By
From the * Haemostasis Research Unit, Max-Planck Institute, Kerckhoff-Klinik, D-61231 Bad
Nauheim, Germany; the Finsen Laboratory, Rigshospitalet, DK-2100 Copenhagen, Denmark; the § Basel Institute for Immunology, CH-4001 Basel, Switzerland; and the
Department of Pathology,
Centre Medical Universitaire, CH-1211 Geneva 4, Switzerland
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Abstract |
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The urokinase receptor (CD87; uPAR) is found in close association with 2 integrins on leukocytes. We studied the functional consequence of this association for leukocyte adhesion and
migration. In vivo, the
2 integrin-dependent recruitment of leukocytes to the inflamed peritoneum of uPAR-deficient mice was significantly reduced as compared with wild-type animals. In vitro,
2 integrin-mediated adhesion of leukocytes to endothelium was lost upon removal of uPAR from the leukocyte surface by phosphatidyl-inositol-specific phospholipase C. Leukocyte adhesion was reconstituted when soluble intact uPAR, but not a truncated form
lacking the uPA-binding domain, was allowed to reassociate with the cell surface. uPAR ligation with a monoclonal antibody induced adhesion of monocytic cells and neutrophils to vascular endothelium by six- to eightfold, whereas ligation with inactivated uPA significantly reduced cell-to-cell adhesion irrespective of the
2 integrin-stimulating pathway. These data
indicate that
2 integrin-mediated leukocyte-endothelial cell interactions and recruitment to
inflamed areas require the presence of uPAR and define a new phenotype for uPAR-deficient mice. Moreover, uPAR ligation differentially modulates leukocyte adhesion to endothelium and
provides novel targets for therapeutic strategies in inflammation-related vascular pathologies.
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Introduction |
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Leukocyte activation and adhesion to the endothelium and the subsequent transendothelial migration are pivotal steps in the recruitment of cells to inflamed tissue. This highly coordinated multistep process requires tight regulation (1, 2). This includes the induction of genes coding for adhesion molecules and the modification of ligand-binding affinities of adhesion receptors on leukocytes as well as their change in avidity due to adhesion receptor clustering of leukocytes. Uncontrolled activation of leukocytes or endothelial cells leads to pathological chronic inflammation causing atherosclerosis, rheumatoid arthritis, and other disease states.
The 2 integrin family of adhesion receptors consists of
the four members, LFA-1 (
L
2, CD11a/CD18),1 Mac-1
(
M
2, CD11b/CD18, CR3), p150,95 (
X
2, CD11c/
CD18), and
D
2 (CD11d/CD18) (3, 4). In acute inflammation, LFA-1 and Mac-1 are the predominant
2 integrins mediating leukocyte adhesion to vascular endothelium. Mac-1 is constitutively expressed on neutrophils and
monocytes, whereas LFA-1 is predominantly expressed on
lymphocytes, but recent data underline its important contribution in neutrophil recruitment (5, 6). Leukocyte activation via cytokines, chemoattractants, or PMA induces
both conformational changes in
2 integrins necessary for
enhanced ligand recognition and translocation of Mac-1 to
the cell surface (2, 7). Likewise, integrin activation is
achieved extracellularly in vitro by the divalent cation
Mn2+ (8). After activation, Mac-1 and LFA-1 firmly bind
to intercellular adhesion molecule (ICAM)-1 (CD54) expressed on vascular endothelial (1) and smooth muscle cells
(9).
2 integrins have been reported to form complexes
with other plasma membrane proteins such as CD63 (10),
the immunoglobulin receptor Fc
RIIIB (CD16b) (11) and
the urokinase receptor (uPAR, CD87) (12, 13) suggesting possible functional interaction.
