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Blood, 15 October 2001, Vol. 98, No. 8, pp. 2448-2455
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
The alternatively spliced  EC domain of human
fibrinogen-420 is a novel ligand for leukocyte integrins
M 2 and X2
Valeryi K. Lishko,
Valentin
P. Yakubenko,
Kathe M. Hertzberg,
Gerd Grieninger, and
Tatiana P. Ugarova
From the Joseph J. Jacobs Center for Thrombosis and
Vascular Biology, Department of Molecular Cardiology, Lerner Research
Institute, Cleveland Clinic Foundation, Cleveland, OH; and the Lindsley
F. Kimball Research Institute of the New York Blood Center, New York,
NY.
 |
Abstract |
The interaction of human plasma fibrinogen with leukocyte integrins
 M 2 (CD11b/CD18, Mac-1) and
X2 (CD11c/CD18, p150,95) is an important
component of the inflammatory response. Previously, it was demonstrated
that binding of fibrinogen to these integrins is mediated by  C, the
globular C-terminal domain of the chain. In this study, evidence
was found of another fibrinogen domain that can serve as a ligand for
the 2 leukocyte integrins: EC, a homologous domain that
extends the chains in a recently discovered subclass of fibrinogen
known as fibrinogen-420. Recombinant EC supported strong
adhesion and migration of cells expressing
M2 and X2,
including nonactivated and activated U937 and THP-1 monocytoid cells,
and neutrophils. Cells transfected with complementary DNA for these
integrins also bound EC. The specificity of interaction was substantiated by inhibition of cell adhesion with antibodies against M, X, and 2
subunits. Also, neutrophil inhibitory factor, a specific inhibitor of
M2 and X2
function, efficiently blocked cell adhesion to EC. In
M2 and X2,
the I domain is the binding site for EC, since
EC bound to recombinant M I and
XI domains in a dose-dependent and saturable manner.
Synthetic peptides that duplicated sequences 190 to 202 and 377
to 395, previously considered putative binding sites in C,
effectively inhibited M2- and X2-mediated adhesion to
EC, suggesting that recognition of EC by
the I domain involves structural features in common with those of C.
These findings identify EC as a second domain in fibrinogen-420 that binds M2 and
X2 and can mediate leukocyte adhesion and migration.
(Blood. 2001;98:2448-2455)
© 2001 by The American Society of Hematology.
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Introduction |
In addition to its function in the
coagulation and hemostatic systems, plasma protein fibrinogen (Fg) also
participates in inflammatory responses. This function of Fg depends on
its ability to interact with leukocyte receptors. On inflammatory
challenge, Fg mediates adhesive and migratory reactions of leukocytes,
such as leukocyte attachment to the vessel wall and subsequent
transmigration through the endothelium into a subendothelial
matrix.1-3 In addition, fibrinogen and fibrin deposited at
sites of vascular injury and within tissues4,5 promote
accumulation of inflammatory cells.6,7 The accessory role
of Fg in inflammation was documented by in vivo studies in which
congenital afibrinogenemia in patients or experimental depletion of Fg
from the circulation in animals altered the manifestation of
inflammatory responses.8-12
The interaction of leukocytes with Fg is mediated by 2 transmembrane
receptors that belong to the integrin gene superfamily: M2 (CD11b/CD18, Mac-1) and
X2 (CD11c/CD18, p150,95). Abundantly expressed on monocytes and neutrophils and induced on lymphocytes during their activation, integrin M2 is
primarily responsible for leukocyte adhesion to Fg.13
Studies in mice deficient in M2
demonstrated that fibrinogen-dependent inflammatory reactions were
significantly curtailed in these animals.14 Specifically, the mice failed to accumulate phagocytes at the site of implantation of
biomaterials, a process that depends entirely on Fg that is spontaneously adsorbed on the implant surfaces.10 The role
of X2, which is enriched on macrophages
and dendritic cells, in binding of Fg is poorly
characterized.15,16
Plasma Fg is a dimer composed of 2 copies of 3 nonidentical polypeptide
