(Received for publication, July 22, 1996, and in revised form, October 14, 1996)
From the Commissariat l'Energie Atomique,
Laboratoire d'Hématologie, Département de Biologie
Moléculaire et Structurale, INSERM U-217, 38054 Grenoble, Cedex
9, France, the § Department of Pathology and the
¶ Molecular Cardiobiology Program, The Boyer Center for Molecular
Medicine, Yale University School of Medicine, New Haven, Connecticut
06536, the
Leukocyte Adhesion Laboratory, Imperial Cancer
Research Fund, Lincoln's Inn Fields, London WC2A 3PX, United Kingdom,
and the ** Molecular Parasitology Group, Institute of Molecular
Medicine, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom
Binding of fibrinogen to intercellular adhesion
molecule 1 (ICAM-1) enhances leukocyte adhesion to endothelium by
acting as a bridging molecule between the two cell types. Here, a panel of four monoclonal antibodies (mAbs) to ICAM-1 was used to dissect the
structure-function requirements of this recognition. All four mAbs
bound to ICAM-1 transfectants and immunoprecipitated and immunoblotted
ICAM-1 from detergent-solubilized JY lymphocyte extracts. Functionally,
mAbs 1G12 and 2D5 inhibited binding of 125I-fibrinogen to
ICAM-1-transfectants and abrogated the enhancing effect of fibrinogen
on mononuclear cell adhesion to endothelium and transendothelial
migration. In contrast, mAbs 3D6 and 6E6 did not affect ICAM-1
recognition of fibrinogen. With respect to other ligands, mAbs 1G12 and
2D5 completely inhibited attachment of Plasmodium
falciparum-infected erythrocytes to immobilized recombinant
ICAM-1-Fc, whereas they had no effect on LFA-1-dependent T
cell binding to ICAM-1-Fc. Conversely, mAbs 3D6 and 6E6 completely abolished LFA-1 binding to ICAM-1-Fc. Epitope assignment using ICAM-1
chimeras and receptor mutants revealed that the fibrinogen-blocking mAbs 1G12 and 2D5 reacted with domain 1 of ICAM-1, and their binding was disrupted by 97 and 70% by mutations of D26 and P70, respectively, whereas mAbs 3D6 and 6E6 bound to domain 2 of ICAM-1. By recognizing a
site distinct from that of 2 integrins Mac-1 or LFA-1,
fibrinogen binding to ICAM-1 may provide an alternative pathway of
intercellular adhesion and/or modulate integrin-dependent
adherence during inflammation and vascular injury.
The regulated adhesion of leukocytes to endothelium followed by
their extravascular emigration and tissue homing form the basis of host
defense mechanisms and immune-inflammatory responses. These processes
depend on a stepwise adhesion cascade coordinated by the sequential
ligand recognition of cell surface receptors expressed on leukocytes
and endothelium, including selectins, integrins, and members of the Ig
superfamily (1, 2). Constitutively detectable on resting endothelial
cells and monocytes, and dramatically up-regulated upon cytokine
stimulation (3), intercellular adhesion molecule-1
(ICAM-1,1 CD54) plays a pivotal role in
both leukocyte-endothelium interaction and leukocyte transendothelial
migration through its recognition of 2 integrin
counterreceptors CD11a/CD18 (LFA-1) (4) and CD11b/CD18 (Mac-1) (5).
ICAM-1-dependent adherence is also exploited during certain
pathogen infections. The major Rhinovirus serogroup and some
Coxsackie viruses use ICAM-1 as an adherence and invasion
receptor on respiratory epithelium (6), whereas recognition of
endothelial ICAM-1 (7) by var gene products expressed on
Plasmodium falciparum-infected erythrocytes (8) contributes
to the severity of cerebral malaria (9).
