Dual Regulation of Ligand Binding by CD11b I Domain

The role of coagulation factor X as a ligand for CD11b/CD18 (Mac-1, αMβ2) in leukocyte adhesion was investigated. A factor X peptide, (G)L238YQAKRFKV246(G), blocked ligand binding to CD11b/CD18 and prevented monocyte procoagulant activity. This peptide also inhibited monocytic THP-1 cell adhesion to tumor necrosis factor α-stimulated endothelium and blocked neutrophil migration through tumor necrosis factor α-activated endothelial cell monolayers. In contrast, other factor X-derived peptides were ineffective. Radiolabeled peptide (G)LYQAKRFKV(G) bound specifically and saturably to isolated recombinant CD11b I domain. Functionally, the factor X sequence (G)LYQAKRFKV(G) dose-dependently inhibited THP-1 cell attachment to intercellular adhesion molecule 1 (ICAM-1) transfectants (IC50 = ∼50 μg/ml), indistinguishably from anti-CD18 monoclonal antibodies 60.3 and IB4. In contrast, peptide (G)LYQAKRFKV(G) failed to reduce binding of125I-fibrinogen to immobilized CD11b I domain, which was abolished by the fibrinogen-derived peptide KYG190WTVFQKRLDGSV202. By Lineweaver-Burke analysis, peptide (G)LYQAKRFKV(G) inhibited factor X binding to CD11b/CD18 in a noncompetitive fashion, and intact factor X did not reduce monocyte-endothelial cell interaction. These data suggest that the factor X sequence (G)LYQAKRFKV(G) defines an ICAM-1-binding site on CD11b I domain physically distinct from and nonoverlapping with the fibrinogen interacting region(s). Engagement of this site induces a conformational change in the holoreceptor, which disrupts a distant factor X-binding site required for monocyte procoagulant activity. These observations demonstrate a dual regulatory role of CD11b I domain in ligand binding and provide a molecular basis for the recently reported anti-inflammatory properties of factor X homologous sequencesin vivo.

lular adhesion molecules (ICAMs) 1 (7,8), and soluble proteins, like complement C3bi (1), fibrinogen and coagulation factor X (9). Blocking monoclonal antibodies (mAbs) (10), direct binding studies to the isolated recombinant protein (11), and mutagenesis experiments (12)(13)(14) converged to identify the ϳ200-amino acid inserted "I" domain in the ␣ subunit as a recognition site for ICAM-1, fibrinogen, and C3bi. However, how these I domain-binding sites for unrelated ligands are structurally and functionally organized has not been completely elucidated. Although differential inhibition by epitope-mapped mAbs proposed the existence of functionally independent I domain subregions (10), receptor mutagenesis studies suggested that the binding of fibrinogen, C3bi, and ICAM-1 can be mediated, at least in part, by functionally overlapping regions (12,14,15).
Differently from other ␤ 2 integrin ligands, binding of factor X to CD11b/CD18 (16) is not mediated by the I domain (11), is inhibited by three spatially distant sequences in the ligand (17), and mediates monocyte procoagulant activity (9). In this study, we sought to reinvestigate the association of factor X with CD11b/CD18 and its potential impact on ␤ 2 integrin-dependent leukocyte adhesion. Using a small factor X peptide (G)L 238 YQAKRFKV 246 (G) (17), we have delineated a discrete region on CD11b I domain that mediates the interaction with ICAM-1 and indirectly modulates a distant binding site for factor X.

MATERIALS AND METHODS
Cells and Cell Cultures-Polymorphonuclear leukocytes (PMN) were isolated from acid-citrate-dextrose anticoagulated blood drawn from informed normal volunteers by Ficoll-Hypaque gradient density centrifugation and dextran sedimentation (18). Human umbilical vein endothelial cells (HUVECs) were prepared by collagenase treatment and used between passages 2 and 4. The monocytic cell line THP-1 and CD11b/CD18 Ϫ T leukemia cell line MLT (ATCC, Rockville, MD) were maintained in culture according to the manufacturer's recommendations. THP-1 cell expression of CD11b/CD18 and recognition of fibrinogen and factor X by these cells have been reported (9).
