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REVIEWS CELLSURFACE ENZYMES IN CONTROL OF LEUKOCYTE TRAFFICKING Marko Salmi*‡§ and Sirpa Jalkanen*‡§ Abstract | 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. EXTRAVASATION CASCADE The multistep process during which a leukocyte migrates from the blood into the tissue through the blood-vessel wall. *MediCity Research Laboratory, University of Turku, Tykistökatu 6A, 20520 Turku, Finland. ‡ Department of Medical Microbiology, University of Turku, Kiinamyllynkatu 13, 20520 Turku, Finland. § Department of Bacterial and Inflammatory Diseases, National Public Health Institute, Tykistökatu 6A, 20520 Turku, Finland. Correspondence to M.S. e-mail: [email protected] doi:10.1038/nri1705 760 | O CTOBER 2005 Lymphocytes continuously circulate between the blood and lymphoid organs, and they, together with other types of leukocyte (such as polymorphonuclear leukocytes and monocytes), rapidly accumulate at sites of inflammation anywhere in the body1,2. Leukocyte trafficking is coordinated by sequential interactions between the leukocyte and vascular endothelial cells. During the EXTRAVASATION CASCADE, blood-borne leukocytes make initial tethering and rolling contacts with the vascular lining, then become activated and firmly adhere to the endothelial cells (FIG. 1; see Supplementary information S1 (movie)). Finally, leukocytes migrate through the vessel wall and undergo chemotaxis towards regions in the tissues, where they carry out their immune function. Many adhesion molecules belonging to the following families are crucial for securing leukocyte–endothelialcell interactions: the selectin family, including CD62L, CD62E and CD62P; the immunoglobulin superfamily, including platelet/endothelial cell-adhesion molecule (PECAM), vascular cell-adhesion molecule 1 (VCAM1) and intercellular adhesion molecule 1 (ICAM1); and the integrin family, including lymphocyte | VOLUME 5 function-associated antigen 1 (LFA1; CD11a–CD18) and very late antigen 4 (VLA4; CD49d–CD29)1–4 (FIG. 1). CHEMOKINES presented by endothelial glycoproteins and matrix molecules, and their G-protein-coupled serpentine receptors at the surface of leukocytes, are then thought to be crucial for the activation and chemotaxis steps5,6. However, in addition to these classical interactions, other molecules from different families are also involved in the different steps of the extravasation cascade. On molecular identification of these, several have, unexpectedly, turned out to be cell-surface-expressed ectoenzymes. Ectoenzymes Ectoenzymes are a large, diverse class of membrane proteins that have their catalytically active sites in the extracellular environment7,8 BOX 1. So, many cellsurface enzymes with well-established roles in cell migration do not belong to this group of enzymes, including receptor-type protein tyrosine kinases and phosphatases (which have their catalytic domains inside cells) and many matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator www.nature.com/reviews/immunol © 2005 Nature Publishing Group REVIEWS Tethering Activation Rolling Transmigration Chemotaxis Firm adhesion Blood Endothelium Tissue Selectins and mucins Integrins and immunoglobulinsuperfamily members Chemokines Chemokines CD73 CD73 CD26 VAP1 CD26 VAP1 Sheddases CD38 CD38 CD157? CD157? ART2? CD39? Autotaxin? CD39? Autotaxin? Figure 1 | Ectoenzymes and the leukocyte-extravasation cascade. The different phases of the multistep adhesion cascade that supports leukocyte exit from the blood into the tissues, and the cell-adhesion and activation molecules that contribute to this cascade, are shown in the top panel. The centre panel shows the main steps at which the bestcharacterized ectoenzymes in the cascade are involved. The bottom panel shows other ectoenzymes that might be involved in the cascade, as determined on the basis of substrate specificity and/or in vitro adhesion data, but the in vivo relevance and/or exact stage at which these ectoenzymes operate remains to be verified. The nucleotidases CD39 and CD73 regulate the balance of ATP and adenosine and the activation of leukocyte integrins and vascular adhesion molecules, as well as the permeability of endothelial cells. CD26 proteolytically modifies the activities of chemokines, and sheddases cleave leukocyte adhesion molecules, such as CD62L and CD44. CD38, CD157 and ART2 (ADPribosyltransferase 2) are involved in the metabolism of NAD and NADP, and they control signals that are triggered by chemokine-receptor engagement. (ART2 also post-translationally modifies adhesion molecules.) The endothelial-cell oxidase VAP1 (vascular adhesion protein 1) is involved at many steps by binding to leukocytes and by producing bioactive endproducts, such as hydrogen peroxide. Autotaxin is involved in the metabolism of extracellular nucleotides and bioactive lipids that can function during integrin activation and chemotaxis. CHEMOKINES Most chemokines are short, soluble peptides that bind serpentine receptors to trigger leukocyte activation and directed movement. Most chemokines belong to the CC-chemokine ligand (CCL) and CXC-chemokine ligand (CXCL) families, which are defined on the basis of their protein sequence. Chemokine receptors are named CC-chemokine receptor (CCR) and CXC-chemokine receptor (CXCR) depending on whether they bind mainly CCL or CXCL chemokines, respectively. (which are soluble enzymes that attach to other cellsurface molecules). The nomenclature for ectoenzymes is confusing. In addition to several original descriptive names, many of them also have CD designations given by immunologists and EC NUMBERS assigned by enzymologists TABLE 1. Here, for clarity, we use the CD designations when they are available, because they refer better to individual molecular species. Research on ectoenzymes has moved forward rapidly during the past few years. The cloning of ectoenzymes, which were previously defined on the basis of their biochemical activity, has shown that ectoenzymes often form families of molecules in which many proteins have indistinguishable or overlapping enzymatic activities9–12. Moreover, gene-targeted animals that lack ectoenzymes have recently been produced, and they have proved to be extremely useful for delineating the effects of individual enzymes in complex biological settings13–16. New data have uncovered several, often NATURE REVIEWS | IMMUNOLOGY unexpected, interactions between ectoenzymes and the adhesion and activation molecules that are involved