In addition to its interaction with cell-associated counterreceptors (2), ICAM-1 recognizes soluble ligands, including hyaluronic acid (10), and fibrinogen (11). Particularly, the functional role of fibrinogen as a novel ICAM-1 ligand has recently received considerable attention. Previous studies demonstrated that through its ICAM-1 recognition, fibrinogen enhanced the adhesion of leukocytes to endothelium (11) and supported transendothelial monocyte migration (12) by acting as a bridging molecule between the two cell types. A similar model was independently proposed for the ability of fibrinogen to increase monocytic cell attachment to mesothelioma cells in a ICAM-1-dependent pathway (13), whereas the ICAM-1-fibrinogen recognition was shown to require intact cytoskeletal organization and topographical receptor distribution on endothelial cells (14). In real-time adhesion experiments in the exposed rabbit mesentery circulation, fibrinogen-ICAM-1 bridging supported firm adhesion of monocytes to endothelium (15), thus suggesting a potential role for this pathway in leukocyte recirculation in vivo (1, 2). Finally, consistent with a role of ICAM-1 in vascular cell signal transduction (16, 17), binding of fibrinogen to ICAM-1 on saphenous rings modulated vascular tone in a non-nitric oxide-dependent mechanism (18). Because elevated concentrations of fibrinogen constitute a major risk factor for atherosclerosis (19), and increased deposition of fibrinogen (20) and expression of ICAM-1 (21) on atherosclerotic endothelium have been demonstrated, these findings suggest that ICAM-1 recognition of fibrinogen may directly contribute to the pathogenesis of vascular injury in vivo.
In order to dissect the structure-function relationship of the ICAM-1-fibrinogen interaction and to begin to elucidate its potential role in vascular cell responses we have generated a panel of monoclonal antibodies (mAbs) to ICAM-1. Using these mAbs to probe the ligand repertoire of ICAM-1, we have identified a discrete region in the first domain (22), which functions as a fibrinogen binding site and is distinct from previously recognized ICAM-1 ligand binding sites (23, 24, 25, 26).
Human umbilical vein endothelial
cells (HUVECs) were established from umbilical cords or purchased from
Clonetics (San Diego, CA). Cells were maintained in growth medium
(Clonetics) containing 10% heat-inactivated fetal bovine serum (FBS;
BioWhittaker, Walkersville, MD), 2 mM
L-glutamine (Irvine Scientific, Santa Ana, CA), 10 mM HEPES, and endothelial cell growth factor (Biomedical
Technologies, Stoughton, MA). In some experiments, HUVECs were
stimulated with 100 units/ml tumor necrosis factor (TNF
, Genzyme
Corp., Cambridge, MA) for 4-6 h at 37 °C (11). The promyelocytic
cell line HL-60 (American Type Culture Collection, Rockville, MD) and
EBV-transformed B lymphoma Daudi (American Type Culture Collection) and
JY were grown in RPMI 1640 (BioWhittaker) containing 10% FBS
(BioWhittaker), 2 mM L-glutamine (Irvine), 10 mM HEPES, and 10
5 M
2-mercaptoethanol (Eastman Kodak Co.). HL-60 cells were terminally differentiated to a monocytic phenotype by culture with 0.1 µM 1,25 dihydroxy vitamin D3 (Biomol,
Plymouth Meeting, PA) and 17.8 µg/ml indomethacin (Calbiochem), as
described (12). Peripheral blood mononuclear cells were isolated from
heparinized blood drawn from normal informed volunteers by differential
centrifugation on a Ficoll-Hypaque gradient, as described (27). T cells
were expanded from unstimulated peripheral blood mononuclear cells by
culture in RPMI 1640 plus 10% FBS following treatment with 1 µg/ml
phytohemagglutinin (Sigma) for 48 h at 37 °C,
followed by addition of 20 ng/ml recombinant interleukin 2 (27).
