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Prepublished online as a Blood First Edition Paper on September 26, 2002; DOI 10.1182/blood-2002-06-1842.
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Blood, 15 February 2003, Vol. 101, No. 4, pp. 1582-1590
PHAGOCYTES
Convergence of the adhesive and fibrinolytic systems: recognition
of urokinase by integrin  M 2 as well as
by the urokinase receptor regulates cell adhesion and
migration
Elzbieta Pluskota,
Dmitry
A. Soloviev, and
Edward F. Plow
From the Joseph J. Jacobs Center for Thrombosis and
Vascular Biology, Department of Molecular Cardiology, Cleveland Clinic
Foundation, Cleveland, OH.
 |
Abstract |
Previous studies demonstrated that integrin
 M 2 (CD11b/18, Mac-1) forms a physical
complex with the urokinase-type plasminogen activator receptor
(uPAR/CD87) on leukocytes. In this study, we used human peripheral
blood neutrophils and transfected cells expressing
M2, uPAR, or both receptors to show that
the integrin can directly interact with urokinase (uPA). We demonstrate
that M2 supported adhesion and
migration of these cells to uPA, and, in each case, blockade of
M2 suppressed the response. Within uPA,
both the kringle and proteolytic domains are recognized by M2, which are distinct from the growth
factor domain that binds to uPAR. Within the M subunit
of the integrin, the I domain interacts with uPA, which is distinct
from the region that interacts with uPAR. On cells expressing uPAR and
M2, both receptors mediated adhesion and
migration. This cooperation was particularly apparent in the responses
of neutrophils to uPA, where blockade of
M2 reduced uPAR-mediated responses and
engagement of uPAR enhanced recognition of uPA by
M2. Thus, recognition of uPA by
M2 allows for formation of a multicontact
trimolecular complex, in which a single uPA ligand may bind
simultaneously to both uPAR and M2. This
complex may play an important role in the control of inflammatory cell
migration and vascular homeostasis.
(Blood. 2003;101:1582-1590)
© 2003 by The American Society of Hematology.
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Introduction |
Cell migration is a complex response that requires
the coordination and cooperation among multiple cell surface receptors, including sensory receptors that detect migratory stimuli, adhesion receptors that mediate interactions of migrating cells with the extracellular matrix, and protease receptors that facilitate movement of cells through their extracellular environment. Members of the integrin family of receptors recognize many extracellular matrix proteins to which cells adhere and de-adhere as they migrate. Binding
partners for proteins of the plasmin(ogen)/fibrinolytic system, such as
plasminogen-binding proteins and urokinase-type plasminogen activator
receptor (uPAR), focus proteolytic activity, particularly to
the leading edge of migrating cells, thus facilitating degradation and
remodeling of the extracellular matrix.1-5 Cooperation between these 2 cell surface systems is particularly evident in the
recruitment of leukocytes during inflammatory responses, whether outside the vasculature or inside the vessel wall. Mice rendered deficient in members of the 2 subfamily of leukocyte
integrins or in components of the plasminogen system exhibit blunted
inflammatory responses in a variety of in vivo
models.6-11
Evidence for direct interplay between integrins and the fibrinolytic
system at cell surfaces has emerged over the past several years. For
example, vitronectin, a matrix protein recognized by several integrins,
was shown to be a ligand for uPAR,12 and urokinase
(uPA) binding to uPAR was shown to change the specificity of several
integrins.13,14 Plasminogen activator inhibitor 1 (PAI-1), an inhibitor of uPA,15 bound to
vitronectin,16,17 thereby modulating cell migration by
inhibiting vitronectin binding to integrins and to
uPAR.18,19 An interaction critical to several of these
functional relationships between the 2 systems is the direct
interaction between uPAR and the integrins.1,20 Several reports of integrin/uPAR interaction have centered on
M2 (Mac-1, CD11b/CD18, CR3) of the
2 subfamily of leukocyte integrins. uPAR and
M2 copurify in monocyte
lysates21 and form a reversible complex on neutrophils as
detected by immunolocalization and fluorescence resonance energy
transfer studies.22,23 Recently, a peptide sequence from
within the M subunit (M25, residues
424-440)24 was shown to bind uPAR and disrupt its
complexes with M2.1,24 The
interaction between uPAR and M2
influences the functions of each other. Complex formation between uPAR
and M2 enhances M2-mediated adhesion to fibrinogen (Fg)
and other ligands of this integrin, as well as degradation of Fg by
human monocytes.14 uPAR engagement of vitronectin also
facilitates M2 function.25 On the other hand, M2 activation
increases uPAR-dependent cell adhesion to
vitronectin.14,25 The importance of the uPA/uPAR system in
the regulation of M2-dependent leukocyte
functions is underscored by several recent observations. uPAR-deficient mice exhibit reduced neutrophil recruitment to inflammatory
stimuli.10 In addition, uPA- and uPAR-deficient mice
exhibit impaired T-cell and macrophage recruitment to
Cryptococcus neoformans, which results in increased
mortality of these animals during infection.6
In the present study, we demonstrate an additional and potentially
important element to the interrelationship between uPAR and
M2. uPA is shown to be a ligand for
M2. Moreover, the region of
M2 that binds to uPA is different from
that which binds to uPAR, and the regions of uPA that bind to
M2 are different from those that bind to
uPAR. Therefore, a trimolecular complex of
uPA/uPAR/M2, in which uPA binds to both
receptors, may form on the leukocyte surface and display unique
functional properties.
