Ectoenzymes in leukocyte migration and their therapeutic potential.

Semin Immunopathol (2014) 36:163–176 DOI 10.1007/s00281-014-0417-9

REVIEW

Ectoenzymes in leukocyte migration and their therapeutic potential Marko Salmi & Sirpa Jalkanen

Received: 20 December 2013 / Accepted: 19 January 2014 / Published online: 18 March 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Inflammation causes or accompanies a huge variety of diseases. Migration of leukocytes from the blood into the tissues, in the tissues, and from the tissues to lymphatic vasculature is crucial in the formation and resolution of inflammatory infiltrates. In addition to classical adhesion and activation molecules, several other molecules are known to contribute to the leukocyte traffic. Several of them belong to ectoenzymes, which are cell surface molecules having catalytically active sites outside the cell. We will review here how several ectoenzymes present on leukocytes or endothelial cell surface function as adhesins and/or modulate the extravasation cascade through their enzymatic activities. Moreover, their therapeutic potential as immune modulators in different experimental inflammation models and in clinical trials will be discussed.

Keywords Ectoenzymes . Leukocytemigration . Endothelium . Inflammation

This article is a contribution to the special issue on New paradigms in leukocyte trafficking, lessons for therapeutics - Guest Editors: F. W. Luscinskas and B. A. Imhof M. Salmi : S. Jalkanen (*) MediCity Research Laboratory, University of Turku, Tykistökatu 6 A, FIN-20520 Turku, Finland e-mail: [email protected] M. Salmi Department of Medical Biochemistry and Genetics, University of Turku, Turku, Finland S. Jalkanen Department of Medical Microbiology and Immunology, University of Turku, Turku, Finland M. Salmi : S. Jalkanen National Institute for Health and Welfare, Turku, Finland

Abbreviations ADAM a disintegrin and metalloproteinase ADP adenine diphosphate AMP adenine monophosphate AOC3 amine oxidase copper-containing 3 ATP adenine triphosphate cADPR cyclic ADP ribose LFA lymphocyte function-associated antigen LPS lipopolysaccharide ICAM intercellular adhesion molecule IL interleukin JAM junctional adhesion molecule Mac macrophage antigen MAdCAM mucosal addressin cell adhesion molecule MT-1membrane bound matrix metalloproteinase 1 MMP NAAD(P) nicotinic acid adenine dinucleotide (phosphate) NAD(P) nicotinamide adenine dinucleotide (phosphate) NF-κB nuclear factor kappa-light-chain-enhancer of activated B cells SSAO semicarbazide sensitive amine oxidase TNF-α tumor necrosis factor-alpha VAP-1 vascular adhesion protein-1 VCAM vascular cell adhesion molecule

Introduction Ectoenzymes are cell surface molecules, which harbor their enzymatically active catalytic sites outside the plasma membrane [1–3]. It has been estimated that up to 4 % of cell surface molecules are ectoenzymes. They form a heterogeneous group of molecules with a range of different enzymatic activities and they serve multiple different functions in the body. Ectoenzymes also show redundancy in function. Thus, the same ectoenzymatic activity can be conferred by different individual molecules, and on the other hand, the same

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ectoenzyme protein may catalyze more than one enzymatic reaction. Moreover, several ectoenzymes have also receptorlike non-enzyme activity-dependent functions in addition to the catalytic activities. During the past three decades, several ectoenzymes have been shown to play a critical role in immune functions, including leukocyte migration. In the early days, the involvement of certain ectoenzymatic activity in leukocyte traffic was often inferred from studies using selective inhibitors, which nevertheless are not entirely specific. More recently, the use of monoclonal antibodies and, in particular, genedeficient mice has allowed dissection of the contribution of individual ectoenzyme molecules in this process. Leukocyte migration from the blood into the tissues under normal conditions and during inflammation follows a multistep extravasation cascade [4–9]. Initially, the blood-borne leukocytes display transient tethering and rolling contacts with the vascular endothelium. If suitable activation signals are available, the leukocytes adhere in a shear-resistant manner to the endothelium. Thereafter, they display intraluminal crawling behavior in search of permissive sites for emigration, and the trans-endothelial migration can then be accomplished either via peri-cellular or trans-cellular routes. The different steps of the extravasation cascade are known to be controlled at the molecular level by well-established adhesion and activation molecules, such as selectins, chemokines, and integrins, and their ligands/receptors on both leukocyte and endothelial cell surface [10–14] (Fig. 1). However, multiple other molecules, including ectoenzymes, are known to be involved at the different steps of the extravasation cascade [15]. Moreover, certain ectoenzymes modulate leukocyte migration in the tissue and, possibly, also their exit from the tissues via the lymphatic vasculature. Ectonucleotidases, such as CD39 and CD73 controlling purinergic signaling, nicotinamide adenine dinucleotide (NAD) metabolizing enzymes, such as CD38, and amine oxidases, such as vascular adhesion protein-1, have been most thoroughly studied in the context of leukocyte migration [15–18]. Other ectoenzymes known to modulate leukocyte migration by trimming cell surface receptors and chemotactic molecules are proteases and peptidases, such as the members of the disintegrin and metalloproteinase (ADAM) family (Fig. 1) [19]. Here, we will review the role of these ectoenzymes in leukocyte migration with a focus on the immunopathogenesis of different inflammatory conditions with an emphasis on the more recent advances. The emerging role of ectoenzymes as diagnostic and predictive targets and as targets for new anti-inflammatory drugs will also be discussed.

