HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Cleaved high molecular weight kininogen binds directly to the integrin
CD11b/CD18 (Mac-1) and blocks adhesion to fibrinogen and ICAM-1
Nijing Sheng, Michael B. Fairbanks, Robert L. Heinrikson, Gabriela Canziani, Irwin M. Chaiken, David M. Mosser,
Hong Zhang, and Robert W. Colman
High molecular weight kininogen (HK)
and its cleaved form (HKa) have been
shown to bind to neutrophils. Based on
studies using monoclonal antibodies
(mAbs), we postulated that CD11b/CD18
(Mac-1) might be the receptor on the
neutrophils for binding to HK/HKa. However, the direct interaction of HK/HKa and
Mac-1 had not been demonstrated. We
therefore transfected HEK 293 cells with
human Mac-1. Cell binding assays using
fluorescein isothiocyanate-labeled HKa
showed increased binding to the Mac-1
transfected cells compared with the control transfected cells. The binding was
specific because unlabeled HKa, Mac-1–
specific antibody, and fibrinogen can
inhibit the binding of biotin-HKa to Mac-1
transfected cells. HKa bound to Mac-1
transfected cells (20 000 molecules/cell)
with a Kd 5 62 nmol/L. To demonstrate
directly the formation of a complex between HKa and Mac-1, we examined the
interaction of HKa and purified Mac-1 in a
cell-free system using an IAsys resonant
mirror optical biosensor. The association
and dissociation rate constants (kon and
koff, respectively) were determined, and
they yielded a dissociation constant (Kd)
of 3.231029 mol/L. The functional significance of direct interaction of HKa to
Mac-1 was investigated by examining the
effect of HKa on cellular adhesion to
fibrinogen and intercellular adhesion molecule-1 (ICAM-1), molecules abundant in
the injured vessel wall. HKa blocked the
adhesion of Mac-1 transfected cells to
fibrinogen and ICAM-1 in a dose-dependent manner. Thus, HKa may interrupt
Mac-1–mediated cell–extracellular matrix
and cell–cell adhesive interactions and
may therefore influence the recruitment
of circulating neutrophils/monocytes to
sites of vessel injury. (Blood. 2000;95:
3788-3795)
r 2000 by The American Society of Hematology
Introduction
High molecular weight kininogen (HK) is an abundant plasma
protein (670 nmol/L) coded for by a gene with 10 exons containing
6 domains (Mr 5 20 kd).1 HK, together with 2 other plasma
proteins, prekallikrein and factor XII, are called the contact system
in the blood coagulation cascade because they have been found to
require contact with artificial, negatively charged surfaces for
activation of the zymogens in vitro. Besides its essential role in the
activation of coagulation when blood contacts foreign surfaces,
such as in cardiopulmonary bypass,2 HK is a multifunctional
protein. HK can interact with blood and vascular cells including
platelets,3 neutrophils,4 monocytes, and endothelial cells.5,6 On
each cell type, HK serves a discrete biologic function. It prevents
the activation of platelets by inhibiting the calcium-activated
cysteine protease calpain,7 and it prevents the binding of thrombin.8
Platelets have been shown to contain HK, which can be expressed
on the exposed membrane surface of activated platelets.9-11 HK that
circulates with plasma prekallikrein in a binary complex serves as
an acquired receptor for kallikrein on the surfaces of neutrophils,12
allowing kallikrein to stimulate neutrophil aggregation13 and
degranulation.14 Endothelial cell-bound HK is a substrate for
plasma kallikrein, which cleaves HK to a 2-chain disulfide-linked
molecule, HKa, and releases the nonapeptide bradykinin. It has
been shown previously that HK/HKa can compete with an adhesive
protein, fibrinogen, for binding to neutrophils,12 and that, as a
consequence, it inhibits the adhesion of neutrophils to fibrinogencoated surfaces under radial flow conditions.15 It has also been
shown that HK/HKa binds specifically, saturably, and reversibly to
neutrophils in the presence of Zn21.4 However, the cell surface
receptor for HK/HKa binding to neutrophils is unclear. Although it
is suggested to be the integrin Mac-1 (CD11b/CD18) based on the
results of antibody blocking studies,12 a direct interaction between
HK/HKa and Mac-1 integrin has not been demonstrated. Moreover,
the occurrence of another receptor on neutrophils for HKa, the
urokinase receptor, made it important to examine further the
hypothesis that HK/HKa directly binds to Mac-1.
Integrins are a family of adhesion molecules serving functions
involved in cell–cell and cell–extracellular matrix interactions.
Mac-1 (CD11b/CD18), LFA-1 (CD11a/CD18), and p150,95
(CD11c/CD18), are leukocyte integrins. Mac-1, also known as
complement receptor type 3 (CR3), is a heterodimeric receptor that
is primarily expressed on monocytes, macrophages, neutrophils,
and natural killer cells. The function of Mac-1 was initially
described as the ability to bind iC3b (a cleaved form of C3b), and
therefore to mediate the phagocytosis and lysis of iC3b-coated
erythrocytes,16 and to contribute to elevated natural killer cell
activity against iC3b-coated target cells.17 In addition to the
From the Sol Sherry Thrombosis Research Center and the Department of
Microbiology and Immunology, Temple University School of Medicine, and the
Department of Medicine, University of Pennsylvania School of Medicine,
Philadelphia, PA; and Pharmacia & Upjohn Inc, Kalamazoo, MI.
Reprints: Nijing Sheng, Sol Sherry Thrombosis Research Center, Temple
University School of Medicine, 3400 North Broad Street, Philadelphia, PA
19140; e-mail: [email protected].
Submitted July 21, 1999; accepted February 7, 2000.