| |
Materials and methods |
Reagents, antibodies, and synthetic peptides
Recombinant human high-molecular-weight (HMW)-tc-uPA
was a generous gift from Jack Henkin (Abbott Laboratories, Chicago,
IL). Neutrophil inhibitory factor (NIF) was provided by Corvas
International (San Diego, CA). The recombinant uPA domains growth
factor domain (GFD; residues 4-43), kringle domain (KD; residues
47-135), and low-molecular-weight (LMW)-tc-uPA (residues
136-411) were purchased from Calbiochem (San Diego, CA). Recombinant
human intercellular adhesion molecule 1 (ICAM-1) was from R & D
Systems (Minneapolis, MN) and human Fg from Enzyme Research Labs (South
Bend, IN). The Cyquant-Cell Proliferation Kit,
phosphatidylinositol-specific phospholipase C (PI-PLC), and
Alexa 488-labeled goat antimouse IgG were purchased from Molecular
Probes (Eugene, OR). The MI domain was
expressed as glutathione-S-transferase (GST)-fusion protein
and purified on glutathione-Sepharose 4B (Pharmacia Biotech, Piscataway, NJ) as previously described.26
Monoclonal antibody (mAb) CBRM1/527 was kindly provided by
Dr T. Springer (Harvard Medical School, Boston, MA). The mAbs 44a, IB4,
and W6/32 were from American Type Culture Collection (Rockville, MD);
mAb P4H9 was from Chemicon International (Temecula, CA); and uPAR mAbs 62022.11 and 3936 were from R & D Systems and American Diagnostica (Greenwich, CT), respectively. M25 (PRYQHIGLVAMFRQNTG)
and control scrambled M25 (M25 SCR;
HQIPGAYRGVNQRFTLM)13,24 peptides were synthesized on an
Applied Biosystems model 430A peptide synthesizer (Foster City, CA)
using N-(9-fluorenyl)methoxycarbonyl chemistry.
Cell lines and neutrophil preparations
Granulocytes were isolated from human peripheral blood of
healthy volunteers drawn into sterile acid-citrate-dextrose
(1:7 volume 145 mM sodium citrate, pH 4.6, and 2% dextrose).
Isolation was performed by density gradient centrifugation onto
Ficoll-Hypaque (Pharmacia, Uppsala, Sweden), followed by dextran
sedimentation of erythrocytes and hypotonic lysis of residual
erythrocytes. The remaining cells were 98% granulocytes, of which more
than 96% were neutrophils and 2% were eosinophils. Contaminating
lymphocytes and monocytes were less than 2% as determined by
Wright staining.
Human epithelial kidney 293 cells transfected with
M2 were maintained as described
previously.28 Nontransfected and
M2 293 cells were stably transfected in
the absence of serum using the LipofectAMINE Plus reagent (Invitrogen,
Carlsbad, CA) with 0.5 to 5 µg pcDNA 3.1 containing the cDNA
for uPAR or vector alone. Cell clones were selected using neomycin
sulfate (Invitrogen), and cells expressing uPAR were detected and
isolated by fluorescence-activated cell sorting (FACS).
Soluble HMW-tc-uPA binding
uPA was labeled with Alexafluor-488 according to manufacturer's
protocol (Molecular Probes). Unstimulated, phorbol myristate acetate (PMA)-treated human neutrophils or HEK293 cells were
suspended in Dulbecco modified Eagle medium (DMEM)/F-12 medium, 1 mM
Mg2+, 0.1% bovine serum albumin (BSA), and 100 nM Alexa
488-HMW-tc-uPA and incubated at 37°C for 0 to 4 hours. Cells were
centrifuged through a cushion of fetal calf serum (FCS) twice and
resuspended in 1% paraformaldehyde/phosphate-buffered saline (PBS).
