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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 96 No. 2 (July 15), 2000:
pp. 506-513
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
VLA-4 ( 4 1) engagement defines a
novel activation pathway for 2 integrin-dependent
leukocyte adhesion involving the urokinase receptor
Andreas E. May,
Franz-Josef Neumann,
Albert Schömig, and
Klaus T. Preissner
From the Deutsches Herzzentrum und 1. Medizinische Klinik des
Klinikums Rechts der Isar, Technische Universität, D-80636
München, Germany; and Institut für Biochemie, Fachbereich
Humanmedizin, Justus-Liebig-Universität, D-35392 Giessen,
Germany.
 |
Abstract |
During acute inflammatory processes,  2 and
1 integrins sequentially mediate leukocyte recruitment
into extravascular tissues. We studied the influence of VLA-4 (very
late antigen-4) ( 41) engagement on 2 integrin activation-dependent
cell-to-cell adhesion. Ligation of VLA-4 by the soluble chimera fusion
product vascular cell adhesion molecule-1 (VCAM-1)-Fc or by 2 anti-CD29 (1 chain) monoclonal antibodies (mAb) rapidly
induced adhesion of myelomonocytic cells (HL60, U937) to human
umbilical vein endothelial cells (HUVECs). Cell adhesion was mediated
via 2 integrin (LFA-1 and Mac-1) activation: induced
adhesion to HUVECs was inhibited by blocking mAbs anti-CD18 (70%-90%), anti-CD11a (50%-60%), or anti-CD11b (60%-70%).
Adhesion to immobilized ligands of 2 integrins
(intercellular adhesion molecule-1 [ICAM-1], fibrinogen, keyhole
limpet hemocyanin) as well as to ICAM-1-transfected Chinese hamster
ovary cells, but not to ligands of 1 integrins (VCAM-1,
fibronectin, laminin, and collagen), was augmented. VCAM-1-Fc binding
provoked the expression of the activation-dependent epitope CBRM1/5 of
Mac-1 on leukocytes. Clustering of VLA-4 through dimeric VCAM-1-Fc was
required for 2 integrin activation and induction of cell
adhesion, whereas monovalent VCAM-1 or Fab fragments of
anti-1 integrin mAb were ineffective. Activation of
2 integrins by 41 integrin
ligation (VCAM-1-Fc or anti-1 mAb) required the
presence of urokinase receptor (uPAR) on leukocytic cells, because the
removal of uPAR from the cell surface by phosphatidylinositol-specific
phospholipase C reduced cell adhesion to less than 40%. Adhesion was
reconstituted when soluble recombinant uPAR was allowed to reassociate
with the cells. Finally, VLA-4 engagement by VCAM-1-Fc or
anti-1 integrin mAb induced uPAR-dependent adhesion to
immobilized vitronectin as well. These results elucidate a novel
activation pathway of 2 integrin-dependent cell-to-cell
adhesion that requires 41 integrin
ligation for initiation and uPAR as activation transducer.
(Blood. 2000;96:506-513)
© 2000 by The American Society of Hematology.
| |
Introduction |
Leukocyte recruitment is a key step in the
pathogenesis of inflammatory diseases such as
atherosclerosis.1,2 It is a highly coordinated multistep
process that requires activation of different adhesion receptors in a
cascade-like fashion.3 The families of selectins and
integrins subsequently become activated to allow adhesion of leukocytes
to endothelium followed by transmigration into the vessel wall. The
integrin family of adhesion receptors is critically involved in this
process by mediating cell-to-cell and cell-to-extracellular matrix
contacts of various strength and duration.4 Integrins are
heterodimeric transmembranous receptors that do not only function as
adhesive proteins but also can transduce cellular signals in a
bidirectional manner4-6: Membrane-bound nonintegrin
components such as integrin-associated protein (IAP/CD47) or the
glycolipid-anchored molecules CD14 as well as urokinase receptor (CD87)
appear to be required for inside-out and outside-in
signaling.7-9 In particular, the integrin VLA-4 (very late antigen-4) ( 41)
has previously been shown to transduce several intracellular activation
signals via tyrosine phosphorylation of intracellular kinases resulting
in nuclear translocation of nuclear factor  B as well as
tissue factor translocation to the cell
surface.10,11
VLA-4 engagement occurs at several stages of cell-to-cell contacts
between various cell types: apart from the selectin family of
adhesion receptors, VLA-4 is engaged in tethering of monocytes, lymphocytes and, potentially, neutrophils to the vessel wall by binding
vascular cell adhesion molecule-1 (VCAM-1) on vascular endothelium.12-15 In addition, VLA-4-VCAM-1 contacts may
occur when leukocytes migrate along or through smooth muscle cell
layers, which highly express VCAM-1 when activated during
atherogenesis.16-18 Within the blood stream, soluble
VCAM-1, which is elevated under inflammatory conditions such as
atherosclerosis,19 myocardial infarction,20 or
stroke,21 may bind to elevated VLA-422 on
leukocytes. Yet, consequences of VLA-4 engagement for adhesive events
have not been reported.