in leukocyte trafficking. This article explores the multiple enzymatic-activitydependent and -independent roles in leukocyte migration of nucleotidases and related enzymes, ADP-ribosyl cyclases, ADP-ribosyltransferases, peptidases, proteases and oxidases that are expressed by leukocytes and endothelial cells TABLE 1. Nucleotidases and related enzymes — such as CD39, CD73 and autotaxin (a member of the CD203 family) — are involved in extracellular metabolism of ATP, and they regulate trafficking by modulating adenosine levels in the body. ADPribosyl cyclases — such as CD38 and CD157 — are important in chemotaxis. ADP-ribosyltransferases — such as ART2 (ADP-ribosyltransferase 2) — can post-translationally modify adhesion molecules. CD10, CD13, CD26, CD156b (also known as ADAM17) and MMPs are examples of peptidases and proteases that can inactivate or activate chemotactic molecules and adhesion molecules that are involved in leukocyte trafficking. Recently, an endothelial cell-surface oxidase, vascular adhesion protein 1 (VAP1; also known as AOC3), has also been shown to have enzymatic and non-enzymatic roles in leukocyte migration. Conceptually, the idea of ectoenzymatic control of leukocyte trafficking allows new insights into adhesive events and offers multiple novel targets for manipulating the movement of immune cells. Nucleotidases and related enzymes ATP is continuously released from cells into the extracellular space. This occurs physiologically through several modes of regulated transport, and additional ATP is released by lytic mechanisms from dying cells at sites of injury17. The binding of ATP and ADP to purinoceptors (that is, purinergic receptors) of the P2X and P2Y families has many pro-thrombotic and pro-inflammatory effects, including induction of cytokine secretion and dendritic cell (DC) activation7,17–20. Several ectoenzymes are involved in the hydrolysis of ATP to ADP then AMP and, finally, adenosine (FIG. 2). Adenosine binds purinoceptors of the P1 family and is an anti-inflammatory molecule21. CD39 is a nucleoside triphosphate diphosphohydrolase that sequentially converts extracellular ATP to AMP by way of ADP 22. It is expressed by many types of leukocyte, such as B cells, T cells and Langerhans cells, as well as by vascular endothelial cells. Migration of CD39-deficient monocytes or macrophages through an endothelial-cell monolayer towards ATP or CC-chemokine ligand 2 (CCL2) is impaired 23 . Moreover, ISCHAEMIAREPERFUSION INJURY causes an abnormally high increase in vascular permeability in the absence of CD39 REF. 24. Most strikingly, CD39-deficient mice show exacerbated leukocyte infiltration after exposure to chemicals that are skin irritants, and this is thought to result from decreased hydrolysis of the pro-inflammatory molecule ATP at the site of injury25. The role of CD39 in inflammation is complex, however, because other VOLUME 5 | O CTOBER 2005 | 761 © 2005 Nature Publishing Group REVIEWS Box 1 | What is an ectoenzyme? Ectoenzymes are membrane proteins that have their enzymatically active site outside the plasma membrane, in the extracellular environment. One of the first ectoenzymes to be identified was cholinesterase133, which degrades acetylcholine at neuronal synapses. Ectoenzymes can be classified according to their enzymatic activities134. Many of them are peptidases (that is, they clip off amino acids from the termini of peptides), proteases (which cleave proteins), hydrolases and nucleotidases (which hydrolyse extracellular nucleotides, NAD and NADP) or oxidases (which oxidize various substrates). Many ectoenzymes are type II integral membrane proteins with a short amino terminus in the cytosol or are glycosylphosphatidylinositol-linked molecules. Many ectoenzymes (such as CD26, CD38, CD73, autotaxin and vascular adhesion protein 1) are also found as soluble forms in biological fluids134. types of inflammatory stimulus lead to unaltered, or even attenuated, responses in CD39-deficient mice as a result of the impaired antigen-presenting functions of CD39-deficient DCs25. Of the ectoenzymes that are involved in purine metabolism, the function of CD73 in leukocyte trafficking has been studied the most extensively. It is a glycosylphosphatidylinositol (GPI)-linked cell-surface molecule that is expressed at high levels by vascular endothelial cells and by 5–15% of peripheral-blood lymphocytes. By contrast, granulocytes and monocytes lack this enzyme26,27. CD73 catalyses the dephosphorylation of AMP to adenosine28 (FIG. 2). Through activation of the adenosine receptors A2AR and A2BR at the surface of neutrophils, adenosine functions as an anti-adhesive signal for the binding of neutrophils to microvascular endothelial cells15,29. This is consistent with the findings that neutrophil–endothelial-cell interactions are inhibited by A2AR agonists and that inflammation is exacerbated in A2AR-deficient animals and in wild-type animals that have been treated with an A2AR antagonist21,30,31. The anti-adhesive functions of adenosine probably result from prevention of leukocyte activation (through inhibiting shedding of CD62L and inhibiting induction of expression of CD18-containing integrins)29. Adenosine also downregulates the expression of vascular adhesion molecules (that is, CD62E and VCAM1) 32. In addition, adenosine decreases leukocyte trafficking by inhibition of cytokine release from the endothelium32. Finally, adenosine Table 1 | Ectoenzymes: nomenclature and catalytic reactions Name EC number Catalytic activity Substrate Products Nucleotidases and related enzymes CD39 EC 3.6.1.5 ATP diphosphohydrolase ATP ATP ADP AMP ADP AMP CD73 EC 3.1.3.5 5′-Nucleotidase AMP Adenosine Autotaxin (CD203) EC 3.1.4.39 EC 3.6.1.9 EC 3.1.4.1 Lysophospholipase Lysophospholipids Nucleotide pyrophosphatase ATP Nucleotide phosphodiesterase cAMP ATP ADP AMP LPA and S1P AMP AMP ADP AMP Adenosine ADP-ribosyl cyclases and ADP-ribosyltransferases CD38 EC 3.2.2.5 ADP-ribosyl cyclase NAD(P) hydrolase cADPR hydrolase Base-exchange catalyst NAD(P) NAD(P) cADPR NAD(P) cADPR(P) and nicotinamide ADPR(P) and nicotinamide ADPR NAAD(P) CD157 EC 3.2.2.5 ADP-ribosyl cyclase NAD(P) hydrolase cADPR hydrolase Base-exchange catalyst NAD(P) NAD(P) cADPR NAD(P) cADPR(P) and nicotinamide ADPR(P) and nicotinamide ADPR NAAD(P) ART2 EC 2.4.2.31 ADP-ribosyltransferase NAD(P) ADP-ribosylated proteins Peptidases and proteases CD10 EC 3.4.24.11 Neutral endopeptidase Peptide bond Cleaved peptide CD13 EC 3.4.11.2 Aminopeptidase N Peptide bond Cleaved peptide CD26 EC 3.4.14.5 Dipeptidyl peptidase X-Pro/Ala Cleaved peptide MT1-MMP EC 3.4.24.80 Matrix metalloproteinase Protein Proteolytic fragments ISCHAEMIAREPERFUSION INJURY CD156b EC 3.4.24.86 Metalloproteinase Protein Proteolytic fragments An injury in which the tissue first suffers from hypoxia as a result of severely decreased, or completely arrested, blood flow. Restoration of normal blood flow then triggers inflammation, which exacerbates the tissue damage. Oxidases EC 1.4.3.6 Amine oxidase Amine Aldehyde, H2O2 and NH3 Oxidase NADPH Superoxide and H2O2 EC NUMBER (Enzyme commission number). A number that belongs to an international classification of enzymes. 