From the Department of Pathology, Boyer Center for Molecular
Medicine, Yale University School of Medicine, New Haven,
Connecticut 06536 and the Division of Vascular Surgery,
Brigham and Women's Hospital, Harvard Institutes of Medicine,
Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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The monoclonal antibody (mAb) 7E3 directed to the
platelet integrin IIb
3 was tested
for its cross-reactivity with the homologous leukocyte integrin
M
2. Nested recombinant fragments of
M I domain were expressed as glutathione
S-transferase fusion proteins and analyzed for antibody
recognition. In enzyme-linked immunosorbent assay, mAb 7E3 bound
M I domain fragments containing the amino-terminal sequence Cys128-Ser172, whereas the
carboxyl-terminal region Leu173-Pro291 was
ineffective. A synthetic peptide designated R1.1 and duplicating the
M sequence
G127CPQEDSDIAFLIDGSGSIIPHDF150 bound mAb 7E3.
In contrast, the adjacent
M region
F150RRMKEFVSTVMEQLKKSKTLFS172 or a
control peptide with a scrambled R1.1 sequence was not recognized by
mAb 7E3. Binding of mAb 7E3 to
M I domain blocked
monocyte and neutrophil adhesion to immobilized fibrinogen and
fibrinogen-dependent leukocyte-endothelium bridging,
indistinguishably from bona fide anti-
2 mAb
IB4. In contrast, leukocyte binding to stable transfectants expressing
intercellular adhesion molecule-1 was not affected by mAb 7E3.
Balloon-mediated injury of iliofemoral arteries in rabbits resulted in
prominent deposition of fibrinogen and increased monocyte adhesion to
the injured vessel, in a reaction inhibited by mAb 7E3, but unaffected
by control mAb 14E11. Through its cross-reactivity between
IIb
3 and
M
2, mAb 7E3 may initiate a new class of
integrin antagonists, capable of simultaneously targeting platelet and leukocyte adhesion mechanisms in vascular injury.
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INTRODUCTION |
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Hemostasis and immune-inflammatory responses (1) are maintained by
the adhesive interactions mediated by integrins
IIb
3 (GPIIb/IIIa) on platelets (2) and
M
2 (Mac-1) on leukocytes (3). Despite
their critical role in vascular cell homeostasis and signaling (4),
platelet and leukocyte adhesion mechanisms participate in the
pathogenesis of vascular injury. This is emphasized by the role of
IIb
3 in platelet aggregation and thrombus
formation (5, 6) and of
M
2 in leukocyte
recruitment (3), procoagulant activity (7), and reperfusion injury.
Considerable effort has been devoted to the identification of molecular
antagonists of
IIb
3 (9) and
M
2 (10), capable of disrupting aberrant platelet and monocyte adherence mechanisms. In this context,
administration of anti-
IIb
3
mAb1 7E3 (11) reduced the
incidence of mortality, myocardial infarction, and other emergency
procedures in patients at risk of cardiovascular ischemic disease (12,
13).
In previous studies, it was also reported that mAb 7E3 unexpectedly
cross-reacted with the active conformation of
M
2, induced on monocytes by inflammatory
stimuli (14) or Mn2+ ions (15). These observations were
recently independently confirmed with direct binding studies of mAb 7E3
to
M
2 transfectants (16), whereas a
~200-amino acid-inserted "I" domain in
M (17) was provisionally implicated in this cross-reactivity (18).
In this study, we sought to reinvestigate the molecular basis of mAb
7E3 cross-reactivity with M
2 and its
potential relevance to leukocyte adhesion, in vivo. We found
that mAb 7E3 recognizes a discrete region in
M I domain
(17), which is critically involved in monocyte adherence to fibrinogen
in vitro and in balloon-injured arteries, ex
vivo.
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MATERIALS AND METHODS |
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Cell Culture and mAbs--
Polymorphonuclear leukocytes (PMN)
were isolated from acid citrate dextrose-anticoagulated blood drawn
from normal informed volunteers by Ficoll-Hypaque (Amersham Pharmacia
Biotech) centrifugation and dextran sedimentation, as described (19).
PMN were suspended in serum-free RPMI 1640 medium (BioWhittaker,
Walkersville, MD) at a concentration of 1 × 106/ml.