Cell-bound HMW-tc-uPA was detected by FACS.
Adhesion assays
The 96-well nontissue culture-treated plates (Falcon, Becton
Dickinson, San Diego, CA) were coated with HMW-tc-uPA, GFD, KD, LMW-tc-uPA, or BSA (200 nM in PBS) for 3 hours at 37°C and then blocked with 0.5% polyvinylpyrrolidone (PVP) for 1 hour at room temperature. The 293 cells and neutrophils were resuspended in the
serum-free DMEM/F-12 medium. Neutrophils were stimulated with 20 nM PMA
(Sigma Chemical, St Louis, MO) for 20 minutes at 37°C. Only about 5%
to 8% of neutrophils were apoptotic as determined by fluorescein
isothiocyanate (FITC)-labeled annexin V binding (R & D Systems). In
inhibition experiments, the cells were pretreated with respective
antibodies or reagents for 30 minutes at 37°C, then seeded at 1 to
2 × 105 cells/well onto the coated plates and incubated
at 37°C for 30 minutes. The plates were washed with PBS, and the
number of adherent cells in each well was quantified using the Cyquant
Cell Proliferation Assay Kit (Molecular Probes), according to the
manufacturer's instructions. The data from cell adhesion and migration
assays are presented as the total number of adherent or migrated cells, determined from standard curves developed with a known number of
Cyquant-labeled cells.
Migration assays
Cell migration assays were performed in serum-free DMEM/F-12
medium using Costar 24-transwell plates with 3 µm (for neutrophils) or 8 µm (for 293 cells) pore polycarbonate filters (Corning, Corning, NY). HMW-tc-uPA and its domains (0-100 nM) were added to the lower chambers in total volume of 600 µL medium, whereas the upper wells contained a final volume of 200 µL after addition of the cells. To
commence the assay, 50 µL cell suspension (2 × 105
cells/well) was added to the upper chambers, and the plates were placed
in a humidified incubator at 37°C and 5% CO2. For
inhibition experiments, the function-blocking mAbs were added to the
upper chambers at 20 µg/mL. Assays were stopped after 6 hours by
removing the upper wells and wiping the inside of upper wells with a
cotton swab to remove nonmigrated cells. The migrated cells were
quantitated using the Cyquant Cell Proliferation Kit.
| |
Results |
Neutrophil responses to uPA are mediated by both uPAR and
M2
Most studies of uPAR/integrin interactions have emphasized the
influence of uPAR on integrin-mediated responses. In view of the
physical association between M2 and uPAR
on the surface of leukocytes,13,21-23 we hypothesized that
the integrin might influence uPAR-dependent responses. Because the
uPA/uPAR system is implicated in cell migration, a process
indispensable in inflammation, we first assessed the influence of
M2 on neutrophil migration toward uPA.
Consistent with previous reports,29 neutrophils migrated
toward HMW-tc-uPA; the increase in migration to uPA was 3-fold greater
than to buffer (Figure 1A). The
function-blocking mAb (clone 62022.11) to uPAR inhibited this migration
to background levels (Figure 1A). When 2 different mAbs to
M2 were added, they were as effective as
anti-uPAR in suppressing the migratory response. One of these mAbs
(44a) was directed to the M and the other (IB4) to the
2 subunit. Each completely blocked neutrophil migration
to uPA, whereas a control mAb, W6/32, to an unrelated surface antigen
(major histocompatability complex class I [MHC-I]), had no
effect.
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| Figure 1.
Recognition of uPA by neutrophils is
M2 integrin- and uPAR-dependent.
(A) Migration to uPA. Human polymorphonuclear cells (PMNs) were
resuspended in a serum-free DMEM/F-12 medium containing PMA (20 nM) and
added (2 × 105 cells/well) to the upper chambers of
transwells. HMW-tc-uPA was added to the lower chambers, and selected
function-blocking mAbs (20 µg/mL) were added to both chambers. PMNs
were allowed to migrate to the lower chamber for 6 hours in a
humidified incubator at 37°C and 5% CO2. The number of
migrated cells was determined as described in "Materials and
methods." The data are expressed as means ± SEM of duplicate
wells from 3 independent experiments. (B) Adhesion to uPA.
PMA-stimulated PMNs (1 × 105 cells/well) were seeded
onto wells of microtiter plates coated with HMW-tc-uPA or BSA and
allowed to adhere for 30 minutes at 37°C. Nonadherent PMNs were
removed by 3 washings with PBS, and representative photomicrographs of
the wells were taken (Original magnification, × 200). (C)
Direct binding of uPA. Unstimulated or PMA-stimulated PMNs (20 nM, 20 minutes at 37°C) were incubated in DMEM/F-12 medium/0.1% BSA with
100 nM Alexa 488-HMW-tc-uPA in the absence or presence of the
indicated blocking mAbs for 30 minutes at 37°C. Cells were
centrifuged through a cushion of FCS twice and resuspended in 1%
paraformaldehyde/PBS. Cell-bound HMW-tc-uPA was detected by FACS
analysis.