Supported by National Institutes of Heart Lung and Blood training grant
5T32HL07777 (R.W.C.) and project 2 of PO1 HL 56914 (R.W.C.) and by
American Heart Association grant 9730241N (N.S.).
3788
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.
r 2000 by The American Society of Hematology
BLOOD, 15 JUNE 2000 • VOLUME 95, NUMBER 12
BLOOD, 15 JUNE 2000 • VOLUME 95, NUMBER 12
function of Mac-1 in the immune defense system through binding
to its ligand, iC3b, Mac-1 mediates a number of cell–cell and
cell–extracellular matrix interactions in which iC3b is not involved,
such as neutrophil aggregation18 and chemotaxis,19 and neutrophil
adhesion to human umbilical vein endothelial cells.20 These
observations suggested that Mac-1 is a multifunctional receptor.
Through its ability to recognize multiple and unrelated ligands,
including iC3b,16,17 fibrinogen,21 factor X,22 and the counter–
receptor intercellular adhesion molecule-1 (ICAM-1),23 Mac-1
plays critical roles in cell adhesion, migration, and invasion, which
are central to inflammation, immune responses, vascular biology,
hemostasis, and thrombosis. The binding of Mac-1 to fibrinogen/
fibrin results in the adhesion of neutrophils/monocytes to the sites
of fibrin deposition, and the binding of Mac-1 to ICAM-1 causes
the adhesion of neutrophils/monocytes to the endothelium. After
binding to the zymogen of factor X, Mac-1 coordinates the
activation of factor X independent of tissue factor and factor VII;
this is followed by rapid fibrin formation.24 Therefore, the ability to
interfere with Mac-1–mediated leukocyte adhesion functions offers
many opportunities for therapeutic intervention in diseases as
diverse as thrombosis, inflammation, and cancer.
In this report, we show, for the first time, that cleaved high
molecular weight kininogen, HKa, binds directly to Mac-1 both on
cells and in a purified system, and we demonstrate that HKa
inhibits Mac-1–mediated adhesion to fibrinogen and ICAM-1. This
study may provide additional information for drug design in
anti-adhesion therapy.
Materials and methods
Materials
Cleaved human high molecular weight kininogen, HKa, and human
fibrinogen were purchased from Enzyme Research Laboratories (South
Bend, IN). Recombinant human ICAM-1 was obtained from R&D Systems
(Minneapolis, MN). Phosphate-buffered saline (PBS) and Hanks’ balanced
salt solution without calcium chloride, magnesium chloride, magnesium
sulfate, and sodium bicarbonate were purchased from GIBCO BRL (Grand
Island, NY). Reactive biotin analog, NHS-LC-biotin, was obtained from
Pierce Chemical (Rockford, IL). Fluorescein isothiocyanate (FITC) on
celite for fluorescent labeling of proteins and FITC-conjugated avidin were
purchased from Sigma (St Louis, MO). 5-Chloromethylfluorescein diacetate for cell labeling was purchased from Molecular Probes. FITCconjugated goat antimouse IgG (heavy and light chains) was purchased
from Jackson ImmunoResearch Laboratories (West Grove, PA). A monoclonal antibody, integrin aMm (44), specific for the integrin aµ subunit, was
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). 2LPM19c, a
monoclonal antibody against the aµ subunit of Mac-1, was purchased from
DAKO (Carpenteria, CA). A monoclonal anti-pan cytokeratin mixture
(clone C-11) and a monoclonal anti-cytokeratin 4.62 were all purchased
from Sigma.
INTERACTION OF KININOGEN WITH MAC-1
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Flow cytometry and cell sorting
Cells were incubated with Mac-1–specific antibody LM2/1 (ascitic fluid,
mouse IgG1, antihuman CD11b)25 for 45 minutes on ice. After they were
washed 3 times with cold Hanks’ balanced salt solution buffer with 1%
bovine serum albumin (BSA), the cells were incubated with FITCconjugated goat antimouse IgG for 30 minutes on ice. After they were
washed 4 times, the cells were fixed with 1% paraformaldehyde in PBS
overnight and then analyzed on a flow cytometer. For cell sorting, the cells
were directly analyzed and sorted on a flow cytometer without fixation with
paraformaldehyde.
Labeling of kininogen
For binding of cleaved human high molecular weight kininogen (HKa) to
HEK 293 cells, HKa was labeled with FITC or biotin. Briefly, a total
reaction volume of 1 mL containing 1 mg HKa, 1 mg celite–FITC (Sigma)
or 1 mg NHS-LC–biotin was adjusted to pH 8.0 with 5% sodium
bicarbonate solution. The reaction mixture was incubated for 20 minutes at
room temperature with intermittent vortexing for labeling with FITC or for
2 hours on ice for labeling with biotin. The labeled HKa (FITC–HKa or
biotin–HKa) was purified by Sephadex G-25 spin column (Bio-Rad
Laboratories, Hercules, CA) and eluted with PBS. The labeled protein
retained more than 95% of its procoagulant activity.
Binding assays
Unstimulated HEK 293 cells (43106/mL), transfected with Mac-1 or
control, were washed with binding buffer (10 mmol/L HEPES, pH 7.2, 137
mmol/L NaCl, 4 mmol/L KCl, 50 µmol/L ZnCl2, and 0.1% gelatin) and
incubated with FITC–HKa at indicated concentrations for 30 minutes at
room temperature. Nonspecific binding was measured by including 10
mmol/L EDTA in the binding assay. After they were washed, the cells were
fixed with 1% paraformaldehyde in PBS and then analyzed on an Epics
Elite flow cytometer (Coulter Diagnostics, Hialeah, FL). Alternatively, the
binding reactions were carried out in wells of a filtration 96-well plate
containing polyvinylidene difluoride (PVDF) membrane (pore size, 1.2 µm;
Millipore, Bedford, MA). After filtration, the fluorescence of cell bound
FITC–HKa was measured on a Cytofluor 2350 fluorescence plate reader
(Millipore) with a 485-nm excitation filter and a 530-nm emission filter.