Vascular adhesion protein-1 Vascular adhesion protein-1 (VAP-1; encoded by the gene amine oxidase copper-containing 3, AOC3) is an ectoenzyme

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belonging to primary amine oxidases (also known as semicarbazide sensitive amine oxidases) [20, 21]. It catalyzes oxidative deamination of primary amines into aldehydes in a reaction, which also produces hydrogen peroxide and ammonium [22]. The identity of the most important physiological substrates of VAP-1 is still unresolved, but at least soluble amines formed during intermediary metabolism (such as methylamine and aminoacetone) and certain cell surface proteins carrying suitable free amine groups (such as Siglec-10) can serve as substrates [23, 24]. VAP-1 is a homodimer of two 90 kD subunits [25]. A relatively narrow channel lined by charged amino acids leads from the surface of the molecule to the catalytically active site, which is buried deep inside the molecule [26]. The substrate channel apparently physically constrains the bulkiness and charge of the amino group presenting molecules, which are able to form contacts with the catalytic center. Numbers of VAP-1 inhibitors have been developed, which show selectivity to VAP-1 when compared to other primary amine oxidases (diamine oxidase (AOC1) and retina-specific amine oxidase (AOC2)), to related copper-containing amine oxidases (such as lysyl oxidase) and to flavin-containing amine oxidases such as monoamine oxidases A and B [23]. Many of the early competitive inhibitors are carbonyl reactive substances like semicarbazide and hydroxylamine. More recently, mechanism-based inhibitors, such as BTT-2052, LJP 1586, and PXS-4861A, with improved specificity, potency, and kinetics have been developed [27–29]. VAP-1 is expressed on the vascular endothelium, smooth muscle cells, and adipocytes [30, 31]. In endothelial cells, it is mainly present in vesicles under resting conditions, whereas inflammatory stimuli in vitro and in vivo induce luminal VAP1 expression [32, 33]. Lymphatic endothelial cells are devoid of VAP-1. Siglec-10 and Siglec-9, sialic acid binding Igfamily proteins, on B cells and granulocytes/monocytes, respectively, have been identified as leukocyte counterreceptors for VAP-1 [2, 24]. Although adipocyte VAP-1 may contribute to cell migration by producing biologically active signaling molecules and controlling glucose uptake [34], the contribution of endothelial VAP-1 in leukocyte traffic is most thoroughly understood.

Deletion or inhibition of VAP-1 attenuates inflammatory reactions Mice lacking VAP-1 are grossly normal and show only minor alterations in constitutive lymphocyte homing to secondary lymphoid organs under normal conditions [35]. In contrast, in the absence of VAP-1, many inflammatory reactions are inhibited in several different in vivo models. Initially, we showed that TNF-α induced peritonitis is milder in VAP-1 deficient mice than in wild-type controls [35]. Anti-collagen


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Fig. 1 Ectoenzymes in the leukocyte extravasation cascade. Involvement of conventional homing-associated molecular families (in cyan) and ectoenzymes (in blue) at different steps mediating leukocyte–endothelial

cell interactions. The steps involved are judged from direct observations or inferred from the known contributions of the classical adhesion and activation molecules modulated by ectoenzymes

antibody induced arthritis, acute lung injury, and immune responses after oral vaccination are also attenuated in mice lacking VAP-1 [36–38]. VAP-1 inhibitors have been extensively used in different models of inflammation. They reduce the numbers of infiltrating leukocytes in carrageen-induced skin inflammation, lipopolysaccharide (LPS)-induced lung damage, sodium-dextran induced colitis, collagen-induced and anti-collagen antibody induced arthritis, peritonitis, allogenic liver transplantation, and cerebral ischemia–reperfusion models in mice and rats [29, 36, 39–44]. Notably, postponed VAP-1 inhibition has also been highly beneficial in reducing inflammation. For instance, in a relapsing EAE model, VAP-1 inhibition after the first relapse almost completely prevented the emergence of subsequent new relapses [40]. Similarly, in acute lung inflammation, PXS486 inhibitor initiated 4 h after LPS instillation markedly reduced neutrophil counts in the bronchoalveolar lavage fluid [29, 42]. Thus, VAP-1 inhibition apparently has anti-inflammatory potential in therapeutic settings mimicking the treatment of clinical inflammatory diseases. Together, these data also imply that the ectoenzymatic activity of VAP-1 is crucial for supporting leukocyte migration.