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Soluble complexes of uPA and uPAR can be sequestered by vitronectin
within the extracellular matrix30 and uPA can bind to uPAR
on the surfaces of cells (for reviews, see Chapman and
Wei1 and Preissner et al4). Such interactions
would present uPA as an immobilized substrate, which could support
adhesion of M2-bearing cells. To test
this possibility, neutrophils were added to plates coated with BSA or
HMW-tc-uPA, and adhesion was measured after 30 minutes at 37°C. As
shown in Figure 1B, when neutrophils were stimulated with PMA, they
attached and spread on HMW-tc-uPA but not BSA. Nonstimulated
neutrophils showed little adhesion to either substratum. The mAbs not
only to uPAR but also to M2 reduced adhesion of the stimulated neutrophils onto HMW-tc-uPA (Figure 1B).
Control mAb W6/32 or nonimmune mouse IgG decreased adhesion by less
than 10% under these conditions (not shown). Taken together, these
data suggest a significant role for both uPAR and
M2 in the adhesive and migratory response
of human neutrophils to uPA.
Migration and adhesion are complex responses, and we sought to more
directly examine the role of the 2 receptors in uPA recognition by
measuring the binding of Alexa 488-labeled HMW-tc-uPA to neutrophils by FACS (Figure 1C). Unstimulated neutrophils showed little binding of
soluble Alexa 488-labeled HMW-tc-uPA (mean fluorescence intensity [MFI] = 10.9); however, on stimulation of the cells with PMA, binding increased by 8-fold (MFI = 92.3). The Alexa 488-HMW-tc-uPA binding was completely inhibited by a 50-fold molar excess of unlabeled
HMW-tc-uPA, verifying specificity (data not shown). Binding of labeled
HMW-tc-uPA to PMA-stimulated cells was significantly abrogated by
blocking mAbs to uPAR (MFI = 32.3) and to the M (MFI = 12.1) and 2 subunits (MFI = 16.8) of
M2. Control mAb W6/32 or nonimmune mouse
IgG did not inhibit uPA binding to neutrophils (data not shown). In
addition, NIF, a high-affinity ligand of the MI domain
of M2,31,32 also blocked
binding of the Alexa 488-labeled HMW-tc-uPA to the cells. Thus,
recognition of uPA by neutrophils and the functional responses of the
cells to uPA clearly are influenced by M2
as well as by uPAR.
M2 recognizes HMW-tc-uPA and uPAR
enhances the interaction
A likely explanation for these observations is that
M2 may directly recognize uPA. This
possibility was pursued using transfected HEK293 cells expressing
M2, uPAR, or both
M2/uPAR. FACS analyses were performed to
estimate the expression levels of the various receptors, and the
results are summarized in Table 1. These
data indicate that the expression level of uPAR or
M2 was not influenced by the presence or
absence of the other receptor. The nontransfected HEK293 cells
exhibited no detectable expression of M2
and very low expression (~2-fold above background) of uPAR. The level
of uPAR expression in the transfected cells was 8- to 9-fold higher than that of the endogenous receptor and was similar in the presence or
absence of M2. The
M2 expression levels were similar in the
presence or absence of uPAR. Thus, functional differences between
M2 in the
M2 cells and the
M2/uPAR cells or between uPAR in the uPAR
cells and M2/uPAR cells could not be
attributed to the expression levels of the receptors.
Adhesion of the various cell lines to different forms of uPA,
enzymatically inactive, single-chain uPA (HMW-sc-uPA), enzymatically active 2-chain uPA (HMW-tc-uPA), and
diisoproprylfluorophosphate-inactivated (DIP)-HMW-tc-uPA, was
measured. All 3 forms of uPA supported adhesion of the
M2 cells, even though uPAR expression was
minimal on these cells (Figure 2A),
indicating that recognition of uPA by M2
does not require proteolytically active uPA. The extent of adhesion to
these 3 uPA forms was similar and was similar to that observed with the
cells expressing uPAR alone. In contrast, mock-transfected cells or
cells expressing a different 2 integrin,
L2, which expressed similarly low levels
of endogenous uPAR as the M2 cells,
failed to adhere to uPA. Additionally, uPA recognition by
M2/uPAR cells was enhanced 2-fold
compared with the cells expressing M2 or
uPAR alone, suggesting additive contributions of the 2 receptors to
recognition of the ligand. Adhesion of all cell lines to a control
protein, BSA, was negligible.
