National Public Health Institute and MediCity Research Laboratory, Turku University, 20520 Turku, Finland
CD73, otherwise known as ecto-5-nucleotidase, is a glycosyl-phosphatidylinositol-linked 70-kD
molecule expressed on different cell types, including
vascular endothelial cells (EC) and certain subtypes of
lymphocytes. There is strong evidence for lymphocyte CD73 having a role in several immunological phenomena such as lymphocyte activation, proliferation, and
adhesion to endothelium, but the physiological role of
CD73 in other cell types is less clear. To compare the
biological characteristics of CD73 in different cell
types, we have studied the structure, function, and surface modulation of CD73 on lymphocytes and EC.
CD73 molecules on lymphocytes are shed from the cell
surface as a consequence of triggering with an antiCD73 mAb, mimicking ligand binding. In contrast, triggering of endothelial CD73 does not have any effect on
its expression. Lymphocyte CD73 is susceptible to
phosphatidylinositol phospholipase, whereas only a
small portion of CD73 on EC could be removed by this
enzyme. Furthermore, CD73 on EC was unable to deliver a tyrosine phosphorylation inducing signal upon
mAb triggering, whereas triggering of lymphocyte
CD73 can induce tyrosine phosphorylation. Despite the
functional differences, CD73 molecules on lymphocytes
and EC were practically identical structurally, when
studied at the protein, mRNA, and cDNA level. Thus,
CD73 is an interesting example of a molecule which
lacks structural variants but yet has a wide diversity of
biological functions. We suggest that the ligand- induced shedding of lymphocyte CD73 represents an
important and novel means of controlling lymphocyte-
EC interactions.
Lymphocyte recirculation is known to be finely regulated by different adhesion molecules expressed on
lymphocytes and endothelial cells (EC)1 and by
chemokines, controlling the activation status of the cells (30). Lymphocytes make the initial contact with EC by
rolling along the vessel wall in the high endothelial venules
of lymphoid tissues or along the flat-walled endothelium
at sites of inflammation. Lymphocyte rolling is a well characterized phenomenon, and it is known to be mediated by
selectins, situated on the tips of microvilli both on the lymphocyte and EC surface (42). More stable adhesion is
achieved through binding of activated integrins to their EC
counterparts, and lymphocytes eventually migrate through the vessel wall using integrins and integrin ligands of the Ig superfamily (8, 30).
CD73/ecto-5 A subpopulation of peripheral blood lymphocytes
(PBL) expresses CD73 on the majority of B cells and
CD8+ T cells but on only about 10% of CD4+ T cells (10,
38, 40). CD73/ecto-5 As the diversity of ecto-5 Cells, Cell Lines, and Antibodies
HUVECs (human umbilical vein endothelial cells) were isolated as described earlier (2). They were cultured on gelatin-coated flasks in EC
basal medium (Clonetics Corp., San Diego, CA) supplemented with 10%
human AB-serum (Finnish Red Cross, Helsinki, Finland), 50 µg/ml EC
growth factor (Boehringer Mannheim GmbH, Mannheim, Germany), 5 U/ml heparin (Sigma Chemical Co., St. Louis, MO), 4 mM L-glutamine,
100 U/ml penicillin, and 100 µg/ml streptomycin. HEC-endothelial cell
line, equivalent to EaHy-926 cell line (11), was a kind gift of Dr. H. Holthöfer (University of Helsinki, Finland). HECs were cultured in RPMI-1640
medium supplemented with 10% FCS, 4 mM L-glutamine, and antibiotics
(referred to as complete medium). Human PBL from healthy, voluntary
donors were isolated using Ficoll-Hypaque (Histopaque-1077; Pharmacia
Fine Chemicals, Uppsala, Sweden). For tyrosine phosphorylation studies, peripheral blood mononuclear cells were enriched for CD73+ cells by depleting CD4+ and CD11b+ cells using anti-CD4 (CRL8002, mouse IgG1,
ATCC) and anti-CD11b (ATCC) antibodies and Dynabeads (Dynal, Inc.,
Oslo, Norway).
mAb 4G4 (mouse IgG1) recognizes CD73 (2, 3). The CD73 workshop
mAb 1E9 (mouse IgG3) was a kind gift of Dr. Linda Thompson (Oklahoma Medical Research Foundation, Oklahoma City, OK). Binding inhibition studies demonstrated that the 4G4 and 1E9 mAbs recognize distinct epitopes of the CD73 molecule (Fig. 1). For epitope mapping, HEC
cells were sequentially incubated with 4G4, 1E9, and an FITC anti-mouse
IgG3 (Southern Biotechnology, Birmingham, AL) or with 1E9, 4G4, and
an FITC anti-mouse IgG1 (Southern Biotechnology) and analyzed by
FACScan®. mAb Hermes-3 recognizes CD44 (16). Anti-phosphotyrosine
mAb 4G10 was purchased from Upstate Biotechnology (Lake Placid,
NY). An irrelevant mAb against chicken T cells (3G6) was used as a negative control.
mAb Treatment of Lymphocytes and HEC
Cells were treated in the following four different ways: (a) lymphocytes
and EC were resuspended in 3G6, 4G4, or 1E9 mAb (either at 37 or 4°C)
containing 10% FCS at a concentration of 1 × 106 cells/ml. Cells were
then incubated at 37 or 4°C for the indicated times, placed on ice, washed
with cold PBS containing 0.1% NaN3 and 2% FCS, and stained by immunofluorescence staining for the expression of CD73, as described previously (2). (b) Lymphocytes and EC were preincubated on ice for 30 min in
the presence of either 3G6, 4G4, or 1E9 mAb and washed with cold RPMI.
