Download CELL SURFACE ENZYMES IN CONTROL OF LEUKOCYTE

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CELLSURFACE 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
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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
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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
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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, ISCHAEMIAREPERFUSION 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
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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
ISCHAEMIAREPERFUSION
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.
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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.
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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
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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
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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
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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
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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
TOPAQUINONE
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
TOPAQUINONE 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
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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
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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
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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
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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.
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