uPAR consists of three homologous domains and is anchored to the plasma membrane by a glycolipid moiety that
is susceptible to dissociation by phosphatidyl-inositol-
specific phospholipase C (piPLC) (14). Intact uPAR binds
the protease uPA (urinary-type plasminogen activator) as
well as the adhesive protein vitronectin with high affinity
(15), and thereby plays a critical role in pericellular proteolysis and modulation of cellular contacts in adhesion and
migration (16, 17). Although uPAR lacks its own transmembrane and cytoplasmic domain, uPA binding has been
reported to transduce signals to the cell interior in leukocytes resulting in calcium mobilization (18), protein kinase
phosphorylation (12, 19), and other cellular effects (18,
21, 22). For some of these functions, it has been suggested
that uPAR uses related transmembrane integrins as signal
transduction devices (23). In fact, uPAR has been localized
together with different integrins in focal adhesion areas
(24), and increased uPAR expression and localization of
the receptor to the leading edge of migrating monocytes
appears to be essential for locomotion or invasiveness of
cells independent of uPA activity (20, 25). It has been shown in vitro that uPAR crucially influences integrin
function (26): the presence of uPAR inhibited 1 integrin-
mediated cell binding to fibronectin, whereas uPAR favored
2 integrin-dependent monocyte adhesion to fibrinogen,
one of the adhesive ligands of Mac-1. Moreover, using
uPA antisense or ligation with uPA inhibited Mac-1 binding to and degradation of fibrinogen (27, 28). Based on
these studies, uPAR has been proposed to form a functional unit with integrins on the cell surface.
These relationships prompted us to investigate the contribution of uPAR in 2 integrin-mediated cell-to-cell interactions in vivo using uPAR knockout mice, as well as in
vitro, to elute the mechanism of receptor cross-talk. Evidence is provided that uPAR is required for
2 integrin-
dependent leukocyte adhesion and recruitment.
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Materials and Methods |
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Materials
Manganese chloride was obtained from Sigma Chemical Co. (Munich, Germany) and PMA from GIBCO BRL (Paisley, Scotland). piPLC was from Oxford Glyco-Systems (Abingdon, UK). Intact recombinant soluble uPAR as well as the chymotrypsin-cleaved truncated form lacking domain 1 were produced as previously described (29, 30) and were provided by Dr. Niels Behrendt (Finsen Laboratory, Copenhagen, Denmark). uPA (Medac, Hamburg, Germany) was inactivated by diisopropyl-fluorophosphate (Serva, Heidelberg, Germany) as previously described (31).
Antibodies
The following mouse anti-human uPAR mAbs were used in
vitro. mAb no. 3936 (IgG2a-type), provided by Dr. Richard Hart
(American Diagnostica, Greenwich, CT), is known to block uPA
binding by recognizing an epitope of uPAR that has not been
clearly identified yet (32). (Fab')2 fragments were generated using
digestion by immobilized pepsin followed by protein A-Sepharose
affinity chromatography (Pierce Chemical Co., Rockford, IL).
Purity was controlled by polyacrylamide gel-electrophoretic analysis. mAbs R3 and R9, which recognize domain 1 and interfere
with uPA binding, and mAbs R2, R4 and R8, which recognize
domain 2 and 3 without influencing uPA binding, were provided
by Dr. Gunilla Hoyer-Hansen (Finsen Laboratory, Copenhagen,
Denmark). The characteristics of mAbs R2, R3, R4, R8, and R9
have been described previously (33). Mouse anti-human 2 chain
(CD18) mAb 60.3 (IgG2a-type) blocks
2 integrin-mediated leukocyte adhesion to endothelium (34) and was provided by Dr.
John Harlan (Harborview Medical Center, Seattle, WA). Mouse
anti-human IgG2a (Sigma Chemical Co.) was used as isotype-matched control antibody. When necessary, fluorescein-conjugated mouse mAbs anti-CD11a (no. 25.3), anti-CD11b (Bear 1),
and anti-CD18 (no. 7E4) (Immunotech, Hamburg, Germany)
were used for flow cytometry.
The following reagents were used in animal experiments. R-PE-labeled streptavidin (Southern Biotechnology Associates, Inc., Birmingham, AL) and biotin-conjugated rat anti-mouse CD11b
(M1/70), Ly-6G (Gr-1) (RB6-8C5), and CD3e (145-2C11) (all
from PharMingen, San Diego, CA). Anti-L
2 integrin (CD11a)
(FD 441.8) (35) and YN1.1 (36) (American Type Culture Collection, Rockville, MD) were affinity purified and dialyzed under
endotoxin-free conditions.