762 | O CTOBER 2005 VAP1 NADPH oxidase EC 1.6.3.1 ADPR, ADP-ribose; ADPR(P), ADPR or ADPR phosphate; ART2, ADP-ribosyltransferase 2; cADPR, cyclic ADP-ribose; cADR(P), cADPR or cADPR phosphate; cAMP, cyclic AMP; H2O2, hydrogen peroxide; LPA, lysophosphatidic acid; MT1-MMP, membrane-type-1 matrix metalloproteinase; NAAD, nicotinic-acid-adenine dinucleotide; NAAD(P), NAAD or NAAD phosphate; NAD(P), NAD or NADP; NH3, ammonia; S1P, sphingosine 1-phosphate; VAP1, vascular adhesion protein 1; X, any amino acid. | VOLUME 5 www.nature.com/reviews/immunol © 2005 Nature Publishing Group REVIEWS increases endothelial-cell permeability by phosphorylation of tight-junction-associated proteins, such as vasodilator-stimulated phosphoprotein33. The expression of CD73 is increased at sites of inflammation, by interferon-α, and in hypoxic conditions, by hypoxia-inducible factor 1 REFS 34,35. This leads to increased adenosine production and is one of the protective mechanisms that increases endothelialcell permeability and limits leukocyte trafficking to sites of inflammation, including ischaemia–reperfusion injury. Ligation of CD73 at the surface of lymphocytes and endothelial cells causes different biological outcomes. Engagement of lymphocyte CD73 with an antibody that mimics the binding of an unknown natural ligand causes rapid shedding of CD73 and simultaneous clustering of LFA1 on the lymphocyte surface36. This leads to increased binding of these lymphocytes to endothelial cells. Most probably, this is independent of the enzymatic activity of CD73. By contrast, no shedding of endothelial-cell CD73 takes place under similar conditions37. However, direct contacts between lymphocytes and endothelial cells lead to inhibition of the enzymatic activity of CD73 by an unknown mechanism38. In addition, lymphocytes have high levels of extracellular adenosine deaminase bound to CD26 molecules at their surface39 , and this quickly degrades the remaining adenosine. The net effect of the decreased adenosine production is promotion of lymphocyte transmigration38 (FIG. 2). Mice that lack CD73 show increased leukocyte attachment to the endothelium in an in vivo ischaemia– reperfusion model and increased monocyte binding Endothelial cell Extracellular space ATP N C Autotaxin? Pro-inflammatory effects C N ADP N Pro-thrombotic effects CD39 C to ex vivo-perfused carotid arteries40 TABLE 2. Under conditions of hypoxia, vascular leakage is increased in several organs in CD73-deficient mice. It is most obvious in the lungs and occurs together with increased neutrophil accumulation around larger pulmonary vessels34,40. Other enzymes that are involved in the degradation and synthesis of extracellular nucleotides can also be involved in leukocyte migration. For example, nucleotide pyrophosphatases and nucleotide phosphodiesterases hydrolyse pyrophosphate and phosphodiester bonds in nucleotides, respectively, which results in the conversion of ATP to AMP41. One of these enzymes, autotaxin, was recently found to be identical to an extracellular form of lysophospholipase D42, which hydrolyses lysophospholipids to produce lysophosphatidic acid (LPA) and hydrolyses sphingosylphosphorylcholine to produce sphingosine 1-phosphate (S1P)43. Both LPA and S1P bind specific G-protein-coupled receptors at the surface of immune cells and other cell types, and they are chemoattractants for certain types of leukocyte44,45. In vivo, S1P and its receptor(s) are required for migration of thymocytes from the thymus, homing of lymphocytes to the peripheral lymph nodes, exit of T cells from the secondary lymphoid organs and migration of B cells within the spleen (from the marginal zone to the follicles)45. It should be noted, however, that there are other biosynthetic pathways for the production of S1P and LPA and that autotaxin is not found in lymphoid tissues41. Therefore, the possibility that autotaxin has a role in leukocyte trafficking awaits confirmation. Finally, there are also ectonucleotide kinases that continuously replenish the extracellular pools of ADP and ATP46. So, both ATP-generating and ATP-consuming pathways coexist on the surface of leukocytes and endothelial cells47, and their dynamic balance regulates local ATP and adenosine levels in this microenvironment. Moreover, autotaxin, which is involved in extracellular nucleotide metabolism, has multiple functions (as perhaps do some of the other enzymes discussed here), some of which increase the levels of bioactive lipids that are involved in leukocyte trafficking. AMP ADP-ribosyl cyclases and chemokines CD73 N Adenosine Anti-inflammatory effects ADA CD26 Lymphocyte Inosine Figure 2 | Extracellular ATP metabolism and leukocyte trafficking. On resting endothelium, extracellular ATP is dephosphorylated to ADP and to AMP by CD39. AMP is dephosphorylated to adenosine by CD73. ATP can also be hydrolysed to AMP by autotaxin, but whether autotaxin is expressed by lymphocytes or endothelial cells is uncertain. ATP, which binds purinoceptors of the P2X and P2Y families, is pro-inflammatory. By contrast, adenosine, which binds purinoceptors of the P1 type, is anti-inflammatory. When lymphocytes bind the endothelium, the enzymatic activity of CD73 is inhibited (by an unknown mechanism), and less adenosine is produced. Moreover, the remaining adenosine is degraded to inosine by adenosine deaminase (ADA) that is bound to lymphocyte-expressed CD26. This results in increased transmigratory activity of the lymphocytes through counteracting the antiinflammatory functions of adenosine. C, carboxyl terminus; N, amino terminus. NATURE REVIEWS | IMMUNOLOGY CD38 is expressed by most lymphoid cells, and its synthesis is regulated by the differentiation and activation stage of the cell48. In humans, it is expressed by most medullary