In this study, we sought to investigate the hypothesis
that engagement of VLA-4 on monocytic cells by its natural ligand
VCAM-1 or by anti-1 integrin monoclonal antibody
(mAb) can trigger enhanced adhesiveness via 2 integrins.
We provide evidence that the 2 major 2 integrins
LFA-1 (leukocyte function-associated-1) and Mac-1 become activated in
response to VLA-4 engagement and contribute equally to the induced
adhesiveness of leukocytic cells to the endothelium. In addition, we
demonstrate that the urokinase receptor directly becomes involved upon
VLA-4 engagement and controls the transdominant activation
between VLA-4 and 2 integrins.
| |
Materials and methods |
Reagents
Phorbol 12-myristate 13-acetate (PMA) was from Gibco (Paisley,
Scotland, UK). Fibrinogen, fibronectin, laminin, collagen, and keyhole
limpet hemocyanin (KLH) were from Sigma (Munich, Germany). Vitronectin
was purified from human plasma as described.23 Soluble human recombinant intercellular adhesion molecule-1 (ICAM-1) was kindly
provided by Dr Carl Figdor (Nijmegen, The Netherlands); soluble human recombinant VCAM-1 was from R & D Systems (Wiesbaden, Germany). Human recombinant VCAM-1-Fc was kindly provided by Dr Dietmar Seiffge (Hoechst, Frankfurt, Germany).
Recombinant phosphatidylinositol-specific phospholipase C was from
Oxford Glyco-Systems (Abingdon, UK). Recombinant soluble
urokinase receptor (uPAR) was produced as described24,25
and was generously provided by Dr Niels Behrendt (Finsen
Laboratory, Copenhagen, Denmark). Recombinant soluble tissue factor
pathway inhibitor was from American Diagnostica (Pfungstadt, Germany).
Mouse antihuman CD29 (1 integrin chain) mAbs K20 and
LIA1/2 as well as mAb antihuman CD49d (4 chain, HP2.1)
were from Immunotech (Hamburg, Germany); HP2.1 was described to block
cell-to-cell adhesion via VLA-4-VCAM-1 interactions. F(ab) fragments
of mAb K20 were generated by digestion with immobilized papain followed
by protein A-Sepharose affinity chromatography (Pierce, Rockford, IL),
and the purity was confirmed by polyacrylamide gel electrophoretic
analysis. Cross-linking of F(ab) fragments of mAb K20 was achieved
using affinipure F(ab)2 fragment goat antimouse
immunoglobulin G (IgG; Dianova, Hamburg, Germany). KIM185 is a mAb that
binds and directly activates the common 2 integrin chain
CD1826 and was kindly provided by Dr Marc
Robinson (Celltech Ltd, Slough, England). Mouse antihuman
2-chain (CD18) mAb 60.3 (IgG2a-type) blocks
2 integrin-mediated leukocyte adhesion to endothelium27 and was generously provided by Dr John
Harlan (Seattle, WA). Murine antihuman LFA-1 (anti-CD11a)
L15 was kindly provided by Dr Carl Figdor. Monoclonal
antibody CBRM1/5, which recognizes only the activated epitope of CD11b
and blocks Mac-1-dependent adhesion,28 was generously
provided by Dr Timothy Springer (Boston, MA). Blocking
mAb anti-v3 (LM609) was from Chemicon (Temecola, CA).
Murine antihuman IgG2a (Sigma) was used as isotype-matched control antibody.
Cells
Human myeloid 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). Twenty-four hours prior to the
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 polymorphonuclear neutrophils were isolated
by discontinuous density gradient centrifugation using Histopaque-1119
and -1077 (Sigma) 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) were isolated as
described29 and cultured (for 2 to 4 passages) in low serum endothelial cell growth medium (PromoCell, Heidelberg, Germany) on
gelatin-coated tissue culture plastic. Chinese hamster ovary (CHO)
cells transfected with ICAM-1 were purchased from American Type Culture
Collection (Rockville, MD) and cultured in RPMI-1640 with
10% fetal calf serum.