Chinese hamster ovary cells were transfected with a full-length
cDNA clone encoding ICAM-1 (22) in the mammalian expression vector
pRC/CMV by electroporation (Invitrogen, San Diego, CA), with selection of stable transfectants in Dulbecco's modified Eagle's medium (BioWhittaker) containing 10% FBS, 1 mM
L-glutamine, nonessential amino acids plus 1 mg/ml G418
(Life Science, Grand Island, NY), as described (12).
BALB/c mice were immunized intraperitoneal with
107 viable Daudi lymphocytes in phosphate-buffered saline
(PBS), pH 7.4. Screening for hybridoma selection included: (i) flow
cytofluorometric reactivity with resting or TNF-stimulated HUVECs;
(ii) effect on 125I-fibrinogen binding to HUVECs; and (iii)
effect on fibrinogen-dependent leukocyte-endothelium
interaction (11, 12). Four hybridomas, all of the IgG1 isotype and
designated 3D6, 6E6, 1G12, and 2D5, were established by two rounds of
subcloning by limiting dilution. Purified IgG of all mAbs was prepared
by affinity chromatography on immobilized protein A (Bio-Rad).
Anti-ICAM-1 mAbs 7.5C2, RR1/1, and 8.4A2 were characterized previously
(23). Anti-ICAM-1 mAbs 7F7 and LB-2 were generously provided by Drs. T. Schulz and E. A. Clark, respectively. Anti-ICAM-1 mAb CBR-IC1/4 was
characterized at the Fifth Leukocyte Typing Workshop (28). Anti-CD11b
mAb OKM1 and anti-effector cell protease receptor-1 mAb 6A11 were used
as controls.
The experimental procedures for the isolation and characterization of fibronectin-depleted (<0.1 ng/mg of protein) human fibrinogen have been reported (29). Fibrinogen was 125I labeled by the IODO-GEN method (30) to a specific activity of 0.3 µCi/µg of protein, with separation of free from protein-bound radioactivity by chromatography on a Sephadex G-25 column (Pharmacia Biotech, Inc.) preequilibrated with PBS, pH 7.4. Transferrin was purchased from Sigma and dissolved in PBS, pH 7.4.
Immunoprecipitation and ImmunoblottingJY lymphocytes (107/ml) were surface labeled with 125I-labeled sodium by the IODO-GEN method (30), washed to remove nonincorporated radioactivity, and lysed in 50 mM Tris-HCl, pH 8.3, 150 mM NaCl, 0.5% Triton X-100, 0.5% Nonidet P-40, 50 mM leupeptin, 100,000 IU/ml Trasylol, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (Boehringer Mannheim) and 100 µg/ml soybean trypsin inhibitor (Sigma) for 30 min on ice. Cell extracts were centrifuged at 15,000 × g for 20 min at 4 °C to remove nuclei and other detergent-insoluble materials, precleared, and incubated with the various primary mAbs for 16 h at 4 °C with agitation. The immune complexes were precipitated by addition of 0.2 ml of Sepharose CL4B-conjugated protein A (0.2 g/ml) (Pharmacia) for 4 h at 4 °C, washed in lysis buffer, boiled for 5 min at 100 °C, and electrophoresed on a 7.5% SDS-polyacrylamide gel followed by autoradiography using a Kodak X-Omat AR x-ray film and intensifying screens (DuPont de Nemours, Wilmington, DE). For immunoblotting, detergent-solubilized JY extracts were electrophoresed on a 7.5% SDS-polyacrylamide gel, transferred onto polyvinylidene difluoride membranes (Immobilon, Millipore Corp., Bedford, MA) for 2 h at 450 mAmp, blocked in 5% nonfat dry milk for 1 h at 4 °C, and incubated with 25 µg/ml aliquots of the various primary mAbs for 1 h at 4 °C in 5% dry milk. After the washes, the transfer membranes were incubated with alkaline phosphatase-conjugated goat anti-mouse IgG (Promega, Madison, WI) for 30 min at 22 °C and washed, and protein bands were visualized using nitro blue tetrazolium (Sigma) as a substrate.