chains (A, B, and ) linked by disulfide bonds.17 Structurally, the molecule is organized into a central E domain and 2 peripheral D domains (Figure 1A). The
COOH-terminal parts of the B and chains in the D domain are
folded into the globular C and C domains. Previous studies found
that the binding site for M2 and
X2 in Fg resides in
C.18-20 Two sequences in C, corresponding to 190
to 202 and 377 to 395, were suggested as the putative recognition
sites for leukocyte 2 integrins.21,22 Analyses of synthetic peptides duplicating 190 to 202 and 377 to
395, designated P1 and P2, respectively, showed that the peptides inhibited adhesion of cells expressing M2
and X2, directly supported cell
adhesion20,22 and migration,23 and promoted accumulation of leukocytes on implanted biomaterials.24 It
was also shown that the sequence 383 to 395 (P2-C) is the major
active site in P2.22 The complementary binding site for P1
and P2 peptides in the heterodimeric M2
receptor was localized in the I domain, a region of about 200 amino
acid residues inserted in the M
subunit.22,25
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| Figure 1.
Schematic representation of the domain structure of the
2 species of human Fg.
(A) Fg-340. (B) Fg-420. The D and E domains are boxed. The locations of
EC (in Fg-420 only), C, and C are
shown.
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In the past decade, it was discovered that normal plasma Fg consists of
2 species differentiated by the length of their A chains a more
abundant form with a molecular weight of 340 kd (Fg-340) and a minor
form with extended chains (E) and hence a higher
molecular weight of 420 kd (Fg-420; Figure 1B).26-28 The globular C-terminal domain of E (EC),
which closely resembles C and C, consists of 236 amino acid
residues that are missing in the common chains of Fg-340. In vivo,
EC originates by alternative splicing of the gene
transcript to include the exon VI sequence.29,30 The
properties that the presence of EC confers on Fg-420,
which is structurally identical to Fg-340 in all other respects, are only beginning to be explored. The properties of the 2 Fg species that
are related to classic functions of Fg (ie, clotting, cross-linking by
factor XIII, and fibrinolysis) are similar, except that degradation of
Fg-420 by plasmin releases a stable product containing the EC domain in addition to the conventional degradation
products X, Y, D, and E.31 However, little is known about
how the presence of EC contributes to recognition of
Fg-420 by leukocyte integrins. It is known that EC and
C share about 40% of amino acid identity,30 and
analyses of x-ray structures revealed that folding of the EC domain closely resembles that of
C.32-34 In this study, we analyzed the interaction of
EC with leukocyte integrins. We found that the
EC domain of Fg-420 is a ligand for
M2 and X2
capable of mediating strong leukocyte adhesion and promoting directed migration of leukocytes.
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Materials and methods |
Proteins, peptides, and monoclonal antibodies
Fraction I-2 Fg was purified from umbilical cord
plasma, and separation of human Fg-340 and Fg-420 was achieved by
ion-exchange chromatography on Mono Q as described
previously.31 Fragment D100 (Mr
100 000) was prepared by digestion of human Fg with plasmin. Fg and plasmin were obtained from Enzyme Research Laboratories (South
Bend, IN). Fragment D100 was purified by ion-exchange
chromatography on CM-Sephadex followed by gel filtration on Sephacryl
S-200.35 A recombinant wild-type EC domain
(EC 610-847) was expressed in Pichia pastoris
and purified as described previously.36 A recombinant
wild-type C domain was expressed in P pastoris by using
an Invitrogen protocol (San Diego, CA). Briefly, a complementary DNA
(cDNA) fragment corresponding to 143 to 411 was generated by
polymerase chain reaction from a template consisting of full-length cDNA encoding the human Fg chain that was provided by Dr S. Lord
(University of North Carolina).37 The amplified cDNA
fragment was ligated into pPIC9 expression vector by means of
SnabI and NotI sites and cloned in DH5 cells.