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| Figure 2.
Integrin M2 binds
HMW-tc-uPA and uPAR enhances the interaction.
(A) Adhesion to uPA. HEK293 cells (2 × 105
cells/well) expressing uPAR or 2-integrin receptors or
both were seeded onto wells of microtiter plates coated with
HMW-sc-uPA, HMW-tc-uPA, DIP-HMW-tc-uPA (HMW-tc-uPA was treated
with 5 mM DFP for 2 hours at room temperature and extensively
dialyzed against PBS), or BSA. Cells were allowed to adhere for 30 minutes at 37°C. After several washings, the number of adherent cells
was quantified using the Cyquant Cell Proliferation Kit as described in
"Materials and methods." The data are means ± SEM of
quadruple measurements from 3 independent experiments. (B) Direct
binding of tc-uPA. HEK M2 and
mock cells were incubated in DMEM/F-12 medium/1 mM
Mg2+/0.1% BSA with 200 nM Alexa 488-HMW-tc-uPA for 0 to 4 hours at 37°C. Cell-bound uPA was detected by FACS. (C) Migration to
uPA. HEK293 cells were added (2 × 105 cells/well) to the
upper chamber of transwells in a serum-free DMEM/F-12 medium.
HMW-sc-uPA or HMW-tc-uPA was added to the lower chamber (100 nM). The
cells were allowed to migrate for 6 hours in a humidified incubator at
37°C and 5% CO2. The number of migrated cells was
determined as described in "Materials and methods." The data are
expressed as means ± SEM of duplicate wells from 3 independent
experiments.
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Interaction of uPA as a soluble ligand with the
M2-transfected cells also was
demonstrated. Binding of the Alexa-labeled HMW-tc-uPA with the
M2-expressing HEK cells was detectable by FACS (Figure 2B). The interaction was time dependent and reached a
plateau within 30 minutes and remained constant for up to 4 hours.
Inactivation of the HMW-tc-uPA with diisopropyl-fluorophosphate (DFP) did not affect its binding to the
M2 cells, and no uPA binding to mock
cells was detected (Figure 2B).
Analyses were also undertaken to determine whether uPA could support
M2-mediated cell migration of the
transfected cells and whether uPAR could influence this response.
HMW-sc-uPA and HMW-tc-uPA supported migration not only of uPAR cells
but also cells expressing M2, whereas
mock-transfected cells failed to migrate to both forms of uPA (Figure
2C). The migration of the M2-transfected
cells to HMW-tc-uPA was blocked by mAbs 44a and IB4 to the
M and 2 subunits, respectively.
Consistent with the data of adhesion experiments, the migration of
cells expressing both receptors was higher than that observed with
transfectants expressing the individual receptors.
M2 and uPAR recognize different
domains of uPA
HMW-tc-uPA is composed of an N-terminal growth factor domain
(GFD), a kringle domain (KD), and LMW-tc-uPA, which contains the
protease domain. These various domains of uPA were used to compare the
recognition specificity of M2 and uPAR.
The analyses were first performed in adhesion assays with the various
HEK293 cell lines. uPAR binds to the GFD of uPA33,34 and,
as expected, the HEK293 cells expressing uPAR adhered to the GFD and to
HMW-tc-uPA, which contains the GFD, but not to the KD and LMW-tc-uPA
(Figure 3A). The
M2 cells adhered to HMW-tc-uPA, KD, and
LMW-tc-uPA, but not to the GFD. Thus, M2
recognizes a site in HMW-uPA distinct from the GFD recognized by uPAR.
Although coexpression of uPAR with M2 on
M2/uPAR cells increased adhesion by 50%
to 100%, M2 did not appear to enhance
HMW-uPA binding mediated by uPAR; that is, adhesion of
M2/uPAR cells to the GFD was similar to that of the cells expressing uPAR alone. However, coexpression of the 2 receptors did enhance M2 recognition of
the KD and LMW-tc-uPA (Figure 3A); the adhesion of the cells expressing
both receptors was enhanced to the uPA domains recognized selectively by M2. Mock-transfected cells did not
adhere to uPA or to any of its fragments. As shown on Figure 3B, PMA
stimulation enhanced adhesion of neutrophils to HMW-tc-uPA by 2.5-fold
compared with nonstimulated cells. The PMA-stimulated neutrophils bound
weakly to the GFD, indicating that uPAR can support a low level of
adhesion under the conditions of the assay. However, the adhesion of
the stimulated neutrophils to KD and LMW-tc-uPA was much more extensive (Figure 3B), consistent with a prominent role of
M2 in the interaction. The adhesion of
neutrophils to KD, as well as the binding of Alexa 488-labeled
HMW-tc-uPA to M2-expressing HEK293 cells
detected by FACS (Figure 2B), was not inhibited by the carboxy-terminal lysine analog, 6-aminohexanoic acid (5 mM), or by another
kringle-containing molecule, plasminogen (2 µM). These results are
consistent with previous data indicating that the KD of uPA lacks a
lysine-binding capacity35 and indicate specificity for
recognition of the uPA kringle by M2.