Synthetic Peptides and mAbs-The experimental procedures for the isolation and purification of human plasma fibrinogen have been described previously (9). Aliquots of fibrinogen or factor X were 125 Ilabeled by the IODO-GEN method to a specific activity of 0.5 and 1-2 Ci/g of protein, respectively. Integrity and specific activity of 125 Ifactor X were as described (16). The factor X-derived peptides G 366 YDTKQED 373 (G) (peptide 1), I 422 DRSMKTRG 430 (peptide 2), and (G)L 238 YQAKRFKV 246 (G) (peptide 16, residues in parentheses added to the natural sequence) were previously characterized for their ability to inhibit 125 I-factor X binding to CD11b/CD18 on chemoattractant-stim-* This work was supported by National Institutes of Health Grants HL-43773 and HL-54131. This work was done during the tenure of an Established Investigatorship Award to Dr. Altieri. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 (17). All peptides were synthesized by the W. M. Keck protein chemistry facility at Yale University, purified by high pressure liquid chromatography, and characterized for amino acid composition by mass spectrometry. The factor X peptide (G)L 73 EGFEGKN 80 (G) (peptide 25) was used as a control. 2 mg of peptide 16 were iodinated with 5 mCi of Na 125 I by the IODO-GEN method for 45 min at 4°C, followed by separation of free from peptidebound radioactivity by gel filtration over a Bio-Gel P-2 column (Bio-Rad) pre-equilibrated with PBS, pH 7.2. Anti-CD18 mAbs 60.3 and IB4 were obtained from ATCC. Nonbinding mAb 14E11 was used as a negative control.
Expression of Recombinant Proteins-The establishment and characterization of Chinese hamster ovary cells stably transfected with the ICAM-1 cDNA has been reported (19). The construction and expression of recombinant CD11b I domain (residues Gly 111 -Ala 318 ) and its interaction with fibrinogen and ICAM-1 have been described (11).
Procoagulant Activity and Binding Reactions-The effect of the various factor X peptides on monocyte procoagulant activity was determined by a one-stage sensitive clotting assay using a factor VII-and factor X-deficient plasma (Sigma) as described (17). 50-l aliquots of purified CD11b I domain at 5 g/ml in Tris-buffered saline, pH 8.0 were immobilized onto 96-well U-bottom Falcon 3911 flexible assay plates (Becton Dickinson, Oxnard, CA) for 18 h at 4°C. Duplicate wells were washed in Tris-buffered saline, pH 8.0, post-coated with 10 mg/ml BSA (Sigma) for 90 min at 37°C, rinsed, and mixed with increasing concentrations of 125 I-peptide 16 (8 -62.5 g/ml) in PBS, pH 7.2, 2.5 mM CaCl 2 and 0.1% BSA for 30 min at 22°C. At the end of the incubation, wells were washed twice in PBS, pH 7.2, amputated, and counted in a ␥ counter. Specific binding was calculated in the presence of a 50-fold molar excess of unlabeled peptide 16 or control peptide 25 added at the start of the incubation. Alternatively, increasing concentrations of 125 I-fibrinogen (10-150 g/ml) were incubated with CD11b I domain-coated wells in the presence of 1 mM CaCl 2 and 0.1% BSA for 30 min at 22°C, before determination of specific binding. For all experiments, nonspecific binding was measured in the presence of a 50-fold molar excess of unlabeled fibrinogen and subtracted from the total to calculate net specific binding. In peptide inhibition experiments, CD11b I domain-coated plates were preincubated with increasing concentrations (0.1-1000 g/ml) of fibrinogen-derived P1 peptide KYG 190 