VAP-1 function has also been blocked by monoclonal antibodies, which do not inhibit the oxidase activity of the molecule. Function blocking antibodies have reduced the number of infiltrating leukocytes in multiple acute inflammatory models including peritonitis, liver rejection, and hepatitis, in which selective inhibition in liver homing of Th2 T cells was observed [45–47]. Anti-VAP-1 antibodies have also alleviated inflammation in chronic disease models. In a spontaneous mouse model of type 1 diabetes, for instance, anti-VAP1 mAbs inhibited infiltration of leukocytes into the pancreatic islets and progression to overt diabetes during a six-month therapy period [46]. Humanized and fully human function blocking anti-VAP-1 mAbs have also been produced, which will pave the way for clinical trials [33, 48]. VAP-1 is also involved in leukocyte migration in nonclassical models of inflammation. In tumors, in VAP-1deficient mice, a defect in migration of proangiogenic myeloid-derived suppressor cells into tumors was observed, which resulted in diminished neoangiogenesis and retarded tumor growth [49]. Similar results in primary and metastatic tumors were later obtained when tumor-bearing wild-type mice were treated with VAP-1 inhibitors [50–52]. Moreover,

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in diabetes and age-dependent macular degeneration, which represent non-malignant models of neoangiogenesis, VAP-1 inhibition by small molecule inhibitors attenuates vessel formation by reducing macrophage recruitment [53–56]. VAP-1 appears to play an important role in the development of fibrosis [57]. In liver fibrosis models, anti-VAP-1 antibodies significantly reduced the formation of connective tissue. Carbon tetrachloride induced liver fibrosis was also significantly more severe in wild-type animals than in VAP-1 deficient mice. In addition, in this murine disease model, VAP-1-dependent leukocyte migration appears to play a major role, inasmuch the inflammatory cell infiltrate, which proceeds induction of fibrosis, was reduced in the absence of functional VAP-1.

The mode of VAP-1 action in leukocyte migration VAP-1 contributes to leukocyte migration in both enzyme activity-independent and enzyme activity-dependent pathways. As mentioned earlier, anti-VAP-1 mAbs do not inhibit the catalytic activity of the molecule, and enzyme-inhibitors, on the other hand, do not affect the expression of the molecule [35, 39, 45]. Moreover, the oxidase activity of VAP-1 is entirely dependent on topaquinone modification of a single amino acid (tyrosine 471), and mutagenesis of this residue abolishes all catalytic activity of VAP-1 [22, 39]. In vitro experiments under flow conditions have shown that both enzyme inhibitors and function blocking antibodies inhibit leukocyte–endothelial cell interactions [39, 58]. Moreover, simultaneous inhibition with the enzyme inhibitors and antibodies does not provide any additional or synergistic effects. In vivo intravital microscopy has confirmed that VAP-1 mainly contributes to the rolling and transmigration steps of the extravasation cascade. Blocking of VAP-1 by mAbs or enzyme inhibitors, as well as genetic deletion of VAP-1 increased the rolling velocity and decreased the number of transmigrating leukocytes [35, 39, 58, 59]. Notably, knockin VAP-1 mice, in which an enzymatically dead point mutant of VAP-1 has been introduced to VAP-1 deficient mice, showed that the catalytic activity is necessary for VAP-1dependent extravasation in vivo [60]. The current hypothesis, thus, is that leukocytes first make VAP-1-dependent contacts with endothelium using epitopes that are blocked by anti-VAP-1 mAbs (Fig. 2). Thereafter, the same or another leukocyte surface molecule is used as a substrate for VAP-1. During the ensuing catalytic reaction, a covalent but transient Schiff base is formed between the substrate on leukocytes and enzyme on endothelial cells. After spontaneous cleavage of the bond, an aldehyde, hydrogen peroxide, and ammonium are formed. VAP-1-generated hydrogen peroxide is known to induce the expression of other endothelial adhesion molecules (P- and E-selectin,


intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1) and mucosal addressin cell adhesion molecule (MadCAM)-1), and chemotactic substances (interleukin (IL)-8) as well as activation of transcription factors such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) [61–63]. Thus, the VAP-1-dependent reaction supports both physical leukocyte–endothelial contacts and alters the microenvironment into a proinflammatory direction via signaling effects.

CD38 as an enzyme and receptor CD38 is an ectoenzyme expressed on most mononuclear leukocyte subtypes, including T and B cells and monocytes/ macrophages [17]. CD38 is a leukocyte activation marker, and its expression, therefore, varies depending on the developmental stage and activation status of the leukocyte. CD38 is a multifunctional enzyme involved in the metabolism of NAD [64, 65]. Crystal structure and mutagenesis studies have shown that a single enzymatically active site within the CD38 molecule harbors at least four distinct enzymatic activities. CD38 possesses an NAD cyclase activity by which it produces cyclic adenosine diphosphoribose (cADPR) from NAD+. Through the hydrolase activity, CD38 further generates adenosine diphosphoribose (ADPR) from cADPR (Fig. 3). Alternatively, CD38 can hydrolyze ADPR directly from NAD+. Moreover, in acidic conditions, in the presence of nicotinic acid, CD38 can catalyze formation of nicotinic acid adenine dinucleotide phosphate (NAADP+) from NADP+. All these reaction products, cADPR, ADPR, and NAADP, alter Ca2+ signaling in cells in an inositol-3-phosphate-independent manner by binding to their own receptors and ion channels. NAADP is the most potent second messenger identified to date involved in Ca2+ release, and it appears to act on acidic lysosome-like Ca2+ storages in addition to endoplasmic reticulum. Moreover, NAADP can also act like an extracellular pro-inflammatory molecule by its ability to serve as an agonist for a purinergic P2Y11 receptor [66]. Finally, CD38 can also further hydrolyze NAADP+ into ADP-ribose-2′-phosphate. NAD+ metabolism by CD38 (and CD157 see below) alters NAD+ levels outside cells, where NAD functions as a dangerassociated molecular pattern. In this context, it is notable that CD38 has been recently proposed to undergo alterations in the membrane orientation. Thus, a fraction of the total CD38 pool in lymphoid cells in vitro penetrates the membrane in type III orientation, in which the catalytically active part of the molecule faces the cytosol [67]. Moreover, CD38 may also be expressed in cytoplasm and nucleus of the cells. Finally, the enzymatic activity of CD38 is also partly controlled via another ectoenzymatic activity, since ADP-ribosyltransferase-2 (ART 2) can ADP-ribosylate, and thereby inactivate, CD38 at