Adhesion assays
Cell-to-cell adhesion.
HUVECs or ICAM-1-transfected CHO (CHO-ICAM-1) cells were seeded onto
gelatin-coated 48-well plates (Costar, Badhoevedorp, The Netherlands)
48 hours prior to the experiment. Confluency was confirmed by
microscopic inspection before each experiment. HL60 or U937 cells were
differentiated to monocytic cells (see "Cells") for 24 hours.
These monocytic cells or freshly isolated neutrophils were washed twice
in adhesion medium (serum-free RPMI-1640/HEPES 25 mM), followed by
various pretreatments (described in figure legends) and mixed with the
chromophore BCECF-AM (Molecular Probes, Eugene, OR). After washing, they were added
(7 × 105/mL adhesion medium) to the
prewashed HUVECs or CHO-ICAM-1 monolayers in the absence or
presence of blocking mAb. After 30 minutes 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-mol/L NaOH and
quantified in a fluorescence plate reader (TEKAN, Crailsheim,
Germany). At least triplicate wells were run per test
substance, and results are expressed as mean values ± SEM;
experiments were repeated at least twice.
Cell adhesion to immobilized integrin ligands.
Ninety-six-well plates were coated with human fibrinogen, fibronectin,
laminin, type I/III collagen, vitronectin (each 20 µg/mL), KLH (100 µg/mL), soluble recombinant ICAM-1 or VCAM-1 (20 µg/mL),
respectively, for 2 hours at 37°C and blocked with 1% (wt/vol)
bovine serum albumin for 30 minutes at 25°C. Pretreated leukocytes were seeded at 70 000 cells/well in the
absence or presence of blocking mAb for 30 minutes. After removal of
nonadherent cells by 2 washing steps, adhesion was quantified by
peroxidase reaction using p-nitrophenol as a substrate in an
enzyme-linked immunosorbent assay reader (Bio-Rad, Munich, Germany).
Flow cytometry
Cells (2.5 × 105) were washed twice with
HEPES-buffered saline and incubated with fluorescein-conjugated mAb (as
specified in "Results") at a dilution of 1:50. Mean fluorescence
of 5000 cells was measured in a flow cytometer (Becton Dickinson,
Heidelberg, Germany). Nonspecific fluorescence was determined using an
isotype-matched mouse IgG.
Statistical analysis
Comparisons between group means were performed using ANOVA. Data
represent mean ± SEM. A value of P < .05 was regarded
as significant.
| |
Results |
VLA-4 engagement by VCAM-1-Fc or anti-1 integrin mAb
induces leukocyte adhesion to human endothelial cells
Preincubation of the myelomonocytic cell line HL60 with an antibody
(mAb K20) directed against the 1 integrin chain of VLA-4 or with human soluble recombinant VCAM-1-Fc, respectively, resulted in
strong cell adhesion to HUVECs (Figure 1A).
A similar maximal level of adhesion was reached with PMA or with the
known 2 integrin-activating mAb KIM185. Control
isotype-matched mAb IgG2a as well as an antibody directed against the
chain of VLA-4 did not induce leukocyte adherence. The specificity
of VCAM-1-Fc binding to VLA-4 was confirmed by competition with mAb
anti-VLA-4 (clone HP2.1), which abrogated cell adhesion induced by
VCAM-1-Fc. When 1 integrins were ligated by the same
procedure on the myelomonocytic cell line U937, HL60, or isolated
neutrophils, comparable cell-to-cell adhesion was achieved (Figure 1B).
Preincubation with mAb anti-1 LIA1/2 had a similar
proadhesive effect as observed with mAb K20. Boiling of mAb K20 prior
to incubation with the cells totally abrogated its stimulatory
capacity. Because all reagents were found to be endotoxin-free, this
excludes the possibility that bacterial contamination may be
responsible for cellular activation. Isolated neutrophils, which do not
express VLA-4, did not respond to VCAM-1-Fc incubation (not shown).
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| Fig 1.
Effect of VCAM-1-Fc and anti-1 integrin
mAbs on leukocyte adhesion to endothelium.
