Molecular Identification of a Novel Fibrinogen Binding Site on the First Domain of ICAM-1 Regulating Leukocyte-Endothelium Bridging*

(Received for publication, July 22, 1996, and in revised form, October 14, 1996)

Alain Duperray Dagger , Lucia R. Languino §, Janet Plescia §, Alison McDowall par , Nancy Hogg par , Alister G. Craig **, Anthony R. Berendt ** and Dario C. Altieri §Dagger Dagger

From the Dagger  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 par  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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

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 beta 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.


INTRODUCTION

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 beta 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).


EXPERIMENTAL PROCEDURES

Cells and Cell Culture

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 alpha  (TNFalpha , 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).

mAbs

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 TNFalpha -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.

Protein Purification and Labeling

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 Immunoblotting

JY 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 Studies

The 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 Interaction

Serum-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 TNFalpha -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 TNFalpha -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.

Adhesion Assay

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 Mapping

A 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 TNFalpha -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).


RESULTS

Establishment of Novel Anti-ICAM-1 mAbs

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 TNFalpha 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).


Fig. 1. Flow cytofluorometric characterization of new anti-ICAM-1 mAbs. Resting HUVECs (A), TNFalpha -stimulated HUVECs (B), or stable ICAM-1 transfectants (C) were blocked in 20% human serum and analyzed for their reactivity by flow cytometry with control anti-CD11b mAb OKM1, anti-ICAM-1 mAb LB-2, or the novel anti-ICAM-1 mAbs 1G12 and 2D5 in A and B or with 1G12, 2D5, 3D6, and 6E6 in C. Background fluorescence obtained with mAb OKM1 is shown for each cell type as a negative control. Horizontal and vertical axes measure fluorescence intensity on a 4-log scale and the cell number, respectively.
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Fig. 2. Characterization of anti-ICAM-1 mAbs by immunoprecipitation and immunoblotting. A, 125I-labeled detergent-solubilized extracts of CD11b/CD18- JY lymphocytes were immunoprecipitated with control mAb OKM1 or anti-ICAM-1 mAb 1G12 2D5, 3D6, 6E6, or LB-2. After addition of protein A-conjugated Sepharose CL4B, immunoprecipitated proteins were washed, electrophoresed on a 7.5% SDS-polyacrylamide gel under reducing conditions, and visualized by autoradiography. B, detergent-solubilized JY lymphocyte extracts were electrophoresed on a 7.5% SDS-polyacrylamide gel under reducing conditions, transferred to Immobilon membranes, and incubated with control mAb OKM1 or anti-ICAM-1 mAb 1G12 or 2D5 for 1 h at 4 °C. The membrane was washed, incubated with alkaline-phosphatase-conjugated goat anti-mouse IgG, and washed again, and protein bands were visualized by using tetrazolium salts.
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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 TNFalpha -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 TNFalpha -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).


Fig. 3. Effect of anti-ICAM-1 mAbs on 125I-fibrinogen binding to ICAM-1-expressing cells. Serum-free suspensions of JY lymphocytes (A) or monolayers of Chinese hamster ovary transfectants expressing ICAM-1 (B) were incubated with 25 µg/ml control mAb 6A11 or the various indicated anti-ICAM-1 mAbs for 45 min at 37 °C, washed, and mixed with 0.44 µM 125I-fibrinogen in the presence of 2.5 mM CaCl2 for an additional 20 min at 22 °C before quantitation of specific binding. Data for both panels are the mean ± S.D. of two independent experiments.
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Fig. 4. Effect of anti-ICAM-1 mAbs on fibrinogen-dependent leukocyte-endothelium interaction. A, resting or TNFalpha -stimulated HUVEC monolayers were incubated with 25 µg/ml of control mAb 6A11 or the various anti-ICAM-1 mAbs for 45 min at 37 °C. Suspensions of 51Cr-labeled JY lymphocytes were preequilibrated with 0.44 µM fibrinogen, 100 µM D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone, and 2.5 mM CaCl2 for 20 min at 22 °C before addition to resting or TNFalpha -stimulated HUVECs and determination of cell attachment after a 30-min incubation at 22 °C. B, HUVEC monolayers were grown to confluence on Transwell membranes, equilibrated with control mAb 6A11 or the various anti-ICAM-1 mAbs for 30 min at 37 °C, and further incubated with formyl-methionyl-leucyl-phenylalanine-stimulated vitamin D3-differentiated HL-60 cells preequilibrated with 0.44 µM fibrinogen. After a 2-h incubation at 37 °C, migrated cells were recovered from the bottom of the well and counted microscopically by vital staining. Background levels of JY adhesion (A) or transendothelial migration of differentiated HL-60 (B) without inhibitors were determined in the presence of comparable concentrations of control protein transferrin, under the same experimental conditions. For both panels, data are the mean ± S.E. of at least three independent experiments.
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The effect of the new mAb panel on ICAM-1 recognition of beta 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).


