From the Department of Pathology, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06536
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ABSTRACT |
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The role of coagulation factor X as a ligand for
CD11b/CD18 (Mac-1, M
2) in leukocyte
adhesion was investigated. A factor X peptide,
(G)L238YQAKRFKV246(G), blocked ligand binding
to CD11b/CD18 and prevented monocyte procoagulant activity. This
peptide also inhibited monocytic THP-1 cell adhesion to tumor necrosis
factor
-stimulated endothelium and blocked neutrophil migration
through tumor necrosis factor
-activated endothelial cell
monolayers. In contrast, other factor X-derived peptides were
ineffective. Radiolabeled peptide (G)LYQAKRFKV(G) bound specifically
and saturably to isolated recombinant CD11b I domain. Functionally, the
factor X sequence (G)LYQAKRFKV(G) dose-dependently
inhibited THP-1 cell attachment to intercellular adhesion molecule 1 (ICAM-1) transfectants (IC50 = ~50 µg/ml), indistinguishably from anti-CD18 monoclonal antibodies 60.3 and IB4. In
contrast, peptide (G)LYQAKRFKV(G) failed to reduce binding of
125I-fibrinogen to immobilized CD11b I domain, which
was abolished by the fibrinogen-derived peptide
KYG190WTVFQKRLDGSV202. By Lineweaver-Burke
analysis, peptide (G)LYQAKRFKV(G) inhibited factor X binding to
CD11b/CD18 in a noncompetitive fashion, and intact factor X did not
reduce monocyte-endothelial cell interaction. These data suggest that
the factor X sequence (G)LYQAKRFKV(G) defines an ICAM-1-binding site on
CD11b I domain physically distinct from and nonoverlapping with the
fibrinogen interacting region(s). Engagement of this site induces a
conformational change in the holoreceptor, which disrupts a distant
factor X-binding site required for monocyte procoagulant activity.
These observations demonstrate a dual regulatory role of CD11b I domain
in ligand binding and provide a molecular basis for the recently
reported anti-inflammatory properties of factor X homologous sequences
in vivo.
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INTRODUCTION |
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Leukocyte 2 integrins CD11a-d/CD18 maintain
adherence mechanisms (1), transmembrane signaling (2, 3), and
intercellular communication (4, 5) in disparate inflammatory responses (5, 6). Specifically, leukocyte adherence mediated by CD11b/CD18 (Mac-1,
M
2) (1) depends on the
recognition of unrelated ligands, including counter-receptors,
i.e. intercellular adhesion molecules (ICAMs)1 (7, 8), and soluble
proteins, like complement C3bi (1), fibrinogen and coagulation factor X
(9). Blocking monoclonal antibodies (mAbs) (10), direct binding studies
to the isolated recombinant protein (11), and mutagenesis experiments
(12-14) converged to identify the ~200-amino acid inserted "I"
domain in the
subunit as a recognition site for ICAM-1, fibrinogen, and C3bi. However, how these I domain-binding sites for unrelated ligands are structurally and functionally organized has not been completely elucidated. Although differential inhibition by
epitope-mapped mAbs proposed the existence of functionally independent
I domain subregions (10), receptor mutagenesis studies suggested that the binding of fibrinogen, C3bi, and ICAM-1 can be mediated, at least
in part, by functionally overlapping regions (12, 14, 15).
Differently from other 2 integrin ligands, binding of
factor X to CD11b/CD18 (16) is not mediated by the I domain (11), is
inhibited by three spatially distant sequences in the ligand (17), and
mediates monocyte procoagulant activity (9). In this study, we sought
to reinvestigate the association of factor X with CD11b/CD18 and its
potential impact on
2 integrin-dependent leukocyte adhesion. Using a small factor X peptide
(G)L238YQAKRFKV246(G) (17), we have delineated
a discrete region on CD11b I domain that mediates the interaction with
ICAM-1 and indirectly modulates a distant binding site for
factor X.
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MATERIALS AND METHODS |
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Cells and Cell Cultures--
Polymorphonuclear leukocytes (PMN)
were isolated from acid-citrate-dextrose anticoagulated blood drawn
from informed normal volunteers by Ficoll-Hypaque gradient density
centrifugation and dextran sedimentation (18). Human umbilical vein
endothelial cells (HUVECs) were prepared by collagenase treatment and
used between passages 2 and 4. The monocytic cell line THP-1 and
CD11b/CD18 T leukemia cell line MLT (ATCC, Rockville, MD)
were maintained in culture according to the manufacturer's
recommendations. THP-1 cell expression of CD11b/CD18 and recognition of
fibrinogen and factor X by these cells have been reported (9).