(A) Myelomonocytic HL60 cells were preincubated with medium alone
(control), VCAM-1-Fc (40 µg/mL) with or without mAb anti-VLA-4
(4 chain, HP2.1, 20 µg/mL), mAbs anti-1
integrin (K20), anti-4 integrin (HP2.1), or the
2 integrin-activating mAb KIM185 (20 µg/mL each) or
PMA (10 ng/mL) for 30 minutes at 37°C. After washing, adhesion to
endothelial cell monolayers was performed. (B) HL60 cells (filled
bars), U937 cells (gray bars), or isolated neutrophils (open bars) were
preincubated with medium alone, VCAM-1-Fc (40 µg/mL), mAbs
anti-1 integrin LIA1/2 or K20, boiled mAb K20,
isotype-matched irrelevant IgG2a, or anti-4 integrin mAb
HP2.1 for 30 minutes at 37°C. After washing, adhesion to
endothelial cell monolayers was performed. Values are displayed as
percentage of control and represent the mean ± SEM of at least 3 independent experiments. *Indicates P < .01 compared with
control.
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The dose- and time-dependent response to antibody ligation
demonstrated that adhesion was rapidly induced by lowconcentrations of
mAb K20, reaching a maximum after 30 to 60 minutes (Figure 2). Thus, engagement of VLA-4 seems to
serve as a fast and effective stimulus of leukocyte adhesion.
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| Fig 2.
Kinetics of mAb anti-CD29-induced leukocyte adhesion to
endothelium.
The mAb K20 (circles) or isotype control mAb IgG2a (squares) were added
to myelomonocytic HL60 cells (A) at different concentrations as
indicated for 30 minutes at 37°C, or (B) for different time
intervals as indicated at a concentration of 10 µg/mL. Following
washing, adhesion to endothelial cell monolayers was performed as
described. Values (mean ± SEM) are displayed as percentage of
control (no antibody added). One representative experiment (of 3) is
shown. *Indicates P < .01 compared with control (no mAb
added).
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VLA-4-induced adhesion requires integrin clustering
Based on these findings, we studied the proadhesive effects of
ligation versus clustering of VLA-4 (Figure
3). Isolated, monovalent Fab fragments of
mAb K20 alone (in contrast to the intact mAb) did not induce adhesion.
However, when these monovalent fragments were cross-linked by goat
(Fab)2 antimouse Fab, strong adhesion was induced (Figure
3A). Secondary (Fab)2 antimouse Fab fragments alone
(without the primary mAb K20) did not affect cell adhesion (not shown).
Likewise, preincubation with soluble recombinant monomeric VCAM-1 did
not affect adhesion of otherwise untreated cells, whereas secondary
clustering by mAb anti-VCAM-1 resulted in a 5-fold increase of
cell-to-cell adhesion (Figure 3B). Consistently, the bivalent human
soluble recombinant VCAM-1-Fc induced a 4-fold increase of monocytic
cell adhesion to HUVECs. Thus, cross-linking of VLA-4 accounted for
enhanced cell adhesion.
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| Fig 3.
Effects of clustering versus ligation of VLA-4 on HL60
cell adhesion to HUVECs.
(A) Myelomonocytic HL60 cells were preincubated with medium alone
(control, not shown), isolated Fab fragments of mAb K20 (5 µg/mL), or
mAb K20 (20 µg/mL) for 30 minutes at 37°C. Cells were
washed, and cells preincubated with K20 Fab were further incubated with
increasing concentrations of a secondary goat
(Fab)2 antimouse Fab for 30 minutes at
37°C. After washing, adhesion to endothelial cell monolayers was
performed. (B) HL60 cells were preincubated (20 minutes, 37°C) with
medium (open bars) or human IgG (20 µg/mL, filled bars) to saturate
Fc receptors. Medium alone (control, not shown), soluble VCAM-1
(sVCAM-1; 40 µg/mL), or soluble VCAM-1-Fc (sVCAM-1-Fc; 40 µg/mL)
was added for 30 minutes at 37°C. Cells were washed, and cells
preincubated with soluble VCAM-1 were further incubated with increasing
concentrations of mAb anti-VCAM-1 for 30 minutes at
37°C. Following washing, adhesion to endothelial cell monolayers
was performed. Values are displayed as percentage of control and
represent the mean ± SEM of at least 3 independent experiments.
*Indicates P < .01 compared with the respective control.
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Possible involvement of Fc receptors in these activation pathways was
excluded: (a) cross-linking of Fab fragments of mAb K20 was
performed by antimouse (Fab)2 in the absence of
any Fc fragments; (b) saturation of Fc receptors by human IgG
(Figure 3B) or mAb anti-CD16 and anti-CD32 (not shown) prior to the
experiment did not affect the results.
VLA-4 clustering induces adhesion via activated 2
integrins LFA-1 and Mac-1
To identify the adhesion receptors that become activated upon VLA-4
clustering, we investigated cell adhesion to HUVECs in the presence of
specific blocking mAbs (Figure 4).