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| Figure 3.
Recognition of uPA domains by M2 and
uPAR.
HEK293 cell lines (A) and unstimulated or PMA-stimulated (20 nM) PMNs
(B) were allowed to adhere to microtiter plates coated with HMW-tc-uPA
or its domains for 30 minutes at 37°C. Nonadherent cells were removed
by washing with PBS, and the number of adherent cells was quantitated
as described in "Materials and methods." HEK293 cells expressing
uPAR or M2 or both (C) and PMA-stimulated
PMNs (D) were added to the transwells (2 × 105
cells/well) and allowed to migrate for 6 hours at 37°C in
5% CO2. HMW-tc-uPA or its domains were added to the lower
chamber of the transwell at 100 nM (C) and 10 nM (D) concentrations.
The data are means ± SEM of triple measurements from 3 independent experiments.
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Migration of HEK293 cells and neutrophils was assessed to determine
which uPA domains supported the response. As shown on Figure 3C,
mock-transfected HEK293 cells migrated neither to HMW-tc-uPA nor its
domains. The background migration of these cells was 4000 to 5000 cells
migrated per transwell in the presence and the absence of all uPA
derivatives. HMW-tc-uPA, KD, and LMW-tc-uPA induced migration of the
M2 cells by 2- to 3-fold compared with
buffer alone, but the GFD failed to support migration of these cells. In contrast, for the uPAR cells (Figure 3C), it was HMW-tc-uPA and the
GFD that supported migration, whereas the KD and LMW-tc-uPA did not.
This response pattern is consistent with the recognition of the GFD by
uPAR and the KD and LMW-tc-uPA by M2.
Finally, in the cells transfected with both
M2 and uPAR, robust migration was
observed with HWM-tc-uPA, GFD, KD, and LMW-tc-uPA (Figure 3C). The
migration of the M2/uPAR to the uPA
derivatives was more extensive than observed with the single
transfectants, again suggesting a combined contribution of the 2 receptors in mounting the response.
As shown in Figure 3D, not only HMW-tc-uPA and its GFD, the uPAR
recognition sites, supported migration of PMA-stimulated neutrophils,
but also the KD and weakly LMW-tc-uPA elicited a response. The
M2 function blocking mAbs 44a and IB4
decreased neutrophil migration to all the uPA domains to background
levels, suggesting that this integrin not only directly mediates
neutrophil migration toward KD and LMW-tc-uPA but also influences the
function of uPAR in mediating cell migration toward the GFD. The
function-blocking mAb directed against uPAR reduced neutrophil
migration toward HMW-tc-uPA and GFD as anticipated, but also fully
suppressed migration toward the KD and partially toward LMW-tc-uPA.
Neither normal mouse IgG nor control mAb W6/32 influenced neutrophil
migration toward uPA or any of its domains (not shown). Thus, on
PMA-stimulated neutrophils, uPAR and M2
appear to be strongly linked in regulating the migratory response to uPA.
M2 recognizes uPA via its I
domain
The mAb 44a and NIF, which blocked HMW-tc-uPA recognition by
M2 on neutrophils and HEK293
transfectants, bind to the MI-(A) domain, the
inserted domain of about 200 amino acids in the M subunit.36,37 The MI domain is involved in
the binding of many protein ligands to
M2.38-40 To determine if uPA
also is an MI domain ligand,
M2 and
M2/uPAR cells were allowed to adhere to
HMW-tc-uPA or its domains in the presence or absence of mAb 44a or NIF.
As shown in Figure 4A, both NIF and mAb
44a, but not mAb W6/32, blocked adhesion of the
M2 (upper panel) and
M2/uPAR (lower panel) cells to intact
HMW-tc-uPA and the KD almost completely. Adhesion of these cells to
LMW-tc-uPA was reduced by about 50% to 80% by these reagents. The
M2 cells did not adhere to the GFD, and
the adhesion of M2/uPAR cells to this
domain was reduced slightly (20%) by the MI
domain-blocking reagents.
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| Figure 4.
