©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structural Recognition of a Novel Fibrinogen Chain Sequence (117133) by Intercellular Adhesion Molecule-1 Mediates Leukocyte-Endothelium Interaction (*)

(Received for publication, October 15, 1994)

Dario C. Altieri (1)(§) Alain Duperray (¶) Janet Plescia (1) George B. Thornton (2) Lucia R. Languino (2)(**)

From the  (1)From The Boyer Center for Molecular Medicine, Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06536 and (2)The R. W. Johnson Pharmaceutical Research Institute, San Diego, California 92121

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

In addition to its role in hemostasis, fibrinogen is obligatorily required to mount competent inflammatory responses in vivo. A molecular prerequisite of fibrinogen-dependent inflammation may reside in its ability to associate with intercellular adhesion molecule-1 (ICAM-1), and enhance monocyte adhesion to endothelium by bridging the two cell types. Structure-function characterization of the novel ICAM-1 recognition of fibrinogen was carried out by synthetic peptidyl mimicry of the fibrinogen chain. A novel peptide sequence, NNQKIVNLKEKVAQLEA, designated 3, dose-dependently inhibited (IC 20-40 µg/ml) binding of I-fibrinogen to endothelial cells or ICAM-1-expressing B lymphoblastoid Daudi cells. In contrast, none of the previously identified vascular cell fibrinogen interacting sequences was effective. Increasing concentrations of 3 completely inhibited fibrinogen-mediated adhesion of peripheral blood mononuclear cells or vitamin D(3)-differentiated monocytic HL-60 cells to endothelium, but did not affect leukocyte-endothelium interaction in the absence of fibrinogen. I-Labeled 3 bound specifically and saturably to genetically engineered ICAM-1 transfectants, but not to control non-transfected cells, and associated with ICAM-1 on cytokine-activated endothelium with a K of 34 µM. Consistent with functional recognition of ICAM-1, immobilized 3 supported adhesion of JY lymphoblasts in a dose-dependent reaction inhibited by monoclonal antibodies to ICAM-1. We conclude that a novel fibrinogen 3 sequence NNQKIVNLKEKVAQLEA binds to ICAM-1 and modulates ICAM-1-dependent adhesion. These findings define the structural basis of fibrinogen:ICAM-1 recognition and provide a potential selective target for inhibiting fibrinogen-dependent inflammatory responses.


INTRODUCTION

Although primarily recognized for preserving hemostasis(1) , fibrinogen plays a prominent role in inflammation. Through the structurally diversified recognition of disparate membrane receptors, regulated assembly of fibrinogen on leukocytes participates in intracellular signal transduction, transcription, and translation of activation-dependent genes, release of inflammatory cytokines, and modulation of various leukocyte effector functions (reviewed in (2) ). In this context, fibrinogen is obligatorily required to organize competent inflammatory responses in vivo. Extending earlier observations on the pathogenetic role of fibrinogen in delayed type hypersensitivity(3) , recent studies have demonstrated that systemically defibrinogenated animals fail to recruit monocytes/phagocytes after intra-abdominal piogenic challenges(4) , or at the site of biomaterial implant(5) , and are partially protected from acute tissue damage mediated by platelet-leukocyte cooperation during immune complex glomerulonephritis(6) . Adhesion of leukocytes to vascular endothelium is one of the earliest molecular prerequisites of all inflammatory responses(7, 8) . Although this process is contributed by a redundant ``adhesion cascade'' maintained by various classes of cell surface receptors(9) , recent evidence suggests that fibrinogen may also participate in this mechanism by acting as a bridging molecule between leukocytes and endothelial cells through a novel recognition of ICAM-1(^1)(10) . In order to elucidate the molecular requirements of fibrinogen-dependent inflammation, we have used synthetic peptidyl mimicry and genetically engineered transfectants to dissect the ICAM-1:fibrinogen recognition. The results indicate that a novel fibrinogen chain sequence 117-133 interacts with ICAM-1 and modulates ICAM-1-dependent cell adhesion(7, 8, 9) .


MATERIALS AND METHODS

Cells and Cell Cultures

Peripheral blood mononuclear cells (PBMC) were isolated by differential centrifugation on Ficoll-Hypaque gradient (Sigma) as described(11) . Promyelocytic HL-60(12) , B-lymphoblastoid JY(13) , and Daudi (10) cells were grown in RPMI 1640 (Whittaker Bioproducts Walkersville, MD) plus 10% fetal calf serum (Whittaker) and 2 mML-glutamine (Irvine Scientific, Santa Ana, CA). HL-60 cells were terminally differentiated to a monocyte-like phenotype in the presence of 0.1 µM 1,25-dihydroxy vitamin D(3) (BioMol, Plymouth Meeting, PA) and 17.8 µg/ml indomethacin (Calbiochem, San Diego, CA), according to published protocols(12) . Human umbilical vein endothelial cells (HUVEC, Clonetics, San Diego, CA) were maintained in culture as described(10) .