The monocytic cell line THP-1 (American Type Culture Collection (ATCC),
Manassas, VA) was maintained in complete RPMI 1640 medium (BioWhittaker) containing 10% heat-inactivated fetal bovine serum (Gemini Bioproducts, Calabasas, CA), 2 mM
L-glutamine (Gemini), and 105
2-mercapthoethanol (Eastman Kodak Co.). Human umbilical vein endothelial cells (HUVEC) were isolated by collagenase treatment, maintained in medium 199 (BioWhittaker) plus 20% fetal bovine serum
and endothelial cell growth factor, and plated onto gelatinized tissue
culture plates (Costar Corp., Cambridge, MA). Cells were used between
passages 2 and 4. Chinese hamster ovary cells stably transfected with
the cDNA of intercellular adhesion molecule-1 (ICAM-1) were
established and characterized previously (20). Anti-
IIb
3 mAb 7E3 was generously provided
by Dr. Barry Coller (Mount Sinai Medical Center, New York) and
characterized previously for its cross-reactivity with
M
2 (14, 16). Anti-
2 mAb
IB4 was from ATCC. Nonbinding mAb 14E11 was used as a control.
Recombinant M I Domain Fragments--
The map of
the various
M I domain fragments used in these studies
is shown in Fig. 1. For these
experiments, the full-length
M cDNA was amplified by
PCR in the presence of a forward oligonucleotide 5'-TGTCCTCAAGAGGATAGTGAC-3' and four distinct reverse oligonucleotides 5'-AGAGAACAAGGTTTTGGAC-3' (R1), 5'-CGTGGCCGTGTGTGTC-3' (R2),
5'-CTCATATCCCAAGGGATCG-3' (R3), and 5'-CGGCTTGGATGCGATG-3' (R4). The
fragment R1(
), lacking the amino-terminal R1 sequence
Cys128-Ser172 (Fig. 1), was generated by PCR
using forward and reverse oligonucleotides 5'-TTGATGCAGTACTCTGAAG-3'
and 5'-CGGCTTGGATGCGATG-3', respectively. The
M I domain
fragments B5 and B4 (Fig. 1) were generated with forward and reverse
primers 5'-TTGATGCAGTACTCTGAAG-3' and 5'-ATTCTTTCGGGCTCCGTTG-3' (B5)
and 5'-GCCTTTAAGATCCTAGTTGTC-3' and 5'-CGGCTTGGATGCGATG-3' (B4),
respectively. Each forward primer contained a BamHI
restriction site, whereas a XhoI site was added at the end
of each reverse oligonucleotide. Amplification was carried out in a
total volume of 100 µl with denaturation at 94 °C for 1 min,
annealing at 52 °C for 1 min, and extension at 72 °C for 1 min.
PCR products were separated on 1% agarose gels, gel-purified by
phenol/chloroform extraction, digested overnight with BamHI
and XhoI, and directionally cloned in the prokaryotic
expression vector pGEX-2T (Amersham Pharmacia Biotech) with
transformation in the BL-21 Escherichia coli strain.
Expression of recombinant proteins was carried out as described (18).
Briefly, E. coli cultures containing the various constructs
were grown to A600 ~0.5, induced with 0.1 mM isopropyl-
-D-thiogalactopyranoside (IPTG,
Calbiochem) and grown for 3 h at 37 °C with constant shaking at
225 rpm. Bacteria were centrifuged at 6,000 rpm for 15 min at 4 °C
and suspended in a lysis buffer containing 10 mM Tris-HCl,
150 mM NaCl, 100 µg/ml lysozyme (Calbiochem), and 1%
Triton X-100. Samples were subjected to three cycles of sonication of
20 s each before centrifugation and dialysis in TBS, pH 7.4. Expression of the various I domain fragments of the expected molecular
weight was confirmed in IPTG-induced bacterial lysates, but not in
noninduced samples, by SDS-gel electrophoresis and Coomassie Blue
staining. Fibrinogen was purified from fresh frozen human plasma by
glycine precipitation (19). The interaction of fibrinogen with
M
2 on stimulated monocytes and PMN has
been described previously (7).