thymocytes, tissue-resident T cells and circulating monocytes, whereas only a subpopulation of circulating T cells express this ectoenzyme. Using NAD(P) (that is, NAD or NADP) as a substrate, CD38 can catalyse the formation of several products: nicotinamide (through its NAD(P)-hydrolase activity and its ADP-ribosyl-cyclase activity), cyclic ADPribose (cADPR; through its ADP-ribosyl-cyclase activity), ADP-ribose (ADPR; through its NAD(P)hydrolase activity and its cADPR-hydrolase activity) and nicotinic-acid-adenine dinucleotide (NAAD(P); through its base-exchange catalyst activity)48. CD38 is an inefficient cyclase, and cADPR constitutes only 1–3% of the final product, although at hypoxic VOLUME 5 | O CTOBER 2005 | 763 © 2005 Nature Publishing Group REVIEWS Table 2 | Ectoenzyme-deficient animals and leukocyte trafficking Rodent model Phenotype References Cd10 knockout Exacerbation of intestinal inflammation Cd26 knockout Improvement in homing of haematopoietic stem cells and increased infiltration of T cells into inflamed joints CD26 deficiency* More severe leukocyte influx to lungs during asthma Cd38 knockout Defective chemotaxis of granulocytes and dendritic cells in severe inflammatory models 14,56 Cd39 knockout Exacerbation of skin inflammation by irritants and defective chemotaxis of monocytes 23,25 Cd73 knockout Increased leukocyte traffic to sites of inflammation in ischaemic areas 15,40 Cd156b knockout Decreased CD62L shedding 89 Vap1 knockout Faster rolling, decreased firm adhesion and transmigration, and diminished infiltration of leukocytes into sites of inflammation 16 85 13,78 77 *Using Fisher 344/CRJ rats, which spontaneously lack most CD26 protein and enzyme activity135. Vap1, vascular adhesion protein 1. fMLP (N-formyl-methionyl-leucylphenylalanine). A bacterial peptide that is a highly potent chemoattractant, especially for granulocytes. 764 | O CTOBER 2005 sites the balance of enzymatic activities can shift from NAD(P) hydrolase to ADP-ribosyl cyclase49. Nevertheless, cADPR has gained most attention in terms of leukocyte migration. By mechanisms that are still incompletely understood, the extracellular production of cADPR can induce the intracellular release of calcium in an inositol-trisphosphate-independent manner, and cADPR also regulates the entry of extracellular calcium to cells50,51. CD38-triggered calcium fluxes can therefore have synergistic effects with signalling through chemokine and fMLP (N-formyl-methionylleucyl-phenylalanine) receptors, which signal, in part, by triggering an increase in cytosolic calcium concentration (FIG. 3). Calcium oscillations have been implicated in the regulation of cytoskeletal rearrangements and directed migration52, although they might not be absolutely required for chemotaxis53. Presumably, the regulation of intracellular calcium concentration by CD38 could also modulate the function of leukocyte integrins through inside-out signalling54,55. Generation of CD38-deficient animals uncovered a crucial role for this enzyme in leukocyte trafficking. Although CD38-deficient mice are mostly normal, they have only background levels of cADPR in haematopoietic tissues14,56. (The concentrations in other tissues are close to the normal.) The migration of neutrophils to bacterially infected lungs, and to chemically inflamed peritoneum, is greatly reduced in these mice. CD38-deficient DCs are inefficiently recruited from the skin to the local lymph nodes after antigenic stimulation56. This results in poor priming of T cells and, consequently, impaired induction of humoral immune responses. Moreover, the recruitment of immature DCs from the blood into inflamed skin requires CD38. CD38-deficient granulocytes show attenuated responses to fMLP, and CD38-deficient DCs have decreased responses to chemokines that bind CC-chemokine receptor 2 (CCR2), CCR7 and | VOLUME 5 CXC-chemokine receptor 4 (CXCR4)14,56. The diminished chemotactic responses can be recapitulated in wild-type cells by using cADPR antagonists14,56. Interestingly, CD38-dependent migration is specific to certain stimuli and cell types. For example, the chemotactic response of mouse granulocytes to CXC-chemokine ligand 8 (CXCL8; also known as IL-8) is not affected by CD38 deficiency14, but for human lymphokine-activated killer cells, CD38 is required to support CXCL8-stimulated migration57. Moreover, only the migration of CD38-deficient DCs to inflamed skin and not to several non-inflamed organs (including normal skin) is affected, and CD38 is not involved in the migration of T cells from the blood to the lymph nodes56. CD38 might also have enzymatic-activityindependent functions in leukocyte–endothelial-cell interactions. Early studies showed that CD38-specific antibody blocks the binding of lymphocytes to endothelial cells irrespective of the capacity of the antibody to trigger signalling through CD38 REF. 58, and PECAM was later identified to be an endothelial-cell ligand for lymphocyte CD38 REF. 59 TABLE 3. CD157 is another member of the CD38 family that is expressed by myeloid cells. Ligation of CD157 with specific antibody elicits calcium signalling and results in defective adhesion to the extracellular matrix and impaired chemotaxis towards fMLP. These effects are independent of the enzymatic activities of CD157. Instead, they might involve lateral association of CD157 with leukocyte CD18containing integrins at the membrane, because these molecules colocalize at the cell surface and CD18specific antibody prevents CD157-triggered changes in cell shape that occur after adhesion60. ADP-ribosyltransferases and adhesion molecules NAD(P) is also a substrate for ecto-ADP-ribosyltransferases, which transfer the ADP-ribose moiety from NAD(P) to an acceptor9. ART2 is a GPI-anchored glycoprotein that is expressed by mouse T cells and some natural killer (NK) cells61. Curiously, human cells lack ART2, although they express other ecto-ADPribosyltransferases9,62. ADP-ribose that is generated from NAD(P) by ART2 can covalently modify several proteins at the surface of T cells — including the leukocyte adhesion molecules LFA1, CD43 and CD44, and ectoenzymes such as CD38 — in a way that inhibits their function63–65 (FIG. 3). Moreover, ART2-dependent ADP-ribosylation of the purinoceptor P2X7 (which is a non-selective cation channel) results in its activation, which in turn leads to influx of calcium and shedding of the peripheral lymph-node homing receptor CD62L66. The homing of NAD(P)-treated T cells to the lymph nodes, spleen