Synthetic Peptides, Protein Labeling, and Monoclonal Antibodies (mAbs)

Synthetic peptides containing various sequences of the fibrinogen alpha or chain were characterized in previous studies (11) . Fibronectin-depleted plasma fibrinogen was purified and I-labeled as described(10) . A peptide duplicating the fibrinogen chain sequence 117-133 NNQKIVNLKEKVAQLEA, and designated 3, was synthesized with Lys-Tyr-Gly residues at its H(2)N, and radiolabeled with NaI by the IODO-GEN method(14) , as described(11) . A control peptide containing the 3 sequence in scrambled order (ALENAEVQNLVKKIQKN) was also synthesized. Anti-CD11b mAb was OKM1(11) . Anti-ICAM-1 mAbs were LB-2 (Becton-Dickinson, Mountain View, CA), and the recently established mAbs 1G12, 2D5, and 6E6 functionally characterized for inhibition of I-fibrinogen binding to HUVEC, and of fibrinogen-mediated leukocyte-HUVEC interaction. (^2)

Transfection Experiments

A full-length cDNA clone encoding human ICAM-1 (15) in pRC/CMV (Invitrogen, San Diego, CA) was transfected in subconfluent cultures of Chinese hamster ovary (CHO) cells by electroporation. CHO cells were selected in Dulbecco's modified Eagle's medium (Whittaker) plus 10% fetal calf serum, 1 mML-glutamine, non-essential amino acids, and 1 mg/ml G418 (Geneticin, Life Technologies, Inc., Grand Island, NY), and cloned by limiting dilution at 0.5-2 cells/well. Phenotypical characterization of wild-type (WT) or ICAM-1 CHO transfectants was carried out by flow cytometry with anti-ICAM-1 mAbs 1G12, 2D5, or LB-2.

Binding Studies and Leukocyte-Endothelium Interaction

The fibrinogen-derived synthetic peptides were tested for their ability to block binding of I-fibrinogen to resting HUVEC monolayers or Daudi lymphoblasts, as described(10, 11) . In some experiments, confluent monolayers of ICAM-1 CHO transfectants or WT CHO cells were incubated in serum-free RPMI 1640 medium with increasing concentrations of I-labeled 3 (6-150 µg/ml) in the presence of 1 mM CaCl(2) and 1 mM MgCl(2) for 45 min at 22 °C. After washes, cells were solubilized in 10% SDS, and radioactivity associated under the various conditions was quantitated in a -counter. Nonspecific binding was assessed in the presence of a 100-fold molar excess of unlabeled 3, or control L10 peptide (402-411) added at the start of the incubation, and was subtracted from the total to calculate net specific binding. In another series of experiments, confluent HUVEC monolayers were stimulated with 100 units/ml tumor necrosis factor-alpha for 6 h at 37 °C, and incubated with increasing concentrations of I-3 or I-fibrinogen for 45 min at 22 °C, before calculation of specific binding as described above. Binding parameters for I-3 or I-fibrinogen association with HUVEC were calculated by the method of Scatchard using the Ligand program based on the algorithm of MacPherson(16) . The experimental procedures for leukocyte-HUVEC interaction mediated by fibrinogen have been described previously(10) . In peptide inhibition experiments, Cr-labeled PBMC or vitamin D(3)-differentiated HL-60 cells were preincubated with increasing concentrations (6-400 µg/ml) of 3 or control scrambled peptide, or with a single dose (400 µg/ml) of L10 (17) for 10 min at 22 °C. Cells were stimulated with 10 µM fMet-Leu-Phe, mixed with 150 µg/ml fibrinogen in the presence of 2.5 mM CaCl(2) and immediately added to confluent resting HUVEC monolayers. After a 30-min incubation at 22 °C, leukocyte adhesion under the various conditions tested was quantitated as described(10) .