Leukocyte adhesion induced by both VCAM-1-Fc and by mAb K20 was
abrogated in the presence of the blocking mAb 60.3, directed against
the common 2 integrin chain. In addition, mAbs directed
against the respective chain of LFA-1
(L2) or Mac-1
(M2) significantly inhibited adhesion to
a similar extent. Thus, both LFA-1 and Mac-1 seem to become activated
upon engagement of VLA-4 and appear to contribute equally to enhanced
leukocyte adhesion to endothelial cells. An antibody directed against
the 4 chain of VLA-4, known to effectively block VLA-4
binding to VCAM-1, did not affect cell adhesion in response to mAb K20
or VCAM-1-Fc. Similar results were obtained when myelomonocyte cell
adhesion to ICAM-1-transfected CHO cells was studied after VLA-4
engagement (5-fold and 3.5-fold increase in response to adhesion of mAb
K20 and VCAM-1-Fc, respectively).
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| Fig 4.
VLA-4-induced adhesion is mediated by 2
integrins LFA-1 and Mac-1.
Myelomonocytic HL60 cells were pretreated with medium (control, not
shown), soluble VCAM-1-Fc (40 µg/mL, open bars), or mAb K20 (20 µg/mL, filled bars) for 60 minutes at 37°C. After washing,
adhesion to endothelial cell monolayers was performed in the presence
or absence of blocking mAbs anti-4 (mAb HP2.1, 50 µg/mL), anti-M (mAb CBRM1/5, 50 µg/mL), anti-L (mAb L15, 20 µg/mL), or anti-2 integrin (mAb 60.3, 20 µg/mL).
Values (mean ± SEM) are displayed as percentage of control adhesion
and represent the mean of at least 3 independent experiments.
*Indicates P < .01 compared with the respective control (no
mAb).
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Consistent with these functional data, flow cytometry measurements
directly revealed Mac-1 activation following VLA-4 engagement: preincubation with VCAM-1-Fc resulted in the expression of a cellular Mac-1 epitope, which was expressed only on activated leukocytes and was
detected by mAb CBRM1/5 (Figure 5). No
quantitative changes in expression of 2
integrin were found when mAbs were used that detect LFA-1 (L15) or
Mac-1 (Bear-1) irrespective of their activation state. Thus,
conformational rather than quantitative changes of the 2
integrins LFA-1 and Mac-1 appear to account for enhanced cell
adhesivity in response to VLA-4 occupancy. No changes of integrin
expression were found after incubation of cells with soluble monomeric
VCAM-1 or human IgG, which was used to exclude the involvement of Fc
receptors. In addition, saturation of Fc receptors by human IgG prior
to the experiment did not influence the activating effect of VCAM-1-Fc
on the expression of epitope CBRM1/5.
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| Fig 5.
Induction of an active 2 integrin
conformation by VCAM-1-Fc binding to VLA-4.
Myelomonocytic HL60 cells were pretreated with medium (thin line) or
soluble VCAM-1-Fc (solid line) (40 µg/mL) for 30 minutes at
37°C. After washing, fluorescence-activated cell sorter analysis
was performed as described in "Materials and methods" using mAbs
anti-L (L15) and anti-M (Bear-1), which both detect the
respective integrin independent of its activation state, as well as mAb
CBRM1/5 (anti-M), which only binds to the activated M chain of
Mac-1.28 The broken line represents the nonspecific control
mAb.
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The specificity of VLA-4-promoted leukocyte adhesion was further
defined by cell adhesion to immobilized components of the extracellular matrix as well as to the immobilized recombinant human integrin counter-receptors ICAM-1 and VCAM-1 (Table
1). After preincubation with mAb K20, cell
adhesion to the immobilized ligands of 1 integrins,
collagen and laminin, as well as to the specific ligands of VLA-4,
fibronectin and VCAM-1, was unaffected. In contrast, adhesion to
ICAM-1, the common ligand of the 2 integrins LFA-1 and
Mac-1, as well as to fibrinogen and KLH, which are specific ligands for
Mac-1, was significantly induced. The specificity of these interactions
for 2 integrin-dependent cell adhesion was corroborated
by the findings that mAb K20-induced adhesion to the latter
2 integrin ligands was totally abolished by the blocking
anti-2 integrin mAb (60.3) but not by anti-VLA-4
(HP2.1).