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Epitope Mapping--
Bacterial lysates of the various
IPTG-induced M I domain constructs, or a control
E. coli lysate, were diluted 1:50 in TBS, pH 8.0, and
immobilized (100 µl/well) on plastic microtiter plates (Immulon-2,
Dynatech Laboratories, Chantilly, VA) for 18 h at 4 °C. Wells
were blocked with 3% gelatin for 1 h at 37 °C, rinsed, and
incubated with 20 µg/ml mAb 7E3, anti-
2 (CD18) mAb
IB4, or control mAb 14E11 for 1 h at 37 °C. After washes in TBS
containing 1% bovine serum albumin, 0.5% Tween 20, binding of the
primary antibodies was revealed by addition of biotin-conjugated rabbit anti-mouse IgG for 1 h at 37 °C followed by
streptavidin-alkaline phosphatase and determination of absorbance at
A405 using p-nitrophenyl phosphate
(Sigma). Peptides duplicating adjacent sequences in the
M R1 region (Fig. 1) R1.1
(G127CPQEDSDIAFLIDGSGSIIPHDF150), R1.2
(F150RRMKEFVSTVMEQLKKSKTLFS172), a control
R1.1-scrambled peptide R1.1S (GILGFDEGPCIDHASPDISDFQIS), and a peptide
reproducing the R1.1-homologous region
(V107EDYPVDIYYLMDLSYSMKDDL128) in
3 integrin were synthesized and characterized by amino
acid composition and mass spectrometry. The various peptides were
dissolved at 5 µg/ml in carbonate buffer, pH 9.5, and immobilized
onto plastic microtiter plates (100 µl/well) before determination of
antibody binding, as described above. An additional unrelated factor
X-derived peptide KDGLGEYG was used as a control.
Cell Adhesion--
PMN or THP-1 cells were metabolically labeled
with 300 µCi of 51CrNaO4 (NEN Life Science
Products) for 1 h at 37 °C, washed, and suspended in serum-free
RPMI 1640 at 1 × 106/ml. Two-hundred-µl aliquots of
cell suspension were stimulated with 1 µM fMLP (Sigma),
treated with the various mAbs at 20 µg/ml for 15 min at 22 °C, and
further incubated with or without fibrinogen (200 µg/ml) in 2.5 mM CaCl2 for 20 min at 22 °C. Cells were
added to monolayers of HUVEC or ICAM-1 transfectants for 45 min at
22 °C, and after washes attached cells were solubilized in 20% SDS
with determination of radioactivity in a scintillation -counter. The number of attached cells was calculated by dividing the counts/min observed by the counts/min/cell (21). Alternatively, HUVEC monolayers were preincubated with mAbs IB4, 7E3, or control mAb 14E11 for 30 min
at 22 °C, washed, and mixed with 51Cr-labeled,
fMLP-stimulated THP-1 cells before determination of fibrinogen-dependent intercellular bridging, as described
above. In other experiments, microtiter wells were coated with human or
rabbit (Sigma) fibrinogen at 10 µg/ml for 18 h at 4 °C and blocked with 3% gelatin for 30 min at 37 °C. Wells were incubated with fMLP-stimulated 51Cr-labeled THP-1 cells (1 × 106/ml) preincubated with 20 µg/ml mAbs 7E3, IB4, or
control mAb 14E11 for 10 min at 22 °C with determination of cell
adhesion after a 45-min incubation at 22 °C.
Animal Procedures-- Adult New Zealand White rabbits (3-5 kg weight) were given free access to water and rabbit chow and were housed in a facility with alternating light and dark cycles. Animal care complied with the "Principles of Laboratory Animal Care" (National Society for Medical Research) and the "Guide for the Care and Use of Laboratory Animals" (NIH Publication No. 80-23, revised 1985). The Harvard Medical Area Standing Committee on Animals approved the experimental protocol. For surgical procedures, animals were anesthetized with ketamine (25 mg/kg, KetalarTM, Parke-Davis) and xylazine (5 mg/kg, RompunTM, Mobay, Shawnee, KS) administered by intramuscular injection, supplemented with intravenous doses of the mixture as needed. The iliofemoral arterial segment was exposed bilaterally via extended groin incisions, and all side branches proximal to the femoral bifurcation were ligated to create an isolated segment (22). The superficial femoral artery was then cannulated, and a 2-French Fogarty balloon embolectomy catheter (American Edwards, Anasco, Puerto Rico) was inserted. The catheter tip was passed into the terminal aorta and the balloon inflated and withdrawn three times to denude the vessel. After removal of the catheter, the superior femoral artery was ligated and antegrade flow re-established via the deep femoral branch. Wounds were closed surgically and the animals allowed to recover. At sacrifice (5-6 days after balloon injury) animals were re-anesthetized as described and systemically heparinized with a 1000-unit intravenous bolus. The groin wounds were reopened and patency of the femoral arteries assessed by direct inspection. The animals were then euthanized with an intravenous overdose of sodium pentobarbital. The abdominal aorta and inferior vena cava were cannulated and the distal arterial tree perfused at 80-120 mm Hg with 500 ml of heparinized (10 units/ml) lactated Ringer's solution (Baxter Healthcare Corp., Deerfield, IL) with continuous venous drainage. The femoral arteries were re-cannulated with 20-gauge stainless steel catheters and the entire aortofemoral arterial segment excised and placed into sterile PBS.