Lymphoblast Adhesion Assays

Ninety-six well polystyrene plates were coated with increasing concentrations (1-50 µg/ml) of 3 or control peptide L10 in phosphate-buffered saline, pH 7.2, for 18 h at 4 °C. Peptide-coated wells were washed, blocked with 3% gelatin, and incubated in duplicates with Cr-labeled JY lymphoblasts (1 times 10^6/ml) for 45 min at 37 °C. After washes, specifically attached cells were solubilized in 10% SDS, and radioactivity was quantitated in a scintillation beta-counter. In mAb inhibition experiments, JY lymphoblasts were preincubated with 25 µg/ml of the recently established anti-ICAM-1 mAbs 1G12, 2D5, or 6E6^2 for 20 min at 22 °C, before quantitation of cell attachment onto peptide-coated plates as described above.


RESULTS

Molecular Mimicry of Fibrinogen-dependent LeukocyteEndothelium Bridging

Fibrinogen-derived synthetic peptides were tested for their ability to affect I-fibrinogen interaction with resting HUVEC monolayers(10) . Inhibitory concentrations of RGD-containing antagonists of alpha(V)beta(3) or alphabeta(3) integrin, GRGDSP, RGDS, or L10 (LGGAKQAGDV)(17) , did not diminish I-fibrinogen binding to HUVEC, in agreement with previous observations (10) (Table 1). Similarly, comparable concentrations of P1 (KYGWTVFQKRLDGSV), P1b (KYGQKRLDGSV), or GPRP, that block fibrinogen association with leukocyte integrins CD11b/CD18 (11) and CD11c/CD18(18) , respectively, had no effect on HUVEC-fibrinogen interaction (Table 1). In contrast, a novel synthetic peptide designated 3, and duplicating the fibrinogen chain sequence 117-133 (NNQKIVNLKEKVAQLEA), consistently inhibited by 40-50% I-fibrinogen binding to HUVEC, under the same experimental conditions (Table 1). None of the other partially overlapping synthetic peptides derived from the fibrinogen chain(11) , reduced I-fibrinogen binding to HUVEC (not shown). The ability of 3 to inhibit binding of I-fibrinogen to Daudi lymphoblasts, which are completely devoid of CD11b/CD18 (2) while they express ICAM-1 and adhere to HUVEC in a fibrinogen-dependent manner(10) , was also investigated. I-Fibrinogen bound specifically and saturably to Daudi lymphoblasts (not shown), in a reaction dose-dependently inhibited by increasing concentrations of 3, and unaffected by control peptides L10, P1, or GPRP, under the same experimental conditions (Fig. 1A). Fifty percent inhibition of I-fibrinogen binding to Daudi lymphoblasts (IC) was obtained for 3 concentrations of 20-40 µg/ml (Fig. 1A). The effect of 3 on monocyte adhesion to HUVEC mediated by fibrinogen (10) was investigated. Increasing concentrations of 3 completely inhibited in a dose-dependent fashion fibrinogen-mediated adhesion of PBMC or vitamin D(3)-differentiated monocytic HL-60 cells to resting HUVEC monolayers (Fig. 1B). In contrast, comparable concentrations of L10 or control 3 scrambled peptides were ineffective under the same experimental conditions (Fig. 1B, and data not shown). In another series of experiments, increasing concentrations of 3 up to 400 µg/ml did not diminish vitamin D(3)-differentiated HL-60 monocytic cell adhesion to resting or tumor necrosis factor-alpha-stimulated HUVEC, or to monolayers of ICAM-1 CHO transfectants, in the absence of fibrinogen (not shown). As judged by trypan blue exclusion, maximally inhibitory concentrations of 3 >1 mg/ml did not affect leukocyte or HUVEC viability, under these experimental conditions (not shown).




Figure 1: A, effect of 3 on I-fibrinogen binding to Daudi. Serum-free suspensions of CD11b/CD18 lymphoblastoid Daudi cells at 1.5 times 10^7/ml were preincubated with the indicated increasing concentrations of 3 (bullet, fibrinogen chain 117-133), or control peptides P1 (, 100 µg/ml), GPRP (box, 100 µg/ml), or L10 (, 50 µg/ml) for 10 min at 22 °C. Cells were mixed with 50 µg/ml I-fibrinogen for 30 min at 22 °C in the presence of 1 mM CaCl(2) and 1 mM MgCl(2), and free from cell-associated radioactivity was separated by centrifugation through mixture of silicone oil, before calculation of specific binding. I-Fibrinogen binding to Daudi in the absence of competitors (100% binding) was 25,532 ± 3,150 molecules/cell. Data are the mean ± S.E. of three independent experiments. B, effect of 3 on fibrinogen-mediated leukocyte-endothelium interaction. Suspensions of PBMC (closed symbols; bullet, 3; , L10) or vitamin D(3)-differentiated monocytic HL-60 cells (open symbols; circle, 3; box, L10) were preincubated with the indicated increasing concentrations of 3 or with 400 µg/ml control peptide L10, stimulated with 10 µM fMet-Leu-Phe, and equilibrated with 150 µg/ml fibrinogen for 20 min at 22 °C, in the presence of 2.5 mM CaCl(2). Leukocyte adhesion under the various conditions tested was quantitated after a 30-min incubation at 22 °C. Data are the mean ± S.E. of two independent experiments.