Effect of MI domain-blocking reagents on the adhesion
of M2 and
M2/uPAR-expressing cells to uPA and its
domains.
(A) Effect of MI domain reagents. The
M2 (upper) or
M2/uPAR (lower) HEK293 cells were
pretreated with NIF (0.1 µM), mAb 44a, or control mAb W6/32 to MHC-I
(20 µg/mL) for 30 minutes at 37°C, and then seeded
(2 × 105 cells) onto HMW-tc-uPA or its domains. (B)
Effect of MI domain. Wells, coated with HMW-tc-uPA or
its derivatives, were preincubated with or without recombinant
MI domain (100 nM) for 1 hour at 37°C, washed once,
and the M2/uPAR cells added. (C) Effect
of MI domain reagents on neutrophil adhesion. The
influence of NIF (0.1 µM; white bars) and MI domain
(100 nM; gray bars), or no treatment (black bars) on the adhesion of
PMA-stimulated neutrophils to HMW-tc-uPA or its domains. In each case,
adhesion was measured after 30 minutes. The data are means ± SEM
of triple measurements from 3 independent experiments.
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In a separate approach to evaluate the role of the MI
domain in uPA recognition, M2/uPAR cells
were allowed to adhere to HMW-tc-uPA or its fragments, which had been
preincubated with recombinant MI domain. As shown in
Figure 4B, this preincubation reduced cell binding to intact HMW-tc-uPA
by 60%, to LMW-tc-uPA by 40%, and completely blocked adhesion of the
cells to the KD. However, preincubation of MI domain had
no effect on cell adhesion to the GFD, which is mediated by uPAR. These
results are entirely consistent with the data in Figure 4A and suggest
that recognition of KD is mediated by the MI domain.
Recognition of LMW-tc-uPA is at least partially dependent on the
MI domain. Similar results were obtained with
PMA-stimulated neutrophils (Figure 4C). The presence of the
MI domain (gray bars) reduced adhesion of the stimulated
neutrophils to intact HMW-tc-uPA and LMW-tc-uPA by 50% and reduced
adhesion to KD to background levels. One distinguishing feature with
neutrophils was the effect of NIF (clear bars) on adhesion to the GFD
(Figure 4C). In contrast to the HEK293
M2/uPAR cells, where NIF had a marginal
effect on uPAR-mediated adhesion to the GFD, it produced substantial
(~80%) inhibition of neutrophil adhesion to this domain. Thus,
M2 may exert significant control on uPAR
function on the blood cells.
To further examine the involvement of the I domain in uPA binding to
M2, we assessed the effects of 2 other
M2 ligands, Fg and ICAM-1, which interact
with the I domain of the integrin.41 Varying
concentrations of these ligands were tested for their effects on the
binding of soluble, Alexa 488-labeled HMW-tc-uPA (DFP-inactivated) to M2 cells by
FACS and on adhesion of these cells to immobilized HMW-tc-uPA. As shown
on Figure 5A, Fg decreased soluble uPA
binding and adhesion of the M2 cells in a
dose-dependent manner; at the highest concentration tested (3.0 mg/mL),
the inhibition of these 2 functions was 50% and 70%, respectively.
With ICAM-1, inhibition of soluble HMW-tc-uPA binding and adhesion to
the ligand was also observed although the inhibition was less extensive
than with Fg. These responses were inhibited by 40% to 50% by ICAM-1 (Figure 5B). In the inverse experiment, wells were coated with ICAM-1,
and the effect of varying concentrations of DFP-inactivated HMW-tc-uPA on the adhesion of the M2
cells to this immobilized ligand was assessed. As shown on Figure 5C,
as little as 1 nM DIP-HMW-tc-uPA reduced
M2 cell adhesion to immobilized ICAM-1 by
70%. These results indicate that the binding sites for these MI domain ligands are overlapping but probably not
identical and their interrelationships are complex.28
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| Figure 5.
Relationship between uPA-, fibrinogen-, and
ICAM-1-binding sites in M2.
The M2 cells were preincubated with
increasing concentrations of Fg (A) or ICAM-1 (B) in DMEM/F-12/1 mM
Mg2+, washed once and allowed to bind soluble
(DFP-inactivated) Alexa 488-HMW-tc-uPA or the mixture was allowed to
adhere to immobilized DIP-HMW-tc-uPA for 30 minutes at 37°C.
Cell-bound uPA was detected by FACS, and the number of adherent cells
was counted as described in "Materials and methods." (C)
M2 cells were preincubated with
DIP-HMW-tc-uPA for 30 minutes at 37°C, washed once, and then
seeded onto plates coated with 10 µg/mL ICAM-1. After 30 minutes at
37°C, plates were washed and adherent cells were counted.