Cells were thereafter resuspended in complete medium or RPMI at 37°C
and incubated for the indicated periods of time at 37°C. After this, the
cells were replaced on ice, washed as described, and labeled using either
both first stage mAb (4G4 or 1E9) with a second stage FITC-conjugated
antibody or only a second stage FITC Ab, and analyzed by FACScan. (c)
Lymphocytes were incubated in presence of 4G4 mAb or 3G6 mAb (as
described in a) and analyzed by FACS®, either before or after acetone
permeabilization. Cells were permeabilized by covering them for 2 min in
acetone at Dot-blot Assay for Detecting Cellular
and Soluble CD73
Lymphocytes and EC were incubated with 4G4 mAb (as described above
in b). Thereafter, either 0.25 × 106, 0.5 × 106, and 1 × 106 lymphocytes or
0.3 × 106, 0.6 × 106, and 0.9 × 106 EC were lysed in 100 µl of a buffer containing 50 mM Labeling and Immunoprecipitation of Cells
Freshly isolated PBL and HECs were iodinated with 125I by the lactoperoxidase method (17). Iodinated cells were lysed in lysis buffer (1% Triton
X-100, 0.15 M NaCl, 1.5 mM MgCl2, and 0.01 M Tris, pH 7.2) containing
1% Aprotinin and 1 mM PMSF, and the lysate was clarified by centrifugation. Immunoprecipitation was performed using CnBr-activated Sepharose 4B beads coupled to mAb 4G4, 3G6, or Hermes-3. Antigens were
eluted from the beads with 50 µl Laemmli's sample buffer and resolved in
5-12.5% SDS-PAGE under reducing conditions.
Northern Blot Analysis, Isolation, and Sequencing of
CD73 cDNA
A cDNA encoding CD73 was amplified by reverse transcription PCR
(RT-PCR) from total RNA isolated from PBL. Standard techniques were
used for the RNA isolation, Northern blot analyses, cDNA synthesis,
PCR amplification, subcloning procedures, and plasmid preparations (29).
2 µg of PBL RNA was reverse transcribed into cDNA using random hexamer primers and MMLV reverse transcriptase. The CD73 primers
5 Phosphatidylinositol-specific Phospholipase C
Treatment of HEC Cells
HEC cells detached with 5 mM EDTA were resuspended in RPMI 1640 in
the presence or absence of Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC; Oxford Glycosystems, Oxon, UK) at 1 U/ ml and incubated for 1 h under rotation at 37°C. Finally, immunofluorescence staining for CD73 expression was performed.
Ecto-5 Activity of ecto-5 Detection of Tyrosine Phosphorylated Proteins after
Triggering of CD73 on EC and Lymphocytes
Confluent monolayers of EC in 25 cm2 cell culture flasks (~5 × 106 cells/
flask) or 0.8 × 106 PBL enriched for CD73+ cells (i.e., CD8+CD11b mAb Triggering of Lymphocyte CD73 but Not
Endothelial CD73 Causes Downregulation of the
Molecule at 37°C
CD73 is expressed on ~15% of freshly isolated PBL and
on all cultured HUVECs and HEC cells (2). To study the
consequences of CD73 ligand binding in lymphocytes and
EC, we used a model in which binding of CD73 to its natural ligand (which is still unknown at present) was mimicked by triggering CD73 with an mAb. In this model, lymphocytes or EC (either the HEC endothelial cell line or
HUVECs, adherent or in suspension after detachment by 5 mM EDTA treatment) were incubated for 2 h at 37°C in
the presence of an anti-CD73 mAb 4G4, anti-CD73 mAb
1E9, or a negative control mAb, after which CD73 expression on the cell surface was analyzed by FACS® (in the
case of adherent EC, detachment was with 5 mM EDTA
before immunofluorescence [IF] staining). On lymphocytes, mAb 4G4 caused a reduction in CD73 expression,
but no alteration in the expression level of CD73 was observed after incubation with a negative control mAb or antiCD73 mAb 1E9 (Fig. 2 A). On EC, no reduction in CD73 expression was seen subsequent to mAb treatments (Fig. 2 A).
Kinetics of Surface CD73 Modulation
To study the rate of CD73 downregulation on lymphocytes, the cells were incubated in the presence of antiCD73 mAb for different time periods and analyzed by
FACS®. The downregulation of CD73 on lymphocytes was
maximal after 5 h incubation (1% positive cells left), and
the expression remained low during the entire 24 h followup period (Fig. 2 B). On EC, a minor reduction in the
mean fluorescence intensity (MFI) was seen as a consequence of anti-CD73 treatment at 37°C, but the percentage of positive cells remained unaltered even after prolonged incubation (Fig. 2, A and B). Downregulation of
the lymphocyte CD73 was temperature dependent, as it
did not occur when the cells were incubated with the 4G4
mAb on ice (Fig. 2 B). It was also dependent on divalent
cations, as no downregulation was observed when the cells
were incubated with anti-CD73 mAb at 37°C without Ca2+
and Mg2+ (data not shown). It also is epitope dependent,
as the two different anti-CD73 mAbs, 4G4 and 1E9, which
recognize distinct epitopes of the molecule (Fig. 1), have
profoundly dissimilar effects.
Modulation of CD73 on EC and Lymphocyte Surface
Continuous recycling between the cell surface and the intracellular pool has been described for ecto-5
When lymphocytes were treated with 4G4 mAb at a saturating concentration at 4°C, washed and then incubated
in complete medium without anti-CD73 mAb at 37°C for
15 min, a rapid loss of the antibody-bound CD73 molecules from the cell surface and appearance of new, nonAb-bound CD73 molecules was seen. This was shown as a
smaller percentage of fluorescent cells when lymphocytes,
after incubation at 37°C, were labeled only with an FITCconjugated anti-mouse Ig and analyzed by FACS®, as
compared to the percentage of positive cells when the cells were labeled with both an anti-CD73 mAb and an FITCanti-mouse Ig (Fig. 3 A). The loss of antibody-bound
CD73 from lymphocyte surfaces was very rapid, as the
percentage of positive cells was already reduced after a
2 min incubation in complete medium (data not shown).
Kinetic studies show that after incubating lymphocytes for
up to 5 h in complete medium, the level of antibodybound, surface CD73 was still reduced (i.e., the antibodybound molecules do not return to the cell surface once
they are removed, Fig. 3 B). Binding of anti-CD73 mAb
1E9, again, causes no reduction in the CD73 expression on
lymphocytes (Fig. 3 A).
mAb Binding to Lymphocyte CD73 but Not to
Endothelial CD73 Causes Shedding of the Molecule
To determine whether CD73 on lymphocytes and EC is
shed or internalized, dot blot assays were performed.