Synthetic Peptides and mAbs-- The experimental procedures for the isolation and purification of human plasma fibrinogen have been described previously (9). Aliquots of fibrinogen or factor X were 125I-labeled by the IODO-GEN method to a specific activity of 0.5 and 1-2 µCi/µg of protein, respectively. Integrity and specific activity of 125I-factor X were as described (16). The factor X-derived peptides G366YDTKQED373(G) (peptide 1), I422DRSMKTRG430 (peptide 2), and (G)L238YQAKRFKV246(G) (peptide 16, residues in parentheses added to the natural sequence) were previously characterized for their ability to inhibit 125I-factor X binding to CD11b/CD18 on chemoattractant-stimulated monocytes or PMN (17). All peptides were synthesized by the W. M. Keck protein chemistry facility at Yale University, purified by high pressure liquid chromatography, and characterized for amino acid composition by mass spectrometry. The factor X peptide (G)L73EGFEGKN80(G) (peptide 25) was used as a control. 2 mg of peptide 16 were iodinated with 5 mCi of Na125I by the IODO-GEN method for 45 min at 4 °C, followed by separation of free from peptide-bound radioactivity by gel filtration over a Bio-Gel P-2 column (Bio-Rad) pre-equilibrated with PBS, pH 7.2. Anti-CD18 mAbs 60.3 and IB4 were obtained from ATCC. Nonbinding mAb 14E11 was used as a negative control.
Expression of Recombinant Proteins-- The establishment and characterization of Chinese hamster ovary cells stably transfected with the ICAM-1 cDNA has been reported (19). The construction and expression of recombinant CD11b I domain (residues Gly111-Ala318) and its interaction with fibrinogen and ICAM-1 have been described (11).
Procoagulant Activity and Binding Reactions--
The effect of
the various factor X peptides on monocyte procoagulant activity was
determined by a one-stage sensitive clotting assay using a factor VII-
and factor X-deficient plasma (Sigma) as described (17). 50-µl
aliquots of purified CD11b I domain at 5 µg/ml in Tris-buffered
saline, pH 8.0 were immobilized onto 96-well U-bottom Falcon 3911 flexible assay plates (Becton Dickinson, Oxnard, CA) for 18 h at
4 °C. Duplicate wells were washed in Tris-buffered saline, pH 8.0, post-coated with 10 mg/ml BSA (Sigma) for 90 min at 37 °C, rinsed,
and mixed with increasing concentrations of 125I-peptide 16 (8-62.5 µg/ml) in PBS, pH 7.2, 2.5 mM CaCl2
and 0.1% BSA for 30 min at 22 °C. At the end of the incubation,
wells were washed twice in PBS, pH 7.2, amputated, and counted in a counter. Specific binding was calculated in the presence of a 50-fold
molar excess of unlabeled peptide 16 or control peptide 25 added
at the start of the incubation. Alternatively, increasing
concentrations of 125I-fibrinogen (10-150 µg/ml) were
incubated with CD11b I domain-coated wells in the presence of 1 mM CaCl2 and 0.1% BSA for 30 min at 22 °C,
before determination of specific binding. For all experiments, nonspecific binding was measured in the presence of a 50-fold molar
excess of unlabeled fibrinogen and subtracted from the total to
calculate net specific binding. In peptide inhibition experiments, CD11b I domain-coated plates were preincubated with increasing concentrations (0.1-1000 µg/ml) of fibrinogen-derived P1
peptide KYG190WTVFQKRLDGSV202 (18) or factor X
control peptide 25 or peptide 16 for 30 min at 22 °C. Wells were
mixed with 125I-fibrinogen (25 µg/ml) in the presence of
1 mM CaCl2 and 0.1% BSA for 30 min at
22 °C, before determination of specific binding. In another series
of experiments, serum-free THP-1 cells (1.5 × 107/ml)
were stimulated with 10 µM fMLP in the presence of
increasing concentrations of 125I-factor X and 2.5 mM CaCl2 and a single concentration of control peptide or the three factor X inhibitory peptides (17). After a 20-min
incubation at 22 °C, free and cell surface-bound radioactivity was
separated by centrifugation through a mixture of silicone oil
(Dow-Corning, New Bedford, MA) at 14,000 × g for 5 min
at 22 °C, and specific binding was determined as described above. Binding data were analyzed by the Lineweaver-Burke plot as
described (16).