The plasmid was linearized by using SalI and transformed
into P pastoris (strain GS115) by electroporation.
Expression was induced by transferring the yeast into medium
containing 0.5% methanol. The recombinant C was purified by
chromatography on Reactive Red-120 CL-6B agarose (Sigma-Aldrich, St
Louis, MO). The yeast supernatant was applied to the column, equilibrated with 20 mM Tris-HCl buffer (pH 7.4) containing 80 mM NaCl.
Bound protein was eluted with 50 mM Tris-HCl buffer (pH 7.4) containing
0.5 M NaCl. The protein was dialyzed against 50 mM Tris-HCl (pH 7.4)
with 0.15 M NaCl, and 45% glycerol was added to prevent aggregation.
The purified C was homogeneous, as revealed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis, and contained an intact
COOH terminus, as demonstrated by Western blot analysis using mAb 4A5,
which recognizes 406 to 411.38 The recombinant
EC and C domains were labeled with iodine 125 (125I) by using Iodobeads according to the manufacturer's
protocol (Pierce, Rockford, IL).
The recombinant M I domain corresponding to the human
M sequence D132 to A318 and the X I
domain (residues E148-A335) were expressed in Escherichia
coli as fusion proteins with glutathione S transferase
(GST) by using the expression vector pGEX-4T-1 (Amersham Pharmacia
Biotech, Piscataway, NJ) as described previously.39 The
GST-I domain fusion proteins from the cell lysate were purified by
affinity chromatography using glutathione-agarose (Sigma-Aldrich). To
obtain the M I domain, the fusion partner was removed by
digestion with thrombin and the mixture of GST and M I
domain was separated by cycling through glutathione-agarose. The Fg
peptides P1 and P2-C corresponding to the -chain sequences 190 to
202 (Gly-Trp-Thr-Val-Phe-Gln-Lys-Arg-Leu-Asp-Gly-Ser-Val) and 383 to
395 (Thr-Met-Lys-Ile-Ile-Pro-Phe-Asn-Arg-Leu-Thr-Ile-Gly), respectively, were described previously.22
The mAb 4A5 directed against 406 to 41138 was provided
by Dr G. Matsueda (Bristol-Meyers Squibb, Princeton, NJ). The
monoclonal antibodies (mAbs) 44a40 and IB441
directed against the M and 2 subunits,
respectively, and mAb w6/32 (antihuman HLA) were obtained from the
American Type Culture Collection (ATCC; Rockville, MD). Serotec
(Raleigh, NC) provided mAb 3.9 directed against the X
subunit. Neutrophil inhibitory factor (NIF) was a gift from Corvas
International (San Diego, CA).
Cells
Human embryonic kidney (HEK) 293 cells expressing
M2 were described
previously.42 Chinese hamster ovary (CHO) cells expressing X2 were provided by Dr D. Golenbock
(Boston University). The cell lines were maintained in Dulbecco
modified Eagle medium (DMEM)-F-12 medium (BioWhittaker, Walkersville,
MD) or Ham medium (Life Technologies, Rockville, MD) for
M2 and X2
transfectants, respectively, and supplemented with 10% fetal-calf
serum (FCS) and 25 mM HEPES. The U937 and THP-1 monocytoid cells were
obtained from the ATCC and cultured in RPMI 1640 medium supplemented
with 10% FCS.
Granulocytes were isolated from peripheral blood obtained from
consenting volunteers and anticoagulated with acid-citrate-dextrose essentially as described previously.43 The cells in the
preparation were 98% granulocytes, of which at least 96% were
neutrophils and 2% eosinophils.