and Peyer’s patches is inhibited, and injection of NAD(P) into mice markedly decreases homing. The effects might be specific, because neither VLA4 nor CD62L is ADP-ribosylated and the homing of B cells, which lack ART2, is not affected64. However, because it has subsequently been reported that treatment with NAD(P) can kill T cells66 and that certain www.nature.com/reviews/immunol © 2005 Nature Publishing Group REVIEWS mouse strains express an allelic variant of P2X7 that is resistant to NAD(P)67, careful in vivo homing assays using ART2-deficient cells68 are needed to verify a role for ART2 in leukocyte trafficking. Ectopeptidases and chemokines The receptor-binding activity of chemokines often depends on the amino (N)-terminal residues of the molecule. Therefore, proteolytic trimming can markedly alter the ability of chemokines to attract leukocytes, and there are many N-terminally truncated chemokines in vivo69. Damage Endothelial cell Nicotinamide + ADP-ribosylated proteins Release of chemokines and fMLP Release of NAD(P) Chemokine ART2 Nicotinamide + cADPR or ADPR CD38 fMLP Molecular inactivation ADPribosylation ADPribosyl group ADP-ribosylation ? cADPR CD44 RYR LFA1 Ca2+ Ca2+ Ca2+ Molecular activation K+ + K K+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Cytoskeletal rearrangement P2X7 Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Lymphocyte CD62L Antimigratory? Cell death CD62L shedding Integrin activation Pro-migratory Figure 3 | Extracellular NAD(P) metabolism and leukocyte trafficking. NAD(P) is released from damaged cells. It can then be metabolized by CD38 in a way that promotes directed cell migration to specific stimuli, such as fMLP (N-formyl-methionyl-leucyl-phenylalanine) and certain chemokines. Cyclic ADP-ribose (cADPR), which is produced from NAD(P) by CD38, triggers the release of calcium ions (Ca2+) from ryanodine receptor (RYR)-regulated intracellular stores, and it also causes sustained influx of extracellular Ca2+. This increase in cytosolic Ca2+ concentration enhances chemokine- and fMLP-triggered signals that lead to cytoskeletal rearrangement (and possibly to integrin activation) and directed movement. For other cells, NAD(P) is a substrate for ADP-ribosyltransferases (such as ADP-ribosyltransferase 2, ART2), which drives covalent modifications of specific cell-surface molecules. ADP-ribosylated celladhesion molecules and CD38 become functionally inactive. By contrast, ADP-ribosylation of the purinoceptor P2X7 activates this molecule and causes opening of Ca2+ channels and shedding of CD62L. ADPR, ADP-ribose; K+, potassium ion; LFA1, lymphocyte functionassociated antigen 1; NAD(P), NAD or NADP. NATURE REVIEWS | IMMUNOLOGY CD26 is an ectoenzyme that cleaves N-terminal dipeptides from polypeptides with either proline or alanine residues in the penultimate position70. Numerous biologically active peptides (including neuropeptides, hormones, chemokines and cytokines) are substrates for CD26. In the immune system, CD26 is expressed in an activation-dependent manner by T cells, B cells and NK cells, as well as endothelial cells. Several chemokines (CCL5, CCL11, CCL22, CXCL9, CXCL10, CXCL11 and CXCL12) are CD26 substrates, at least in vitro69–71. Of these, CXCL12 seems to be the most effective target for CD26 REF. 71, and inhibition of the endogenous activity of CD26 increases chemotactic responses towards CXCL12 REF. 72. N-terminally truncated chemokines typically show reduced binding to their cognate receptor and reduced triggering of receptor internalization and desensitization73,74. They can also have altered chemokine-receptor specificity. For example, full-length CCL5 triggers robust chemotactic migration after binding its receptors CCR1 or CCR5. By contrast, truncated CCL5 that lacks the two amino acids at the N terminus cannot induce efficient calcium signalling in human monocytes (which express CCR1), but it still has activity on stimulated macrophages (which express CCR1 and CCR5)75. However, truncated chemokines can also trigger more efficient chemotaxis of certain cell types than do the intact forms. For example, T cells undergo more marked chemotaxis towards CCL5 that has been cleaved by soluble CD26 than towards the unprocessed form of CCL5 REF. 76. These examples show that ectopeptidase-mediated chemokine processing markedly alters the specificity of chemokine function and that it operates, in a responder-cell-typeselective manner, to downregulate75, terminate74 or even potentiate76 the inflammatory reaction. CD26 activity also regulates leukocyte trafficking in vivo. Rats that spontaneously lack CD26 have increased infiltration of T cells in the lungs in models of asthma77. In addition, CD26-deficient mice show increased extravasation of inflammatory cells into the joints78. Moreover, in these mice, the homing of intravenously administered haematopoietic stem cells to the bone marrow is increased, and chemical inhibitors of CD26 increase the engraftment of these cells13. For several cell types, CD26 increases migration by binding extracellular-matrix proteins (such as fibronectin and collagen) and soluble molecules (such as type II plasminogen and extracellular adenosine deaminase) and by laterally associating with other proteins (such as sodium–hydrogen exchangers) at the cell surface39,79–82. The contribution to leukocyte trafficking of these enzymatic-activity-independent receptor functions of CD26 remains poorly characterized. Moreover, the existence of numerous functional and/or structural homologues of CD26 REF. 11 renders the analysis of CD26-dependent cell trafficking even more challenging. CD10 and CD13 are ectopeptidases that are expressed by leukocytes. They have broad specificity and cleave several cytokines and chemoattractants (such as fMLP and CXCL8)83,84. Disruption of the gene encoding VOLUME 5 | O CTOBER 2005 | 765 © 2005 Nature Publishing Group REVIEWS Table 3 | Non-enzymatic functions of ectoenzymes Ectoenzyme Ligand Biological activity References CD26 Adenosine deaminase Reduces adenosine concentrations 39 CD38 PECAM Promotes leukocyte binding to endothelial cells 59 CD73 ND Promotes leukocyte binding to endothelial cells 26,27,36 CD157 ND Reduces chemotaxis and cell adhesion VAP1 ND Involved in leukocyte trafficking 60 101,105–113 ND, not determined; PECAM, platelet/endothelial cell-adhesion molecule; VAP1, vascular adhesion protein 1. CD10 causes exacerbation of intestinal inflammation, owing to increased infiltration of granulocytes85. These