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Fig. 2 VAP-1 in leukocyte–endothelial cell interaction. According to the current model, leukocytes first make VAP-1-dependent contacts with endothelium using epitopes of Siglec-9 on granulocytes and monocytes and Siglec-10 on lymphocytes or other as yet unknown ligands, which are blocked by anti-VAP-1 mAbs. Thereafter, the ligands, or other suitable leukocyte surface molecules, are used as a substrate for VAP-1. During the following catalytic reaction, a covalent but transient Schiff base is

formed between the substrate on leukocytes and VAP-1 enzyme on endothelial cells. After spontaneous cleavage of the bond, the ligand is modified by an aldehyde and hydrogen peroxide and ammonium are released. VAP-1-generated hydrogen peroxide is known to induce the expression of other endothelial adhesion molecules and chemotactic substances as well as activation of transcription factors

two distinct amino acids, which alter the cyclase and hydrolase activities differently [68]. CD38 also has enzyme activity-independent activities [64]. Central for leukocyte migration is the ability of CD38 to bind

to CD31 [69], which is a major endothelial adhesion molecule involved in transendothelial migration. Ligation of CD38 by CD31 triggers intracellular signals, which augment transmigration. The receptor function of CD38 is also regulated by several mechanisms. CD38 can be expressed on the cell surface as a monomer, but more commonly it exists as a dimer or multimer [70]. Moreover, CD38 is enriched in lipid enriched microdomains on the cell surface and it associates laterally with other proteins [71]. Both of these modulations affect the signals delivered by CD38 ligation.

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Migration Integrin activation Fig. 3 CD38 in leukocyte migration. NAD(P) released from stressed cells is catalyzed by CD38 to cyclic ADP-ribose (cADPR), ADPR, and/or NAADP. All these trigger the release of Ca2+ from different intracellular stores and/or influx of Ca2+ from extracellular space. Ca2+-mediated signaling then regulates migration of the cell towards certain chemokines and fMLP and, possibly, also the activation of integrins. Note that CD38 may also be expressed intracellularly

CD38 deficient mice display several abnormalities in leukocyte trafficking. Migration of CD38-deficient neutrophils towards chemotactic signals, which trigger receptors such as CXCR4, CCR7, and fMLP-receptors, is reduced [72, 73]. This is likely caused by the defective Ca2+ signaling required during chemotactic migration [74]. Moreover, migration of CD38-deficient dendritic cells from the skin to draining lymph nodes is suboptimal in the absence of CD38 due to defects in chemokine receptor-CD38 co-operation [75]. Interestingly, CD38 deficiency results in a selective reduction of neutrophil and inflammatory monocyte migration to spleen in Listeriainfected mice, whereas leukocyte traffic to other organs like liver and bone marrow remains comparable to wild-type mice

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[76]. CD38 deficiency also results in blunted leukocyte immigration to areas of post-ischemic brain damage and to joints in collagen-induced arthritis [77, 78]. However, absence of CD38 does not impair inflammatory cell recruitment in all models, e.g., in cytokine induced asthma [79], suggesting that the contribution of CD38 differs depending on the pathogenesis and tissue, which may be related to the triggering of unique chemotactic signals. Moreover, it is known that the activating stimuli triggered via CD38 by CD31 are defective in mice lacking CD38, which may contribute to impaired homing as well as to suboptimal lymphocyte proliferation and immune responses. Finally, it should be noted that CD38 may have other indirect effects on the immune system, since CD38 is a major regulator of oxytocin secretion and social behavior [80]. CD38 may also control adenosinergic signaling, since it can provide an alternative CD39-independent (see below) route for adenine monophosphate (AMP) production. Extracellular NAD can be converted to ADPR by CD38. Another ectoenzyme, CD203a with nucleotide pyrophosphatase/ phosphodiesterase activity, can then convert ADPR into AMP, from which immunoregulatory adenosine is finally produced by CD73 [81].