Cell-adhesion assays
The wells of polystyrene microtiter plates (Costar,
Cambridge, MA; or Immulon 4HBX, Dynex Technologies, Chantilly, VA) were coated with various concentrations of protein ligands for 3 hours at
37°C or overnight at 4°C. The wells were postcoated with 1% polyvinylpyrrolidone (PVP) for 1 hour at 37°C. The cells expressing M2 and X2
were harvested from the flasks with a cell-dissociating buffer (Gibco,
Grand Island, NY) and washed twice in Hanks balanced salt solution
(HBSS). The U937 cells, THP-1 cells, and isolated neutrophils were
harvested by centrifugation. The cells were labeled with Calcein
AM (Molecular Probes, Eugene, OR) for 30 minutes at 37°C,
washed with HBSS and resuspended in the same medium at a concentration
of 2.5 × 105 cells/mL. Aliquots (100 µL) of the
labeled cells were added to each well. For inhibition experiments,
cells were mixed with either antibodies, function-blocking peptides, or
NIF and incubated for 20 minutes at 22°C before they were added to
the coated wells. After 25 minutes of incubation at 37°C in a 3%
CO2 humidified atmosphere, the nonadherent cells were
removed and the plates were washed once with HBSS. Fluorescence was
measured in a fluorescence plate reader (Perseptive Biosystems,
Framingham, MA), and the number of adherent cells was determined from a
standard curve constructed by using the fluorescence of 100-µL
aliquots with a known number of labeled cells.
Cell-migration assays
Chemotactic motility experiments were done under sterile
conditions by using transwell chambers with a pore size 8 µm in
diameter (Costar; Corning, Corning, NY). Here, 600 µL DMEM-F-12
medium containing either the D100 fragment or the
EC domain was placed in the lower chamber. The
M2-expressing cells or U937 cells harvested as described above were resuspended at a concentration of
2 × 106/mL in DMEM-F-12 and RPMI 1640, respectively.
Cells (150 µL) with or without inhibitory antibodies were placed in
the upper chamber and incubated for 5 to 15 hours at 37°C in a 3%
CO2 humidified atmosphere. Cells on the upper surface of
the polycarbonate membrane were then removed by wiping the surface
twice with a cotton-tipped applicator. The membranes were fixed with
formaldehyde and stained with hematoxylin 7211 (Richard-Allan
Scientific, Kalamazoo, MI). The migrated cells on the lower surface of
the filter in 6 randomly chosen low-power fields (×20 magnification)
were counted and the values obtained were averaged. No migration was
observed in chambers with no added proteins.
Solid-phase binding assays
Twelve-well microtiter strips (Immulon 2HB; Dynex Technologies)
were coated with 100 µL of 10 µg/mL recombinant M I
domain in Tris-buffered saline (TBS) containing 1 mM CaCl2
and 1 mM MgCl2 overnight at 4°C and postcoated with 1%
bovine serum albumin (BSA) for 1 hour at 22°C. Increasing
concentrations of 125I-labeled recombinant
EC or C in TBS and 0.05% Tween 20 containing 1 mM
CaCl2 and 1 mM MgCl2 were added to the wells,
which were incubated for 3 hours at 37°C. After washing with TBS and
0.05% Tween 20, bound radioactivity was measured and the amount of
bound proteins was calculated, with correction for nonspecific binding to BSA-coated wells.
To test the interaction of the X I domain, 96-well
plates (Immulon 4BX; Dynex Technologies) were coated with 50 µg/mL
EC and C overnight at 4°C and postcoated with 3%
BSA. Different concentrations of the GST-X I domain in
20 mM TBS (containing 1 mM MgCl2, 1 mM CaCl2,
0.05% Tween 20, and 5% glycerol) were added to the wells and
incubated for 3 hours at 22°C. After washing, bound I domain was
detected with an anti-GST mAb (Upstate Biotechnology, Lake Placid, NY)
at a 1:5000 dilution. After another washing, goat anti-mouse IgG
conjugated to alkaline phosphatase was added for 1 hour, and binding of
the I domain was measured by reaction with p-nitrophenyl phosphate. The
control binding of GST to immobilized EC and C was
typically about 5% to 10% that of the M I domain. Background binding to BSA was subtracted.