Inhibition of biotin–HKa binding to Mac-1–HEK 293 cells
Mac-1–HEK 293 cells were eluted from a confluent monolayer culture dish
with PBS containing 5 mmol/L EDTA and washed with binding buffer (10
mmol/L HEPES, pH 7.2, 137 mmol/L NaCl, 4 mmol/L KCl, 50 µmol/L
ZnCl2, 1 mmol/L MgCl2, 1 mmol/L CaCl2, and 0.1% gelatin). Cells
(43106/mL) in the same buffer were incubated with biotin–HKa (10
nmol/L) for 45 minutes at room temperature in the absence or presence of
unlabeled HKa, antibodies, or fibrinogen at indicated concentrations. After
the addition of avidin–FITC and continued incubation for 10 minutes, 3
aliquots from each reaction were transferred to the wells of a filtration
96-well plate containing PVDF membrane (pore size, 1.2 µm; Millipore).
The wells were pre-wet with binding buffer for 2 hours. After filtration, the
fluorescence of cell bound biotin–HKa was measured on a Cytofluor 2350
system (Millipore). The relative amount of biotin–HKa bound to the cells in
the presence of inhibitors was determined by comparison with biotin–HKa alone.
Cell culture and transfection
To generate human embryonic kidney 293 cells (HEK 293) stably
expressing Mac-1 (Mac-1–HEK 293), wild-type CD11b and CD18 subunits
of Mac-1 in pCDM8 and plasmid pRSVneo were co-transfected into HEK
293 cells using lipofectin reagent according to the manufacturer’s instructions (GIBCO BRL). Plasmid pRSVneo allowed for the selection of stable
transfectants in medium supplemented with G418 sulfate. A single G418resistant clone was selected, cloned, and screened for cell surface Mac-1
expression by flow cytometry. Stable transfected cell lines (Mac-1–HEK
293) were maintained in complete Dulbecco’s modified eagle’s medium
containing 500 µg/mL G418. For control transfection, plasmid pRSVneo
alone was transfected into HEK 293 cells using lipofectin reagent according
to the manufacturer’s instructions.
Adhesion assays
HEK 293 cells transfected with Mac-1 used for adhesion assays were
labeled with 5-chloromethylfluorescein diacetate (CMFDA). Briefly, confluent cells were eluted from dishes and resuspended in serum-free medium
containing CMFDA (10 µmol/L) and incubated at 37°C for 30 minutes.
Free probes were removed by washing with buffer used for adhesion assays
(10 mmol/L HEPES, pH 7.2, 137 mmol/L NaCl, 4 mmol/L KCl, 50 µmol/L
ZnCl2, 1 mmol/L MgCl2, 1 mmol/L CaCl2, and 0.1% gelatin). Ligands,
fibrinogen (50 µg/mL), ICAM-1 (10 µg/mL), or fibronectin (15 µg/mL)
were immobilized on 96-well Immulon II microtiter plates (Dynatech
Laboratories, Chantilly, VA) at room temperature for 2 hours. The wells
were then blocked with gelatin. Labeled cells (3-4 3 105/well) were added
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SHENG et al
to wells and incubated in the presence or absence of indicated HKa for 60
minutes. Unbound cells were removed by washing with adhesion buffer 3
times. Adherent cells were lysed in 0.1 N NaOH, 0.2% sodium dodecyl
sulfate (SDS), and 0.5% Triton X-100. The plates were read on a Cytofluor
2350 system (Millipore).
Purification of Mac-1 from human neutrophils
Mac-1 was purified from human neutrophils with slight modifications to the
immunoaffinity purification procedure previously described.26 Briefly,
human granulocytes were obtained from normal, healthy volunteers by
leukopheresis using a Fenwal CS3000 blood cell separator (Baxter Healthcare, Deerfield, IL) followed by dextran density gradient. The enriched
neutrophil product was then lysed with 0.05 mol/L Tris, pH 8.0, 2 mmol/L
MgCl2, 1.0% Triton X-100, 0.15 mol/L NaCl, 5 mmol/L DFP, and 0.2
TIU/mL aprotinin for 1 hour at 4°C by gentle stirring before the resultant
lysate was centrifuged at 50 000g to remove insoluble material. The
clarified lysate was loaded by batch onto LM2/1 immunoaffinity resin (10
mL CNBr-activated Sepharose (Pharmacia Biotech, Piscataway, NJ) coupled
with 5 mg LM2/1/mL resin) and incubated 16 hours at 4°C with gentle
rotation. The resin was then loaded into a column (1.0313 cm), and
sequentially washed with 20 column volumes (CV) lysis buffer, 10 CV lysis
buffer containing 1.0 mol/L NaCl, and 10 CV lysis buffer substituting 1.0%
n-octyl glucoside for the Triton X-100. Mac-1 was eluted with 5 CV 0.1
mol/L sodium acetate, pH 4.0, 2 mmol/L MgCl2, 0.15 mol/L NaCl, and
1.0% n-octyl glucoside by collecting 1.0-mL fractions in polypropylene
microcentrifuge tubes containing neutralizing buffer (10% by volume of 2
mol/L Tris, pH 9.0). Aliquots of the eluate fractions were immediately
assessed by SDS–polyacrylamide gel electrophoresis (4%-15% gradient
gels), and they showed 2 bands of equal density (MWt, 160 000 and
95,000). Peak fractions were pooled and underwent dialysis against 10
mmol/L HEPES, 0.137 mol/L NaCl, 4 mmol/L KCl, 11 mmol/L D-glucose,
1 mmol/L MgCl2, 1 mmol/L CaCl2, 0.005% Tween-20 for 16 hours at 4°C.
