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

Alain Duperray,Lucia R Languino,Janet Plescia,Alison Mcdowall,Nancy Hogg,Alister G Craig,Anthony R Berendt,Dario C Altieri

doi:10.1074/jbc.272.1.435

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 beta2 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.

Highlights

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
We used a novel monoclonal antibodies (mAbs) 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

Summary

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 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 ϫ 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 ␮g/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 fluoresceinconjugated goat F(abЈ) 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

Inhibition of adhesion

DISCUSSION