data therefore reinforce the concept that the magnitude and specificity of chemoattractant signalling is regulated in a crucial manner by ectoenzymes. domain of the multifunctional adhesion molecule CD44, thereby destabilizing cell–cell and cell–extracellularmatrix adhesion96,97. This mechanism seems to guide thymocyte migration in the thymic medulla, because an extracellular-matrix molecule, laminin-5, induces MT1-MMP-mediated release of a soluble fragment of CD44 from the surface of thymocytes98. MT1-MMP also binds and activates other members of the MMP family (such as pro-MMP2 and pro-MMP13) and thereby coordinates proteins with a broad proteolytic repertoire at the cell surface. In addition, it regulates leukocyte migration by cleaving and inactivating chemokines such as CCL7 REF. 99. Interestingly, ADAMs can also regulate cell–cell adhesion by cleaving chemokines100. Proteases therefore have a fundamental role in regulating the expression of adhesion molecules, in trimming chemokines and, probably, in degrading the extracellular matrix during leukocyte movement in tissues. Ecto-oxidases and leukocyte traffic Sheddases and adhesion molecules TOPAQUINONE A modified tyrosine residue (2,4,5-trihydroxyphenylalanyl quinone) that is required for the enzymatic activity of certain amine oxidases. 766 | O CTOBER 2005 Almost all adhesion molecules that are involved in leukocyte trafficking are present in soluble, biologically active forms in the blood. They are produced by alternative splicing and/or by shedding from the cell surface through the action of proteases86,87, some of which are ectoenzymes. The shedding of the leukocyte rolling receptor CD62L, which occurs on cell activation, has been well studied88. CD156b — a disintegrin and metalloproteinase (ADAM) protein that sheds tumournecrosis factor from the cell surface — proteolytically cleaves CD62L from the surface of thymocytes 89 . However, CD156b inefficiently cleaves a peptide that corresponds to the CD62L membrane-proximal cutting site90. Moreover, there is residual CD62L cleavage in the absence of CD156b91. These data indicate that, although CD156b is clearly involved in CD62L shedding in response to certain stimuli, it might activate other proteins that are required for CD62L cleavage rather than cleaving CD62L directly; therefore, other sheddases that are uncharacterized at present might be involved. The use of inhibitors has shown that MMPs mediate CD62L shedding and affect leukocyte traffic, but the identity of the individual sheddases that are involved remains to be determined, owing to the broad specificity of the reagents that were used92. Moreover, gene-targeting experiments have shown that the inability to shed CD62L does not affect the homing of naive T cells, but it leads to increased homing of activated T cells to the peripheral lymph nodes93,94. These data indicate that the main function of CD62L shedding from lymphocytes is to prevent the re-entry of activated T cells to the secondary lymphoid organs. Membrane-type-1 MMP (MT1-MMP; also known as MMP14) is another ectoenzyme that is involved in the shedding of adhesion molecules95. MT1-MMP (and, less efficiently, MT2-MMP, MT3-MMP and MT5-MMP) cleaves the extracellular ligand-binding | VOLUME 5 VAP1 is an ecto-oxidase that is expressed mainly in the cytosolic vesicles of endothelial cells in several tissues101. Under inflammatory conditions, the expression of VAP1 is induced, and VAP1 translocates to the luminal surface of blood vessels101,102. Cloning of VAP1 showed that the gene encodes a semicarbazidesensitive amine oxidase (SSAO)103. This type of ectoenzyme catalyses oxidative deamination of primary amines in the following reaction: R-CH2-NH2 + H2O + O2 → R-CHO + NH3 + H2O2 (where R denotes an aliphatic or aromatic group). SSAOs are characterized by a unique modification of a tyrosine residue into TOPAQUINONE at the catalytic centre, and their enzymatic activity is inhibited by semicarbazide, hydroxylamine and other carbonyl-reactive compounds104. The role of VAP1 in leukocyte trafficking has mostly been characterized using monoclonal antibodies, which do not inhibit its oxidase activity105,106. So, antibody that is specific for VAP1 blocks lymphocyte binding to venules in frozen-tissue sections and reduces the number of rolling and adherent leukocytes on VAP1expressing endothelial cells in in vitro flow-chamber assays105,107–109. In vivo, treatment with VAP1-specific antibody increases the velocity of rolling granulocytes and decreases the number of firmly adherent cells and cells that have undergone transmigration in inflamed venules110. In the liver, VAP1 seems to be the main adhesion molecule that mediates the binding of CD4+ T helper 2 cells to the endothelial lining106. Inhibition of the different steps of the extravasation cascade by VAP1-specific antibody results in attenuated inflammatory responses in several animal models, including peritonitis, allograft rejection, air-pouch inflammation and liver inflammation106,110–112. The enzymatic activity of VAP1 can be completely abolished by mutating the crucial tyrosine residue, without affecting the expression of antibody-defined epitopes on the molecule. Using SSAO inhibitors, and wild-type and enzymatically inactive VAP1 molecules, it was shown that VAP1 molecules that lack SSAO www.nature.com/reviews/immunol © 2005 Nature Publishing Group REVIEWS Leukocyte Signalling H2O + O2 NH3 + H2O2 Schiff base NH2 O– NH2 O VAP1 ligand or substrate O– O– O OH NH+ O O O O HO VAP1 Endothelial cell Step 1 Step 2 Step 3 Step 4 Aldehyde O Figure 4 | A model of the action of vascular adhesion protein 1 during leukocyte–endothelial-cell interaction. A four-step model of the action of vascular adhesion protein 1 (VAP1) in the interaction between leukocytes and endothelial cells is shown. In step 1, VAP1 is present at the surface of the endothelial cell, and a freely flowing leukocyte expresses a VAP1 ligand or substrate. In step 2, VAP1 binds the leukocyte through a cell-surface epitope, the function of which can be inhibited by specific antibody. In step 3, this interaction guides the leukocyte substrate into the catalytic centre of VAP1, and in the presence of water (H2O) and oxygen (O2), a covalent, but transient, SCHIFF BASE is formed. In step 4, the catalytic reaction leads to an aldehyde modification of the leukocyte substrate and to the release of hydrogen peroxide (H2O2) and ammonia (NH3), which can trigger various signalling pathways