The final protein preparation was sectioned into aliquots and stored at 280°C
until used. Protein concentration was determined using amino acid analysis.
Real-time bimolecular interaction assay
Analysis of Hka–Mac-1 interaction was carried out with purified human
Mac-1 on an IAsys resonant mirror optical biosensor (Affinity Sensors,
Cambridge, UK). In this assay, biotinylated HKa was immobilized on the
sensor chip surface by using an IAsys biotin cuvette (FCB-0401; Affinity
Sensors) and the accompanying protocol. Briefly, the biotin surface of the
cuvette was washed with PBS/Tween-20 buffer, and streptavidin was added
at a concentration of 10 µg/mL. After streptavidin was captured on the
surface, Mac-1 was added to test whether there was any nonspecific binding
of Mac-1 to streptavidin. No binding of Mac-1 to streptavidin was observed.
Biotinylated HKa was then added to the streptavidin-captured surface, and the
amount of immobilized HKa was equivalent to a signal of 400 arc seconds.
The maximum signal (Rmax) expected on Mac-1 binding for the saturation
of 90% of HKa binding sites was calculated from the molecular weight ratio
of analyte (Mac-1)/captured ligand (HKa) and a stoichiometry of 1. The
calculated Rmax is 800 arc seconds. Because of the random biotinylation and
steric hindrance factors that may reduce the stoichiometry of 1 to a fraction,
a lower effective Rmax is expected (see ‘‘Results’’). This cuvette immobilized with HKa was stored refrigerated in binding buffer and used in
subsequent experiments for examining the interaction of Mac-1 and HKa.
Purified Mac-1 was added at the indicated concentration, and the association was monitored. The binding buffer used was: 10 mmol/L HEPES, pH
7.2, 137 mmol/L NaCl, 4 mmol/L KCl, 50 µmol/L ZnCl2, and 0.05%
Tween-20. In the dissociation phase, Mac-1 solution was replaced with the
binding buffer, and the dissociation of the binding was monitored. The HKa
surface was regenerated by the addition of 10 mmol/L EDTA and 0.5 mol/L
NaCl and was followed by washing with binding buffer.
The kinetics of binding was evaluated from linear transformations of the
binding signal.27,28 For a bimolecular interaction,
kon
A 1 B t AB
koff
(1)
The rate equation for above reaction is shown in equation 2.
d[AB]/dt 5 [A][B]kon 2 [AB]koff
(2)
The signal from the biosensor (R) is proportional to the amount of
complex (AB), and the maximum signal (Rmax) is proportional to the
concentration of immobilized protein B, so equation 2 can be rewritten as:
dR/dt 5 kon[A]Rmax 2 (kon[A] 1 koff)R
(3)
Here, kon and koff are on and off rate constants, respectively, and [A] is
the concentration of analyte injected into the sensor cuvette. A plot of dR/dt
versus R has a slope (2ks) equal to 2(kon[A] 1 koff). Straight lines were fit
to the initial linear portion of the transformed association phase data to
obtain ks. A second association phase is evident at high [A]. This phase,
which could not be interpreted with the single bimolecular kinetic model,
may be a consequence of increasing mass (aggregates) of analyte on the
saturated surface. Replot of ks versus [A] yields a slope of kon. During the
dissociation phase [A] 5 0, so from equation 3:
dR/dt 5 2koffR
(4)
Integrating equation 4 gives:
R 5 R0e2kofft
(5)
The dissociation phase of the highest concentration of analyte [A] was
transformed by plotting ln(R0/R) versus time, and koff was calculated from
the slope of a straight line over the initial dissociation phase.
Results
Binding of HKa to HEK 293 cells transfected with Mac-1
The direct interaction between HKa and Mac-1 was examined
using HEK 293 cells stably transfected with a vector containing the
cDNA for human Mac-1. Little signal was detected on control
transfected HEK 293 cells with monoclonal antibody LM2/1,
specific for Mac-1, as demonstrated by flow cytometry (Figure
1A). In contrast, the transfected HEK 293 cells contained 2
populations of cell surface Mac-1 expression (Figure 1B). HEK
293 cells expressing high and low levels of surface human Mac-1
were separated using FACSorting technique, as shown in Figure
1C,D. HEK 293 cells expressing high levels of surface human Mac-1
were used in subsequent experiments, and control transfected HEK
293 cells were used in control experiments. A concentrationdependent binding of FITC–Hka to the Mac-1 transfected HEK
293 cells was observed but was not found using the control
transfected cells when varying concentrations of FITC–HKa were
used in a cell-binding assay (Figure 2). FITC–HKa specifically
bound 3 times more to the Mac-1 transfected HEK 293 cells than to
the control transfected cells (Figure 2). As a control, FITC-labeled
BSA showed no binding to HEK 293 cells transfected with Mac-1
in the same type of binding assays (data not shown). In the presence
of 10 mmol/L EDTA, the binding of FITC–HKa to Mac-1 transfected
HEK 293 cells was inhibited 70% (Figure 2), indicating that the
binding was metal dependent. Using the Scatchard plot of bound/
free versus bound, we found evidence for a single class of binding
site with Kd 5 62 nmol/L and Bmax 5 3.1 fmoles/105 cells (20 000
molecules per cell) in the Mac-1 transfected HEK 293 cell system.