Analytical procedures
To determine the amounts of recombinant EC and
C immobilized on the wells of the microtiter plates, each domain was
adsorbed on the plastic for 3 hours at 37°C at a concentration of 10 or 20 µg/mL and then washed with phosphate-buffered saline. The
concentration of bound proteins was determined by the bicinchoninic
acid method, according to the manufacturer's protocol (Pierce).
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Results |
Adhesion of cells expressing M2 and
X2 to the EC domain
of Fg-420
The binding site for M2 in Fg-340
was shown previously to reside in C, a constituent subdomain of the
D domain (Figure 1).18,21,22 Because C shares about
40% amino acid identity with the EC domain of
Fg-420,30 and the 2 domains are folded into almost
identical structures,33 we tested adhesion of
M2-expressing cells to recombinant
EC. As shown in Figure 2A,
when recombinant proteins were immobilized on tissue culture-treated
polystyrene plates, both proteins supported efficient adhesion of
M2-transfected HEK 293 cells. Adhesion
depended on the concentration used, and similar numbers of cells
adhered to each protein. The concentrations of EC and
C required for half-maximal adhesion were 0.6 µM and 2.1 µM,
respectively. The appearance of the cells adherent to the 2 substrates
was similar (Figure 2B).
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| Figure 2.
Adhesion of cells transfected with
M2 to recombinant EC and
C.
(A) M2-transfected HEK 293 cells
(5 × 104) labeled with Calcein AM in HBSS
and HEPES were added to the wells of 48-well, tissue culture-treated
microtiter plates coated with different concentrations of the
recombinant EC ( ) and C ( ) domains and
postcoated with 1% PVP. After 25 minutes at 37°C, nonadherent cells
were removed by 3 washes with phosphate-buffered saline. Fluorescence
of adherent cells was measured in a fluorescence plate reader and
converted to cell number. Adhesion of mock-transfected cells ( ) to
EC is shown for comparison. Results are the mean ± SE values from 3 individual experiments done in triplicate.
(B) Microscopical study (original magnification, × 20)
of the cells adherent to the control (PVP), C, and
EC.
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The following results supported the idea that integrin
M2 on the surface of
M2-transfected cells is responsible for
recognition of the EC domain. First, mock-transfected
cells adhered poorly to EC (Figure 2A). Second, mAb 44a
and mAb IB4 directed to the M and 2
subunits, respectively, inhibited adhesion to EC (Figure 3A and 3B). Both antibodies inhibited the
M2-mediated adhesion to both
EC and C almost completely (at 2.5 µg/mL mAb 44A
and 10 µg/mL mAb IB4). A control mAb (w6/32) against the class I
major histocompatibility complex did not produce inhibition (data not shown). Third, specificity of the interaction between
M2 and EC was confirmed by
the finding that NIF, a specific inhibitor of
M2,44,45 abolished adhesion
to EC completely (data not shown).
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| Figure 3.
Effect of mAbs on adhesion of
M2-transfected cells to EC
and C.
Calcein-labeled cells were preincubated with different concentrations
of mAb 44a (A) or mAb IB4 (B) for 20 minutes at 22°C with constant
agitation. Then, aliquots (0.1 mL) containing 5 × 104
cells were added to the wells of microtiter plates coated with 5 µg/mL recombinant EC () or C () domains.
After 25 minutes at 37°C, nonadherent cells were removed and cell
fluorescence was measured.