Cells
Human myelo-monocytic HL60 and U937 cell lines (German
Collection of Microorganisms and Cell Cultures, Braunschweig,
Germany) were cultured in RPMI 1640 supplemented with 10%
(vol/vol) fetal calf serum, 1% sodium pyruvate, 1 mM L-glutamine,
100 U/ml penicillin, and 100 µg/ml streptomycin (all from
GIBCO BRL). 24 h before experiments, monocytic differentiation was induced by addition of 50 ng/ml 1,25-dihydroxyvitamin D3 and 1 ng/ml transforming growth factor
1 (Biomol,
Hamburg, Germany). Peripheral blood PMNs were isolated by
discontinuous density gradient centrifugation using Histopaque-1119 and -1077 (Sigma Chemical Co.) as described by the manufacturer. An enrichment of at least 95% neutrophils was obtained
as controlled by flow cytometry using forward and side scatter
analysis and staining for CD15. Human umbilical vein endothelial
cells (HUVECs), provided by Dr. Bernd Pötzsch (Kerckhoff-Klinik), were isolated as previously described (37) and cultured
(for 2-4 passages) in low serum endothelial cell growth medium
(PromoCell, Heidelberg, Germany) on gelatin-coated tissue-culture plastic. Human vascular smooth muscle cells (HVSMCs)
were isolated from aorta or saphenous vein by the explant method
and characterized as previously described (38). Early passage cells
were cultured in smooth muscle cell medium (PromoCell).
Peritonitis Assay
Peritonitis was induced in female uPAR/
(39) or wild-type
mice by intraperitoneal injection of a solution of 4% (wt/vol) in
Bacto Fluid Thioglycollate Medium (Difco Labs., Detroit, MI). Endotoxin-free stock solutions were prepared at 100°C and subsequently heat-sterilized.
Mice obtained by shipping were generally kept (at the Basel Institute for Immunology) for at least 2 wk before the start of experiments to relieve stress. PBS alone or antibodies against ICAM-1, LFA-1 (200 µl of a 1 mg/ml solution each) were injected intravenously into the tail vein 30 min before induction of peritonitis. All reagents were endotoxin-free. After 4 or 24 h, respectively, mice were killed and peritoneal cells were suspended by injection of 5 ml PBS containing 2 mM EDTA and 50 µg/ml heparin into the peritoneum. The loaded mouse was shortly massaged and 4 ml of this lavage were collected and leukocytes were counted on a Coulter counter ZM equipped with a channelizer 256 (Coulter Corp., Miami, FL). Animal studies were approved by the Institutional Review Board.
Adhesion Assays
HUVECs were seeded in gelatin-coated 48-well plates (Costar, Badhoevedorp, The Netherlands) 48 h before the experiment. Confluency was confirmed by microscopic inspection before each experiment. HL60 or U937 cells were radiolabeled
with 1 µCi/ml methyl-[3H]thymidine (Nycomed Amersham,
Little Chalfont, Buckinghamshire, UK) for 24 h and differentiated to monocytic cells (see above) for another 24 h. These
monocytic cells or freshly isolated neutrophils were washed twice
in adhesion medium (serum-free RPMI 1640/Hepes 25 mM),
followed by different pretreatments (see figure legends for details),
and were added (7 × 105/ml adhesion medium) to the prewashed
HUVEC monolayers in the presence or absence of the blocking
mAb anti-2 integrin or isotype IgG (final concentration 10 µg/
ml). After 30 min of coincubation (37°C, 5% CO2, 90% humidity), the plates were gently washed twice with adhesion buffer to
remove nonadherent cells. Remaining adherent cells were lysed
with 1 M NaOH and quantitated in a beta counter. When flow
cytometry was performed in parallel to neutrophil adhesion, the
number of adherent cells in 10 high power fields was counted using light microscopy. At least triplicate wells were run per test
substance, and results are expressed as mean values ± SEM. The
experimental protocol for cell adhesion to HVSMCs, which were seeded 7-9 d before the assay, was identical.
Flow Cytometry
Animal Model.
The mouse leukocyte subpopulations were further analyzed by flow cytometry (Becton Dickinson, Heidelberg, Germany) using the biotin-coupled anti-mouse CD11b (integrinIn Vitro System.
Cells (2.5 × 105) were washed twice with Hepes-buffered saline and incubated with primary mouse anti- human antibodies for 30 min on ice (for CD11b detection at 21°C). Cells were washed again and resuspended in Hepes buffer containing fluorescein-conjugated (Fab')2 fragment of goat anti- mouse IgG (Dianova, Hamburg, Germany). To test the effect of mouse antibodies onStatistical Analysis
Comparisons between group means were performed using multivariant analysis (ANOVA). Data represent mean ± SEM; P < 0.05 was regarded as significant.