WTVFQKRLDGSV 202 (18) or factor X control peptide 25 or peptide 16 for 30 min at 22°C. Wells were mixed with 125 I-fibrinogen (25 g/ml) in the presence of 1 mM CaCl 2 and 0.1% BSA for 30 min at 22°C, before determination of specific binding. In another series of experiments, serum-free THP-1 cells (1.5 ϫ 10 7 /ml) were stimulated with 10 M fMLP in the presence of increasing concentrations of 125 I-factor X and 2.5 mM CaCl 2 and a single concentration of control peptide or the three factor X inhibitory peptides (17). After a 20-min incubation at 22°C, free and cell surface-bound radioactivity was separated by centrifugation through a mixture of silicone oil (Dow-Corning, New Bedford, MA) at 14,000 ϫ g for 5 min at 22°C, and specific binding was determined as described above. Binding data were analyzed by the Lineweaver-Burke plot as described (16). Leukocyte-Endothelium Interaction-Serum-free suspensions of THP-1 or MLT cells (5 ϫ 10 6 /ml) were labeled with 0.5 mCi of 51 Cr (Na 2 CrO 4 , du Pont de Nemours, Wilmington, DE) for 2 h at 37°C with a final incorporation of 0.7-2.1 cpm/cell, washed in PBS, pH 7.4, and suspended in serum-free RPMI 1640. MLT or 10 M fMLP-stimulated THP-1 cells were preincubated with increasing concentrations (60 -1000 g/ml) of factor X peptide 1, 2, or 16 or control peptide 25. After a 30-min incubation at 22°C, 51 Cr-labeled cells (1 ϫ 10 5 ) were added to resting or cytokine-activated (100 units/ml TNF␣ for 4 h at 37°C) HUVEC monolayers for 1 h at 22°C in the presence of 1 mM CaCl 2 . After washes, attached cells were solubilized in 15% SDS, and radioactivity associated under the various conditions was determined in a scintillation ␤-counter. The number of attached cells was calculated by dividing the cpm harvested by the cpm/cell. In other experiments, 51 Cr-labeled THP-1 cells were preincubated with increasing concentrations of factor X peptides (10 -1000 g/ml) or 25 g/ml anti CD18 mAbs 60.3 and IB4 or control mAb 14E11. After a 30-min incubation at 4°C, control or anti-CD18-treated cells were added to confluent monolayers of ICAM-1 transfectants or wild-type Chinese hamster ovary cells, with determination of cell adhesion after a 1-h incubation at 22°C. For transendothelial cell migration, HUVEC were grown to confluency onto gelatin-coated porous transwell membranes (8-m diameter; Costar, Cambridge, MA) and stimulated with 100 units/ml TNF␣ for 4 h at 37°C prior to the experiment. PMN (1 ϫ 10 7 /ml) were preincubated with 500 g/ml factor X peptide 16 or control peptide 25 for 30 min at 22°C, stimulated with 10 M fMLP in the presence of 1 mM CaCl 2 , and added (1 ϫ 10 6 ) to HUVEC for increasing time intervals (30 -120 min) at 37°C. At the end of each incubation, migrated PMN were recovered from the bottom well, washed, stained with 0.2% trypan blue, and counted microscopically. Before each experiment, the integrity of the endothelial cell monolayer was confirmed by methyl green staining and fluorescence microscopy.

Anticoagulant Properties of Factor X Interacting Sequences-
Previous data suggested that binding of factor X to CD11b/ CD18-expressing monocytes is mediated by three spatially distant sites in the ligand catalytic domain (17). Preincubation of THP-1 cells with a saturating concentration of each of the three implicated sequences inhibited factor X-dependent monocyte procoagulant activity by 60 -70% in a time-dependent reaction (not shown) and in agreement with previous observations (17,20).