in both cell types. The molecular interactions between a lymphocyte cell-surface amine (NH2) and the TOPA-quinone (that is, the modified tyrosine residue) in VAP1 are highlighted. SCHIFF BASE The functional group or compound that contains a carbon–nitrogen double bond. HYDROGEN PEROXIDE A potent signalling molecule and inflammatory mediator that is a reactive oxygen species. activity result in impaired rolling, firm adhesion and transmigration of leukocytes under laminar shear flow in vitro105,108,113. Using gene-targeted animals, it has been shown that VAP1 is important for leukocyte trafficking in vivo. Mice that overexpress VAP1 at the surface of endothelial cells show increased binding of lymphocytes to these endothelial cells114. Most importantly, VAP1-deficient animals have clear defects in cell migration during inflammation, although they appear to be normal under non-challenged conditions16. In these animals, lymphocytes and granulocytes roll much faster, and there are fewer firmly adherent cells and cells that have undergone transmigration. Moreover, VAP1-deficient animals manifest with attenuated influx of inflammatory cells during peritonitis and autoimmune diabetes. VAP1 also seems to have a role in physiological lymphocyte recirculation, because lymphocyte homing to the mesenteric lymph nodes and the spleen is impaired in the absence of this molecule. Although the enzymatic-activity dependence of the observed phenotype in VAP1-deficient animals remains to be determined, these findings highlight the importance of VAP1 in normal trafficking of leukocytes under physiological and inflammatory settings in vivo. Because VAP1-specific antibody, SSAO inhibitors and inactivating mutations (and the combination of either antibody and inhibitors or antibody and inactivating mutations) block leukocyte–endothelial-cell interactions to the same extent, VAP1 is thought to bind leukocytes using, first, the adhesive epitope (that is, the VAP1-specific-antibody-dependent epitope) and, second, its enzymatic activity (FIG. 4). Although the leukocyte-expressed ligand or substrate of VAP1 NATURE REVIEWS | IMMUNOLOGY remains to be identified, certain amino sugars and peptides (such as galactosamine and amino groups in the side chains of lysine-containing peptides) have suitable free amino groups that can physically fit in the constrained, narrow and deep, enzymatic pocket of VAP1 REFS 105,115,116, and these are therefore feasible candidates. Notably, the end-products of the SSAO reaction are biologically active compounds. HYDROGEN PEROXIDE (H2O2), in particular, is increasingly being recognized to be an important signalling molecule117,118. Alteration of the redox balance by H2O2, which is freely cell permeable, induces the expression of CD62P and VCAM1 by endothelial cells119,120, and of chemokine receptors121 and MMPs122 by many cell types, thereby increasing cell migration. Because NADPH oxidase has also been suggested to participate in leukocyte transmigration123, several ecto-oxidases might turn out to have versatile roles in leukocyte trafficking by producing reactive oxygen species. Enzymes as rheostats of leukocyte traffic Many ectoenzymes support cell migration in an enzymatic-activity-independent manner TABLE 3. In this case, their function can be regulated at transcriptional, translational and post-translational levels, similar to that of other adhesion molecules. For example, the expression of ectoenzymes is often tightly controlled in a cell-type and activation-dependent manner or by subcellular localization (that is, intracellular expression versus cell-surface expression). The newly recognized importance of the catalytic activity of ectoenzymes in leukocyte extravasation introduces new levels of control to this dynamic process. So, availability of the substrate(s), activity and affinity of the enzyme, and secondary effects of the reaction VOLUME 5 | O CTOBER 2005 | 767 © 2005 Nature Publishing Group REVIEWS products together define the biological outcome. The bioavailability of the substrate in the extracellular space depends on its production, release, uptake, sequestration and degradation. For example, regulated release of the substrate after inflammatory-cell damage, dynamically tunes up or tunes down ectoenzymedriven inflammatory responses. In that sense, these substrates, independent of their molecular identity, are analogous to typical pro-inflammatory mediators (such as hormones, cytokines, chemokines and lipids). For example, NAD(P) is normally almost absent from the extracellular environment. At sites of injury, release of NAD(P) from intracellular sources56,64 might allow CD38-dependent generation of cADPR and priming of cellular responsiveness towards chemoattractants. When the cellular damage is controlled, the decreasing levels of NAD(P) would retune the system to an inactive state56. Similarly, the release of extracellular ATP can regulate the activity of the autotaxin–CD39–CD73 axis. Depending on the local availability of competing substrates, the activity of an ectoenzyme such as autotaxin can even shift from being pro-migratory to being antimigratory 43. The activity and affinity of the ectoenzymes can be reversibly or irreversibly regulated by natural inhibitors — such as ATP and ADP, which inhibit CD73 REF. 124, and galactosamine, which inhibits VAP1 REF. 116 — in a competitive or non-competitive manner. Moreover, the catalytic activity often depends on cofactors, such as copper for the SSAO activity of VAP1 REF. 125. Both inhibitors and cofactors can themselves be subject to other control circuits. Finally, all enzymatic reactions result in the production of potentially active end-products. These often have signalling functions and can be used to propagate (or downmodulate) leukocyte traffic. As the enzymatic reactions are extremely fast and as the enzymes are regenerated during the catalytic cycle, the enzymatic reactions can effectively amplify the signals. There is also crosstalk between different ectoenzymes. Competition for the same substrate is one example, such as occurs in the case of CD38 and ART2 for NAD(P). So, depending on the balance between the CD38 (pro-migratory) and ART2 (possibly antimigratory) activities at the surface of the responding cells, the biological outcome can differ markedly. Moreover, ADP-ribosylation and CD38 inactivation by ART2 might exert a feedback loop65,126. Alternatively, the product of one enzyme can be a substrate for the next enzyme. For example, AMP that is produced by CD39 can be further hydrolysed by CD73. Enzymes from different pathways can also interact. Ligation of CD38 induces a rapid export of CD73 from an intracellular pool to the surface of T cells127. Another example is that induction of CD38 expression coincides with the stimulation of nucleotide pyrophosphatase and nucleotidase activity at the surface of leukaemic cells, which could result in the generation of AMP and adenosine from NAD(P)128. So, ectoenzymes might form networks and pathways in a similar manner to intracellular enzymes. 