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Results |
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Transendothelial migration of leukocytes to inflamed tissue depends
on the interaction of the leukocyte with the vascular endothelium by 2 integrins and ICAM-1. Thioglycollate-
induced peritonitis is a reliable model to test leukocyte emigration into sites of acute inflammation. Disruption of the
mouse ICAM-1-
2 integrin interactions resulted in reduced leukocyte emigration in this model when compared
with wild-type animals (40). Both uPAR-deficient and
wild-type animals of the identical genotype (129 × C57/
BL6 F1) were compared for leukocyte emigration in the
peritonitis model. The number and types of leukocytes in
the peripheral blood were identical in both sets of mice
(data not shown). Lavages performed 4 (Fig. 1) and 24 h
(data not shown) after induction of peritonitis showed
~50% reduction in counts of the total leukocyte population in uPAR-deficient mice when compared with wild-type animals (Fig. 1). When animals were treated with
anti-ICAM-1 or anti-LFA-1 antibodies at the time of induction of peritonitis, the number of emigrating leukocytes
was further reduced by 50% in wild-type mice, but by only
30% in uPAR-deficient animals, suggesting that a major
part of the initial lack of emigration was due to a perturbed
2 integrin/ICAM-1 function. Analysis of the leukocyte
subpopulations by flow cytometry using specific markers as
indicated in Materials and Methods revealed that in uPAR-deficient mice granulocytes almost totally lost their ability to migrate into the peritoneum after 4 and 24 h of inflammation (Fig. 2). Myeloid lineage cells showed significant
reduction in recruitment after 4 h (~55%) and 24 h
(~70%), whereas T lineage cells were hardly affected by
the absence of uPAR after 4 h, but showed significant inhibition in emigration (~60%) after 24 h (Fig. 2). Consistently, administration of mAbs demonstrated that lymphocyte
recruitment after 4 h was largely independent of LFA-1-ICAM-1 interactions in contrast to recruitment after 24 h
of inflammation.
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To further specify those granulocytic subpopulations that
were mostly affected, a differential cell staining (May-Grünwald-Giemsa) was performed (Fig. 3). In uPAR-deficient mice, after 4 h, neutrophil and eosinophil recruitment
was inhibited by >70% or 90%, respectively, and residual
emigration was marginally affected by the administration of
mAbs against ICAM-1 or LFA-1, respectively. In contrast,
in wild-type mice the recruitment of these two cell types
was effectively blocked by these antibodies down to the
level of emigrated cells in uPAR/
mice, suggesting that
leukocyte recruitment in uPAR-deficient mice is diminished through impaired function of the
2 integrin/ICAM-1 system. Basophil emigration into the inflamed peritoneum
was not significantly affected in uPAR-deficient mice but
was comparable to that in wild-type mice receiving anti-
ICAM-1 or anti-LFA-1 mAb, respectively. Thus, the definitive role of uPAR for basophil recruitment is not yet
clear and requires further investigation. Comparable findings for granulocyte subpopulations were noted after 24 h of
inflammation (data not shown). These data provide in vivo
evidence for a functional consequence of the uPAR/
2 integrin system in leukocyte adhesion/migration and present
a new phenotype for uPAR-deficient mice.
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uPAR is expressed on circulating human blood
cells, such as granulocytes, monocytes, and activated T
cells. The human myelo-monocytic cell lines HL60 and
U937 differentiate into mononuclear phagocyte-like cells
after treatment with vitamin D3 and transforming growth factor 1 (31). This in vitro differentiation induced the expression of a monocyte-specific antigen pattern including
the molecules CD14, CD11b, CD18, and CD87 (uPAR)
as measured by flow cytometry (data not shown). Adhesion
of these differentiated monocytic cells to endothelial cell
monolayers was induced six- to eightfold by PMA or
Mn2+, both reagents known for their activation of
2 integrins. The mAb 60.3 directed against the
2 integrin chain
blocked adhesion by 75-85% in both cases, indicating that
leukocyte adhesion to endothelium was
2 integrin dependent (Fig. 4).
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Pretreatment of leukocytes with piPLC removed ~80%
of uPAR from the cell surface as assessed by flow cytometry (data not shown). This resulted in a 70-80% reduction
of PMA-induced or a 65-70% reduction of Mn2+-induced
leukocyte adhesion (Fig. 4). Reconstitution of the uPAR-depleted cells with intact soluble uPAR for 10 min restored
adhesion to the level as observed with untreated cells. This
restoration of uPAR-induced adhesion was abrogated by anti-CD18 antibody, indicating that the 2 integrin-dependent
adhesion was inducible by uPAR.