Lymphocytes and EC were incubated on ice in the presence of anti-CD73 mAb 4G4, washed twice, and then incubated in RPMI at 37°C for 1 h. After the incubation, CD73
was detected either from the supernatant or from the cell lysate using 4G4 mAb as a first stage mAb and peroxidase-conjugated anti-mouse Ig as a second stage mAb, or
by performing the detection using only the second stage
Ab. As can be seen in Fig. 3 C, CD73 could be detected in
the lymphocyte supernatant using only the second stage
Ab in the dot blot assay, indicating that the molecule initially present on the cell surface had been shed into the supernatant after mAb binding. Signals from lysed lymphocytes are weaker, suggesting that only low amounts of
CD73 are left in the cell after mAb treatment at 37°C. On
the other hand, no CD73 could be detected from the EC
supernatant, but the CD73 signals from the EC lysate were
strong (Fig. 3 C). Thus, CD73 on EC is not removed from
the cell surface due to mAb binding, and EC do not spontaneously secrete CD73. Instead, the reduction of mAbbound CD73 on EC surface after resuspending the cells in
medium at 37°C, is probably due to internalization of the
molecule, as in the dot blot experiment in which signals
are much stronger when detection is performed using both
first stage and second stage mAb as compared to detection
only with the second stage mAb (Fig. 3 C). To further confirm that continuous exposure of lymphocytes to anti-CD73
Ab at 37°C causes shedding instead of internalization of
CD73, lymphocytes were treated with mAb, permeabilized with acetone, and then stained with anti-CD73 mAb
to study whether intracellular CD73 could be detected. As
can be seen in Fig. 4, after anti-CD73 preincubation at
37°C, no CD73 could be detected on either nonpermeabilized or acetone-permeabilized lymphocytes. If the cells
were incubated in the presence of a negative control mAb,
CD73 was present both on the non-permeabilized cells
and in the acetone-permeabilized cells.
We thus conclude that CD73 molecules are shed from
the lymphocyte surface upon antibody binding, and new
CD73 molecules from inside the cell replace those which
are lost. When extra Ab is available at 37°C to bind newly
surface-located molecules, the internal pools of CD73
molecule are depleted (or the synthesis of CD73 is downregulated). Thus, no molecules are available to replace
those which are shed, and the cells become completely CD73 negative.
Cross-linking of CD73 on Lymphocytes Causes
Patching, Whereas Cross-linking of CD73 on EC
Causes Capping
When PBL were incubated sequentially with an anti-CD73
mAb and a cross-linking FITC-conjugated second stage
Ab, at 4°C, patching of CD73 was observed on the lymphocyte surface when cells were analyzed by fluorescent
microscopy (Fig. 5 A). When the same procedure was performed at 37°C, a substantial number of the cells became CD73 negative, and the remaining CD73 reactivity was
found in large clusters (Fig. 5 B). On EC, partial capping
of CD73 can be seen after antibody cross-linking at 4°C,
but substantial reactivity can still be found all around the
cell (Fig. 5 C). After cross-linking at 37°C, the capping of
CD73 to one pole is complete, and practically no CD73 reactivity can be detected on other parts of the cell (Fig. 5 D).
Adherent HEC cells display a granular staining pattern after CD73 cross-linking both at 4°C (Fig. 5 E) and at 37°C
(Fig. 5 F).
Immunoprecipitation of CD73 from Lymphocytes
and EC
We have shown above, by several complementary methods, how CD73 surface expression is differentially modulated following Ab binding and incubation at 37°C in
lymphocytes and EC. To elucidate whether structural divergence between lymphocyte and endothelial CD73 could
eplain the differences, we immunoprecipitated the CD73 molecule from 125I-labeled lymphocytes and EC. However, no significant differences in the size of the CD73
molecule were observed (Fig. 6). The protein migrates as a
single, 70-kD band in 5-12.5% SDS-PAGE gels under reducing conditions, whether it is derived from EC or PBL.
Northern Blot Analyses, Cloning, and Sequencing of
Lymphocyte and Endothelial CD73 cDNA
Although no gross differences at the protein level were detected in CD73 expressed on different cell types, we examined polyA+ RNA isolated from both lymphocytes and
HEC cells by Northern blot analysis to determine if there
is any size divergence at the mRNA level. In both HEC
and PBL mRNAs, a strong band of ~3.9 kb was detected
(Fig. 7). Despite similar amounts of polyA+ RNA (10 µg/
lane), the signal detected from lymphocyte RNA was severalfold stronger, possibly indicating a much higher turnover rate of lymphocyte CD73 as compared to the EC
CD73. Prolonged exposure of the blot revealed the presence of smaller, hybridizing bands in both samples which
may represent minor CD73 transcripts, prematurely truncated mRNAs, or degradation products. To compare the
sequences of CD73 HEC and PBL cDNAs, we isolated a
full length CD73 cDNA from lymphocytes by PCR and
completely sequenced both this and the previously cloned
endothelial form from HEC cells (2). No significant differences were found in the nucleotide sequences, which were
almost identical to the previously published human CD73
cDNA sequence from placental tissue (21). A single nucleotide change, A > G1175, was found in both HEC and PBL
cDNAs, resulting in a threonine to alanine change, and an
A > C730 in HEC cDNA, resulting in a lysine to asparagine substitution. These may most likely be attributed to
PCR amplification errors or polymorphism due to the different tissue materials used.