C

CD39

CD73

Fig. 4 Extracellular ATP metabolism regulates the inflammatory status. The key enzymes dephosphorylating ATP, ADP, and AMP, and those responsible for reverse phosphorylating reactions are indicated. The adenosine is degraded by adenosine deaminase, which associates with CD26. ADA=adenosine deaminase, ADO=adenosine, INO=inosine

CD157 in leukocyte traffic CD157 is a paralog of CD38 [82]. It is a GPI-linked ectoenzyme, which can produce ADPR and NAADP, and it also harbors weak ADPR cyclase activity. CD157 is expressed on myeloid and endothelial cells among other cell types. Ligation of CD157 also confers intracellular signals into leukocytes, which alter cell migration. In in vitro studies, CD157 has been shown to participate in monocyte adhesion to endothelial cells, as well as in leukocyte migration and transmigration through the endothelium [83, 84]. CD157 can alter cellular signaling involved in adhesion and migration by lateral associations on the plasma membrane with beta1- and beta2integrins, which are major adhesion molecules mediating the firm adhesion and transmigration steps [85].

Peptidases modify the homing-associated chemokines CD26 (dipeptidyl-peptidase IV) is abundantly expressed on several cell types. In the immune system both endothelial cells and leukocytes are CD26 positive. CD26 modulates leukocyte traffic via several mechanisms. CD26 serves as a docking site for adenosine deaminase and, thereby, controls purinergic signaling (see Fig. 4). CD26 also has a fundamental role in modulating leukocyte migration by truncating a wide variety of chemokines and other cytokines. Cleavage of N-terminal dipeptides

may either activate or inhibit the chemokines or even change their specificity. For example, CD26-dependent cleavage of CCL5 increases its activity, whereas removal of dipeptides from CXCL12 leads to loss of its function. There are also examples from collaborative activity of different peptidases. For instance, CD26 truncates CXCL11 and eliminates its capacity to act as a lymphocyte chemoattractant, and further processing of CXCL11 by CD13 aminopeptidase reduces its anti-angiogenic properties [86]. CD26 deficiency aggravates lung inflammation in both rats and mice. CD26 knockout mice have more severe arthritis and EAE than their wild-type controls [87–89]. In contrast, several pre-clinical experiments have shown beneficial effects of CD26 inhibitors on ischemia–reperfusion injuries in the heart and lungs (reviewed in [90]). Moreover, long-term treatment with high-affinity CD26 inhibitor, alogliptin, reduces atherosclerosis and inflammation by decreasing monocyte recruitment and chemotaxis in low-density lipoprotein receptor deficient mice [91]. Due to the wide variety of substrates of CD26, the consequences from targeting CD26 may vary depending on the disease and the organ affected. Future studies are, thus, needed to better dissect the contributions of different CD26 substrates in the pathophysiology of different diseases.


Proteases generate soluble adhesion molecules Low quantities of soluble homing-associated molecules are present in serum in physiological conditions. However, during inflammation and cancer, their concentrations may increase several fold and they may then actively regulate inflammatory processes. Soluble forms of adhesion molecules may serve as decoy competitors for the corresponding cell surface adhesion molecules. Alternatively, ligation of a counter-receptor by the soluble adhesion molecule may trigger pro-inflammatory signals in the receptor bearing cells. Thus, soluble forms may either inhibit or enhance the inflammatory response [92, 93]. Many of them are shed from the membrane bound forms by membrane bound matrix metalloproteinases or members of the ADAM family [19]. Certain adhesion receptors like Lselectin are physiologically released from the cell surface for optimal leukocyte–endothelial cell interactions to take place, while others are mainly shed in pathological conditions. Membrane type-1 matrix metalloproteinase (MT-1-MMP) is perhaps the most important member of the group of six membrane-bound MMPs in the context of leukocyte migration, since it cleaves central adhesion molecules such as CD44 and ICAM-1. Shedding of CD44 makes the cells more motile, and release of ICAM-1 regulates transmigration of the leukocytes [15, 94]. Besides modulating adhesion molecules, MT1-MMP can also inactivate chemokines such as CCL7 and CXCL12 by cleaving them [19]. ADAM8 (CD156a), ADAM10 (CD156c), and ADAM17 (TACE, CD156b) are the members of the ADAM family mostly responsible for shedding of homing-associated molecules from both leukocytes and endothelium. Each of them has a multitude of substrates, some of which are even shared. ADAM8 cleaves L-selectin and VCAM-1, while ADAM10 can cleave for example CD44, a membrane bound chemokine CX3CL1 and VE-cadherin. Shedding of CX3CL1 deliberates endothelium-bound leukocytes and facilitates their subsequent transmigration. The shedding of VE-cadherin also promotes leukocyte transmigration by increasing the permeability of the vasculature (reviewed in [95, 96]). ADAM17 is well-known for its activity to shed tumor necrosis factor-alpha (TNF-α). The activity of ADAM17 is negatively regulated by tetraspanin CD9 [97]. However, besides TNF-α, ADAM17 has several other substrates such as macrophage antigen (Mac)-1, ICAM-1, and junctional adhesion molecule A (JAM-A). Monocyte ADAM17 is thought to be responsible for the cleavage of Mac-1 that facilitates endothelial transmigration by accelerating the rate of diapedesis [98]. Together with ADAM10, ADAM17 generates soluble JAM-A that reduces neutrophil infiltration to sites of inflammation [92]. Conditional ADAM17 knockout mice lacking ADAM17 on leukocytes show more rapid leukocyte recruitment to sites of inflammation than wild-type mice and accelerated bacterial clearance [99]. On the other hand, mice with

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dramatically reduced ADAM17 levels in all tissues have eye, heart, and skin defects and increased susceptibility to intestinal inflammation demonstrating an important role for ADAM17 in controlling inflammation in several organ systems [100]. In summary, sheddases are, thus, pivotal in trimming both endothelial and leukocyte adhesion molecules and chemokines needed for optimal extravasation of the leukocytes from the blood into the tissues.