Inhibition of biotin–HKa binding to Mac-1 transfected
HEK 293 cells
The specificity of biotin–HKa binding to Mac-1 transfected cells
was examined using nonbiotinylated HKa in a competition binding
assay. Figure 3 showed a concentration-dependent inhibition of
biotin–HKa binding to Mac-1 transfected cells by unlabeled HKa.
BLOOD, 15 JUNE 2000 • VOLUME 95, NUMBER 12
INTERACTION OF KININOGEN WITH MAC-1
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Figure 2. Specific binding of HKa to HEK 293 cells transfected with Mac-1
(Mac-1–HEK 293). FITC–HKa (at indicated concentrations) was incubated with HEK
293 cells either transfected with Mac-1 or control transfected in binding buffer
containing 50 µmol/L ZnCl2 for 30 minutes at room temperature, as described in
‘‘Materials and methods.’’ The same reaction was also performed in the presence of
10 mmol/L EDTA to determine the nonspecific binding. Concentrations of FITC–HKa
used were 0, 15, 30, 60, and 120 nmol/L. Reactions were carried out in wells of a
filtration 96-well plate containing polyvinylidene difluoride membrane. After filtration,
the fluorescence of cell-bound FITC–HKa was measured on a Cytofluor 2350
system. (d) HKa binding to Mac-1 transfected HEK 293 cells. (.) HKa binding to
untransfected HEK 293 cells. (s) HKa binding to Mac-1 transfected HEK 293 cells in
the presence of 10 mmol/L EDTA. (,) HKa binding to untransfected HEK 293 cells in
the presence of 10 mmol/L EDTA. The data are the mean 6 SE of triplicate reactions.
that the interaction between HKa and Mac-1 transfected cells was
mediated primarily by the aµ subunit of Mac-1.
Effect of divalent cations on the HKa binding to Mac-1
To determine the effect of divalent cations on the HKa and Mac-1
interaction, binding assays of FITC–HKa to Mac-1 transfected or
untransfected HEK 293 cells were performed in the presence of
different divalent cations as indicated. In each binding assay, cells
were preincubated with buffer containing 10 mmol/L EDTA for 10
minutes at 37°C and then washed with binding buffer without any
divalent cation. As shown in Figure 5, in the presence of Zn21, the
Figure 1. Transfection of HEK 293 with Mac-1. Flow cytometry analysis and
FACSorting of HEK 293 cells transfected with Mac-1 (A to D). (A) Immunofluorescence assay using mAb LM2/1 with control transfected HEK 293 cells as a control.
(B) Immunofluorescence assay using mAb LM2/1 with Mac-1 transfected HEK 293
cells. (C) Low level of expression of Mac-1, after FACSorting of Mac-1 transfected
HEK cells. (D) High level of expression of Mac-1, after FACSorting of Mac-1
transfected HEK cells.
In the presence of 50-fold excess of unlabeled HKa, the binding of
biotin–HKa to Mac-1 transfected cells was reduced to 20%.
Fibrinogen is a known ligand for binding to Mac-1.21 A concentration-dependent inhibition of biotin–HKa binding to Mac-1 transfected cells by fibrinogen was observed (Figure 4), indicating that
fibrinogen competed with biotin–HKa in binding to Mac-1 transfected cells. Antibody 2LPM19c, a monoclonal antibody against
the aµ subunit of Mac-1, inhibited the binding of biotin–HKa (10
nmol/L) to Mac-1–HEK cells by more than 90% with a 50%
inhibitory concentration of 10 6 3 µg/mL (Figure 4), indicating
Figure 3. Inhibition of biotin-HKa binding to Mac-1 transfected cells by HKa.
Biotin–HKa (10 nmol/L) was incubated with Mac-1 transfected HEK 293 cells
(4 3 106/mL) in binding buffer containing 50 µmol/L ZnCl2 for 45 minutes at room
temperature in the presence of indicated HKa concentrations. The fluorescence of
cell-bound biotin–HKa was measured, and the percentage of biotin–HKa bound to
the cells in the presence of HKa was determined by comparing that with biotin–HKa
alone, which was 100%. Data are the mean 6 SE of 3 separate experiments.
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SHENG et al
Figure 4. Inhibition of biotin–HKa binding to Mac-1 transfected cells by
antibody and fibrinogen. Biotin–HKa (10 nmol/L) was incubated with Mac-1
transfected or control transfected HEK 293 cells (4 3 106/mL) in binding buffer containing
50 µmol/L ZnCl2, 1 mmol/L MgCl2, and 1 mmol/L CaCl2 for 45 minutes at room
temperature. The reactions were carried out either in the presence or absence of antibody,
2LPM19c, or fibrinogen as indicated. The relative amount of biotin–HKa bound to the
Mac-1 transfected cells in the presence of inhibitors was determined by comparing
that with biotin–HKa alone. Data are the mean 6 SE of 3 separate experiments.
binding of HKa to Mac-1 transfected HEK 293 cells increased
2-fold. In the presence of Mn21, the binding of HKa to Mac-1
transfected HEK 293 cells was increased; however, the binding of
HKa to untransfected cells was also increased. There is no
significant difference in HKa binding to Mac-1 transfected cells
with or without the presence of Mg21.
Interaction of HKa and purified Mac-1
We have examined the interaction of HKa and purified Mac-1 using
an IAsys resonant mirror optical biosensor (Affinity Sensors). In
this type of binding assay, biotinylated HKa was immobilized on
the sensor chip surface by binding to streptavidin that had been
pre-captured on the surface of an IAsys biotin cuvette (Affinity
Sensors). Before HKa was immobilized on the surface of the
cuvette, a control experiment was performed in which purified
Mac-1 was added to the streptavidin surface. No interaction was
detected between the streptavidin surface and Mac-1 at the highest
concentration used in this experiment (data not shown). Purified
Figure 5. Effect of divalent cation on the HK-Mac-1 interaction. To determine the
effect of divalent cations on the HK–Mac-1 interaction, binding assays of FITC–HKa
(60 nmol/L) to Mac-1 transfected (open bars) or untransfected (black bars) HEK cells
were performed in the presence of different divalent cations as indicated. Cells were
preincubated with 10 mmol/L EDTA for 10 minutes at 37°C, then washed with binding
buffer without any divalent cation (2mol/L). One of the divalent cations was added to
each assay as follows: MgCl2, MnCl2, and CaCl2 at 1 mmol/L, and ZnCl2 was added at
50 µmol/L.