Leukocyte-Endothelium Interaction--
Serum-free suspensions of
THP-1 or MLT cells (5 × 106/ml) were labeled with 0.5 mCi of 51Cr (Na2CrO4, du
Pont de Nemours, Wilmington, DE) for 2 h at 37 °C with a final
incorporation of 0.7-2.1 cpm/cell, washed in PBS, pH 7.4, and
suspended in serum-free RPMI 1640. MLT or 10 µM
fMLP-stimulated THP-1 cells were preincubated with increasing
concentrations (60-1000 µg/ml) of factor X peptide 1, 2, or 16 or
control peptide 25. After a 30-min incubation at 22 °C,
51Cr-labeled cells (1 × 105) were added
to resting or cytokine-activated (100 units/ml TNF for 4 h at
37 °C) HUVEC monolayers for 1 h at 22 °C in the presence of
1 mM CaCl2. After washes, attached cells were
solubilized in 15% SDS, and radioactivity associated under the various
conditions was determined in a scintillation
-counter. The number of
attached cells was calculated by dividing the cpm harvested by the
cpm/cell. In other experiments, 51Cr-labeled THP-1 cells
were preincubated with increasing concentrations of factor X peptides
(10-1000 µg/ml) or 25 µg/ml anti CD18 mAbs 60.3 and IB4 or control
mAb 14E11. After a 30-min incubation at 4 °C, control or
anti-CD18-treated cells were added to confluent monolayers of ICAM-1
transfectants or wild-type Chinese hamster ovary cells, with
determination of cell adhesion after a 1-h incubation at 22 °C. For
transendothelial cell migration, HUVEC were grown to confluency onto
gelatin-coated porous transwell membranes (8-µm diameter; Costar,
Cambridge, MA) and stimulated with 100 units/ml TNF
for 4 h at
37 °C prior to the experiment. PMN (1 × 107/ml)
were preincubated with 500 µg/ml factor X peptide 16 or control peptide 25 for 30 min at 22 °C, stimulated with 10 µM
fMLP in the presence of 1 mM CaCl2, and added
(1 × 106) to HUVEC for increasing time intervals
(30-120 min) at 37 °C. At the end of each incubation, migrated PMN
were recovered from the bottom well, washed, stained with 0.2% trypan
blue, and counted microscopically. Before each experiment, the
integrity of the endothelial cell monolayer was confirmed by methyl
green staining and fluorescence microscopy.
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RESULTS |
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Anticoagulant Properties of Factor X Interacting Sequences-- Previous data suggested that binding of factor X to CD11b/CD18-expressing monocytes is mediated by three spatially distant sites in the ligand catalytic domain (17). Preincubation of THP-1 cells with a saturating concentration of each of the three implicated sequences inhibited factor X-dependent monocyte procoagulant activity by 60-70% in a time-dependent reaction (not shown) and in agreement with previous observations (17, 20).
Effect of Factor X Peptides on Leukocyte-Endothelium
Interaction--
Preincubation of monocytic THP-1 cells with an
inhibitory concentration (500 µM) (17) of factor X
peptide 16 (G)L238YQAKRFKV246(G) significantly
inhibited monocyte attachment to TNF-activated HUVEC from 67 ± 7.5 to 31 ± 6.6% (Fig.
1A). In contrast, the factor X
peptides G366YDTKQED373(G) (peptide 1) or
I422DRSMKTRG430 (peptide 2), which have been
demonstrated (17) to block ligand binding to CD11b/CD18 or control
factor X peptide (G)L73EGFEGKN80(G) (peptide
25), did not reduce THP-1 cell adhesion to HUVEC (Fig. 1A).
At variance with the effect on monocytic cell adhesion, none of the
factor X peptides, including peptide 16, decreased attachment of
CD11b/CD18
T cells (MLT) to TNF
-activated endothelium
under the same experimental conditions (Fig. 1B). Inhibition
of monocytic cell adhesion to HUVEC by the factor X peptide
(G)LYQAKRFKV(G) was specific and dose-dependent (Fig.