PI-PLC Treatment of HEC Cells
We next addressed the question of whether differences in
the anchoring of CD73 to the plasma membrane of the cell
could be observed between lymphocytes and EC. CD73 is
known to be a GPI-linked molecule, and it has been previously shown that lymphocyte CD73 can be cleaved off by
PI-PLC treatment of cells (3, 40). To study the susceptibility of CD73 to PI-PLC in EC, HEC cells were treated with
PI-PLC, immunofluorescence stained with anti-CD73 mAb,
and analyzed by FACScan® (Fig. 8). A significant proportion of CD73 on HEC cells was resistant to PI-PLC as the
MFI was only slightly decreased by enzyme treatment. It is
probable that each individual cell has both PI-PLC-susceptible and PI-PLC-resistant CD73 molecules on its surface as, although the MFI of the cells was reduced by PIPLC-treatment, no significant changes were seen in the
number of positive cells. Internalization of CD73 on HEC
cells at 37°C does not seem to be dependent on the PIPLC susceptibility of the molecule because the internalization could also be observed after the PI-PLC treatment
(data not shown).
Inhibition of EC Ecto-5 To demonstrate the nucleotidase activity of CD73 expressed on EC, i.e., the ability of CD73 to hydrolyze extracellular mononucleotides to their corresponding nucleosides, we performed enzyme assays by adding 14C-labeled
AMP as a substrate to HEC cell cultures and then detecting [14C]adenosine and [14C]inosine as products of the
enzyme activity. High amounts of [14C]adenosine and
[14C]inosine were generated in the presence of a negative
control antibody, whereas when an anti-CD73 workshop
Ab 1E9 was present, the nucleoside production was inhibited by 60% (Fig. 9 A). This demonstrates that CD73 on
EC has high ecto-5
Antibody Triggering of CD73 Induces Tyrosine
Phosphorylation of Certain Proteins in Lymphocytes
but Not in EC
Tyrosine phosphorylation and dephosphorylation of several proteins has been demonstrated due to mAb triggering of lymphocyte CD73 (9). To compare the signal transducing abilities of CD73 in lymphocytes versus EC, we
triggered CD73 on both cell types with mAb's, and analyzed the alterations in patterns of tyrosine phosphorylated proteins by western blotting total cellular lysates. For
lymphocyte triggering we used a population of freshly isolated, CD8+CD11b To elucidate the properties of CD73 in lymphocytes and
EC, the key effector cells in lymphocyte homing and in
many other important immunological phenomena, we have
studied the endothelial and lymphocyte CD73 molecules
in terms of (a) expression and its modulation; (b) structure
at the protein, mRNA, and cDNA levels; and (c) signaling and enzymatic functions. The differences we observed between lymphocyte and endothelial CD73 imply that it has
profoundly dissimilar functional properties in these different cell types.
As the natural ligand of CD73 is not known, mAb binding to CD73 was used to mimic ligand engagement in this
study. It was shown that CD73 is downregulated from the
lymphocyte surface due to mAb 4G4 binding at 37°C (see
Fig. 2). When lymphocytes are cultured in fresh medium at
37°C, after being labeled at 4°C with anti-CD73 mAb, the
antibody-bound CD73 molecules are shed from the cell
surface into the supernatant, as CD73 can be detected in
the supernatant using only a secondary Ab in the dot blot
assay. The shed molecules are rapidly replaced by new
CD73 molecules, which probably come from intracellular
stores, or are newly synthesized (see Fig. 3). If there is new
anti-CD73 Ab available, the shedding continues and the
cells become completely depleted of both surface and intracellular CD73. Partial downregulation (both endocytosis and shedding) of other GPI-linked molecules, like decay accelerating factor, Thy-1, and Ly6A from the leukocyte
surface due to mAb binding at 37°C, has been described
earlier (5, 34). No alteration was seen in the expression of
lymphocyte CD31 after similar antibody triggering (data
not shown), indicating that surface modulation as a consequence of ligand binding is a specific property of only certain molecules. In the case of the GPI-linked molecules,
either endogenous phospholipase C or vesiculation might
be responsible for the release of the molecule from the
plasma membrane. In sharp contrast to lymphocytes, no
downregulation of CD73 due to mAb binding at 37°C was
observed in EC. However, if EC were labeled with antiCD73 mAb at 4°C, and subsequently resuspended in fresh, warm medium, a reduction in the fluorescence intensity
was observed after staining the cells with only a secondary
Ab rather than with both an anti-CD73 mAb and a secondary Ab. This indicates that some of the CD73 molecules initially present on the endothelial surface are removed after placing the cells in warm medium, and that
these molecules are replaced by new molecules coming
from intracellular stores. The reduction of CD73 on EC,
however, is not caused by mAb binding but is probably an
indication of spontaneous recycling of the molecule between intracellular pools and the cell surface, as has been
described for ecto-5 No major differences were observed at the protein level
when lymphocyte and endothelial CD73 molecules were
compared, i.e., both molecules have a molecular mass of
~70 kD. Previously reported immunoprecipitations of
CD73 have been performed from lysates of lymphocytic
cell lines expressing CD73, and the molecular masses reported have been comparable to that now detected on
lymphocytes (10, 13, 40). Northern blot analyses on HEC
and PBL polyA+ RNA were also performed to detect the
CD73 mRNA in both cell types. The size of the mRNA
detected was 3.9-4.0 kb, which corresponds well to the reported 4.1-kb size of CD73 mRNA from human placenta (21). At the nucleotide sequence level, the cDNAs originating from lymphocytes and EC were practically identical
to each other and to the previously published placental form.
Several GPI-linked molecules, including CD73, have
been shown to be capable of transmitting activation signals to leukocytes (27). The mechanisms of signal transduction mediated by these molecules lacking intracellular
domains have been difficult to understand. But recent
studies have shown that GPI-linked molecules can be expressed on the cell surface in clusters closely associated with other molecules, which often include tyrosine kinases
of the src family (12, 19, 32). Thus, interaction with the src
family protein kinases is one possible means for the GPIanchored molecules to participate in the signal transduction system of the cell (22, 35). We have now shown that
CD73 on EC was unable to deliver a tyrosine phosphorylation inducing signal upon mAb triggering, whereas triggering of lymphocyte CD73 induced tyrosine phosphorylation (see Fig. 9; 9). It is possible that the association of
CD73 in clusters with other signaling molecules is strong in lymphocytes and less frequent in EC, and this is responsible for the different signal transducing abilities. The different clustering/capping tendencies of CD73 on lymphocytes versus EC, demonstrated by fluorescence microscopy
(Fig. 5), also support the view that the subcellular localization and association with other cellular molecules is critical
for the physiological responses mediated by the molecule.