BLOOD, 15 JUNE 2000 • VOLUME 95, NUMBER 12
Mac-1 was then added at indicated concentrations to the Hkacaptured surface, and the response (in arc seconds) versus time (in
seconds) was recorded. An overlay of sensor-grams for the binding
of Mac-1 to the HKa surface is shown in Figure 6A. Sensor-grams
show 2 phases: an association phase, detected when purified human
Mac-1 was added and was allowed to bind to the immobilized HKa
(20-300 seconds), and a dissociation phase, in which the Mac-1
solution was replaced with buffer (300-400 seconds). The association phase was linearized according to equation 3, and a plot of
dR/dt versus R is shown in Figure 6B. A replot of the slope of these
lines (ks) against Mac-1 concentration gives a straight line (Figure
6C). The slope of this straight line equals the association rate
constant (kon) of 5.6 3 106 M21s21. The dissociation phase was
analyzed according to equation 5. Figure 6D shows a plot of
ln(R1/Rn) versus time using the dissociation phase data of the
highest concentration of Mac-1. This plot did not fit the model of
single exponential decay. To estimate the dissociation rate constants, the plot ln(R1/Rn) versus time was divided into fast and slow
phases (phases 1 and 2, respectively). The dissociation rate
constant (koff) calculated from the slope of the first 20 seconds of
this plot (phase 1) was 18.131023 s21. Then the equilibrium
dissociation constant (Kd), calculated from the ratio of the rate
constants, was 3.2 nmol/L. Independent evaluation of Kd from the
steady state binding signal resulted in an estimated value of 5.26
nmol/L (R2 5 0.95; Scatchard plot not shown).
Mac-1 transfected HEK 293 cells binding
to HKa-immobilized surface
Specific binding of HKa to Mac-1 transfected HEK 293 cells was
also observed using the IAsys resonant mirror optical biosensor
(Affinity Sensors) (Figure 7). In this assay, biotinylated HKa was
immobilized on the surface of a cuvette as described in ‘‘Materials
and methods.’’ The binding of Mac-1 transfected HEK cells to HKa
was significantly greater than that of untransfected cells to HKa at
equivalent cell numbers. The binding was Zn21 dependent because,
Figure 6. A real-time observation of HK-Mac-1 interaction. (A) Overlay sensorgram from the IAsys showing the binding of Mac-1 to immobilized HKa. At ‘‘1’’ purified
human Mac-1 was added to the cuvette at the following concentrations: 5, 10, 20, 40,
60, 80, 120, and 160 nmol/L. At ‘‘2’’ the cuvette was washed with buffer (10 mmol/L
HEPES, 137 mmol/L NaCl, 4 mmol/L KCl, 50 µmol/L ZnCl2, and 0.05% Tween-20).
(B) dR/dt plots of association data according to equation 3. Straight lines were fitted to
the initial linear portion of the association phase data to obtain ks. (C) Plot of ks versus
Mac-1 concentration. The slope of the line gives the association rate constant. (D)
Dissociation phase data from 160 nmol/L Mac-1 sensor-gram plotted according to
equation 5. A straight line was fitted to the first 20 seconds of the plot for phase 1, and
the other straight line was fitted to the data between 20 and 70 seconds for phase 2.
BLOOD, 15 JUNE 2000 • VOLUME 95, NUMBER 12
INTERACTION OF KININOGEN WITH MAC-1
3793
of Mac-1 transfected HEK cells to fibrinogen and ICAM-1 by 62%
and 85%, respectively. HKa has no effect on the adhesion of Mac-1
transfected HEK cells to fibronectin, which is mediated by b1
integrin, indicating that HKa specifically inhibits the Mac-1–
mediated adhesion. Moreover, untransfected HEK cells failed to
adhere to fibrinogen or ICAM-1.
Discussion
Figure 7. HEK 293 cells transfected with Mac-1 or untransfected binding to
immobilized HKa using the IAsys optical sensor. At ‘‘1’’ cells (105) were added,
and at ‘‘2’’ they were washed with buffer (10 mmol/L HEPES, 137 mmol/L NaCl, 4
mmol/L KCl, 50 µmol/L ZnCl2, and 0.5% BSA). (A) Mac-1 transfected HEK 293 cells.
(B) Untransfected HEK 293 cells. (C, D) Untransfected and Mac-1 transfected HEK
293 cells in the presence of 10 mmol/L EDTA, respectively. Data are representative of
3 separate experiments.
in the presence of 10 mmol/L EDTA, the binding of Mac-1
transfected HEK cells to HKa was decreased by 50%.
HKa inhibits Mac-1–mediated cell adhesion to fibrinogen
and ICAM-1
The functional relevance of HKa directly interacting with Mac-1
was investigated by examining the effect of HKa on cellular
adhesion to fibrinogen and ICAM-1, molecules abundant in the
injured vessel wall. As shown in Figure 8, HKa blocked adhesion
Figure 8. Effect of HKa on cellular adhesion to fibrinogen and ICAM-1. HEK 293
cells transfected with Mac-1 were labeled with CMFDA, added to wells precoated
with the ligands ICAM-1 (.), fibrinogen, (s) or fibronectin (d), and incubated in the
presence or absence of indicated HKa for 60 minutes. As a control, untransfected
HEK 293 cells labeled with CMFDA were added to fibrinogen-coated (j) or
ICAM-1–coated (h) wells. After they were washed 3 times, adherent cells were lysed.