Extracellular ATP metabolism regulates inflammation Extracellular adenosine triphosphate (ATP) is degraded to adenine diphosphate (ADP), adenine monophosphate (AMP), and further to adenosine and inosine. ATP is highly inflammatory, ADP pro-thrombotic, and adenosine antiinflammatory. CD39 is mainly responsible for degrading ATP to ADP and further to AMP, while CD73 dephosphorylates AMP to adenosine [101, 102]. Kindlin-2, an integrin activator regulates the expression of both of these enzymes via a clathrin-dependent mechanism [103]. The enzymatic activity of CD39 (ATP disphosphohydrolase) and CD73 (ecto-5-nucleotidase) controls the inflammatory status and integrity of vasculature by determining the balance between pro-inflammatory and anti-inflammatory substances. The key players in the cascade are depicted in Fig. 4. ATP and ADP bind to P2X and P2Y members of the purino-receptor families. Adenosine, on the other hand, has four receptors (A1, A2A, A2B, and A3), from which A1, A2A, and A2B are evolutionarily conserved and share significant homology, whereas A3 vary markedly in different species. In addition, adenosine has receptor-independent effects, because it can diffuse through the cell membrane into the cytosol. CD39 is expressed on many cell types such as vascular endothelial cells and different leukocyte subtypes including dendritic cells, monocytes, lymphocytes, and neutrophils. CD39 decreases expression of CD11b/CD18. Lack of CD39 leads to increased trafficking of monocytes and neutrophils seen as exacerbated cerebral ischemic inflammation and acute lung injury in CD39 deficient mice [104, 105]. Paradoxically, CD39 knockout mice have attenuated allergic airway inflammation, which is thought to be connected to the limited capacity of CD39 negative dendritic cells to induce Th2 type immunity [106]. Besides blood vasculature, CD73 is expressed on afferent, but not on efferent lymphatic vessels, on a subset of lymphocytes including regulatory T cells and on certain epithelial cells. Expression of CD73 is regulated by several ways. CD73 promoter contains a cyclic adenosine monophosphate (cAMP) response element and hypoxia-inducible factor 1 (HIF1) binding site [107]. Interestingly, adenosine increases intracellular cAMP concentrations via the triggering of A2B

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receptor and further up-regulates CD73. Thus, CD73dependent adenosine production increases CD73 transcription in a form of a positive feedback loop. In addition, inflammation often renders tissues hypoxic and, thereby, induces local expression of the transcriptional regulator HIF1. Since the CD73 gene contains a HIF1-binding site, hypoxia can induce CD73 expression and concomitant adenosine signaling as well [101]. When leukocytes bind to vascular endothelium, enzymatic activity of endothelial CD73 is inhibited and the dephosphorylation cascade reverts to an opposite direction, in which different ectokinases utilize adenosine to produce AMP, ADP, and ATP (Fig. 4). The remaining adenosine is simultaneously degraded by adenosine deaminase resulting in low adenosine concentrations and, thus, helping the leukocytes extravasate from the blood into the tissues [108]. CD73-generated adenosine also diminishes the expression of E-selectin, ICAM-1, and VCAM-1 [109], which further contributes to the reduction of leukocyte–endothelial cell interaction and transmigration. The role of CD73 in leukocyte trafficking is clearly demonstrated in CD73 knockout mice. In normal conditions, they do not show marked aberrancies in leukocyte extravasation, but in inflammatory conditions lymphocyte homing to lymph nodes via high endothelial venules is significantly increased [110]. Similarly, in several other inflammation models, CD73 deficient mice have increased leukocyte adhesion to endothelium and massive leukocyte accumulation into the tissues due to their leaky vasculature [111–114]. The expression of CD73 protein and its catalytic activity on afferent lymphatics vessels is high. Surprisingly, however, on lymphatic endothelium, CD73 is apparently not involved in the regulation of permeability or leukocyte trafficking. This conclusion has been made based on the studies utilizing CD73 deficient mice, in which the wild-type lymphocytes home via the afferent lymphatics into the lymph nodes as efficiently as in the wild-type mice. Instead, CD73 on lymphocytes contributes to lymphocyte migration via the afferent lymphatic vessels as lymphocytes from CD73 knockout mice have diminished capacity to travel via the lymphatics in wild-type mice [115]. CD73 on regulatory T cells is thought to contribute to the suppressive function of these cells [116]. Moreover, it may also contribute to binding of these cells to vasculature. Engagement of CD73 on lymphocytes causes clustering of lymphocyte function-associated antigen (LFA)-1 (CD11a/CD18) that results in increased adhesiveness in lymphocytes to endothelial cells [117]. This is in line with the observation that CD73 knockout mice have a reduced number of intra-tumoral regulatory T cells in a murine model of melanoma without alterations in total numbers of tumor infiltrating leukocytes. Reduced numbers of regulatory T cells in the tumors may help to tilt the balance of anti-tumor response to pro-inflammatory


and cytotoxic direction and, thereby, contribute to the retarded tumor growth seen in the absence of CD73 in multiple cancer models [118, 119].