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We found previously that P1 and P2-C peptides from C blocked
adhesion of M2-bearing cells to the
D100 fragment, with P2-C being a stronger inhibitor of
adhesion.22 Because P1 (190-202) and P2-C (383-395)
sequences are similar to corresponding regions 652 to 664 and 838 to
847 in EC, we tested the effect of the P1 and P2-C
peptides on adhesion of M2-transfected
cells to immobilized EC. As shown in Figure
4, both P1 and P2-C blocked adhesion to
EC and D100 in a dose-dependent manner. On a
molar basis, P2-C was more effective than P1 peptide. For
D100, 50% inhibition (IC50) was attained with
210 µM P2-C and 350 µM P1. The peptides were less potent inhibitors
of adhesion of the cells to EC than to immobilized
D100 (IC50, 65 µM for P2-C and 195 µM for
P1). The competence of both P1 and P2-C to inhibit cell adhesion to
EC seems to be consistent with their ability to
cross-inhibit each other's activity22; ie, P2 could
inhibit adhesion of the M2-expressing
cells to immobilized P1 and vice versa. The ability of P1 and P2-C to
inhibit cell adhesion to EC and C suggests that these
Fg domains may share common molecular or structural determinants
required for recognition by M2.
Alternatively, the peptides may inhibit cell adhesion to
EC and C by a steric or allosteric mechanism.
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| Figure 4.
Inhibition of cell adhesion to the immobilized
EC and D100 by P1 and P2-C peptides.
Calcein-labeled M2-transfected cells were
preincubated with increasing concentrations of P1 (open symbols) and
P2-C (solid symbols) peptides for 20 minutes at 22°C. Aliquots (0.1 mL; 2.5 × 105 cells/mL) were added to wells coated with
10 µg/mL EC (circles) or 2 µg/mL D100
(triangles), and adhesion was quantitated. Values are the numbers of
adherent cells. Data from a representative experiment are
shown.
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The second 2 integrin, X2,
can recognize C in the D domain of fibrinogen.20 To
test the capacity of EC to support
X2-mediated adhesion, we used CHO cells
expressing X2. Adhesion of these cells to
several fibrinogen derivatives, including D100, was
characterized previously.20 Although D100
includes a second homologous subdomain, C, earlier studies found
that C does not contribute significantly to the adhesion-promoting
activity of D100 (Merkulov S. and Ugarova T., unpublished
data, 2000). As shown in Figure
5A, this study found that
X2-expressing CHO cells adhered to
EC in a dose-dependent manner. Cell adhesion to
EC was similar to that to D100 when the
coating concentrations used were in the range of 0 to 2 µg/mL. However, at concentrations greater than 2 µg/mL, the cells continued to show dose-dependent adhesion to EC but adhesion to
D100 declined. Wild-type CHO cells adhered poorly to all 3 substances (EC, C, and D100 ligands; 5%
adhesion; data not shown). The anti-2 mAb IB4,
anti-X mAb 3.9, and NIF produced 80%, 62%, and 62%
inhibition, respectively, of the
X2-expressing cells (Figure 5B).
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| Figure 5.
Adhesion of X2-expressing
CHO cells to EC and D100.
(A) Calcein-labeled cells (0.1 mL; 5 × 105
cells/mL) were added to the wells of 96-well microtiter plates coated
with various concentrations of EC () and
D100 ( ) and postcoated with 1% PVP. After the cells
were allowed to adhere for 25 minutes at 37°C, nonadherent cells were
removed by 3 washes with phosphate-buffered saline and the number of
adherent cells was determined by measuring the fluorescence. (B)
Effect of mAbs and NIF on adhesion of cells to wells coated with 10 µg/mL EC. The cells were preincubated with 10 µg/mL
mAb IB4, 10 µg/mL mAb 3.9, or 2 µg/mL NIF for 15 minutes at 22°C.
Aliquots of cells were then added to wells coated with
EC. Results are the total numbers of adherent cells and
the mean ± SE values from 2 individual experiments.