Effect of Factor X Peptide 16 on CD11b I Domain Ligand Recognition-The possibility that the factor X peptide 16 may affect intercellular adhesion mediated by CD11b/CD18-ICAM-1 interaction was first investigated. Increasing concentrations of peptide 16 blocked the adhesion of fMLP-stimulated THP-1 cells to monolayers of ICAM-1 transfectants in a concentration-dependent manner, with an IC 50 value of ϳ50 g/ml (Fig. 3A). A comparable degree of inhibition was observed in control experiments after THP-1 cell preincubation with anti-CD18 mAbs IB4 and 60.3 but not with control mAb 14E11 (Fig. 3B). In direct binding studies, 125 I-peptide 16 bound to immobilized recombinant CD11b I domain in a specific and concentration-dependent reaction was competitively inhibited by Ͼ90% by a 50-fold molar excess of unlabeled peptide 16 but not by control peptide 25 (Fig. 4). The effect of factor X sequence (G)LYQAKRFKV(G) on CD11b I domain recognition of fibrinogen was also investigated (11). Binding of 125 I-fibrinogen to isolated recombinant I domain was specific and saturable, in agreement with previous observations (11). Under these experimental conditions, increasing inhibitory concentrations of peptide 16 or control factor X peptide 25 failed to reduce 125 I-fibrinogen binding to isolated CD11b I domain (Fig. 5). In contrast, increasing concentrations of fibrinogen-derived P1 peptide (␥Gly 190 -Val 202 ) inhibited binding of 125 I-fibrinogen to isolated I domain in a dose-dependent man-ner with an IC 50 value of ϳ4 -6 M (Fig. 5), in agreement with previous observations (18).
Molecular Dissection of Factor X Binding to CD11b/ CD18 -The reciprocal relationship between the three factor X peptides inhibiting ligand binding to CD11b/CD18 (17) was investigated. All three factor X peptides (G)LYQAKRFKV(G) (peptide 16), GYDTKQED(G) (peptide 1), and (G)DRSMK-TRG (peptide 2) inhibited binding of 125 I-factor X to fMLPstimulated THP-1 cells equally well in a concentration-dependent manner (not shown) and in agreement with previous observations (17). However, analysis of binding data by the Lineweaver-Burke plot revealed that only the factor X sequence (G)DRSMKTRG inhibited ligand binding in a genuine competitive manner, as judged by the nearly identical yЈ intercept values determined in the presence (y ϭ 4.8) or in the absence (y ϭ 4.32) of antagonist (Fig. 6). In contrast, the factor X sequences GYDTKQEDG and (G)LYQAKRFKV(G) inhibited factor X binding to CD11b/CD18 in noncompetitive manner, with 4 -5-fold differences in the predicted y intercept values in control curves (y ϭ 4.32) or in the presence (y ϭ 21.7; Ref. 18) of these inhibitors (Fig. 6).

DISCUSSION
In this study, we have shown that a factor X sequence, (G)L 238 YQAKRFKV 246 (G), exerted a dual anticoagulant and anti-inflammatory effect by blocking monocyte procoagulant activity and inhibiting leukocyte-endothelium interaction. This pathway involved peptide engagement of a discrete ICAM-1binding site on CD11b I domain with indirect disruption of a distant factor X-binding region in the holoreceptor.
Through its promiscuous ligand repertoire (2), CD11b/CD18 mediates a variety of cell adhesive interactions and functions as a procoagulant receptor via its high affinity recognition of factor X (9). The possibility that this interaction may also participate in leukocyte adhesion mechanisms has been postulated. Previously, interruption of CD11b/CD18-dependent coagulation prevented thrombin-dependent chemotaxis (21) and monocyte adhesion (22,23) to virally infected endothelium (17). Moreover, an adhesive determinant of Bordetella pertussis, filamentous hemagglutinin, was recently shown to contain sequences homologous to the three factor X regions inhibiting ligand binding to CD11b/CD18 (20). Synthetic peptidyl mimicry of these regions produced a potent anti-adhesive and antiinflammatory effect by blocking leukocyte-endothelium interaction in vitro and reducing neutrophil accumulation in the colony-stimulating factor in an in vivo model of bacterial meningitis (20). However, the molecular basis of this anti-inflammatory pathway was not elucidated.