As CD73 has been shown to be involved in controlling
lymphocyte binding, it is possible that concentrating several CD73 molecules together in large clusters facilitates
the adhesion mediated by this molecule. Alternatively,
CD73 on lymphocytes could function as a co-regulatory
molecule, in which case signals mediated by ligand binding
to CD73 might lead to activation of other adhesion molecules. Interestingly, co-localization of Several adhesion molecules are known to have alternative forms which are GPI linked to the cell surface, but not
much is known about the functional importance of these
modifications. We believe that GPI-linked molecules, being very mobile, can provide an efficient way of controlling
the migratory process of lymphocytes, and we suggest that
the rapid shedding of lymphocyte CD73 due to ligand binding represents a novel mechanism for regulating lymphocyte-EC contacts in the high endothelial venule. The
fact that the two anti-CD73 mAbs which recognize different epitopes of the molecule (i.e., 4G4 and 1E9), have different effects on the shedding of CD73, also suggests that
the physiological functions mediated through CD73 are
dependent on the binding of the ligand(s) to distinct epitopes of CD73. Ligand-induced shedding, mimicked by mAb binding, has also been previously described for the
CD44 molecule both in vitro and in vivo, and, interestingly, only certain anti-CD44 mAb's are able to induce the
release of the molecule. In vivo, the shedding of CD44 has
been suggested to have importance in controlling extravasation of lymphocytes at inflammatory sites (6). Furthermore, L-selectin shedding is a well known phenomenon, and, recently, inhibition of the shedding was shown to impair neutrophil rolling (43).
Indicative of the data presented, CD73 in EC might
have a completely different function from CD73 in PBL.
Electron microscopy studies have revealed ecto-5 In conclusion, these studies support the view that structurally identical CD73 molecules expressed on different
cells and tissues have different functions. The enzymatic
function of CD73 (or ecto-5-nucleotidase (ecto-5
-NT), is a 70-kD glycosyl-phosphatidylinositol (GPI)-linked molecule which
can be detected in several different mammalian tissues
and cell types (46). Ecto-5
-NT enzyme activity catalyzes
the extracellular dephosphorylation of nucleoside monophosphates to their corresponding nucleosides. This enables the uptake of adenosine, inosine, and guanosine into the cell and their subsequent reconversion into ATP and
GTP in the purine salvage pathway (36). The physiological
role of ecto-5
-NT, however, probably differs in various
organisms and tissues, and it most likely extends beyond
its enzymatic activity (46). Plasma membrane-bound ecto5
-NT (CD73) has been shown to be involved in controlling lymphocyte-EC interactions, as binding of lymphocytes to cultured EC can be inhibited by an anti-CD73
mAb (2, 3). Ecto-5
-NT has also been implicated in cell-
matrix interactions in chicken fibroblasts (33) and as a signal transducing molecule in the human immune system (9,
24, 37). In particular, its role as a costimulatory molecule
in T cell activation has been well established (14, 24).
Transient expression of CD73 on neuronal cells has been
described during developmental processes and, on lymphocytes, CD73 serves as a maturation marker, being absent from the surface of both immature B and T cells (15, 37).
-NT has also been detected in nervous tissue: on venules in various tissues and on follicular dendritic cells in the secondary lymphoid tissue (2, 10, 40).
The subcellular expression of ecto-5
-NT has been studied intensively in rat liver tissue where the molecule is expressed both intracellularly and on the surface of hepatocytes. A similar localization has been observed in rat fibroblasts, guinea pig neutrophils, and capillary EC (18, 23, 28,
45, 46). Continuous recycling of ecto-5
-NT between the
cell surface and the intracellular pools has been described
in hepatocytes, fibroblasts, and rat hepatoma cells (31, 41,
45). The natural ligand(s) of CD73 are not known at
present.
-NT-expressing cells and tissues is substantial and the molecule has several putative
roles, it is important to clarify its functional and structural
properties on different cell types. Thus, we studied properties of CD73 expressed on both cell types having importance in lymphocyte homing, PBL and vascular EC. Significant differences were found in the function and cell
surface modulation of CD73 on these cell types.
Materials and Methods
Fig. 1.
1E9 and 4G4 mAbs recognize distinct epitopes of the
CD73 molecule. 4G4 is a mouse IgG1, and 1E9 is a mouse IgG3
mAb. (a) FACS® staining of HEC cells using 1E9 and FITC anti-
mouse IgG1 (anti-mouse IgG1 faintly cross-reacts with IgG3 causing the low positivity). (b) FACS® staining of HEC cells using
4G4 and FITC anti-mouse IgG1. (c) HEC cells were sequentially
treated with 1E9 mAb, 4G4 mAb, and FITC anti-mouse IgG1.
(d) FACS® staining of HEC cells using 4G4 mAb and FITC anti-
mouse IgG3. (e) FACS® staining of HEC cells using 1E9 mAb
and FITC anti-mouse IgG3. (f) HEC cells were sequentially
treated with 4G4 mAb, 1E9 mAb, and FITC anti-mouse IgG3.
Percentage of positive cells and the mean fluorescence intensity
are indicated in the upper right hand corner of each panel.
[View Larger Version of this Image (22K GIF file)]
20°C. Cells were then pelleted, washed once with cold PBS
containing 0.1% NaN3 and 2% FCS and stained for the expression of
CD73. (d) To study the ability of 4G4 mAb to induce patching or capping of the CD73 molecule on lymphocyte and HEC cell surface, cells were
first incubated with 4G4 mAb for 20 min at 37°C and then washed twice
with RPMI 1640. After this, cells were incubated at 37°C for 20 min in
presence of an FITC-conjugated anti-mouse Ig diluted in RPMI 1640, washed twice with RPMI, and fixed with 1% paraformaldehyde. For comparison, lymphocytes and HEC cells were stained on ice in presence of
0.1% NaN3. Cytocentrifuge preparations of stained lymphocytes and
HEC cells were analyzed under a fluorescence microscope.