ICAM-1-dependent Recognition of the Fibrinogen 3 Sequence

To investigate the potential interaction of the 3 sequence with the bridging receptor ICAM-1(10) , stable clonal cell lines of ICAM-1 transfectants were genetically engineered and phenotypically characterized for their reactivity with established anti-ICAM-1 mAb LB-2(9, 10) , or with the recently generated mAbs 1G12, 2D5, or 6E6^2 (Fig. 2A). I-Labeled 3 bound to ICAM-1 CHO transfectants, but not to WT CHO cells, in a concentration-dependent reaction, approaching saturation at 100-150 µg/ml doses of added ligand (Fig. 2B), and inhibited by a 100-fold molar excess of unlabeled 3, while comparable concentrations of control peptide L10 were ineffective (Fig. 2, B and C, and data not shown). The affinity of 3 for ICAM-1 was calculated by Scatchard plot analysis (16) of peptide binding isotherms. As shown in Fig. 3, I-3 bound to a single class of receptors on cytokine-activated HUVEC, with a K(d) of 34 µM (Fig. 3), while I-fibrinogen binding to these cells was regulated by a K(d) of 0.1 µM (Fig. 3), in agreement with previous observations (10) . Finally, fractionation of I-surface-labeled JY lymphoblast extracts by affinity chromatography on 3-Sepharose, resulted in a divalent cation-dependent elution of a major 95-kDa band, consistent with the size and molecular organization of ICAM-1 (9) (not shown).


Figure 2: A, characterization of ICAM-1 transfectants. CHO cells were transfected with a full-length ICAM-1 cDNA, selected in 1 mg/ml G418, and cloned by limiting dilution. Quantitation of ICAM-1 surface expression on WT CHO cells or on ICAM-1 transfectants (clone 1) was carried out by flow cytometry with anti-ICAM-1 mAbs LB-2 or 2D5 (Footnote 2). Similar results not shown in the figure were obtained with anti-ICAM-1 mAbs 1G12 and 6E6 (Footnote 2). Background fluorescence was determined in the presence of control mAb OKM1. B and C, binding of I-3 to ICAM-1 transfectants. Increasing concentrations (6-150 µg/ml) (B) or 50 µg/ml (C) of I-3 were added to ICAM-1 transfectants, or control WT CHO cells, for 45 min at 22 °C in the presence of 1 mM CaCl(2) and 1 mM MgCl(2). Nonspecific binding was assessed in the presence of a 100-fold molar excess of unlabeled 3 and was subtracted from the total to calculate net specific binding. Data are the mean ± S.E. of the replicates of a representative experiment.




Figure 3: Scatchard plot analysis of ligand binding to ICAM-1. Confluent HUVEC monolayers were stimulated with 100 units/ml tumor necrosis factor-alpha for 6 h at 37 °C, and incubated with increasing concentrations of I-fibrinogen (1-50 µg/ml) or I-3 (6-150 µg/ml) for 45 min at 22 °C. After washes, specific binding was calculated as described in the legend to Fig. 2. Scatchard plot analysis and quantitative parameters of binding isotherms were generated with the Ligand program based on the algorithm of MacPherson(16) . Data are representative of at least four independent experiments.



The functional effect of 3 on ICAM-1-dependent adhesion was investigated. As shown in Fig. 4, immobilized 3 specifically supported adhesion of JY lymphoblasts in a concentration-dependent reaction (Fig. 4A), that was abrogated by cell preincubation with mAbs 1G12 and 2D5, previously selected for inhibiting ICAM-1-fibrinogen interaction^2 (Fig. 4B).


Figure 4: A, 3-mediated lymphoblast adhesion. Plastic assay plates were coated with the indicated increasing concentrations of 3 or control peptide L10, blocked with 3% gelatin, and incubated with Cr-labeled JY lymphoblasts at 1 times 10^6/ml for 45 min at 37 °C, before quantitation of attached cells. B, ICAM-1-dependent lymphoblast adhesion to 3. The experimental conditions are the same as in A, except that JY lymphoblasts were preincubated with or without the various anti-ICAM-1 mAbs 2D5, 1G12, 6E6 (Footnote 2) at 25 µg/ml before addition to peptide-coated plates and quantitation of cell attachment. Anti-ICAM-1 mAb 6E6 does not inhibit ICAM-1-fibrinogen interaction (Footnote 2).