VLA-4 engagement induces uPAR-mediated adhesion
The uPAR forms functional complexes with integrins on the cell
surface and has been shown to control integrin
activities.30-32 Thus, the involvement of uPAR in
VLA-4-induced adhesion was studied: preincubation with mAb K20 (Figure
6A) or soluble VCAM-1-Fc (but not
unclustered soluble VCAM-1, not shown) directly resulted in significant
monocytic cell adhesion to immobilized vitronectin, an extracellular
matrix protein that specifically binds to uPAR. Induced cell adhesion
was inhibited in the presence of a blocking anti-uPAR mAb (R3), whereas
the nonblocking anti-uPAR mAb R4 or the integrin-blocking mAb
anti-v3 (LM609) had no effect. Flow cytometric
analysis showed no quantitative changes of uPAR surface expression (not
shown), indicating that enhanced adhesion was due to conformational
activation of uPAR rather than quantitative up-regulation.
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| Fig 6.
Requirement of uPAR for leukocyte adhesion in response to
VLA-4 ligation.
(A) Myelomonocytic HL60 cells were treated with medium alone (open bar)
or with mAb K20 (20 µg/mL, filled bars) for 30 minutes at 37°C.
Following washing, adhesion to immobilized vitronectin was performed in
the presence or absence of mAbs anti-uPAR R3 (blocking), R4
(nonblocking), or anti-v3 LM609 (20 µg/mL) as
described. Values represent the mean ± SEM of 3 independent
experiments. (B) Myelomonocytic HL60 cells were treated with medium
alone (open bars) or pretreated with phosphatidylinositol-specific
phospholipase C (0.5 U/mL) for 90 minutes at 37°C
(filled bars), washed, and incubated for 10 minutes in the absence or
presence of soluble intact uPAR (D1-D3, 16 nmol/L), the
truncated form of uPAR (D2/D3, 20 nmol/L) lacking domain
1, or with soluble tissue factor pathway inhibitor (sTFPI; 16 nmol/L), followed by incubation for 20 minutes with
medium, soluble VCAM-1-Fc (40 µg/mL), or mAb K20 (15 µg/mL) as
indicated. Following washing, adhesion to endothelial cell monolayers
was performed as described. Values are displayed as percentage of
control (no pretreatment, 100%) and represent the mean ± SEM of 3 independent experiments. *Indicates P < .01
compared with the respective medium control; n.s., not significant
(P > .05).
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We further tested the relevance of the uPAR on integrin-mediated
myelomonocyte adhesion in response to VLA-4 engagement by both mAb K20
or VCAM-1-Fc, respectively (Figure 6B). Removal of uPAR from the
leukocyte surface by preincubation with phosphatidylinositol-specific phospholipase C resulted in a significant inhibition of cell adhesion stimulated by VCAM-1-Fc (64% inhibition) or mAb K20 (62%
inhibition). Adhesion was almost completely restored when soluble
recombinant uPAR was added prior to the cell adhesion assay.
Control proteins such as the truncated form of uPAR, which lacks
domain 1, or recombinant tissue factor pathway inhibitor, which
represents another glycolipid-linked protein on
monocytes, did not restore adhesion (Figure 6B). These findings
demonstrate that, upon VLA-4 engagement, uPAR is activated and can
either directly bind to vitronectin or serve a superior regulatory
function particularly in the inductionof 2
integrin-dependent cellular adhesive interactions provoked by VLA-4 occupancy.
| |
Discussion |
The established chain reaction of adhesive events necessary for
leukocyte adhesion and transmigration includes directed activation between selectins, 2 integrins, and
1/4 integrin. The present study
demonstrates that clustering of VLA-4 on the surface of monocytic cells
by VCAM-1 enhances cell adhesion to human vascular endothelium via
2 integrins. VLA-4-mediated cell adhesion was a result
of activation of the 2 major 2 integrins, LFA-1 and Mac-1. The activated urokinase receptor (uPAR) is substantially involved in this activation pathway. Thus, bidirectional integrin transactivation including the action of uPAR appears to be plausible for VLA-4-VCAM-1 interactions, particularly during the fast process of
firm leukocyte adhesion and migration, and may stabilize and promote
leukocyte infiltration via rapid activation of 2 integrins.
The specificity of cell-to-cell adhesion was confirmed by a variety of
approaches, including specific extracellular matrix ligands,
ICAM-1-transfected CHO cells, and blocking antibodies. In particular,
cellular interactions induced by VLA-4 occupancy were blocked by mAb
against the common 2 integrin chain CD18 as well as by
mAbs against the respective chain of LFA-1 (CD11a) or Mac-1
(CD11b). Thus, LFA-1 and Mac-1 appear to contribute equally to the
adhesive strength of leukocytes, comparably to physiologic situations.33 Three additional findings suggest that VLA-4
engagement may activate 2 integrins via conformational
changes rather than quantitative up-regulation: (1) flow cytometry did
not show any changes in quantitative surface expression of LFA-1 or
Mac-1 in response to VLA-4 engagement; (2) direct Mac-1 activation was confirmed by flow cytometry using mAb CBRM1/5 that only detects Mac-1
in its activated, binding state28; (3) functional relevance of this activation-dependent epitope on Mac-1 and of an activated LFA-1
was proven in adhesion assays, because mAb CBRM1/5 or mAb L15
effectively blocked cell adhesion in response to VLA-4 engagement.