Plates were read on a Cytofluor 2350 system. Percentage of adherent cells to each
ligand in the presence of HKa was determined by comparing that with in the absence
of HKa, which was calculated as 100%. Data are the mean 6 SE of 3 separate
experiments.
Plasma kallikrein activates human neutrophils,14,15 and, in plasma,
prekallikrein circulates in a binary complex with high molecular
weight kininogen (HK).29 Bradykinin is released from HK by
plasma kallikrein cleavage. Cleaved HK (HKa) consists of a heavy
chain and a light chain that remain linked by a single interchain
disulfide bond. On the basis of studies of a patient with HK
deficiency,12 we suggested that HK might serve as a cofactor for
kallikrein binding to neutrophils. Later, we demonstrated that
human neutrophils contain and bind HK,4 and further studies of HK
binding to neutrophils indicated that both heavy chain (domain 3)
and light chain (domain 5) of HK were involved in the binding to
neutrophils.30 We have shown that fibrinogen acts as a noncompetitive inhibitor of HK binding to neutrophils.12 Inhibition studies
with monoclonal antibodies suggested that Mac-1 might be the HK
binding site on neutrophils. However, a direct interaction between
HK/HKa and Mac-1 has not been demonstrated. Although both HK
and HKa have been used previously for studies of the binding of
kininogen to neutrophils, the contamination of HKa in HK
preparation is not excluded. In this study, we used HKa and showed
for the first time that HKa binds directly to cell surface human
Mac-1 integrin and, consistent with this, forms a complex with
Mac-1 in a purified system.
To examine the direct interaction of HKa and Mac-1 integrin,
we initially used Chinese hamster ovary (CHO) cells stably
transfected with human Mac-1 in the binding studies. However, we
found no significant difference in HKa binding to Mac-1 transfected CHO cells or control transfected CHO cells (data not
shown), indicating that other receptor(s) may be present on the
surface of CHO cells and may be bound to HKa. Because it has
been shown that 1 binding site for HKa on human endothelial cells
is urokinase plasminogen activator receptor (uPAR),31 we postulated that the binding of HKa to control transfected or untransfected
CHO cells is probably caused by the binding of HKa to uPAR.
Recently, human prourokinase was demonstrated to bind to 50 000
sites on CHO cells with Kd 5 1.1 nmol/L. Cloning of the receptor
showed a 63% identity with human uPAR,32 consistent with
our hypothesis.
Because human embryonic kidney 293 cells (HEK 293) normally do not express uPAR or Mac-1 receptor, we used this cell line
to transfect with the human Mac-1 receptor. Our data showed that
the binding of HKa to Mac-1 transfected HEK 293 cells was 3-fold
greater than the binding of HKa to control transfected HEK 293
cells. The following evidence from this report indicated that the
binding of HKa to Mac-1 was specific. Unlabeled HKa inhibited
the binding of biotin-HKa to Mac-1 transfected HEK 293 cells in a
concentration-dependent manner. A known Mac-1 ligand, fibrinogen, also inhibited the binding of biotin–HKa to Mac-1 transfected
cells, but with lower binding affinity to Mac-1 than HKa (Figures 3,
4). This agrees with our previous finding that fibrinogen has a lower
binding affinity to human neutrophils than kininogen.12 Antibody
against the a subunit of Mac-1 inhibited the binding of biotin–HKa
to Mac-1 transfected HEK 293 cells by 50%.
3794
SHENG et al
In the studies of HKa binding to Mac-1 transfected HEK 293
cells, we have found that there was some residual binding (20%) of
HKa to untransfected HEK 293 cells (data not shown). Recently,
Hasan et al33 reported that cytokeratin 1 is a major endothelial cell
receptor for kininogens. To examine whether cytokeratin 1 is
present on HEK 293 cells, we performed an immunofluorescence
assay using the collections of monoclonal anti-cytokeratins because a cytokeratin 1-specific monoclonal antibody was unavailable. We used a monoclonal anti-pan cytokeratin mixture that
recognizes human cytokeratins 1, 4, 5, 6, 8, 10, 13, 18, and 19; a
monoclonal anti-pan cytokeratin (clone C-11) that recognizes
cytokeratins 4, 5, 6, 8, 10, 13, and 18; and monoclonal anticytokeratin 4.62, which is immunospecific for cytokeratin 19. Flow
cytometry results showed that the first of these antibody mixtures,
the only 1 containing cytokeratin 1, was positive, indicating that
cytokeratin 1 is expressed on HEK 293 cells (data not shown).
Therefore, a possibility for the residual binding is cytokeratin 1. In
addition, Herwald et al34 isolated a 33-kd protein, which was
identified as gC1q receptor, 35 on an HK affinity column from
EA.hy926 cells, a human umbilical vein endothelial cell line. The
binding of HK to gC1q receptor did not require Zn21, though other
investigators36 report that Zn21 is required for ligand blots. Another
explanation for the residual binding is the presence of the
gC1q receptor.