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Because of the findings showing that EC contains the
binding site for M2 and
X2, we investigated whether the presence of additional recognition sites in Fg-420 confers greater adhesive potency than that observed with Fg-340. Various concentrations of
purified Fg-340 and Fg-420 were deposited on plastic and adhesion of
M2-transfected cells was tested. As
expected, cells adhered to both Fg forms; however, adhesion to Fg-420
was not significantly greater than that to Fg-340 (data not shown). It
is not known whether adhesion to Fg-420, with its multiple binding
sites, differs from that of Fg-340 with respect to the cellular
activities it triggers.
We next studied the ability of EC to support adhesion of
U937 and THP-1 monocytoid cells. As shown in Figure
6A, U937 cells stimulated with phorbol
12-myristate 12-acetate (PMA) recognized and attached to
EC strongly, and the adhesion was more extensive than
that to D100. Similar results were obtained for adhesion of
THP-1 (data not shown). Adhesion of both cell lines in the absence of
PMA to both immobilized ligands was only slightly less than with PMA
stimulation. Adhesion of U937 and THP-1 to EC was inhibited by anti-M mAb 44a and anti-2
mAb IB4 (Figure 6B). However, in contrast to the results with
M2-transfected cells, inhibition was not
complete. The maximal inhibition level achieved at a concentration of
20 µg/mL was 58% with mAb 44a and 77% with mAb IB4.
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| Figure 6.
Adhesion of U937 cells to EC and
D100.
(A) U937 cells in HBSS were labeled with Calcein and activated with 10 nM PMA. Aliquots of 5 × 105cells/mL were then added to
the wells coated with EC () and D100
(). (B) Effect of mAbs on adhesion to EC. Activated
U937 cells were preincubated with either mAb 44a, mAb 3.9, or mAb IB4
alone (20 µg/mL), with a combination of 44a and 3.9 or with 4 mM EDTA
for 15 minutes at 22°C. Then, aliquots of 5 × 104
cells were added to microtiter plates coated with 10 µg/mL
EC.
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Because U937 and THP-1 cells express both
M2 and X2,
the effect of mAb 3.9 against the X subunit was tested
alone and in combination with mAb 44a. We found that mAb 3.9 inhibited
adhesion of U937 cells by 48% and that adding mAb 3.9 to mAb 44a
resulted in 75% inhibition. Similar results were obtained with THP-1
cells. Thus, an additional interaction of EC with other
receptors on the surface of U937 and THP-1 cells may account for the
incomplete inhibition of adhesion. It is notable that EDTA, a specific
inhibitor of integrin-mediated interaction, completely inhibited
adhesion of monocytoid cells to immobilized EC, whereas
mAb w6/32 against HLA did not produce inhibition (data not shown).
To extend our findings obtained with transfected cells and monocytoid
cells, we examined the ability of neutrophils, which are known to
express high levels of M2, to bind to
EC. We found that nonstimulated neutrophils isolated
from fresh human blood attached readily to immobilized
EC and D100 in a dose-dependent manner
(Figure 7A) and that mAb IB4, which
recognizes the common 2 subunit of
M2 and X2
on the surface of neutrophils, effectively inhibited adhesion (Figure
7B). In addition, mAbs against M (44a) and
X (3.9) inhibited neutrophil adhesion to
EC by 48% and 33%, respectively (data not shown). The
combination of both anti--subunit mAbs decreased adhesion by about
70%, similar to the mAb IB4 inhibition. Taken together, these results
clearly show that EC can support strong adhesion of
leukocytes that depends to a large extent on integrins
M2 and X2.
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| Figure 7.
Adhesion of activated neutrophils to surfaces coated
with EC and D100.
(A) Neutrophils were isolated from human blood, resuspended in HBSS,
and labeled with Calcein. Cells (0.1 mL;
2 × 105cells/mL) were activated with 10 nM PMA and then
distributed into wells of plates coated with various concentrations of
EC () and D100 (). Adhesion was done
as described in the legends for Figures 2 to 5. (B) Adhesion of
activated neutrophils to wells coated with 10 µg/mL EC
(solid bars) and 10 µg/mL D100 (open bars) in the absence
or presence of 10 µg/mL mAb IB4.