The role of VLA-4 was elucidated by using specific ligands such as
soluble VCAM-1 as well as a function blocking mAb anti-VLA-4. When mAb
anti-VLA-4 was allowed to compete with soluble VCAM-1-binding to the
cells, binding of VCAM-1 and subsequent cell adhesion were blocked. In
contrast, mAb anti-VLA-4 did not affect induced adhesion when added
following VLA-4 clustering by VCAM-1. Therefore, in the
described activation pathway, adhesion to HUVECs is induced via VCAM-1-VLA-4 interaction and mediated via 2
integrin (LFA-1 and Mac-1)-ICAM-1 interaction.
The specificity of this transdominant activation of integrins was
further confirmed by the finding that cell adhesion to the immobilized
2 integrin ligand ICAM-1, but not to the VLA-4-ligand VCAM-1, was stimulated by antibody ligation of VLA-4. Consistently, adhesion to immobilized fibrinogen, an adhesive extracellular matrix
protein that selectively binds Mac-1, was induced, whereas adhesion to
specific 1-integrin ligands such as fibronectin, laminin, or collagen remained unaffected. The fact that freshly isolated neutrophils as well as myelomonocytic cells (U937, HL60) reacted with 2 different ICAM-1-expressing cell types (HUVEC and CHO-ICAM-1) in a similar manner after 1 integrin
engagement suggests that the functional interaction of 1
and 2 integrins is not restricted to a particular cell
type. Consistently, activation of LFA-1 via VLA-4-VCAM-1 interactions
has recently been described on T cells.34 Circulating
neutrophils strongly express 1 integrins 51 (VLA-5) and
61 (VLA-6) but no VLA-4
(41). Consistent with published results,
fluorescence-activated cell sorter analysis showed strong expression of
the 1 integrin chain as well as 5 and
6 (not shown) but no surface expression of
4 (mAb HP2.1). As expected, VCAM-1-Fc did not induce
neutrophil adhesion (not shown), whereas mAb anti-1
(K20) effectively induced neutrophil adhesion to HUVECs (Figure 1B). We
were able to identify the natural receptor-ligand pair (VLA-4-VCAM-1)
that activates 2-integrin function on monocytic cells.
Which natural ligands of 1 integrins are able to
activate neutrophil 2 integrins remains to be clarified. Because VLA-4 is also expressed on neutrophils under certain
circumstances (eg, after transmigration)35 the described
activation pathway may be operative on these cells in vivo.
Relative surface expression as well as the activation status of the
involved integrins may influence the respective adhesive response of
different leukocyte subtypes. For example, LFA-1 activation may
dominate on T cells,36 whereas Mac-1 activation may be of importance on monocytes. In addition, chemokines may selectively activate VLA-4 on a specific cell type, such as
eosinophils,37 thereby inducing an activation cascade that
results in 2 integrin-mediated adhesion.
Engagement of VLA-4 was achieved by soluble forms of VCAM-1 as well as
mAb directed against VLA-4. To distinguish between effects of binding
only versus integrin cross-linking, bivalent VCAM-1-Fc as well as Fab
fragments of mAb K20 were used. In addition, VLA-4-bound VCAM-1 was
secondarily clustered by mAb anti-VCAM-1. Evidence is provided that
intracellular signals following VLA-4 cross-linking may account for
2 integrin activation and cell-to-cell adhesion:
clustering of VLA-4 was required for the observed cell-to-cell adhesion, because Fab fragments of mAb K20 induced adhesion only if
cross-linked by secondary mAb. Similarly, soluble monovalent VCAM-1 on
its own had no effect, whereas cross-linking by secondary mAb
anti-VCAM-1 strongly enhanced adhesion. Consistently, soluble bivalent
VCAM-1-Fc effectively induced the expression of an
activation-inducible epitope on Mac-1 and enhanced cell adhesion via
2 integrin activation. Intracellular signal transduction
in response to VLA-4 cross-linking has been previously
described.10,11 Further studies need to elucidate the role
of intracellular and extracellular molecular crosstalks that are
required for 1 integrin-dependent transdominant activation of 2 integrins. At present, the putative
signaling mechanism leading to 2 integrin activation in
response to VLA-4 engagement seems to differ from previously published
pathways because preliminary experiments revealed that tyrosine
phosphorylation was not involved (unpublished observations). We could
rule out a potential involvement of Fc receptors in this activation
pathway, because 1 integrin clustering was achieved in
the absence of Fc fragments (Figure 3A) and saturation of Fc receptors
prior to clustering of VLA-4 by VCAM-1-Fc or VCAM-1/anti-VCAM-1 did not affect adhesion (Figure 3B).