Ex Vivo Monocyte Adhesion-- The freshly excised arterial segments were flushed gently with PBS prewarmed to 37 °C, and a microvascular clamp was placed at the origin of each external iliac artery. 51Cr-Labeled THP-1 cells (107/ml) were stimulated with 1 µM fMLP in the presence of 2.5 mM CaCl2, equilibrated with control mAb 14E11 or mAb 7E3 (10-25 µg/ml) for 30 min at 22 °C, and infused to fill each vessel segment. Paired arteries in each case were treated with mAb 7E3 versus control mAb 14E11. The external surface of the filled arteries was rinsed copiously with PBS and the vessels placed in PBS at 37 °C for 1 h. At the end of the incubation, a 5-mm segment of proximal aorta (not exposed to THP-1 cells intraluminally) was excised for measurement of background counts. The clamps were then removed, and each vessel was flushed gently with 3 ml of PBS at 1 ml/min by timed hand injection. The external surface was again rinsed and 5-mm segments cut (two from each iliofemoral segment) for scintillation counting. Segments of the remaining tissue were embedded in optimal cutting temperature compound (Bayer, West Haven, CT) and snap-frozen in 2-methylbutane cooled in liquid nitrogen for immunohistochemical analysis. Tissue was prepared for scintillation counting by carefully mincing each specimen and solubilizing in 1% Triton X-100, 10% SDS overnight at 22 °C. The following day an equal volume of scintillant was added, and radioactivity was determined in a scintillation counter.
Immunohistochemistry--
Snap-frozen tissue was cut into
6-µm-thick cross-sections and adhered to glass slides coated with
0.25% gelatin and 0.025% chromium potassium sulfate
(CrK(SO4)2, Sigma). Tissue was fixed in acetone
at 20 °C for 10 min, quenched in 3% H2O2,
and blocked with 10% normal horse serum. Following each step, slides
were washed with TBS containing 135 mM NaCl, 25 mM Tris, 2.6 mM KCl, pH 7.4, plus 1% fetal
bovine serum. A mouse anti-fibrinogen antibody characterized in
previous studies (19) was applied for 1 h at 22 °C, followed by
a secondary biotinylated horse anti-mouse antibody (Vector Laboratories
Inc. Burlingame, CA) for 2 h at 22 °C. Negative controls were
prepared in the absence of primary antibody or with nonbinding isotype
control (IgG1). Sections were incubated with an avidin-biotin
peroxidase complex (ABC kit, Vector) followed by 3-amino-9-carbazole.
Slides were counterstained with Mayer`s hematoxylin (Sigma) followed
by 30% NH3OH.
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RESULTS |
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Epitope Mapping of mAb 7E3 on M I Domain--
In
enzyme-linked immunosorbent assay, mAb 7E3 bound all recombinant I
domain fragments containing the
M sequence
Cys128-Pro291 (Fig.
2A). The minimal
M fragment reacting with mAb 7E3 contained the R1
sequence Cys128-Ser172 (Fig. 2A).
Consistent with these findings, mAb 7E3 failed to react with three
carboxyl terminus
M I domain fragments lacking the R1
region and designated R1(
) (Leu173-Pro291),
B5 (Leu173-Asn232), and B4
(Ala233-Pro291), or with control,
non-IPTG-induced, bacterial lysate (Fig. 2A). In
peptide mapping experiments, mAb 7E3 bound the R1.1 sequence G127CPQEDSDIAFLIDGSGSIIPHDF150 corresponding to
the amino-terminal half of the R1 region in
M I domain,
whereas a peptide with the R1.1 sequence in scrambled order (R1.1S) was
not recognized by mAb 7E3 (Fig. 2B). Similarly, synthetic
peptides duplicating the adjacent I domain region
F150RRMKEFVSTVMEQLKKSKTLFS172 (R1.2), the
R1.1-homologous sequence
V107EDYPVDIYYLMDLSYSMKDDL128 in
3 integrin (Beta3), or an unrelated factor X sequence
KDGLGEYG (control) did not associate with mAb 7E3 (Fig.