In a dose-dependent manner, intact soluble uPAR increased adhesion of piPLC-treated leukocytes to endothelium to maximal levels, whereas addition of the truncated
form of uPAR lacking the uPA binding domain D1 did
not, indicating a specific structural requirement for 2 integrin activation (Fig. 5). In contrast to PMA, which induces
both increased surface expression and activation of
2 integrins, flow cytometric analysis revealed no change in integrin expression after Mn2+ treatment (data not shown), as
previously described (8). Likewise, removal of uPAR did
not affect surface expression of the
L,
M, or
2 chain of
the integrins in resting or stimulated cells as analyzed by
flow cytometry (data not shown). Thus, the presence of
uPAR appears to support adhesion by regulating integrin
function rather than by quantitatively changing
2 integrin
levels on the cell surface.
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Since uPAR
is directly involved in 2 integrin-mediated leukocyte adhesion to the endothelium, we examined the consequences of uPAR occupancy on leukocyte adhesion to endothelial
cells using different mAbs against uPAR. Preincubation
with the anti-uPAR mAb no. 3936 resulted in the six- to
eightfold increase of cell adhesion reaching the same maximal level as achieved with PMA or Mn2+ (Fig. 6). For undifferentiated HL60 or U937 cells, which show low surface
expression of
2 integrins and uPAR, respectively, the activating anti-uPAR mAb increased adhesion only 1.5-2-fold (data not shown). Boiling of the antibody totally abrogated
its adhesion-stimulating effect. In addition, (Fab')2 fragments of mAb no. 3936 had a very similar proadhesive effect as compared with the intact mAb. Control isotype-
matched mAb IgG2a as well as other anti-uPAR mAbs directed against different epitopes of uPAR did not induce leukocyte adherence. The dose- and time-dependent kinetics
of antibody ligation suggested that the uPAR-mAb complex serves as a fast and effective trigger of
2 integrin activation (Fig. 7). In contrast, the anti-uPAR mAb R3 that
has the same inhibitory effect on uPA binding to the receptor as mAb no. 3936 was not able to promote leukocyte
adhesion. The anti-uPAR mAbs did not significantly
change
2 integrin expression (data not shown), indicating
that a functional conformational change of the adhesion receptor is responsible for the proadhesive effect.
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Analogous to PMA- or Mn2+-induced 2 integrin-dependent cell adhesion, mAb 60.3 could abrogate mAb no.
3936-induced leukocyte adhesion to cultured endothelial
and smooth muscle cells, indicating
2 integrin dependency
(Fig. 8). In addition, vitamin D3/transforming growth factor
1 differentiated U937 cells and freshly isolated peripheral blood neutrophils responded to mAb no. 3936 in an
identical manner, emphasizing that this functional interaction of uPAR with
2 integrins occurs in different cell
populations such as monocytes and neutrophils.
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Although the addition of exogenous uPA, the natural ligand of uPAR, had
no effect on background adhesion, uPA significantly inhibited 2 integrin-mediated adhesion in response to any of the three described stimulating pathways (Fig. 9); adhesion
of leukocytes to endothelium achieved by cell activation
via PMA, direct extracellular integrin activation via Mn2+,
or uPAR ligation by the activating mAb no. 3936 was inhibited by active (data not shown) as well as by enzymatically inactive uPA. Thus, uPA binding to uPAR independent of its catalytic activity appears to control
2 integrin
activation irrespective of the integrin-activating stimulus.
Since uPA or its inactivated isoform did not alter the surface expression of the integrin
L,
M, or
2 chain in either resting or stimulated cells (data not shown), we propose that the binding of uPA to uPAR prevents the
induction of conformational change(s) leading to
2 integrin activation.
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![]() |
Discussion |
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The major contribution of 2 integrins in immune defense and inflammatory processes relates to their pivotal
role in mediating cellular contacts between leukocytes and
endothelium as a prerequisite for subsequent transmigration
towards a chemotactic stimulus. Previous in vitro studies
have demonstrated that uPAR forms complexes with integrins (12, 13) and thereby modulates integrin-mediated binding to extracellular matrix proteins (26).