Fig. 5. Effect of anti-ICAM-1 mAbs on ICAM-1 recognition of beta 2 integrin LFA-1 (A) or P. falciparum-infected erythrocytes (B). Ninety-six-well plastic microtiter plates were coated with recombinant ICAM-1-Fc, washed, and postcoated with assay buffer containing 2.5% albumin. A, ICAM-1-Fc-coated plates (0.24 µg/well) were incubated with the indicated increasing concentrations of the various anti-ICAM-1 mAbs or control anti-CD11a mAb 38 and further mixed with [3H]-labeled PHA-activated human T cells (2 × 105/well) for 30 min at 37 °C before washing and quantitation of specific cell attachment. B, the experimental conditions were essentially as in A, except that immobilized ICAM-1-Fc (50 µg/ml) was incubated with the various mAbs (10 µg/ml) for 30-60 min at 22 °C before addition of a suspension of trophozoite-infected erythrocytes at 8% parasitemia, 2% hematocrit for 1 h at 37 °C, and quantitation of PRBC adhesion by Giemsa staining. For both panels, data are the mean ± S.D. of 2 (A) or 4 (B) determinations.
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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).


Fig. 6. Epitope mapping of novel anti-ICAM-1 mAbs. The construction and nomenclature of ICAM-1 human/mouse chimeras and of targeted point mutations in ICAM-1 domain 1 were as described (23). Binding of anti-ICAM-1 mAbs 1G12, 2D5, 3D6, and 6E6 to the various recombinant ICAM-1 constructs was determined by flow cytometry and calculated as percent epitope expression as compared with control mAb binding. T20, T20CS/DCK; D26, D26QPKL/KEDLS; L43, L43LPGN/E-SGP; P70, P70DG/GTV; h1 m2345, human domain 1, murine domains 2-5; m1 h2345, murine domain 1, human domains 2-5; h12, human domains 1 and 2; h123, human domains 1-3; h345, human domains 3-5. Data are the means of at least two independent determinations.
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Table I.

Effect of anti-ICAM-1 mAbs on fibrinogen-dependent JY lymphocyte adhesion to resting HUVECs.

The experimental conditions are the same as in Fig. 4A. Control JY lymphocyte adhesion to HUVECs was 18.5 and 32.1% in the absence and in the presence of fibrinogen, respectively. The epitope assignment (23, 28) of the various anti-ICAM-1 mAbs used is as follows: mAbs 7.5C2 and CBR-IC1/4, domain 1, D26-sensitive; mAb RR1/1, domain 1, P70-sensitive; mAb 7F7, domain 1, L42-sensitive; mAb 8.4A6, domain 2. Data are expressed as the mean ± S.D. of two independent determinations.
mAb Inhibition of adhesion

% of control
2D5 93.2  ± 14.2
7.5C2 95.6  ± 18.5
CBR-IC1/4 43.0  ± 10.2
RR1/1 85.3  ± 13.3
8.4A6 11.2  ± 9.2
7F7 3.5  ± 7.2


DISCUSSION

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 beta 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 beta 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 beta 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.