Binding StudiesThe experimental procedures for the binding of 125I-fibrinogen to ICAM-1-expressing cells have been described (11). Briefly, serum-free suspensions of JY lymphocytes (1.5 × 107/ml) were mixed with 0.44 µM 125I-fibrinogen in the presence of 2.5 mM CaCl2 for 20 min at 22 °C. At the end of the incubation, cell surface-associated radioactivity was separated from unbound material by centrifugation of 300-µl aliquots of the JY incubation reaction through a mixture of silicone oil (Dow Corning, New Bedford, MA) at 15,000 × g for 5 min, and radioactivity was determined in a gamma counter. Alternatively, confluent monolayers of ICAM-1 transfectants were incubated with 0.44 µM 125I-fibrinogen for 20 min at 22 °C as described above, washed three times in serum-free RPMI 1640, solubilized in 20% SDS, and counted in a gamma counter. Nonspecific binding (10-30%) was assessed in the presence of a 50-fold molar excess of unlabeled fibrinogen added at the start of the incubation and was subtracted from the total to calculate net specific binding. In mAb inhibition experiments, JY lymphocytes or ICAM-1 transfectants were incubated with 25 µg/ml control mAb 6A11 or the various anti-ICAM-1 mAbs for 45 min at 37 °C, washed, and incubated with 0.44 µM 125I-fibrinogen as described above before determination of specific binding.
Fibrinogen-dependent Leukocyte-Endothelium InteractionSerum-free suspensions of JY lymphocytes at 5 × 106/ml were labeled with 0.5 mCi 51Cr
(Na2CrO4; specific activity, 487.4 mCi/mg; DuPont NEN) for 2 h at 37 °C with incorporation of
~2-6 cpm/cell. After being washed in PBS, pH 7.4, cells were
equilibrated with 0.44 µM fibrinogen or control protein
transferrin in the presence of 2.5 mM CaCl2 and
100 µM
D-phenylalanyl-L-prolyl-L-arginine
chloromethyl ketone for 20 min at 22 °C before addition (1.5-3 × 105 cells/well) to resting or TNF-stimulated HUVEC
monolayers. After a 30-min incubation at 22 °C, wells were washed
and attached cells were solubilized in 20% SDS with determination of
radioactivity in a beta counter. For mAb inhibition experiments,
resting or TNF
-stimulated HUVECs were incubated with 25 µg/ml
control mAb 6A11 or the various anti-ICAM-1 mAbs for 45 min at
37 °C, washed, and mixed with 51Cr-labeled JY
lymphocytes preequilibrated with 0.44 µM fibrinogen before determination of specific cell adhesion under the various conditions tested, as described above. Fibrinogen-dependent
monocyte transendothelial cell migration was assessed as described
(12), using HUVEC monolayers grown to confluency on gelatin-coated
porous Transwell membranes (diameter 3-8 µm, Costar). Cells were
incubated with 25 µg/ml control mAb 6A11 or the various anti-ICAM-1
mAbs for 30 min at 37 °C before addition of vitamin
D3-differentiated HL-60 cells, stimulated with 10 µM formyl-methionyl-leucyl-phenylalanine (Sigma) and preequilibrated with 0.44 µM
fibrinogen or control protein transferrin, and 2.5 mM
CaCl2. After a 2-h incubation at 37 °C, HL-60 cells
transmigrated under the various conditions tested were recovered from
the bottom of the well and counted microscopically by vital
staining.
