria of the gut to execute their effector functions. In inflammation, the recirculation routes of lymphocytes are altered, and these may explain the pathogenesis of certain extra‐intestinal manifestations of gut infections and inflammatory bowel diseases. The increased knowledge on the mechanisms that regulate lymphocyte homing and imprinting has clear applicability in designing more effective vaccination regimens. A detailed understanding of the mucosal homing has recently led to the development of the first successful anti‐adhesive therapeutics in human.

The same endothelial receptor controls lymphocyte traffic both in vascular and lymphatic vessels

The mechanisms controlling the exit of lymphocytes from tissues via lymphatics are practically unknown. We have now identified a 270–300‐kDa molecule designated common lymphatic endothelial and vascular endothelial receptor‐1 (CLEVER‐1) on human lymphatic endothelium and high endothelial venules. We show that it mediates binding of lymphocytes both to high endothelial venules and to lymphatic vessels. Moreover, blocking of the function of CLEVER‐1 results in significant reduction of lymphocyte traffic in vivo. Notably, CLEVER‐1 is also an inducible vascular adhesion molecule for other classes of leukocytes at sites of inflammation in peripheral tissues. These findings suggest that CLEVER‐1 is involved in regulation of lymphocyte recirculation and migration of leukocytes to sites of inflammation and is a potential new target to control inflammation.

Clever‐1/Stabilin‐1 regulates lymphocyte migration within lymphatics and leukocyte entrance to sites of inflammation

Clever‐1/Stabilin‐1 is a scavenger receptor present on lymphatic and sinusoidal endothelium as well as on a subset of type II macrophages. It is also induced on vasculature at sites of inflammation. However, its in vivo function has remained practically unknown and this work addresses those unknown aspects. We demonstrate using in vivo models that Clever‐1/Stabilin‐1 mediates migration of T and B lymphocytes to the draining lymph nodes in vivo and identify the adhesive epitope of the Clever‐1/Stabilin‐1 molecule responsible for the interaction between lymphocytes and lymphatic endothelium. Moreover, we demonstrate that Ab blocking of Clever‐1/Stabilin‐1 efficiently inhibits peritonitis in mice by decreasing the entrance of granulocytes by 50%, while migration of monocytes and lymphocytes into the inflamed peritoneum is prevented almost completely. Despite efficient anti‐inflammatory activity the Ab therapy does not dramatically dampen immune responses against the bacterial and foreign protein Ag tested and bacterial clearance. These results indicate that anti‐Clever‐1/Stabilin‐1 treatment can target two different arms of the vasculature – traffic via lymphatics and inflamed blood vessels.

Blockade of Vascular Adhesion Protein-1 Inhibits Lymphocyte Infiltration in Rat Liver Allograft Rejection

Vascular adhesion protein-1 (VAP-1) has been shown to mediate lymphocyte adhesion to endothelia at sites of inflammation, but its functional role in vivo has not been tested in any rodent model. Here we report the effects of VAP-1 blockade on rat liver allograft rejection. BN recipients of PVG liver allografts (known to develop acute rejection by day 7) were treated with 2 mg/kg anti-VAP-1 (a new anti-rat VAP-1 mAb 174-5) or isotype-matched irrelevant antibody (NS1) every other day (n = 6/group) and one group with anti-VAP-1 2 mg/kg daily (n = 7). On day 7, samples were collected for transplant aspiration cytology, histology, and immunohistochemistry. Lymphocyte infiltration to the graft was clearly affected by VAP-blockade. The total inflammation, mainly the number of active lymphoid cells, in transplant aspiration cytology was significantly decreased in animals treated with anti-VAP-1 (4.7 +/- 1.0 and 2.4 +/- 1.0 corrected increment units, respectively) compared to control (6.6 +/- 1.0) (P < 0.05). In histology, the intensity of portal inflammation was significantly decreased (P < 0.05). The amount of T cells expressing activation markers diminished. This is the first demonstration in any prolonged in vivo model that VAP-1 plays an important role in lymphocyte infiltration to sites of inflammation, and, in particular, liver allograft rejection.

Loss of vascular adhesion protein-1 expression in intratumoral microvessels of human skin melanoma

Intratumoral vessels are different both structurally and phenotypically, and this may have clinical significance. In this study we analysed the expression of an adhesion molecule – vascular adhesion protein-1 (VAP-1) – in melanoma-associated blood vessels in 28 primary skin melanoma cases using immunocytochemistry and immunoelectron microscopy. We have found that VAP-1 protein expression is significantly decreased in intratumoral vessels compared with peritumoral ones; this difference was independent of the tumour thickness. Loss of VAP-1 protein expression occurred in both endothelial and smooth muscle cell components. Unlike in other cancer types, the VAP-1 protein expression of intratumoral vessels did not correlate with the density of cytotoxic T-lymphocytes or dendritic cells. On the other hand, the 5-year survival of melanoma patients with low VAP-1 protein expression in intratumoral blood vessels (≤25%) was lower (26.3%) than in patients whose VAP-1 expression was higher (42.6%, P=0.0632). These results support the idea that the phenotype of intratumoral blood vessels is important in the progression of malignant melanoma.

A Novel GGA-Binding Site Is Required for Intracellular Sorting Mediated by Stabilin-1

ABSTRACT Stabilin-1 is a unique scavenger receptor that combines endocytic and intracellular sorting functions in macrophages. Stabilin-1 mediates the endocytosis of acetylated low-density lipoprotein (acLDL), SPARC, and growth hormone family member placental lactogen (PL). At the same time, stabilin-1 is involved in trans-Golgi network-to-endosome routing of the endogenous chitinase-like protein SI-CLP (stabilin-interacting chitinase-like protein). A DDSLL motif in the cytoplasmic tail of stabilin-1 interacts with GGA adaptors; however, the deletion of DDSLL reduces but does not abrogate this interaction. Here, we identified a novel GGA-binding site, EDDADDD, in the cytoplasmic tail of stabilin-1. The deletion of EDDADDD impaired and the deletion of both the DDSLL and EDDADDD sites abrogated the interaction of stabilin-1 with GGAs. The surface exposure of stabilin-1 and stabilin-1-mediated endocytosis of acLDL, SPARC, and PL were not affected by the deletion either of DDSLL or EDDADDD or both. At the same time, both GGA-binding sites were necessary for the intracellular sorting of SI-CLP performed by stabilin-1. Our data indicate that the novel GGA-binding site EDDADDD is essential for stabilin-1-mediated intracellular sorting but is not required for endocytosis.

Vascular adhesion protein-1 (VAP-1)

In 1980s the leukocyte adhesion molecules and their ligands on the vascular endothelium were thought to explain tissue-selective, or even tissue-specific, leukocyte traffic. At the same time it became apparent that vessels in inflamed joints displayed binding characteristics clearly distinct from those in peripheral lymph nodes, gut and skin. In search of joint-selective endothelial adhesion molecules we therefore isolated vascular fragments from inflamed human synovial samples to raise monoclonal antibodies (mAbs) against endothelial antigens [1]. From this screen, we identified an mAb, 1B2, that readily stained synovial blood vessels and inhibited lymphocyte adhesion in classical frozen section binding assays. Differential screening against other known endothelial adhesion molecules allowed us to conclude that we had identified a new adhesion molecule. This antigen was named vascular adhesion protein-1 (VAP-1; nowadays also known by the gene name amine oxidase copper containing -3, AOC3).