2B). In control experiments, mAb 14E11 or
anti-
2 (CD18) mAb IB4 did not recognize any
M I domain fragments and did not associate with
2-,
3-, or factor X-derived peptides in
enzyme-linked immunosorbent assay (Fig. 2, A and
B).
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Differential Regulation of Leukocyte Adhesion by mAb 7E3--
The
effect of mAb 7E3 on M
2 recognition of
ICAM-1 (1) was first investigated. Preincubation of fMLP-stimulated PMN
or monocytic THP-1 with mAb 7E3 or control mAb 14E11 did not reduce the
attachment of these cells to monolayers of resting HUVEC or ICAM-1
transfectants (Fig. 3). In contrast,
anti-
2 mAb IB4 nearly completely abrogated PMN adhesion
to HUVEC or ICAM-1 transfectants, and significantly inhibited THP-1
cell attachment to either cell type, under the same experimental
conditions (Fig. 3).
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Effect of mAb 7E3 on Monocyte Adhesion to Balloon-injured Arteries-- Balloon-mediated injury of external iliac arteries in rabbits resulted in prominent attachment of fMLP-stimulated, 51Cr-labeled THP-1 cells to the de-endothelialized injured vessel, as compared with noninjured arteries (Fig. 5 and data not shown). Under these experimental conditions, equilibration of THP-1 cells with mAb 7E3 ex vivo nearly completely inhibited the attachment of these cells to balloon-injured vessels, whereas control mAb 14E11 was ineffective (Fig. 5). In four of four animals, mAb 7E3 inhibited monocyte THP-1 cell adhesion to balloon-injured vessels by 53.6 ± 10.6% at 10 µg/ml and by 85.9 ± 3.9% at 25 µg/ml (n = 2). In immunohistochemical analysis, an anti-fibrinogen antibody strongly reacted with the luminal and medial aspects of the balloon-injured rabbit iliac arteries (Fig. 6), whereas no specific fibrinogen staining was observed in noninjured arteries (not shown). In control experiments, no specific staining of injured vessels was observed in the absence of a primary antibody, under the same experimental conditions (Fig. 6).
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DISCUSSION |
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In this study, we have shown that
anti-IIb
3 mAb 7E3, an integrin antagonist
currently used in clinical practice (11), binds a discrete region of
M I domain and inhibits fibrinogen-mediated leukocyte
adhesion in vitro, and in balloon-injured arteries of rabbits, ex vivo.
Although of paramount importance for normal hemostasis (23), platelet
aggregation mechanisms maintained by IIb
3
may precipitate thrombus formation and acute ischemic cardiovascular
emergencies (5, 6). Among integrin antagonists capable of targeting platelet adherence mechanisms, anti-
IIb
3
mAb 7E3 significantly reduced mortality and emergency procedures in
patients undergoing acute coronary intervention (12, 13). At the
molecular level, it was also shown that mAb 7E3 possessed an unusual
pattern of antigen recognition, which included, in addition to
IIb
3, the related integrin
v
3 (9), and the active form of the
leukocyte integrin
M
2 (14, 16). This was
potentially relevant to the beneficial effect of mAb 7E3 in
vivo, because inhibition of
v
3 reduced neointimal hyperplasia in models of vascular injury (24), and
M
2-dependent leukocyte
adherence contributed to monocyte recruitment and reperfusion injury
(8).
Here, mAb 7E3 bound a cross-reacting epitope in the amino-terminal R1
region Cys128-Ser172 of M
I-domain, which was further narrowed to the R1.1 peptide sequence G127CPQEDSDIAFLIDGSGSIIPHDF150.
The most salient feature of this motif is the presence of the amino acids Asp140-Ser142-Ser144,
which comprise the first group of oxygenated residues of the metal
ion-dependent adhesion site (MIDAS) on
M I
domain (17). Experimental evidence obtained with homology models,
mutagenesis, and divalent ion binding studies suggests that a
MIDAS-like motif containing the DXSXS motif, in
which X is a nonconserved amino acid, is structurally and
functionally present in all integrin
subunits (25-28).