FOOTNOTES

*   This work was supported by National Institutes of Health Grants HL43773, HL54131 (to D. C. A.), and CA71870 (to L. R. L.) and by Donaghue Medical Research Foundation Grant 95-006 (to L. R. L.) This work was done during the tenure of an American Heart Association Established Investigatorship Award to Dr. Altieri. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger Dagger    To whom correspondence should be addressed: Yale University School of Medicine, BCMM 436B, 295 Congress Ave., New Haven, CT 06536. Tel.: 203-737-2869; Fax: 203-737-2290.
1    The abbreviations used are: ICAM-1, intercellular adhesion molecule-1; FBS, fetal bovine serum; HUVEC, human umbilical vein endothelial cell; mAb, monoclonal antibody; PRBC, parasitized red blood cell; TNFalpha , tumor necrosis factor alpha ; PBS, phosphate-buffered saline.

Acknowledgments

We thank Drs. T. Shulz, R. Rothlein, and E. A. Clark for providing anti-ICAM-1 mAbs.


REFERENCES

  1. Springer, T. A. (1994) Cell 76, 301-314 [Medline] [Order article via Infotrieve]
  2. Carlos, T. M., and Harlan, J. M. (1994) Blood 84, 2068-2101 [Abstract/Free Full Text]
  3. Pober, J. S., Gimbrone, M. A., Jr., Lapierre, L. A., Mendrick, D. L., Fiers, W., Rothlein, R., and Springer, T. A. (1986) J. Immunol. 137, 1893-1896 [Abstract/Free Full Text]
  4. Smith, C. W., Marlin, S. D., Rothlein, R., Toman, C., and Anderson, D. C. (1989) J. Clin. Invest. 83, 2008-2017 [Medline] [Order article via Infotrieve]
  5. Diamond, M. S., Staunton, D. E., de Fougerolles, A. R., Stacker, S. A., Garcia-Aguilar, J., Hibbs, M. L., and Springer, T. A. (1990) J. Cell Biol. 111, 3129-3139 [Abstract]
  6. Greve, J. M., Davis, G., Meyer, A. M., Forte, C. P., Yost, S. C., Marlor, C. W., Kamarck, M. E., and McClelland, A. (1989) Cell 56, 839-847 [Medline] [Order article via Infotrieve]
  7. Berendt, A. R., Simmons, D. L., Tansey, J., Newbold, C. I., and Marsh, K. (1989) Nature 341, 57-59 [CrossRef][Medline] [Order article via Infotrieve]
  8. Smith, J. D., Chitnis, C. E., Craig, A. G., Roberts, D. J., Hudson-Taylor, D. E., Peterson, D. S., Pinches, R., Newbold, C. I., and Miller, L. H. (1995) Cell 82, 101-110 [Medline] [Order article via Infotrieve]
  9. Turner, G. D. H., Morrison, H., Jones, M., Davis, T. M. E., Looareesuwan, S., Buley, I. D., Gatter, K. C., Newbold, C. I., Pukritayakamee, S., Nagachinta, B., White, N. J., and Berendt, A. R. (1994) Am. J. Pathol. 145, 1057-1069 [Abstract]
  10. McCourt, P. A. G., Ek, B., Forsberg, N., and Gustafson, S. (1994) J. Biol. Chem. 269, 30081-30084 [Abstract/Free Full Text]
  11. Languino, L. R., Plescia, J., Duperray, A., Brian, A. A., Plow, E. F., Geltosky, J. E., and Altieri, D. C. (1993) Cell 73, 1423-1434 [Medline] [Order article via Infotrieve]
  12. Languino, L. R., Duperray, A., Joganic, K. J., Fornaro, M., Thornton, G. B., and Altieri, D. C. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1505-1509 [Abstract]
  13. Shetty, S., Kumar, A., Pueblitz, S., Emri, S., Gungen, Y., Johnson, A. R., and Idell, S. (1996) Thromb. Haemostasis 75, 782-790 [Medline] [Order article via Infotrieve]
  14. van de Stolpe, A., Jacobs, N., Hage, W. J., Tertoolen, L., Y., v. K., Novakova, I. R. O., and de Witte, T. (1996) Thromb. Haemostasis 75, 182-189 [Medline] [Order article via Infotrieve]