2A), whereas control peptide
25 was ineffective (Fig. 2A). In other experiments, the
factor X peptide 16 specifically inhibited fMLP-stimulated PMN
transendothelial cell migration at all time intervals tested (Fig.
2B).
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Effect of Factor X Peptide 16 on CD11b I Domain Ligand
Recognition--
The possibility that the factor X peptide 16 may
affect intercellular adhesion mediated by CD11b/CD18-ICAM-1 interaction
was first investigated. Increasing concentrations of peptide 16 blocked the adhesion of fMLP-stimulated THP-1 cells to monolayers of ICAM-1 transfectants in a concentration-dependent manner, with
an IC50 value of ~50 µg/ml (Fig.
3A). A comparable degree of
inhibition was observed in control experiments after THP-1 cell
preincubation with anti-CD18 mAbs IB4 and 60.3 but not with control mAb
14E11 (Fig. 3B). In direct binding studies,
125I-peptide 16 bound to immobilized recombinant CD11b I
domain in a specific and concentration-dependent reaction
was competitively inhibited by >90% by a 50-fold molar excess of
unlabeled peptide 16 but not by control peptide 25 (Fig.
4). The effect of factor X sequence
(G)LYQAKRFKV(G) on CD11b I domain recognition of fibrinogen was also
investigated (11). Binding of 125I-fibrinogen to isolated
recombinant I domain was specific and saturable, in agreement with
previous observations (11). Under these experimental conditions,
increasing inhibitory concentrations of peptide 16 or control factor X
peptide 25 failed to reduce 125I-fibrinogen binding to
isolated CD11b I domain (Fig. 5). In
contrast, increasing concentrations of fibrinogen-derived P1 peptide
(Gly190-Val202) inhibited binding of
125I-fibrinogen to isolated I domain in a
dose-dependent manner with an IC50 value of
~4-6 µM (Fig. 5), in agreement with previous observations (18).
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Molecular Dissection of Factor X Binding to
CD11b/CD18--
The reciprocal relationship between the three factor X
peptides inhibiting ligand binding to CD11b/CD18 (17) was investigated. All three factor X peptides (G)LYQAKRFKV(G) (peptide 16), GYDTKQED(G) (peptide 1), and (G)DRSMKTRG (peptide 2) inhibited binding of 125I-factor X to fMLP-stimulated THP-1 cells equally well
in a concentration-dependent manner (not shown) and in
agreement with previous observations (17). However, analysis of binding
data by the Lineweaver-Burke plot revealed that only the factor X
sequence (G)DRSMKTRG inhibited ligand binding in a genuine competitive
manner, as judged by the nearly identical y intercept
values determined in the presence (y = 4.8) or in the
absence (y = 4.32) of antagonist (Fig.
6). In contrast, the factor X sequences
GYDTKQEDG and (G)LYQAKRFKV(G) inhibited factor X binding to CD11b/CD18
in noncompetitive manner, with 4-5-fold differences in the predicted
y intercept values in control curves (y = 4.32) or in the presence (y = 21.7; Ref. 18) of these
inhibitors (Fig. 6).
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DISCUSSION |
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In this study, we have shown that a factor X sequence, (G)L238YQAKRFKV246(G), exerted a dual anticoagulant and anti-inflammatory effect by blocking monocyte procoagulant activity and inhibiting leukocyte-endothelium interaction. This pathway involved peptide engagement of a discrete ICAM-1-binding site on CD11b I domain with indirect disruption of a distant factor X-binding region in the holoreceptor.
Through its promiscuous ligand repertoire (2), CD11b/CD18 mediates a variety of cell adhesive interactions and functions as a procoagulant receptor via its high affinity recognition of factor X (9). The possibility that this interaction may also participate in leukocyte adhesion mechanisms has been postulated. Previously, interruption of CD11b/CD18-dependent coagulation prevented thrombin-dependent chemotaxis (21) and monocyte adhesion (22, 23) to virally infected endothelium (17). Moreover, an adhesive determinant of Bordetella pertussis, filamentous hemagglutinin, was recently shown to contain sequences homologous to the three factor X regions inhibiting ligand binding to CD11b/CD18 (20). Synthetic peptidyl mimicry of these regions produced a potent anti-adhesive and anti-inflammatory effect by blocking leukocyte-endothelium interaction in vitro and reducing neutrophil accumulation in the colony-stimulating factor in an in vivo model of bacterial meningitis (20). However, the molecular basis of this anti-inflammatory pathway was not elucidated.