Ectoenzymes as predictive and prognostic markers Proteolytic or phospholipase activity, or the usage of alternatively spliced messenger RNA (mRNA), can produce soluble forms from the membrane-bound ectoenzymes. Analyses of membrane bound and soluble forms of several ectoenzymes have turned out to have diagnostic, predictive, and prognostic value in many immune-related diseases. A soluble form of VAP-1 (sVAP-1) is constitutively produced and detected in plasma [93, 120]. It is enzymatically active and may, thus, modulate biological processes by competing with the cell surface bound VAP-1 and by exerting signaling functions through the catalytic end-products. sVAP1 is up-regulated in certain inflammatory diseases, such as type 1 and 2 diabetes, hepatitis, and stroke, but not in many other inflammatory conditions [121, 122]. In diabetes, VAP-1dependent formation of advanced glycation end-products, which represent non-enzymatic glycosylation reactions, may contribute to vascular damage seen in advanced disease forms [123–125]. Notably, increased levels of sVAP-1 is also an independent predictive marker of cardiovascular calcifications, adverse events, and mortality [126, 127], implying that increased VAP-1 expression/activity may be related to increased macrophage and lymphocyte traffic to vascular wall and oxidative damage typically seen in atherosclerotic lesions. In line with this scenario, transgenic overexpression of VAP-1 in vascular endothelium aggravates atherosclerotic lesions [128]. In malignancies, increased sVAP-1 levels have been reported to be independent prognostic markers in liver, gastric, and colorectal cancer [129–131]. Moreover, decreased expression of VAP-1 mRNA has been shown to correlate with more aggressive forms of prostate cancer [56]. CD38 has been widely used in immunophenotyping of chronic lymphocytic leukemia [132]. Increased CD38 expression is associated with aggressive forms of disease. CD38 has been proposed to alter the migration of transformed leukocytes and to favor their lodging to bone marrow and lymphoid tissues. This effect may be partly dependent on CD38–CD31 interaction and partly mediated via altered responses to chemotactic signals such as CXCL12 and CXCR4. CD38 induction may also play a harmful role in acute promyelogenic leukemias. Retinoic acid induced differentiation therapy, which is routinely used to differentiate the promyelogenic leukemia cells into more mature granulocytes, can induce CD38 expression on the differentiated cells [133]. The aberrantly expressed CD38 may then enhance homing of the leukemic cells to the lungs and activate them, which can contribute to the development of inflammatory retinoic acid


syndrome. CD38 is also found in a soluble form and in exosomes in the blood, but the correlation of these forms to pathologies have not been analyzed in detail [134, 135]. High expression of CD73 is variably reported to be a poor [136, 137] or good [138] prognostic marker in different cancers, which probably reflects different contributions of CD73 on different (normal vs. tumor) cell types. In hematological malignancies, low CD39 activity has been correlated with worsening of the disease in chronic lymphatic leukemia [139]. Low soluble CD73 activity in serum also serves as an independent negative prognostic factor in acute pancreatitis (our unpublished results).

Potential clinical use of ectoenzymes as drug targets The multifunctional nature of VAP-1/semicarbazide sensitive amine oxidase (SSAO) mediating leukocyte traffic to sites of inflammation, inducing other adhesion molecules, and causing endothelial cell damage via formation of advanced glycation end-products makes VAP-1 a potential drug target. Supported by encouraging results from experimental models (see above), clinical trials with VAP-1 blockade have been launched. VAP-1 has been targeted with a mouse monoclonal and, later, with fully human monoclonal antibodies in skin inflammations, ulcerative colitis, and rheumatoid arthritis. Anti-VAP-1 antibodies have been shown to be safe without any serious side effects [140]. In rheumatoid arthritis patients, repeated administration of an anti-VAP-1 antibody has alleviated inflammatory symptoms, which is the first clinical evidence suggestive of potential therapeutic effects http://www. biotie.com/en/product_and_development/idevelopment_ pipeline/vap1_antibody. Other potential diseases to target are a wide variety of liver inflammations, in which VAP-1mediated leukocyte–endothelial cell interactions are important [141]. Although multiple VAP-1 selective enzyme inhibitors have been developed (see above), none of them has yet entered clinical trials. A multitude of studies using CD73 knockout animals have unambiguously shown that CD73-dependent adenosine production is necessary to maintain endothelial cell integrity and its absence results in severe inflammations in various organ systems [111–114]. Therefore, up-regulation of endogenous CD73 would serve as an option to alleviate inflammation. Acute lung inflammation/acute respiratory distress syndrome (ARDS) is a life-threatening condition without an efficient cure. This disease can occur, for example, subsequent to pneumonia, sepsis, influenza, or trauma and lethality is high—around 35–40 %. Encouraged by the preclinical studies using a mouse model for acute lung injury [113], phase I and II clinical studies in patients suffering from acute lung injury/ ARDS were just recently completed [142]. The patients received interferon-beta formulated for intravenous use for six