768 | O CTOBER 2005 | VOLUME 5 Enzymes as therapeutic targets Ectoenzymes are attractive candidates for designing new means to interfere with undesirable leukocyte trafficking. This can be achieved by inhibiting the ectoenzymes that promote leukocyte trafficking (such as CD38 and VAP1) or by inducing the activity of ectoenzymes that normally inhibit leukocyte trafficking (such as CD26 and endothelial-cell-expressed CD73). In many cases, ectoenzymes can be inactivated either by monoclonal antibodies that block function or by small-molecule enzyme inhibitors. The development of small-molecule inhibitors of adhesion molecules and chemokine receptors has proved challenging. By contrast, the catalytic centres of enzymes are often relatively easy to target, because analogues of known substrates and inhibitors, together with crystal structures, are good starting points for the rational design of drugs. Moreover, in the case of ectoenzymes, the inhibitors do not need to be cell permeable. Many of the enzyme inhibitors are water soluble and can be administered orally. Small-molecule inhibitors have been successfully used to treat several inflammatory diseases in animal models. For example, the tripeptide Ile-Pro-Ile, which is a specific inhibitor of CD26, increases homing of haematopoietic stem cells to bone marrow13, and 8-bromoadenine cADPR, which is an antagonist of cADPR, inhibits chemokinetriggered migration of leukocytes56. Some ectoenzymes have already been targeted in early clinical trials. For example, VAP1 is being neutralized with antibody, to block inflammation129. As mentioned earlier, in other cases, it is desirable to promote the activity of ectoenzymes to control inflammation. The nucleotidases that regulate extracellular ATP metabolism offer several possibilities to reduce harmful inflammation130. For example, by increasing the expression of CD73, using interferons35, and/or by providing additional substrate in the form of AMP, more adenosine can be endogenously produced to render the endothelium more anti-adhesive. Future prospects Research on ectoenzymatic control of leukocyte trafficking has advanced rapidly during the past few years. Nevertheless, the field still faces several challenges. Despite impressive lists of soluble and cell-surface-bound in vitro substrates, we still lack information about the relevant physiological substrates for many ectoenzymes. There is an urgent need for the development of new and more specific chemical inhibitors of individual ectoenzymes that are suitable for in vivo experimentation. Many ectoenzymes form a family of related molecules. Dissection of the roles of individual molecules in the total enzymatic activity and in the biological outcomes therefore needs further work. Up to 4% of leukocyte cellsurface antigens are expected to be ectoenzymes60, and many ectoenzymes are also expressed on the endothelium. At present, we know nothing about the function of most of these enzymes in terms of leukocyte trafficking. In fact, there are several examples in which an ectoenzyme has a well-established role in the migration www.nature.com/reviews/immunol © 2005 Nature Publishing Group REVIEWS of tumour cells, fibroblasts or epithelial cells, yet its role in immune-cell trafficking remains unexplored. Moreover, ectoenzymes such as endothelial-cell-expressed angiotensin-converting enzyme and its reaction product angiotensin II, which mainly regulate blood pressure, also seem to affect leukocyte–endothelial-cell contacts131,132. The potential of ectoenzyme-deficient animals that have been generated for other purposes should be fully exploited in research on leukocyte trafficking. The dual nature of ectoenzymes also needs to be rigorously tested, because some of their functions seem to be independent of their enzymatic activity. It is feasible that the large extracellular domains of ectoenzymes and their association with other membrane proteins can mediate responses without involvement of their catalytic activity. However, many of the postulated (non-substrate) ligands of ectoenzymes remain to be isolated. Combined use of specific enzyme inhibitors and point mutations that 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. von Andrian, U. H. & Mempel, T. R. Homing and cellular traffic in lymph nodes. Nature Rev. Immunol. 3, 867–878 (2003). Muller, W. A. Leukocyte–endothelial-cell interactions in leukocyte transmigration and the inflammatory response. Trends Immunol. 24, 327–334 (2003). Ley, K. & Kansas, G. S. Selectins in T-cell recruitment to non-lymphoid tissues and sites of inflammation. Nature Rev. Immunol. 4, 325–335 (2004). Pribila, J. T., Quale, A. C., Mueller, K. L. & Shimizu, Y. Integrins and T cell-mediated immunity. Annu. Rev. Immunol. 22, 157–180 (2004). Kunkel, E. J. & Butcher, E. C. 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Is the Fischer 344/CRJ rat a protein-knock-out model for dipeptidyl peptidase IV-mediated lung metastasis of breast cancer? Clin. Exp. Metastasis 17, 609–615 (1999). Acknowledgements We thank F. Marttila-Ischihara for the intravital-microscopy video and G. Yegutkin for critical reading of the manuscript. Competing interests statement The authors declare competing financial interests: see web version for details. Online links DATABASES The following terms in this article are linked online to: Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene adenosine deaminase | ART2 | autotaxin | CD10 | CD13 | CD26 | CD38 | CD39 | CD73 | CD156b | CD157 | VAP1 FURTHER INFORMATION Department of Bacterial and Inflammatory Diseases: http://www.ktl.fi/portal/english/osiot/research,_people___ programs/bacterial_and_inflammatory_diseases/ Sirpa Jalkanen’s homepage: http://research.utu.fi/receptor/projects/sjalkane/sjalkane.html SUPPLEMENTARY INFORMATION See online article: S1 (movie) Access to this links box is available online. VOLUME 5 | O CTOBER 2005 | 771 © 2005 Nature Publishing Group