Here, at variance with the anti-adhesive functions of all three factor
X homologous sequences (20), a single factor X peptide (G)L238YQAKRFKV246(G) (peptide 16) acted as a
potent antagonist of leukocyte-endothelium interaction and
transendothelial cell migration. Three independent lines of evidence
indicate that this anti-adhesive effect resulted from peptide
engagement of an ICAM-1-binding site on CD11b I domain (10-13). First,
this peptide had no effect on CD11b/CD18 T cell (MLT)
adhesion to endothelium. Second, binding of monocyte THP-1 cells to
monolayers of ICAM-1 transfectants was equally well inhibited by factor
X peptide 16 or by anti-CD18 mAbs. Thirdly, direct radiolabeled peptide
binding studies demonstrated that this sequence physically interacted
with CD11b I domain. The use of a small synthetic peptide, like peptide
16, to probe the CD11b/CD18 ligand repertoire allowed delineating a
more precise functional map of CD11b I domain. Consistent with the
differential pattern of inhibition obtained with I domain
epitope-mapped mAbs (10), the ability of this peptide to disrupt an
ICAM-1-binding site without affecting the recognition of fibrinogen
postulates the existence of physically separate subdomains mediating a
nonoverlapping recognition of these two ligands. This is at variance
with previous studies of site-directed mutagenesis, in which single
amino acid changes in the CD11b metal ion-dependent
adhesion site (15) suppressed the receptor recognition of fibrinogen,
ICAM-1, and C3bi (12, 14, 15). This may be explained by the ability of
the metal ion-dependent adhesion site to transduce
conformational effects in spatially distant ligand-binding sites (see
below) without affecting subunit assembly or receptor surface
expression.
A potential model of how perturbation of a discrete I domain region,
i.e. ICAM-1 site, could indirectly modulate
ligand binding to the holoreceptor was provided by dissecting the
factor X interaction with CD11b/CD1. Although three spatially distant
sites in factor X indistinguishably blocked ligand binding and monocyte
procoagulant activity (17), only one of these regions,
I422DRSMKTRG430, was a genuine competitive
inhibitor of this interaction. The other two factor X peptides,
including peptide 16, were noncompetitive antagonists, thus potentially
disrupting a secondary docking during receptor-ligand interaction. This
suggests that binding of the anti-adhesive sequence (G)LYQAKRFKV(G) to
an ICAM-1 recognition site on CD11b I domain may induce a
conformational change in the holoreceptor with disruption of a distant
factor X-binding pocket. Consistent with this hypothesis,
125I-factor X did not specifically associate with
recombinant isolated CD11b I domain (11), and in the present studies,
the intact macromolecule did not affect leukocyte-endothelium
interaction.2 Altogether,
these data suggest a more dynamic role of 2 integrin I
domains, not only in providing physical interacting site(s) for ligand
recognition, but also in regulating the state of receptor activation
(24) and ligand binding affinity (25).
In summary, these data underscore the role of the CD11b I domain in multiple regulation of ligand binding and identify a factor X sequence that, similarly to its prokaryotic homologue (20), displayed potent anticoagulant and anti-inflammatory properties. The availability of a small and high affinity probe like peptide 16 should facilitate the identification of the complementary ICAM-1-binding site on CD11b and define its role in leukocyte traffic and recirculation (4, 5).
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL-43773 and HL-54131. This work was done during the tenure of an Established Investigatorship Award to Dr. Altieri.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.
To whom correspondence should be addressed: Yale University School
of Medicine, BCMM 436B, 295 Congress Ave., New Haven, CT 06536. Tel.:
203-737-2869; Fax: 203-737-2290; E-mail: Dario.Altieri{at}yale.edu.
1 The abbreviations used are: ICAM, intercellular adhesion molecule; mAb, monoclonal antibody; PMN, polymorphonuclear leukocytes; TNF, tumor necrosis factor; HUVEC, human umbilical vein endothelial cell; PBS, phosphate-buffered saline; BSA, bovine serum albumin; fMLP, formylmethionylleucylphenylalanine.
2 M. Mesri, J. Plescia, and D. C. Altieri, unpublished observations.
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REFERENCES |
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