The glycolipid-anchored cell surface glycoprotein uPAR plays a central
role for both proteolytic and adhesive cellular
functions.7,32,38 By forming a functional unit in a yet
poorly understood manner, uPAR modulates 2-integrin
function.31,32,39,40 Specific binding of soluble uPAR to a
variety of human hematopoietic cells (including HL60 cells, monocytes,
and neutrophils) has been previously demonstrated.41
Functional relevance of soluble uPAR for cell activation has been shown
by others30,42 and our group.32,40 In the
present study, a central role for uPAR in VLA-4-induced cell
adhesivity is emphasized, because uPAR was required for
2 integrin-mediated leukocyte adhesion to endothelium
following VLA-4 engagement: while the removal of uPAR hardly allowed
adhesion, full cell adhesivity was regained by addition of soluble
uPAR. In addition, apart from 2 integrin activation,
direct activation of uPAR was noted after VLA-4 clustering, as measured
by enhanced specific monocytic cell adhesion to immobilized
vitronectin, an extracellular matrix protein that specifically binds to
uPAR. Thus, clustering of VLA-4 by VCAM-1 activates leukocyte adhesion to extracellular matrix or vitronectin via activated uPAR as well as to
endothelial cells via activated 2 integrins. Apart from uPAR activation and direct binding to vitronectin, uPAR appears to
control the described integrin crosstalk. These findings confirm the
concept that uPAR plays a key role for integrin regulation and is
essential for an adequate function of both LFA-1 and
Mac-1.32
The relevance of monocyte rolling for lesion development in early
atherogenesis has recently been reviewed.43 VLA-4
interaction with VCAM-1 was shown to stabilize rolling and prolonged
transit time of monocytes in early atherosclerotic
lesions.44 Our study demonstrates that this stabilization
may not only be due to VLA-4-VCAM-1 binding, but also through the
exchange of subsequent activation signals leading to engagement of
2 integrins LFA-1 and Mac-1. Subsequent 2
integrin-dependent firm cell adhesion and transmigration could thus be
regulated by anti-1-integrin strategies. Interruption of signals that result in "downstream" cell activation and
enhanced cell adhesivity via 2 integrins may, therefore,
additionally account for protective effects of direct VLA-4-VCAM-1
blockade. Apart from atherogenesis, leukocyte recruitment is
fundamental for the development of restenosis following percutaneous
coronary interventions45-47 as well as for myocardial
damage after ischemia and reperfusion.48 These entities are
characterized by enhanced surface expression of adhesion molecules on
monocytes and neutrophils as well as on vascular endothelium. Our
findings can assist in the design of new therapeutic strategies for
prevention of unwanted leukocytic cell recruitment during inflammatory
cardiovascular processes by employing specific antagonists against the
VLA-4-VCAM-1 and the urokinase receptor systems.
| |
Acknowledgments |
We gratefully acknowledge the excellent technical assistance of
Monika Hölderle and Thomas Schmidt. We thank Drs Marc
Robinson, Carl Figdor, John Harlan, and Timothy Springer for the
generous supply of antibodies. We are grateful to Dr Dietmar Seiffge
for kindly providing VCAM-1-Fc and Dr Niels Behrendt for soluble
recombinant urokinase receptor. We also thank Dr Triantefyllos Chavakis
(Giessen, Germany) for critically reading the manuscript.
| |
Footnotes |
Submitted September 14, 1999; accepted February 29, 2000.
Supported in part by grants from Novartis-Foundation (Nürnberg,
Germany), the Deutsche Forschungsgemeinschaft (Ne 540/1-2, Bonn,
Germany), and the GTH (Gesellschaft für Thrombose und
Hämostaseforschung).
Reprints: Andreas E. May, Deutsches Herzzentrum, Technische
Universität München, Lazarettstr. 36, D-80636
München, Germany; e-mail: may{at}dhm.mhn.de.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Top
Abstract
Introduction