This study demonstrates that uPAR is needed for 2 integrin-dependent leukocyte recruitment into sites of acute
inflammation. Migration of neutrophils and monocytes
into the inflamed peritoneum was drastically reduced after
4 h in uPAR-deficient mice. Consistently,
2 integrin-
dependent cell adhesion of leukocytes to endothelial cells
was abrogated after depletion of uPAR from the cell surface, whereas reconstitution with soluble intact, but not
truncated, uPAR could totally rescue
2 integrin-mediated
adhesion. Regulation of
2 integrin activity probably involves uPAR domain 1, since occupancy by a mAb that
blocks uPA-binding to this domain strongly induced leukocyte adhesion to vascular endothelial cells, whereas uPA
itself inhibited
2 integrin-dependent adhesion.
The adhesion of leukocytes to inflamed vascular endothelium and the transendothelial migration largely depend
on the activation of 2 integrins and binding to its counter-receptor ICAM-1 (41, 42), as evidenced by inhibition and
gene-targeting studies (40, 43, 44). Although
2 integrin
complexes are essential to neutrophil emigration, recent results from
2-deficient mice demonstrated that Mac-1/
LFA-1-independent pathways for cell recruitment can be
used during acute inflammation in the peritoneum and the
lung (45).
In this study, a new phenotype for uPAR-deficient mice
is described that is similar to that of wild-type animals after
treatment with inhibiting antibodies against the vascular 2
integrin LFA-1 and its ligand ICAM-1. Further blockade
with mAbs of leukocyte migration in uPAR
/
mice was
marginal, suggesting that the function of the
2 integrin/ ICAM-1 adhesion system was blocked by the absence of
uPAR. This strongly suggests that leukocyte recruitment to
the acutely inflamed peritoneum requires the uPAR. In
fact, under conditions of acute inflammation the phenotype
of uPAR mice resembles that of ICAM-1- or
L-deficient
mice (40, 46).
In accordance with the known role of 2 integrins for
the acute and early inflammatory responses, reduced leukocyte infiltration in uPAR
/
mice was observed after short-term inflammation for 4 or 24 h. It was shown previously
that the total number of migrated leukocytes in uPAR
/
mice was not affected after long-term inflammation for 3 d
(47). This may be due to the fact that prolonged inflammation upregulates and activates several other adhesion receptor systems on leukocytes and vascular cells apart from
2
integrins, such as
1 and
7 integrins, vascular cell adhesion
molecule 1, or addressins, all of which contribute to leukocyte recruitment (1). Especially after long-term inflammation, the
4
1 integrin may substitute for
2 integrins. The
finding that migration of granulocytes that do not express
4
1 integrin was mainly affected by the absence of the
uPAR further supports this concept.
Recent in vitro studies have shown that the presence of
uPAR is needed for Mac-1 binding to fibrinogen (27), and
it also regulates fibrinogen degradation by forming a functional unit with the 2 integrin (28). Consistently,
2 integrin-dependent cell-to-cell adhesion required the presence
of endogenous or exogenously added intact (soluble) uPAR
in vitro. In the absence of uPAR, neither PMA nor Mn2+
induction of piPLC-treated leukocytes was sufficient to allow adhesion, indicating a superior regulatory function for
uPAR regardless of the stimulatory pathway. Moreover,
the intact (soluble) uPAR appears to be crucial in the cross-talk with
2 integrins (Fig. 10), implying direct binding interactions between these two receptors as was demonstrated in an isolated system for Mac-1 (26). In contrast, the
truncated form of uPAR was ineffective, and uPA itself significantly inhibited cell-to-cell adhesion, suggesting that
domain 1 of uPAR is predominantly involved in
2 integrin activation. A similar inhibitory effect of uPA on
2 integrin function has previously been proposed with regard
to Mac-1-mediated fibrinogen binding and degradation
(27, 28).