Intriguingly, mAbs raised against this region in
3-inhibited fibrinogen binding to the receptor (29), and a synthetic peptide duplicating the MIDAS-like motif bound ligand and
divalent ions (30). Under our experimental conditions, a
3-derived peptide containing the MIDAS homology motif
failed to associate with mAb 7E3. This is consistent with the inability of mAb 7E3 to recognize the isolated
3 subunit (31) and
suggests that conformational and/or divalent ion-dependent
changes in
IIb
3 are required to form a
high affinity antibody binding interface (17). Whether or not mAb 7E3
recognizes MIDAS-like structures in other integrin
subunits is
currently not known. However the data presented here suggest that the
antigenic accessibility of this shared motif may be modulated by
receptor-specific conformational changes and/or the requirement of
additional contact site(s) in the
/
heterodimer. This complex
pattern of multiple integrin recognition may not be unique of mAb 7E3,
because other function blocking anti-
IIb
3
mAbs, i.e. 25E11, have been shown to cross-react with
M
2 (32).
The next question addressed by this study was the potential physiologic
relevance of mAb 7E3 cross-reactivity with
M
2. Consistent with the critical role of
M I domain in ligand binding (17, 18, 33, 34),
engagement of the mAb 7E3 cross-reacting epitope suppressed the
receptor recognition of fibrinogen, in agreement with recent
observations (16). This resulted in inhibition of leukocyte adherence
to immobilized fibrinogen and of fibrinogen-dependent leukocyte-endothelium bridging (21), indistinguishably from bona
fide anti-
2 integrin mAb IB4. In contrast, at
variance with a recent study (16), mAb 7E3 failed to reduce the
M
2 recognition of ICAM-1 (1, 3). Although
differences in protocol may account for this discrepancy, experimental
evidence with epitope-mapped mAbs (33), and peptide inhibition studies
(35), suggests that the ICAM-1- and fibrinogen-binding sites on
M I domain are physically distinct and
nonoverlapping.
An in vivo model of vascular damage further underscored the
relevance of mAb 7E3 targeting of monocyte-fibrinogen interaction. In
these studies, balloon-mediated injury of the iliofemoral arteries in
rabbits resulted in prominent deposition of fibrinogen as detected by
immunohistochemistry and in agreement with previous studies (36). In
addition to promoting increased procoagulant activity (37), this
translated in our study in prominent monocyte attachment to the injured
vessel, in a reaction specifically inhibited by mAb 7E3. Consistent
with the importance of leukocyte adherence in vascular disease (38),
this suggests that fibrinogen deposited on atherosclerotic lesions
(39), and balloon-injured arteries (36), may provide an ideal substrate
for leukocyte recruitment. In turn, this may further exacerbate
vascular damage by promoting increased IL-1 (40) and tissue factor (41)
gene expression, chemotaxis (42), and release of oxidative radicals
(43). Whether or not the inhibition of monocyte adhesion by mAb 7E3
contributes to its protective effect in ischemic disease (12, 13) is
currently not known. However, the data presented here are consistent
with a model in which mAb 7E3 blockade of 3 integrins on
platelets and endothelium, and
M
2 on
leukocytes may simultaneously inhibit multiple cell adherence pathways
at the interface between thrombosis and inflammation (44).
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ACKNOWLEDGEMENTS |
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We thank Dr. Coller for kindly providing mAb 7E3 and for critically reading the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants RO1 HL43773 and HL54131.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Fellow of the Leukemia Research Foundation.
¶ This work was done during the tenure of an American Heart Association Established Investigatorship Award. To whom correspondence should be addressed: Yale University School of Medicine, BCMM 436B, 295 Congress Ave., New Haven, CT 06536. E-mail: dario.altieri{at}yale.edu.
The abbreviations used are:
mAb, monoclonal
antibody; HUVEC, human umbilical vein endothelial cells; ICAM-1, intercellular adhesion molecule-1; IPTG, isopropyl--D-thiogalactopyranosideMIDAS, metal
ion-dependent adhesion sitePMN, polymorphonuclear
leukocytesPCR, polymerase chain reactionTBS, Tris-buffered salinefMLP, formylmethionylleucylphenylalanine.
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REFERENCES |
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