The experimental procedures for the preparation and purification of recombinant ICAM-1-Fc from COS7 cells transiently transfected with the ICAM-1 construct were reported previously (23, 27). The effect of various anti-ICAM-1 mAbs on T cell adhesion to immobilized recombinant ICAM-1-Fc was investigated as described (27). Briefly, 5 × 107 PHA/interleukin 2-activated T cells were labeled with 25 µCi of [3H]thymidine overnight and washed three times in assay medium. Following the LFA-1 activation protocol in 2 mM MgCl2 and 1 mM EGTA in 20 mM HEPES buffer containing 140 mM NaCl and 2 mg/ml D-glucose (31), T cells were briefly spun at 75 × g for 1 min onto 96-well microtiter plates preparatively coated with recombinant ICAM-1-Fc at 0.24 µg/well overnight at 4 °C and postcoated with 2.5% bovine serum albumin for 1 h at 22 °C (31). In inhibition experiments, ICAM-1-Fc-coated plates were incubated with increasing concentrations (0.5-10 µg/ml) of the various anti-ICAM-1 mAbs before addition of [3H]-labeled T cells (2 × 105/well) for a 30-min incubation at 37 °C. After the wells were washed, radioactivity associated with the wells under the various conditions tested was determined in a beta counter.
In another series of experiments, ICAM-1-Fc was spotted onto plastic microtiter plates at a concentration of 50 µg/ml and allowed to adsorb for 2 h at 37 °C. Spots were aspirated to dryness and wells were postcoated with PBS, pH 7.4, plus 1% bovine serum albumin. Plates were incubated with 10 µg/ml of control or the various anti-ICAM-1 mAbs for 30-60 min before addition of aliquots of a suspension of trophozoite-infected erythrocytes at 8% parasitemia, 2% hematocrit, for 1 h at 37 °C, resuspending the mixture every 10 min, as described previously (23). Plates were washed with binding medium containing 1% glutaraldehyde to fix cells to the plate, prior to Giemsa staining and drying. The number of parasitized red blood cells (PRBCs) adherent per square millimeter was determined under high power light microscopy.
Epitope MappingA panel of domain expression and
homolog-scanning mutants of ICAM-1 was utilized as described (23). The
panel permits the assignment of mAb epitopes to individual domains or
combinations of domains and sublocalization within domain 1 of ICAM-1
(23). cDNAs encoding ICAM-1 mutants into the pCDM8 expression
vector were transfected into COS7 cells using the DEAE-dextran method. Transfection was allowed to proceed for 2-4 h in the presence of
chloroquine before washes and a 2-min treatment with 10% DMSO (23).
After culture in fresh Dulbecco's modified Eagle's medium plus 10%
newborn calf serum, 2 mM L-glutamine, and
penicillin/streptomycin for 24 h at 37 °C, transfected cells
were detached by trypsin-EDTA treatment and replated. Forty-eight to
72 h after transfection, cells were detached with 2 mM
EDTA, washed, and suspended in PBS, pH 7.4, containing 1% bovine serum
albumin, 1% newborn calf serum at a concentration of 5 × 105/ml. Cells were added to 96-well microtiter plates
(105 cells/well) and stained with control or with the
various anti-ICAM-1 mAbs for 30 min on ice before being washed and
incubated with fluorescein-conjugated polyvalent goat anti-mouse IgG
(Sigma) for 30 min on ice. Cells were washed, fixed in
PBS plus 1% FBS and 1% formalin, and stored at 4 °C before flow
cytofluorometric analysis on a Coulter EPICS cell sorter as described
(23). In another series of experiments, aliquots (1 × 107/ml) of resting or TNF-stimulated HUVECs or wild-type
ICAM-1 transfectants were harvested, washed once in PBS/EDTA and twice in PBS, pH 7.4, blocked in 20% human serum for 30 min at 0 °C, and
incubated with 25 µg/ml of the various primary mAbs in PBS, pH 7.4, plus 2% bovine serum albumin for 1 h on ice. After the washes,
the cells were stained with a 1:20 dilution of fluorescein-conjugated goat F(ab
)2 anti-mouse IgG (Tago Inc., Burlingame, CA) for
45 min on ice, washed, and immediately analyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA).
Four murine mAbs
raised against ICAM-1+ Daudi cells were analyzed for
reactivity with ICAM-1-expressing cells by flow cytometry. The newly
generated mAbs 1G12 and 2D5 bound to resting HUVECs with a broad and
heterogeneous pattern of reactivity, indistinguishable from that
observed with anti-ICAM-1 mAb LB-2 (Fig. 1A).