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Binding of EC to M2
and X2 is mediated by the corresponding
I domains
In the heterodimeric M2 receptor,
the I domain, a region of about 200 amino acid residues inserted in the
M subunit, contributes importantly to recognition of
several ligands, including Fg.46 It was shown previously
that synthetic peptides P1 and P2, which duplicate recognition
sequences in the C domain, specifically bound to the recombinant I
domain.22,25 Also, in earlier studies, we found that the
X I domain of X2 is
responsible for the binding of P2-C.39 Therefore, in this
study, we examined whether EC can directly interact with
the M I and X I domains. The recombinant M I domain was immobilized on the wells of microtiter
plates, and the binding of 125I-labeled EC
and C was measured. To examine the interaction between
EC and the X I domain, binding of the
X I domain as a fusion protein with GST was assessed.
Figure 8 shows that EC bound to the M I and X I domains in a
dose-dependent and saturable manner. The interaction of
EC and C with the corresponding I domains seemed to
be similar in affinity because the molar concentrations of each ligand
required for half-maximal binding were about the same.
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| Figure 8.
Binding of EC and C to the
M I and X I domains.
(A) Different concentrations of 125I-labeled ligands
EC () and C () in TBS, 1 mM CaCl2,
and 1 mM MgCl2 were added to wells coated with 10 µg/mL
of the recombinant M I domain and incubated for 3 hours
at 37°C. After washing with TBS and 0.05% Tween 20, bound
radioactivity was measured and the amounts of bound ligands were
calculated, with correction for nonspecific binding to BSA-coated
wells. (B) Different concentrations of recombinant X I
domain produced as a fusion protein with GST in TBS containing 1 mM
MgCl2, 1 mM CaCl2, 0.05% Tween 20, and 5%
glycerol were added to microtiter plates coated with 50 µg/mL
EC () and C () and postcoated with 3% BSA.
After incubation for 3 hours at 22°C, anti-GST mAb (1:5000) was added
to the wells for an additional 1.5 hours. Binding of the I domains was
then detected with a secondary goat anti-mouse IgG conjugated to
alkaline phosphatase, with subsequent development of the reaction with
p-nitrophenyl phosphate.
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EC supports migration of cells expressing
M2
Fg was shown previously to promote
M2-mediated migration of leukocytes in an
in vivo animal model,3 and D100 and Fg
recognition peptides P1 and P2 supported a chemotactic response of
leukocytes in vitro.2,23,47 Therefore, we tested the
ability of EC to mediate migration of
M2-transfected HEK 293 cells and
monocytoid cells. The M2-transfected
cells were allowed to migrate in a transwell toward a gradient of
EC or D100 at 37°C for 15 hours, after
which the cells that migrated through and attached to the underside of
the membrane were fixed, stained, and counted. Different concentrations
of each protein (range, 1-100 µg/mL) placed in the lower chamber of
the transwell system supported efficient migration of the cells, and on
a molar basis, D100 was a somewhat more efficient inducer
of migration than was EC (Figure
9A). The migration toward
EC depended on M2, since
mAb 44a against the M subunit and mAb IB4 against the
2 subunit blocked the response completely (Figure 9A).
Also, EC and D100 promoted efficient migration of nonstimulated U937 cells (Figure 9B). As with the M2-transfected cells, D100
was a more potent migratory agent: at a concentration of 50 µg/mL of
each protein in the lower chamber, 40 ± 8 cells/field migrated to
D100, whereas 22 ± 5 cells/field migrated to
EC. Migration of U937 cells to both ligands was strongly inhibited by mAb 44a, but mAb IB4, the anti-2-specific
mAb, impeded migration by only 30%. The ability of EC
to mimic C in vivo in promoting
M2-mediated migration of leukocytes
remains to be tested and the advantage of such apparent
redundancy explored.
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Abstract
Introduction