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Although uPAR-dependent proteolytic activity did not
seem to be critical for leukocyte recruitment (47), the focalized proteolytic activity of the uPAR-uPA complex is
tightly correlated with the migratory or invasive potential
of cells in a variety of biological systems (48). On the other
hand, active uPA has been found to support cell migration
in vitro via proteolysis or cell-activating processes and was
required for adequate leukocyte recruitment into the lungs
and a subsequent inflammatory response after 3 wk of fungal infection (49). Collectively, our combined in vivo and
in vitro studies together with previous reports strongly indicate that proteolysis-independent cross-talk of uPAR
with 2 integrins occurs in the initial phase of leukocyte interaction with the vessel wall, whereas subsequent recruitment into inflamed tissue may require proteolysis-dependent mechanisms including plasmin action.
uPAR appeared to induce and facilitate 2 integrin activation on its own when stimulated by the specific anti-uPAR mAb no. 3936 or its (Fab')2 fragment. Although
cross-linking of uPAR cannot be ruled out, this antibody
provides an additional stimulatory pathway for
2 integrin
engagement. In accordance with this concept, uPA was
found to interfere by either competing for mAb no. 3936 binding or by not allowing integrin activation to occur as
pointed out before. Since removal of cell surface-associated
uPA by acidic wash (data not shown) and the use of other
antibodies that also block uPA binding to uPAR (such as
R3) did not affect cell adhesion, we attribute the proadhesive capability of mAb no. 3936 to its specific ligation of
uPAR rather than to competition with endogenous uPA.
In contrast to our findings that the mAb no. 3936 induces
2 integrin-dependent cell-cell adhesion, Mac-1-mediated leukocyte adhesion to fibrinogen has been shown to
be inhibited by the anti-uPAR mAb 3B10 (27). These diverse findings may be due to the experimental systems used
and/or to the different integrins or ligands involved. Although vitronectin and PAI-1 are known to regulate
uPAR as well as
v integrin-dependent cell-substrate adhesion (50), both factors did not interfere with
2 integrin-dependent cell-cell adhesion (data not shown), underlining the specificity of the uPAR system in this regard.
Our findings indicate that uPAR plays a major role for
leukocyte adhesion. Nevertheless, patients with paroxysmal
nocturnal hemoglobinuria (PNH) lacking cell-bound uPAR
and other cell surface proteins due to a defective production
of glycolipid anchors do not necessarily show an enhanced
susceptibility for infections, whereas isolated neutrophils
from these patients are impaired in their transendothelial
migration in vitro (53). Possible explanations might be that
(a) in PNH, which is an acquired clonal disease, a portion
of the cells remains unaffected and may therefore be sufficient for host defense as demonstrated in the experiments with uPAR-deficient mice; or (b) circulating soluble uPAR
is enhanced three- to fourfold in PNH patients (54) and
might be sufficient for 2 integrin function in vivo in these
patients, as documented here in vitro. The present observations indicate that uPAR controls integrin-mediated interactions in vitro as well as in vivo, and these findings might
have therapeutic consequences for the treatment of hyperinflammatory or invasive processes related to vascular or
immune diseases.
![]() |
Footnotes |
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Address correspondence to Klaus T. Preissner, Haemostasis Research Unit, Max-Planck-Institute, Kerckhoff-Klinik, Sprudelhof 11, D-61231 Bad Nauheim, Germany. Phone: 49-603-299-6719; Fax: 49-603-299-6707; E-mail: klaus.t.preissner{at}kerckhoff.med.uni-giessen.de
Received for publication 5 February 1998 and in revised form 12 May 1998.
A.E. May was supported by a postdoctoral fellowship grant from the Deutsche Gesellschaft für Kardiologie, Herz- und Kreislaufforschung. Part of this work was supported by a grant (Pr 327/1-3) from the Deutsche Forschungsgemeinschaft and by a grant (no. 31-49241.96) from the Swiss Science Foundation.We are grateful to Barbara Eccabert (Basel Institute for Immunology) for her skilful expert assistance in the
animal experiments and Drs. Niels Behrendt and Gunilla Hoyer-Hansen for their generous supply of soluble
intact and truncated uPAR as well as anti-uPAR antibodies. We appreciate the kind gift of anti-2 integrin
antibody 60.3 from Dr. John Harlan. We also thank our colleagues Dr. Bernd Pötzsch and Bettina Kropp for
providing HUVEC and Drs. Matthias Germer and Karl-Dieter Wohn for very helpful discussions.
Abbreviations used in this paper
HUVEC, human umbilical vein endothelial cells;
HVSMC, human vascular smooth muscle cells;
ICAM-1, intercellular adhesion molecule 1;
LFA-1, L
2 integrin (CD11a/CD18);
Mac-1,
M
2 integrin (CD11b/CD18);
ns, not significant;
piPLC, phosphatidyl-inositol-specific phospholipase C;
uPA, urokinase (urinary-type
plasminogen activator);
uPAR, urokinase receptor.
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