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M2 binds HMW-tc-uPA independently of
uPAR but uPAR enhances the interaction
To exclude that low levels of uPAR intrinsic to the HEK293 cells
might be necessary for M2 to bind uPA, 2 reagents were used: phosphatidylinositol-specific phospholipase C
(PI-PLC), which releases uPAR and other
glycosylphosphatidylinositol (GPI)-anchored proteins from cell
surfaces42 and the M25 peptide, corresponding to the
uPAR-binding sequence in the M subunit, which disrupts M2/uPAR complexes.13,24
HEK293-transfected cells were pretreated with PI-PLC, M25 peptide, or a
sequenced scrambled peptide (M25 SCR) and then tested for adhesion to
the various tc-uPA fragments (Figure 6A).
Adhesion of uPAR cells (upper panel) to HMW-tc-uPA and the GFD was
completely blocked by PI-PLC treatment, verifying that the reagent was
functional. As expected, the M25 peptide or its scrambled (SCR) control
did not affect uPAR-mediated adhesion to HMW-tc-uPA of the GFD. PI-PLC
treatment and the M25 peptide did not affect adhesion of
M2 cells (middle panel) to HMW-tc-uPA, KD, and LMW-tc-uPA, verifying that the
M2-mediated adhesion of these cells to
these derivatives was uPAR independent. Treatment of
M2/uPAR cells (lower panel) with PI-PLC
or M25 peptide, but not with SCR, reduced cell adhesion to HMW-tc-uPA,
KD, and LMW-tc-uPA by 70%, 60%, and 40%, respectively, to levels
similar to that observed for cells expressing only
M2. Recognition of GFD was abolished by
PI-PLC, whereas M25 and SCR had a negligible effect on adhesion,
consistent with the exclusive role of uPAR in recognition of the GFD.
With PMA-stimulated neutrophils (not shown), the M25 peptide, but not
M25 SCR, reduced their adhesion to KD (as well as to HMW-tc-uPA and
LMW-tc-uPA) but not to GFD. PI-PLC reduced their adhesion by more than
80% not only to the GFD but also to the KD, confirming the extensive
involvement of uPAR in M2-mediated recognition of uPA.
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| Figure 6.
Influence of uPAR on
M2-mediated adhesion to uPA.
(A) uPAR (upper panel), M2 (middle
panel), or M2/uPAR (lower panel)
transfected HEK293 cells were treated with 0.5 U/mL PI-PLC (black
bars), 100 µM M25 peptide (white bars), the control-scrambled M25
peptide SCR (striped bars), or untreated (gray bars) for 30 minutes at
37°C, and then allowed to adhere HMW-tc-uPA or its domains for 30 minutes at 37°C. (B) The indicated transfected cells were
preincubated with GFD (0.4 µM) for 30 minutes and then seeded onto
kringle domain (KD)-coated wells for 30 minutes at 37°C.
After 4 washings with PBS, the adherent cells were quantitated. The
results are expressed as means ± SEM of quadruplets from 3 independent experiments.
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Based on the observation that considerably more (2- to 3-fold)
M2/uPAR cells adhered and migrated to
HMW-tc-uPA than cells expressing either
M2 or uPAR alone, we hypothesized that
uPAR may enhance M2-mediated recognition
of uPA. To test this possibility, the various HEK293-transfected cells
were preincubated with soluble GFD to allow occupancy of uPAR and then
seeded onto KD (Figure 6B). Pretreatment of the mock-transfected 293 and uPAR cells with the GFD did not induce adhesion to KD and did not
affect adhesion of the M2 cells to the
KD. However, the presence of GFD increased the binding of
M2/uPAR cells to KD by 2-fold. In the
inverse experiment (data not shown), pretreatment of the
M2/uPAR cells with KD did not enhance
their adhesion to the GFD. These results suggest that binding of GFD to
uPAR may alter the function of M2 to
enhance its recognition of KD; on the other hand, recognition of KD of
uPA by M2 does not appear to stimulate
uPAR recognition of the GFD of uPA.
| |
Discussion |
In this study, we have examined the relationship between 2 cell
surface receptors, uPAR and M2, and the
proteolytic enzyme, uPA. Although these 3 molecules and their
interrelationship have been the subject of numerous analyses over the
past decade (for reviews, see Chapman and Wei,1 Preissner
et al,4 and Chapman et al20), our studies
have led to a novel data set. The underpinning of these findings is
that uPA is a ligand for M2. This
interaction was demonstrated with transfected cells expressing
M2 and with cells that express this
integrin naturally, peripheral blood neutrophils. The interaction of
uPA with M |
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Abstract
Introduction