HUVEC stimulation with TNF resulted in a coordinated 5-10-fold
increased mAb reactivity (Fig. 1B), in agreement with
previous observations (3). In parallel experiments, all four newly
generated mAbs (1G12, 2D5, 3D6, and 6E6), plus control anti-ICAM-1 mAb
LB-2, strongly and homogeneously bound to stable ICAM-1 transfectants (Fig. 1C). In immunoprecipitation and immunoblotting, all
four new mAbs recognized a band of ~90 kDa, indistinguishable from that resolved by control anti-ICAM-1 mAb LB-2 (Fig. 2
and data not shown) and consistent with the molecular size and
structural organization of ICAM-1 (2). In contrast, no specific bands were immunoprecipitated or immunoblotted by control mAb OKM1 under the
same experimental conditions (Fig. 2).
Differential Inhibition of ICAM-1-dependent Adherence by Novel Anti-ICAM-1 mAbs
The effect of the new mAb panel on
ICAM-1-fibrinogen recognition was investigated. Saturating
concentrations of anti-ICAM-1 mAb 2D5 inhibited the binding of
125I-fibrinogen to JY lymphocytes (Fig.
3A) or ICAM-1 transfectants (Fig.
3B) by 70-90%, whereas control mAb 6A11 and anti-ICAM-1 mAbs 3D6 and 6E6 were ineffective under the same experimental conditions (Fig. 3). Anti-ICAM-1 mAb 1G12 partially inhibited 125I-fibrinogen binding to JY lymphocytes by 60% and to
ICAM-1 transfectants by 30-45% (Fig. 3). In parallel experiments,
anti-ICAM-1 mAb 1G12 or 2D5 completely inhibited the
fibrinogen-dependent increase in JY lymphocyte adhesion to
resting or TNF-stimulated HUVEC (Fig. 4A)
and abrogated transendothelial cell migration of vitamin D3-differentiated HL-60 cells mediated by fibrinogen (Fig.
4B), in agreement with previous observations (12). In
contrast, control mAb 6A11 and anti-ICAM-1 mAbs 3D6 and 6E6 did not
reduce fibrinogen-dependent adhesion of JY lymphocytes to
resting or TNF
-stimulated HUVEC (Fig. 4A) or
transendothelial migration of differentiated HL-60 cells mediated by
fibrinogen (Fig. 4B) under the same experimental conditions.
No disruption of the HUVEC monolayer was microscopically observed under
the various conditions tested (not shown).
The effect of the new mAb panel on ICAM-1 recognition of
2 integrin LFA-1 (32) or P. falciparum-infected erythrocytes (7) was next investigated.
Consistent with the inability of mAbs 1G12 and 2D5 to diminish binding
of vitamin D3-differentiated HL-60 cells to ICAM-1
transfectants (12), these mAbs did not significantly reduce the binding
of PHA-activated human T cells to immobilized recombinant ICAM-1-Fc at
any concentration tested (Fig. 5A). In contrast, increasing concentrations of mAb 3D6 or 6E6 completely blocked T cell adhesion to ICAM-1-Fc-coated plates in a
dose-dependent manner (Fig. 5A). In control
experiments, anti-CD11a mAb 38 and anti-ICAM-1 mAbs 15.2 and 7.5C2 also
inhibited T cell attachment to immobilized ICAM-1-Fc in a
dose-dependent fashion (Fig. 5A), in agreement
with previous observations (23). Finally, the fibrinogen-blocking, LFA-1-nonblocking mAbs 1G12 and 2D5 both completely inhibited adhesion
of P. falciparum-infected erythrocytes to ICAM-1-Fc-coated plates (Fig. 5B), whereas the LFA-1-blocking,
fibrinogen-nonblocking mAbs 3D6 and 6E6 produced only a partial and
variable degree of inhibition of PRBC binding to immobilized ICAM-1-Fc
under the same experimental conditions (not shown).