With the use of EDTA, the binding of HKa to Mac-1 transfected
cells was greatly reduced, indicating that the binding was metal
dependent. Examining the effect of divalent cations on the HKa
binding to Mac-1 transfected cells showed that, in the presence of
Zn21, the binding of HKa to Mac-1 transfected HEK 293 cells
increased 2-fold (Figure 5). The trace element Zn21 is an important
cofactor of several proteins and enzymes, such as transcription
factors,37,38 focal adhesion molecules,39 or matrix metalloproteinases.40 Previous studies have shown that the binding of HKa to
neutrophils, platelets, and human umbilical vein endothelial cells
requires Zn21, and Zn21 is the only divalent cation that appears to
be essential.3-5,31 In plasma, the total Zn21 concentration is 10 to 25
µmol/L.41 The actual free Zn21 concentration is significantly lower
(1-3 µmol/L) because most of the ion is bound to albumin.42 Mg21
or Ca11 alone does not support the binding of HKa to Mac-1
transfected HEK 293 cells. Altieri43 has reported that Mn21 ions
can dramatically stimulate the adhesive functions of the leukocyte
integrin Mac-1. In our experiment, Mn21 stimulated the binding of
HKa to Mac-1 transfected HEK 293 cells 2-fold; however, the
nonspecific binding of HKa to untransfected HEK 293 cells was
also increased (Figure 5). A structural feature of leukocyte integrins
is that each a subunit contains 3 homologous repeats that have
putative divalent cation-binding sites. Ca11 and Mg21 are required
for stabilizing the interaction of integrin a and b chains44 and,
therefore, are required for the function of integrins. It is unclear
whether Zn21 can play a similar role in stabilizing the interaction of
integrin a and b domains.
Using an IAsys resonant mirror optical biosensor (Affinity
Sensors), we examined the interaction of HKa and purified Mac-1.
With this technique, it is possible to monitor both the association
and the dissociation between HKa and Mac-1 in real-time and,
therefore, to measure rate constants. Linearization analysis of
Hka–Mac-1 binding data showed a good fit to a theoretical 1:1
interaction model for the lower concentration range of Mac-1. The
dissociation phase analysis did not fit the model of single exponen-
BLOOD, 15 JUNE 2000 • VOLUME 95, NUMBER 12
tial decay, especially at high concentrations of Mac-1, as shown by
the curvature of the plot in Figure 6D. The Scatchard plots of
Req/C (bound/free) versus Req (bound), which were estimated
from the extrapolated values of R (response) at dR/dt 5 0, showed
a single slope with Kd 5 21/slope 5 5.26 nmol/L. At higher Mac-1
concentrations, the deviation from the 1:1 model may result from
the binding of Mac-1 to the lower affinity sites of HKa molecules
generated by random biotinylation or from an aggregation of
Mac-1 molecules. The equilibrium dissociation constant (Kd),
calculated by using the dissociation rate constant for phase 1 and
the calculated kon, was 3.2 nmol/L. This Kd agrees reasonably well
with the Kd of 9 to18 nmol/L for the binding of HK to neutrophils
reported previously by Gustafson et al.4 The equilibrium dissociation constant (Kd) calculated from the slowest dissociation step
was 0.33 nmol/L. This value is approximately 1 order lower than
that of HK binding to neutrophils.4 However, interactions at a
surface with the receptor as the soluble ligand may account for the
discrepancy.
Leukocyte integrin Mac-1 recognizes multiple and unrelated
ligands, including iC3b, fibrinogen, factor X, and ICAM-1.16,17,21-23
The engagement of Mac-1 with its ligands fibrinogen, factor X, or
serum-opsonized zymosan triggered monocyte degranulation and
cathepsin G activation of factor X.45 Activated platelets express not
only P-selectin but also different b2-integrin ligands, including
fibrinogen and ICAM-2. Polymorphonuclear adhesion to activated
platelets is initiated with a P-selectin–dependent recognition step
and is followed by adhesion-strengthening interactions mediated
by b2-integrin Mac-1.46 HKa could potentially inhibit these
functions of the neutrophils or monocytes. However, since there are
multiple receptors on neutrophils or monocytes, it is difficult to
prove that inhibition with HKa is brought about by the interaction
of HKa with Mac-1.
Here, we report that cleaved high molecular weight kininogen
(HKa) is another ligand for Mac-1. HKa can bind directly to
Mac-1, both on cells and in a purified system. To investigate the
functional significance of Mac-1-HKa interaction, we demonstrated that HKa inhibited the Mac-1–dependent cell adhesion to
fibrinogen and ICAM-1, molecules abundant in the acutely injured
vessel wall.47 The interaction of Mac-1 to fibrin(ogen) and
ICAM-1 results in the adhesion of neutrophils and monocytes to
the sites of fibrin deposition and to the endothelium, respectively.
In fact, the early response to vascular injury is characterized
by the migration of platelets and inflammatory cells, including
neutrophils and monocytes, to the injured vessel wall.48,49 Therefore, the finding that HKa directly interacts with Mac-1 and
inhibits Mac-1–dependent adhesion may offer an opportunity for
the discovery of peptides that can be expected to decrease
neutrophil/monocyte activation and adhesion to perturbed endothelial cells. Efforts to uncover such peptides have focused on
identifying the binding sites of kininogen on endothelial cells.34,50
More recently, fine mapping has revealed 3 noncontiguous binding
regions on kininogen for neutrophils.51 Neutrophils express both
Mac-1 and uPAR, and it is known that HKa binds to uPAR.31 Thus,
some of the sequences on HKa could be caused by binding to uPAR
rather than to Mac-1. Therefore, further characterization of the
regions in HKa involved in Mac-1 binding, using direct binding of
HKa to Mac-1 on transfected cells or in a purified system, may
well provide new approaches to interrupt Mac-1 mediated neutrophil/monocyte adhesion. These studies will ultimately help in
designing peptides that could serve as leads for antiadhesive
therapeutic intervention.
BLOOD, 15 JUNE 2000 • VOLUME 95, NUMBER 12
INTERACTION OF KININOGEN WITH MAC-1
3795
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