DISCUSSION

In this study we have identified a novel fibrinogen chain sequence (NNQKIVNLKEKVAQLEA) that mediates ligand binding to ICAM-1 and potentially contributes to fibrinogen-dependent inflammation(3, 4, 5, 6) . As anticipated in previous studies(10) , the ability of fibrinogen to enhance monocyte adhesion to endothelium by bridging the two cell types (10) may represent one of the earliest molecular prerequisites for its obligatory role in preserving competent inflammatory responses in vivo(3, 4, 5, 6) .

Initial screening of a library of fibrinogen-derived peptides synthesized and characterized in previous studies(11) , identified the 3 sequence for its ability to block binding of I-fibrinogen to HUVEC and other ICAM-1-expressing cells, i.e. Daudi, and to inhibit monocyte-endothelium interaction mediated by fibrinogen(10) . While none of the other previously identified fibrinogen interacting motifs with vascular cell receptors was effective in this system, the 3 sequence may act in concert with the previously described CD11b/CD18 recognition on the fibrinogen chain Gly-Val(11) , to facilitate leukocyte anchoring to endothelium in the bridging model of intercellular adhesion(10) . Physical association of the 3 sequence with ICAM-1 (10) was demonstrated in direct I-labeled peptide binding studies to HUVEC and genetically engineered ICAM-1 transfectants. As judged by Scatchard plot analysis of binding isotherms, I-3 association with ICAM-1 was regulated by a K(d) of 34 µM, in excellent agreement with the IC concentrations of 20-40 µg/ml required for peptide inhibition of fibrinogen binding and leukocyte-endothelium bridging. This suggests that 3-mediated inhibition of ICAM-1 adhesive recognitions is mediated by genuine peptidyl mimicry, with disruption of the ligand binding groove as a consequence of receptor occupancy(19) . It is noteworthy that 3 had no inhibitory effect in experiments that directly investigated the molecular association between ICAM-1 and CD11a/CD18, as exemplified by HL-60 cell adhesion to cytokine-activated HUVEC or to monolayers of ICAM-1 transfectants in the absence of fibrinogen. This suggests that the fibrinogen binding site on ICAM-1 may be spatially and topographically distinct from the CD11a/CD18 recognition on the first Ig-like domain of the receptor(9) .

While other regions in fibrinogen may cooperate in ligand docking with ICAM-1, the data reported here suggest that the 3 motif functions as a dominant recognition sequence for ICAM-1 in cell adhesion reactions. In this context, similar to the ability of other linear peptidyl sequences in fibrinogen(11) , CS1 fibronectin(20) , or ICAM-2 (21) to function as competent cell adhesion-promoting ligands irrespective of their conformational state, immobilized 3 supported attachment of JY lymphoblasts in a reaction inhibited by anti-ICAM-1 mAbs.

In summary, the identification of the 3 sequence as a minimal ligand recognition for ICAM-1 offers a novel potential target for highly selective inhibition of fibrinogen-dependent cellular inflammatory responses in vivo(3, 4, 5, 6) . In this context, peptidomimetics derived from the 3 sequence and structure would have the required specificity to interfere with fibrinogen/ICAM1-1-dependent leukocyte-endothelium bridging, without affecting the hemostatic functions of other vascular cell fibrinogen receptors in vivo(2) .


FOOTNOTES

*
This work was supported by National Institutes of Health Grants RO1 HL-43773 and HL-54131. This work was done during the tenure of an Established Investigatorship award from the American Heart Association (to D. C. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be sent: The Boyer Center for Molecular Medicine, Dept. of Pathology, Yale University School of Medicine, 295 Congress Avenue, New Haven, CT 06536. Tel.: 203-737-2869; Fax: 203-737-2290.

Present address: DBMS/Laboratory of Hematology, Unité 217, Institut National de la Santé et de la Recherche Medicale, F38041, Grenoble Cedex, France.

**
Present address: Dept of Pathology, Yale University School of Medicine, New Haven, CT 06520.

(^1)
The abbreviations used are: ICAM-1, intercellular adhesion molecule-1; CHO, Chinese hamster ovary; HUVEC, human umbilical vein endothelial cells; mAb, monoclonal antibody; PBMC, peripheral blood mononuclear cells; WT, wild type.

(^2)
A. Duperray, J. Plescia, L. R. Languino, and D. C. Altieri, unpublished observations.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.