Epitope Mapping and Residue Assignment of Novel Anti-ICAM-1 mAbs
We initially tested the four new mAbs on domain expression
constructs. It was apparent from direct mAb binding experiments to
recombinant human/mouse ICAM-1 chimeras that mAbs 1G12 and 2D5 both
recognized constructs containing human domain 1, as expressed in the
wild-type ICAM-1 sequence or in the h123, h1234, or h1 m2345 construct
(Fig. 6). In contrast, mAbs 3D6 and 6E6 both recognized ICAM-1 constructs containing human domain 2, as expressed in wild-type ICAM-1, h123, h1234, and m1 h2345 but not in h345. Thus, mAbs 1G12 and
2D5 map to domain 1 of ICAM-1, whereas mAbs 3D6 and 6E6 recognize
domain 2 (Fig. 6). Within domain 1, the binding of mAbs 1G12 and 2D5
was completely disrupted by the mutation D26QPKL/KEDLS (Fig. 6), a
region predicted to lie at the amino-terminal apex of ICAM-1 (23), and
was reduced by 60-70% as a result of the mutation P70DG/GTV (Fig. 6)
in a region predicted to form a neighboring loop in the first ICAM-1
domain (23). Established epitope-mapped anti-ICAM-1 mAbs were also
tested for their ability to affect fibrinogen-dependent
adhesion of JY lymphocytes to HUVECs. In agreement with the epitope
prediction studies reported above, two D26-sensitive mAbs, 7.5C2 and
CBR-IC1/4, (23, 28) inhibited lymphocyte-endothelium bridging mediated
by fibrinogen by 97 and 43%, respectively (Table I).
Consistent with the requirement of the neighboring loop in the
formation of the fibrinogen binding pocket on ICAM-1, mAb RR1/1, whose
epitope(s) was disrupted by P70 mutation (23), also inhibited
fibrinogen-dependent JY lymphocyte adhesion to HUVECs by
85% (Table I). In contrast, mAbs 7F7, which maps to another area of
domain 1 (23), or 8.4A6, recognizing domain 2 of ICAM-1, failed to
decrease fibrinogen-dependent intercellular adhesion under
the same experimental conditions (Table I).
|
In this study, we used a novel mAb panel specific for distinct ICAM-1 epitopes to dissect the ICAM-1 recognition for fibrinogen and its relationship with the binding site(s) for LFA-1 integrin and P. falciparum-infected erythrocytes. Based on inhibition studies and epitope mapping with recombinant ICAM-1 chimeras and receptor mutants, we have identified a novel fibrinogen binding site on the first Ig-like domain of ICAM-1, completely disrupted by mutation of D26 and partially affected by mutation of P70. This region was distinct from the LFA-1 interacting site (26), whereas it more completely overlapped the ICAM-1 recognition of P. falciparum-infected erythrocytes (23). Interestingly, the same residues, D26 and P70, have been previously shown to contribute part of the binding site of the major Rhinovirus serogroup on domain 1 of ICAM-1 (25).