-octyl glucoside, 1 mM PMSF, and 1% Aprotinin (Sigma
Chemical Co.) in PBS. The 100 µl aliquots of lysates were cleared by centrifugation and transferred onto a wet nitrocellulose membrane (HybondECL; Amersham Intl., Buckinghamshire, UK) using a dot blot apparatus (The Convertible, Filtration Manifold System; GIBCO BRL, Gaithersburg, MD). Antigen detection on nitrocellulose was performed as described before (25), using either mAb 4G4 at 2 µg/ml as a first stage reagent and a peroxidase-conjugated rabbit anti-mouse Ig (Dakopatts A/S,
Glostrup, Denmark) at 1:3,000 dilution (containing 5% FCS) as a second
stage reagent, or only the peroxidase-conjugated rabbit anti-mouse Ig.
Immunodetection was performed using an ECL detection kit for Western
blotting (Amersham Intl.), following the manufacturer's instructions.
Light emission was detected using Hyperfilm MP (Amersham Intl.).
GGGGATCCAGTTCACGCGCCACAG3
and 5
CCCTCGAGGCAAGGAGAATTTTTGG3
, based on the sequence of the published placental CD73 cDNA (20), were used to amplify a full length CD73 fragment at amplification conditions of 94°C for 1 min, 54°C for 1 min, and
72°C for 1 min with 30 amplification cycles. The resulting 1.75-kb amplified fragment was blunt-end cloned into pUC18 using a SureClone kit (Pharmacia Fine Chemicals) and sequenced on both strands using genespecific primers on an automated sequencer (Prism 377; Applied Biosystems, Foster City, CA).
-NT Inhibition
-NT was analyzed as reported previously (3). [14C]AMP
(ICN Biomedicals, Inc., Irvine, CA) was converted into 14C-adenosine and
14C-inosine by HEC ecto-5
NT in presence of 30 µg/ml of purified antiCD73 mAb 1E9 or negative control mAb 3G6. Substrate and product were separated by thin-layer chromatography, and the corresponding radioactivity was detected by autoradiography. Quantitation was performed
by measuring the intensity of the dots, using the Microcomputer Imaging
Device (MCID; Imaging Research Inc., Ontario, Canada).
T
cells and B cells) were washed twice with PBS and then incubated for 30 min at 4°C in the presence of anti-CD73 mAb 4G4, anti-CD73 mAb 1E9,
or a negative control mAb 3G6. After two washes with PBS, a rabbit anti-
mouse Ig (Dako, Santa Barbara, CA), at a 20 µg/ml concentration, was
added, and incubation continued for another 30 min. After washing, the
cells were incubated for variable time periods at 37°C in RPMI 1640 to
allow signaling, and then the cells were lysed in lysis buffer containing
10 mM EDTA and 1 mM orthovanadate (Sigma Chemical Co.; for EC,
1 ml of buffer was used and for lymphocytes, 200 µl of buffer was used).
The cell lysate was centrifuged at 13,000 rpm for 15 min at 4°C and treated with protein G-Sepharose to remove immunoglobulins. Total cellular proteins in 45 µl of lysate were separated by SDS-PAGE and transferred onto nitrocellulose. To detect tyrosine phosphorylated proteins, the filters
were first incubated for 1 h in 5% BSA in TBS-T (0.05% Tween in TBS)
and then immunoblotted with an anti-phosphotyrosine mAb (4G10; Upstate Biotechnology). An irrelevant mAb was used as a negative control at
an equivalent concentration (0.2 µg/ml). The second-stage reagent was a
biotinylated anti-mouse IgG (Vector Laboratories, Burlingame, CA)
which was finally detected by a horseradish peroxidase biotin streptavidin
conjugate (Amersham Intl.). The filters were developed using enhanced
chemiluminescence.
Results
Fig. 2.
Triggering of CD73 at 37°C results in CD73 downregulation in lymphocytes but not in EC. (A) Lymphocytes, HECs,
and HUVECs were incubated for 2 h in presence of an antiCD73 mAb 4G4, anti-CD73 mAb 1E9, or a negative control
mAb 3G6 at 37°C, and the expression of CD73 on the cell surface
was analyzed by IF and FACS®. The arrows in the middle and
bottom panels point at the CD73 positive subpopulation of PBL.
(B) Downregulation of CD73 on lymphocyte surface is temperature dependent. CD73 expression was analyzed by IF staining
and FACS® after incubating lymphocytes and HECs in presence
of an anti-CD73 (
, continuous line) or in presence of a negative control mAb (
, dashed line) for various time periods at
37° or 4°C. Percentage of positive cells is shown in the graphs on
the left, and the MFI of positive cells is shown in the graphs on
the right.
[View Larger Versions of these Images (39 + 31K GIF file)]
-NT in rat fibroblasts and hepatocytes (31, 41, 45). We studied whether
similar spontaneous recycling of CD73 would take place in
EC. Triggering of CD73 with an mAb does not seem to
greatly affect the level of surface expression of the molecule on EC, as shown in Fig. 2. However, when EC, which
had been surface labeled with anti-CD73 mAb on ice,
were incubated at 37°C in fresh medium, it was seen that a
considerable portion of the CD73 molecules was removed from the cell surface, and new CD73 molecules appeared
on the surface as a replacement. This was seen as a markedly lower net MFI after detection with only an FITC
anti-mouse Ig as compared to the net MFI after detection
with both anti-CD73 mAb and a second stage Ab (Fig. 3 A).
The percentage of positive cells was only slightly reduced,
suggesting that only a portion of CD73 molecules on each
individual cell is removed from the surface, while the rest
are unaffected (Fig. 3 A). Thus, in the case of the EC,
binding of CD73 does not seem to affect its surface expression, but rather, the results suggest that the CD73 molecules are recycling.
Fig. 3.