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consecutive days. During the therapy, their soluble CD73 concentration significantly increased, IL-6 and IL-8 concentrations decreased, and, most remarkably, the odds of mortality decreased 81 %. Thus, CD73 may be a key target to alleviate lung inflammation, although beneficial effects can most likely be seen in other organ systems such as in kidneys often affected in these patients as well. In addition, statins have been reported to increase CD73 expression and adenosine production. However, the effect seems to be transient as a four-month therapy with pravastatin did not increase CD73 activity in serum [143]. This can be explained by the inability of statins to induce new protein synthesis. Instead, they block Rho-GTPase-dependent endocytosis of CD73 that results in marked increase of CD73 expression on the cell surface [144]. Therefore, statins may only be suitable for short-term therapy as needed for example in preconditioning before major operations. Several agonists and antagonists of adenosine receptors are or have been in clinical trials targeting various diseases including inflammatory diseases, autoimmune disorders, and cancer [145]. As an example, A3 receptor agonist, CF101, treatment showed beneficial effects in 20 % of rheumatoid arthritis patients. Although the adenosine receptors are potential drug targets, their development for clinical use is challenging due to their complex and time-dependent expression and signaling patterns in different cell types. For example, high extracellular adenosine concentration is beneficial in ischemic injuries of different organs, whereas long-lasting elevated levels of adenosine may contribute to the development of fibrosis [146]. Moreover, other ectoenzymes are targets for drugs already available or in clinical trials. Even though they have not been principally developed to inhibit leukocyte migration, they may also have an effect on that due to the multifunctional properties of these ectoenzymes. Several drugs inhibiting the enzyme activity of CD26 are already widely used to treat patients suffering from type II diabetes. These drugs prevent CD26 to degrade incretin, a gastrointestinal hormone that increases insulin secretion from the pancreas [147]. Based on the wide variety of different CD26 targets including chemokines, it is highly likely that the CD26 inhibitors also modify migration/homing of leukocytes in the patients. Several clinical trials are ongoing to test whether CD26 inhibitors capable of attenuating inflammation and plaque development in experimental models of atherosclerosis can decrease cardiovascular events in patients with atherosclerosis (reviewed in [148]). Recently, CD26 inhibitor sitagliptin has been administered to patients suffering from hematological malignancies to enhance engraftment of cells from a single-unit umbilical cord blood. Systemic CD26 inhibition was well-tolerated and the results were encouraging. However, optimizing sitagliptin dosing to achieve more

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sustained CD26 inhibition is needed to improve outcome [149]. Human and humanized antibodies against CD38 have been produced to treat hematopoietic malignancies [150]. One of them, daratumumab, has already shown efficacy in phase II clinical trials in multiple myeloma patients, of which 90 % are highly CD38 positive. The antibodies are thought to exert their effects via complement-mediated killing and preventing multiple myeloma cell adhesion to bone marrow. Besides multiple myeloma, other hematopoietic malignancies expressing high amounts of CD38 are also potential diseases to target with anti-CD38 antibodies [151].

Conclusions Ectoenzymes have unequivocally been shown to contribute to leukocyte homing and migration in in vitro and in vivo settings. They are an interesting group of molecules which often harbor both receptor-like and catalytic functions. This dual nature makes them especially attractive targets for drug development. Before the full spectrum of ectoenzyme-mediated functions can be delineated, several hurdles have to be overcome. Firstly, the broad expression pattern of many of the ectoenzymes necessitates the use of cell-type specific knockout models in future in vivo studies. Secondly, the dual adhesin/enzyme nature of the molecules would be dramatically better understood, if pointmutants only affecting the catalytic activity would be analyzed side-by-side with the full knockout molecules. Thirdly, dissection of the contribution of multiple individual enzymatic activities carried within one enzyme molecule, and thereby the effect of the different endproducts, to the functional outcome is a formidable challenge in the future. Fourth, a more global view of ATP and NAD as extracellular danger signals and their complex and intermingled signaling cascades should be delineated. Fifth, while the role of certain ectoenzymes, such as VAP-1, has been mainly studied from the leukocyte migration point of view, most others have mainly been studied in other contexts. Therefore, more directed efforts are needed to separate ectoenzyme-mediated secondary effects in general signaling modules from primary involvement in leukocyte migration. On the other hand, the inflammation-focused studies should always also be interpreted from a more general perspective due to the inherent multifunctional nature of the ectoenzymes. Finally, the unique potential of targeting ectoenzymes with specific small molecule inhibitors and function-modulating antibodies should be fully exploited in inflammatory disease models and ultimately, hopefully, in clinical trials.


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