The initial suggestion that the association of fibrinogen with ICAM-1 involved structurally distinct requirements from LFA-1 recognition (26) came from the limited inhibition of ligand binding obtained with mAb LB-2 (11), which maps to the LFA-1 binding site at K40 and L43 on domain 1 (23). Consistent with this prediction, the novel anti-ICAM-1 mAbs 2D5 and 1G12 described here completely suppressed the recognition of fibrinogen without affecting the LFA-1 binding site, and conversely, mAbs 3D6 and 6E6 failed to reduce fibrinogen binding but completely abolished the ICAM-1-LFA-1 interaction. Previous studies demonstrated that the binding sites for PRBCs and LFA-1 are spatially distinct (23) and could be located on essentially opposite sides of domain 1 of ICAM-1 by molecular modeling (23). The fact that mAbs 1G12 and 2D5 inhibit equally well both fibrinogen and PRBC binding to ICAM-1 places the fibrinogen binding site in a structural region separate from the LFA-1 site (23, 32). Three of the four described mAbs sensitive to the mutation D26QPKL/KEDS (1G12, 2D5, and 7.5C2) block fibrinogen binding, whereas the fourth mAb (CBR-IC1/4) has a partial inhibitory effect. Intriguingly, mAbs 7.5C2 and CBR-IC1/4 also block LFA-1 access to ICAM-1 but not PRBC binding (23, 28). Thus, four mAbs have now been shown to recognize a very limited region of the domain but have quite distinct functional effects. The simplest explanation for this finding, which has previously been demonstrated for CD4 (33), is that the two sets of mAbs bind to a closely related region of the molecule but approach it in quite different directions. This may also mean that the direct binding site(s) for fibrinogen, PRBCs, or LFA-1 may not involve these specific residues, but an adjacent region, dependent on the precise footprint of the mAb and the positioning of the Fc portion once it is bound. We were unable to test this possibility using direct binding of 125I-fibrinogen to human/murine ICAM-1 chimeras because it has been previously demonstrated that murine ICAM-1 recognizes human fibrinogen (11).
The main implication of these observations is that the
ICAM-1-fibrinogen pathway of intercellular bridging (11, 12, 13, 15)
operates structurally independently of 2 integrins LFA-1 and Mac-1, whose binding sites on ICAM-1 have been previously localized
to domains 1 (see above), and 3 (34), respectively. This suggests that
leukocyte-endothelium interaction mediated by fibrinogen may be also
independently regulated from the stepwise adhesion cascade contributed
by selectins and
2 integrins (1, 2). In this context,
fibrinogen supported firm adhesion of monocytic cells to rabbit
mesentery endothelium in vivo, even in the absence of an
initial selectin-dependent component of leukocyte tethering
and rolling (15). Alternatively, the membrane-distal location of the
fibrinogen-recognition site on ICAM-1, at the apex of the protein,
makes it ideally positioned to act in an accessory fashion to
cooperatively potentiate LFA-1- or Mac-1-dependent leukocyte adherence, bridging the distance between cells while the
steric inhibition of the cellular glycocalyx is overcome. Along these
lines, it is noteworthy that although the fibrinogen binding site may
have an impact on the LFA-1 site, as judged by the effect of
cross-blocking mAbs and P70 mutations, the ICAM-1-LFA-1 interaction,
conversely, can be entirely abrogated by mAbs to domain 2, which have
no effect on fibrinogen recognition, including the novel mAbs 3D6 and
6E6 and mAbs 8.4A6 and R6.5D6, characterized in previous studies (23).
This opens the attractive possibility that fibrinogen occupancy, while
initiating intercellular bridging per se, may shift the
adhesive balance between
2 integrins and ICAM-1 by
primarily cooperating with Mac-1-dependent interactions. The potential pathophysiological implications of these observations are
underscored by the prominent role of fibrinogen as a major risk factor
for atherosclerosis and vascular diseases, invariably characterized by
increased leukocyte adhesion to endothelium and infiltration of the
arterial intima (19). Indeed, elevated plasma concentrations of
fibrinogen have been shown to correlate with an increased adhesion of
leukocytes to endothelium in patients with advanced atherosclerosis
(35).
In summary, the identification of a novel fibrinogen binding site on ICAM-1 may help in elucidating the pleiotropic contribution of this adhesive receptor to inflammation and vascular injury (36). The differential inhibitory properties of the mAb panel described here may be beneficial to selectively targeting specific aspects of ICAM-1-dependent adherence and leukocyte recruitment at inflammatory sites in vivo.
We thank Drs. T. Shulz, R. Rothlein, and E. A. Clark for providing anti-ICAM-1 mAbs.