Downregulated CD73 is rapidly replaced by new CD73
molecules. (A) Lymphocytes, HECs, and HUVECs in suspension
were preincubated with negative control mAb, anti-CD73 mAb
4G4, or anti-CD73 mAb 1E9, at 4°C; washed twice and incubated
in complete medium at 37°C for 15 min; and then stained with either anti-CD73 mAb and a second stage FITC-conjugated Ab, or
only with a second stage Ab. The arrows in the middle and bottom panels point at the CD73 positive subpopulation of PBL. (B)
FACScan® analysis of lymphocytes and HECs after a saturating
preincubation on ice with an anti-CD73 mAb and a subsequent
incubation in complete medium at 37°C for various periods of
time. After incubation, cells were either labeled with both anti-CD73 mAb and a second stage, FITC-conjugated Ab (
, continuous
line) or only with a second stage, FITC-conjugated Ab (
, dashed line). MFI is measured for all cells. (C) CD73 can be detected from
the lymphocyte supernatant but not from the EC supernatant. Lymphocytes, HECs, and HUVECs were incubated with the anti-CD73
mAb as described above, and the cell lysate and the incubation supernatant were analyzed for presence of CD73 using either both a primary and a secondary Ab or only a secondary Ab in a dot blot assay, as described in Materials and Methods. The three spots in each
panel represent different numbers of cells. For lymphocytes: left spot, 1 × 106 cells, middle spot, 0.5 × 106 cells, and right spot, 0.25 × 106
cells except that the right spot of the cell lysate detected with both first and second stage Abs has 1.5 × 106 cells. For EC: left spot, 0.9 × 106 cells, middle spot, 0.6 × 106 cells, and right spot, 0.3 × 106 cells.
[View Larger Versions of these Images (38 + 18 + 22K GIF file)]
Fig. 4.
CD73 is not detectable in permeabilized lymphocytes
after mAb 4G4 treatment at 37°C. FACS® analysis was performed with permeabilized and non-permeabilized lymphocytes
after 2 h incubation at 37°C in presence of either anti-CD73 mAb
4G4 or negative control mAb. The arrows in the middle panel
point at the CD73-positive subpopulation of PBL.
[View Larger Version of this Image (29K GIF file)]
Fig. 5.
Cross-linking of CD73 results in different staining patterns on lymphocyte and endothelial surfaces. Lymphocytes and EC
were stained with mAb 4G4 and a second stage FITC-conjugated Ab, as described in Materials and Methods. (a) Patching of CD73 on
lymphocyte surface after cross-linking at 4°C. (b) At 37°C, the majority of lymphocytes becomes CD73 negative due to CD73 cross-linking, and the remaining CD73-reactivity is found in large patches (arrow). (c) Capping of CD73 on EC after cross-linking at 4°C (arrow). (d) Capping of CD73 on EC after cross-linking at 37°C (arrows). (e) Punctate staining pattern of adherent HEC cells stained with antiCD73 mAb at 4°C. (f) Punctate staining pattern of adherent HEC cells stained with anti-CD73 mAb at 37°C. (g) Adherent HEC cells
stained with a negative control mAb 3G6 at 37°C. In a, b, c and d, cells were stained in suspension and analyzed microscopically on
cytospin preparations. Bar, 10 µm.
[View Larger Version of this Image (92K GIF file)]
Fig. 6.
Molecular mass of CD73 from lymphocytes and EC.
(A) 125I-labeled HECs were lysed, and immunoprecipitations
were performed with a negative control mAb (lane 1), mAb 4G4
(lane 2, arrow), and mAb Hermes-3, which recognizes the CD44
molecule (lane 3) as described in Materials and Methods. (B) Lysates from freshly isolated 125I-labeled PBL were immunoprecipitated with a negative control mAb (lane 1), mAb 4G4 (lane 2, arrow), and mAb Hermes-3. No significant size differences were
observed between CD73 molecules from lymphocytes and EC.
[View Larger Version of this Image (45K GIF file)]
Fig. 7.
Northern blot analysis of lymphocyte and endothelial CD73 mRNA.
PolyA+ RNA (10 µg/lane),
prepared from human PBL
and from cultured HECs, was denatured and separated
by electrophoresis on a 1.1%
agarose gel. The RNA was
blotted onto a Hybond N
membrane and hybridized with a 32P-labeled CD73
cDNA probe. Posthybridization wash conditions were 0.1× SSC, 0.1% SDS at 65°C
for 1 h. SSC is 0.15 M NaCl,
0.015 M Na citrate. The signal detected from lymphocyte RNA was several times
stronger than that from HEC
RNA, and thus, the exposure time for the PBL blot is 12 h
and the exposure time for the
HEC blot is two weeks.
[View Larger Version of this Image (32K GIF file)]
Fig. 8.
PI-PLC treatment of HEC cells. HEC cells in suspension were treated with PI-PLC and analyzed by immunofluorescence staining and FACS® for the expression of CD73. Negative
control staining was performed using a class-matched, nonbinding mAb as the first stage antibody.
[View Larger Version of this Image (10K GIF file)]
-NT Activity
by an Anti-CD73 mAb
-NT activity. Ecto-5
-NT activity of
CD73 expressed on human PBL has been shown earlier (3, 40).
Fig. 9.
Characterization of function of endothelial CD73 molecule. (A) Endothelial CD73 has an intact ecto-5-NT activity
that is inhibitable by anti-CD73 Ab but not with a negative
control mAb. Conversion of [14C]AMP to [14C]adenosine and
[14C]inosine by endothelial ecto-5
-NT was quantitated, as described in Materials and Methods. The results shown are the
mean of duplicates from two independent experiments ± SEM.
(B) mAb triggering of CD73 on lymphocyte surface causes tyrosine phosphorylation of certain protein substrates. PBL enriched for CD73+ cells were treated with a negative control, 1E9
and 4G4 mAb's for different periods of time, and lysed. Tyrosine
phosphorylation was detected from the lysates by tyrosine blotting, as described in the Materials and Methods. Arrows point at
the 26- and 28-kD tyrosine-phosphorylated products induced by
anti-CD73 triggering. (C) mAb triggering of CD73 on the endothelial surface does not cause tyrosine phosphorylation. Adherent HECs were triggered with either an anti-CD73 mAb or a negative control mAb for 15, 30, or 45 min, cells were lysed, and
tyrosine phosphorylation of intracellular proteins was detected
from the lysates by tyrosine blotting, as described in Materials
and Methods.