  15. Sriramarao, P., Languino, L. R., and Altieri, D. C. (1996) Blood 88, 3416-3423 [Abstract/Free Full Text]
  16. Durieu-Trautmann, O., Chaverot, N., Cazaubon, S., Strosberg, A. D., and Couraud, P.-O. (1994) J. Biol. Chem. 269, 12536-12540 [Abstract/Free Full Text]
  17. Chirathaworn, C., Tibbetts, S. A., Chan, M. A., and Benedict, S. H. (1995) J. Immunol. 155, 5479-5482 [Abstract]
  18. Hicks, R. C. J., Golledge, J., Mir-Hasseine, R., and Powell, J. T. (1996) Nature 379, 818-820 [Medline] [Order article via Infotrieve]
  19. Ross, R. (1993) Nature 362, 801-809 [CrossRef][Medline] [Order article via Infotrieve]
  20. Bini, A., Fenoglio, J., Jr., Mesa-Tejada, R., Kudryk, B., and Kaplan, K. L. (1989) Arteriosclerosis 9, 109-121 [Abstract]
  21. Poston, R. N., Haskard, D. O., Coucher, J. R., Gall, N. P., and Johnson-Tidey, R. R. (1992) Am. J. Pathol. 140, 665-673 [Abstract]
  22. Simmons, D., Makgoba, M. W., and Seed, B. (1988) Nature 331, 624-627 [CrossRef][Medline] [Order article via Infotrieve]
  23. Berendt, A. R., Mc Dowall, A., Craig, A. G., Bates, P. A., Sternberg, M. J. E., Marsh, K., Newbold, C. I., and Hogg, N. (1992) Cell 68, 71-81 [Medline] [Order article via Infotrieve]
  24. Ockenhouse, C. F., Betageri, R., Springer, T. A., and Staunton, D. E. (1992) Cell 68, 63-69 [Medline] [Order article via Infotrieve]
  25. McClelland, A., deBear, J., Connolly Yost, S., Meyer, A. M., Marlor, C. W., and Greve, J. M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7993-7997 [Abstract]
  26. Staunton, D. E., Marlin, S. D., Stratowa, C., Dustin, M. L., and Springer, T. A. (1990) Cell 61, 243-254 [Medline] [Order article via Infotrieve]
  27. Dransfield, I., Cbanas, C., Craig, A., and Hogg, N. (1992) J. Cell Biol. 116, 219-226 [Abstract]
  28. Craig, A. G., Turner, G. D. H., Pinches, R. A., Newbold, C. I., and Berendt, A. R. (1995) in Leukocyte Typing V (Schlossman, S., ed), pp. 1554-1557, Oxford University Press, Oxford
  29. Altieri, D. C., Bader, R., Mannucci, P. M., and Edgington, T. S. (1988) J. Cell Biol. 107, 1893-1900 [Abstract]
  30. Fraker, P. J., and Speck, J. C., Jr. (1978) Biochem. Biophys. Res. Commun. 80, 849-857 [Medline] [Order article via Infotrieve]
  31. Stewart, M. P., Cabanas, C., and Hogg, N. (1996) J. Immunol. 156, 1810-1817 [Abstract]
  32. Staunton, D. E., Dustin, M. L., Erickson, H. P., and Springer, T. A. (1990) Cell 61, 243-254 [Medline] [Order article via Infotrieve]
  33. Wang, J., Yan, Y., Garrett, T. P. J., Liu, J., Rodgers, D. W., Garlick, R. L., Tarr, G. E., Husain, Y., Reinherz, F. I., and Harrison, S. C. (1990) Nature 348, 411-418 [CrossRef][Medline] [Order article via Infotrieve]
  34. Diamond, M. S., Staunton, D. E., Marlin, S. D., and Springer, T. A. (1991) Cell 65, 961-971 [Medline] [Order article via Infotrieve]
  35. Duplaa, C., Couffinhal, T., Labat, L., Fawaz, J., Moreau, C., Bietz, I., and Bonnet, J. (1993) Eur. J. Clin. Invest. 23, 474-479 [Medline] [Order article via Infotrieve]
  36. Sligh, J. E., Jr., Ballantyne, C. M., Rich, S. S., Hawkins, H. K., Smith, C. W., Bradley, A., and Beaudet, A. L. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8529-8533 [Abstract/Free Full Text]

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