[View Larger Versions of these Images (38 + 32 + 51K GIF file)]
lymphocytes, among which CD73+ cells
are highly enriched; and thus, the detection of CD73-
mediated signals is facilitated. In lymphocytes the appearance of an ~26-kD phosphorylated substrate was observed 5 min after beginning triggering with anti-CD73
mAb 1E9 (time point zero is when the unbound antibody is washed away and the cells are resuspended in warm medium to allow signaling). The phosphorylation peaked at
15 min and was still clearly visible at 45 min. At the 5 min
time point, 1E9 also triggered stronger phosphorylation of
a 28-kD protein, as compared to negative control mAb.
The phosphorylation of the 28-kD protein was strongest at
30 min and weaker at the longest triggering time of 45 min.
Induction of a tyrosine phosphorylated protein of the
same size and with similar kinetics has also been reported earlier due to anti-CD73 triggering (9). The appearance of these 26- and 28-kD bands was specifically due to triggering with 1E9 mAb since no such induction was observed
after triggering with an irrelevant control antibody 3G6 or
mAb 4G4 under the same conditions (Fig. 9 B). In addition, induction of an ~50-kD phosphorylated protein was
seen at shorter time points (1 and 5 min). A 50-kD phosphorylated band was also visible 15 min after commencing the triggering with 4G4 mAb. When EC were
triggered under similar conditions, no differences in the
phosphotyrosine blots were seen when the triggering was
performed using either an irrelevant control Ab or an antiCD73 mAb 1E9 for 15, 30, or 45 min (Fig. 9 C). Triggering of EC with an anti-CD73 or a negative control mAb was
also performed for shorter periods of time (data not
shown), but this also had no effect. This suggests that the
CD73 molecule has different signal transducing properties
in lymphocytes and EC. In lymphocytes, a phosphotyrosine pathway is involved, but whether CD73-mediated signaling plays any role in EC remains to be seen.
Discussion
-NT in rat fibroblasts, hepatocytes,
and a hepatoma cell line (31, 41, 45).
2-integrins in GPIlinked molecule rich clusters has been described, and thus,
activation of LFA-1 may be controlled by interaction with
some of the cluster-associated molecules (7). Accordingly,
1-integrin-mediated cell adhesion was recently shown to
be regulated by the urokinase receptor, a GPI-linked cell
surface protein (44). We have previously suggested a connection between LFA-1 and CD73, because no additive inhibition in lymphocyte binding to EC was seen when both
anti-LFA-1 and 4G4 mAbs were present in the adhesion
assay, as compared to either of them alone (2). It is thus
possible that CD73 signalling plays a role in the regulation
of LFA-1 activity and in this way, has importance in regulating lymphocyte emigration. This is supported by our recent results, which show that triggering of lymphocytes with anti-CD73 mAb 4G4 increases lymphocyte adhesion
to cultured EC by ~50% in an LFA-1-inhibitable manner,
suggesting that CD73 has an important role in regulating
LFA-1-mediated lymphocyte binding (Airas, L., J. Niemelä, and S. Jalkanen, manuscript in preparation). Shedding of CD73 from the lymphocyte surface is likely to have
profound importance for the regulation of LFA-1 activity,
as triggering with the anti-CD73 mAb 1E9, which does not
cause shedding of CD73, also fails to have the LFA-1-activating effect. Furthermore, we have recently shown that
CD73 on lymphocytes plays an important role in mediating binding of lymphocytes to venules in inflamed skin,
whereas CD73 on EC has minimal importance in this (4).
-NT activity both at the basal and the luminal side of EC in intact
venules (26). As ecto-5
-NT has been shown to bind to
laminin and fibronectin, it is possible that ecto-5
-NT facilitates the attachment of EC to the basement membrane
(33). The ecto-5
-NT enzyme activity in EC is also quite
high (Fig. 9 A), and thus the ecto-enzyme function in the vessels probably also has physiological significance.
-NT) is to hydrolyze extracellular nucleotides to their corresponding nucleosides, but
there is an increasing amount of evidence for other functions for the CD73 molecule (46). In particular, a role in
mediating adhesion of different cell types and a co-signaling role in T cell activation and proliferation are of interest. Importantly, the suggested functions of CD73 do not
mutually exclude each other; instead, they give a clear example of how the physiological role of a molecule can vary
between different cells and organs. The differential modulation of CD73 in various cell types likely has fundamental
importance in the regulation of these divergent functions.
Received for publication 6 March 1996 and in revised form 11 November 1996.
Please address all correspondence to Laura Airas, MediCity Research Laboratory, Tykistökatu 6A, 20520 Turku, Finland. Tel.: 358-2-333-7001; Fax: 358-2-333-7000.We thank Ms. Merja Mäkitalo for excellent technical assistance; Dr. Erkki Nieminen for help with the figures, and Dr. Kaisa Silander for DNA sequencing. Dr. Linda Thompson (Oklahoma Medical Research Foundation, Oklahoma City, OK) is greatly acknowledged for sending us the 1E9 mAb.
This work was supported by the Finnish Academy, the Finnish Cancer Union, the Sigrid Juselius Foundation, the Life and Pension Insurance Companies, the Paulo Foundation, and the Research and Science Foundation of Farmos. Dr. David J. Smith is a fellow of the European Community Human Capital and Mobility program.
EC, vascular endothelial cells;
ecto-5NT, CD73/ecto-5
-nucleotidase;
GPI, glycosyl-phosphatidylinositol;
HUVEC, human umbilical vein endothelial cells;
IF, immunofluorescence;
MFI, mean fluorescence intensity;
PBL, peripheral blood lymphocytes;
PI-PLC, phosphatidylinositol-specific phospholipase C.