Here, at variance with the anti-adhesive functions of all three factor X homologous sequences (20), a single factor X FIG. 6. Molecular dissection of factor X binding to CD11b/CD18. THP-1 cells (1.5 ϫ 10 7 /ml) were incubated with the indicated increasing concentrations of 125 I-factor X and 2.5 mM CaCl 2 in the presence or in the absence of a single concentration of control peptide or the various inhibitory factor X peptides for 15 min at 22°C, before determination of specific binding. Competitive/noncompetitive peptide inhibition of 125 I-factor X binding to THP-1 cells was determined by analysis of binding data by the Lineweaver-Burke plot. The variant (G)DRSMKTRG for peptide 2 was used (17). peptide (G)L 238 YQAKRFKV 246 (G) (peptide 16) acted as a potent antagonist of leukocyte-endothelium interaction and transendothelial cell migration. Three independent lines of evidence indicate that this anti-adhesive effect resulted from peptide engagement of an ICAM-1-binding site on CD11b I domain (10 -13). First, this peptide had no effect on CD11b/ CD18 Ϫ T cell (MLT) adhesion to endothelium. Second, binding of monocyte THP-1 cells to monolayers of ICAM-1 transfectants was equally well inhibited by factor X peptide 16 or by anti-CD18 mAbs. Thirdly, direct radiolabeled peptide binding studies demonstrated that this sequence physically interacted with CD11b I domain. The use of a small synthetic peptide, like peptide 16, to probe the CD11b/CD18 ligand repertoire allowed delineating a more precise functional map of CD11b I domain. Consistent with the differential pattern of inhibition obtained with I domain epitope-mapped mAbs (10), the ability of this peptide to disrupt an ICAM-1-binding site without affecting the recognition of fibrinogen postulates the existence of physically separate subdomains mediating a nonoverlapping recognition of these two ligands. This is at variance with previous studies of site-directed mutagenesis, in which single amino acid changes in the CD11b metal ion-dependent adhesion site (15) suppressed the receptor recognition of fibrinogen, ICAM-1, and C3bi (12,14,15). This may be explained by the ability of the metal ion-dependent adhesion site to transduce conformational effects in spatially distant ligand-binding sites (see below) without affecting subunit assembly or receptor surface expression.
A potential model of how perturbation of a discrete I domain region, i.e. ICAM-1 site, could indirectly modulate ligand binding to the holoreceptor was provided by dissecting the factor X interaction with CD11b/CD1. Although three spatially distant sites in factor X indistinguishably blocked ligand binding and monocyte procoagulant activity (17), only one of these regions, I 422 DRSMKTRG 430 , was a genuine competitive inhibitor of this interaction. The other two factor X peptides, including peptide 16, were noncompetitive antagonists, thus potentially disrupting a secondary docking during receptor-ligand interaction. This suggests that binding of the anti-adhesive sequence (G)LYQAKRFKV(G) to an ICAM-1 recognition site on CD11b I domain may induce a conformational change in the holoreceptor with disruption of a distant factor X-binding pocket. Consistent with this hypothesis, 125 I-factor X did not specifically associate with recombinant isolated CD11b I domain (11), and in the present studies, the intact macromolecule did not affect leukocyte-endothelium interaction. 2 Altogether, these data suggest a more dynamic role of ␤ 2 integrin I domains, not only in providing physical interacting site(s) for ligand recognition, but also in regulating the state of receptor activation (24) and ligand binding affinity (25).
In summary, these data underscore the role of the CD11b I domain in multiple regulation of ligand binding and identify a factor X sequence that, similarly to its prokaryotic homologue (20), displayed potent anticoagulant and anti-inflammatory properties. The availability of a small and high affinity probe like peptide 16 should facilitate the identification of the complementary ICAM-1-binding site on CD11b and define its role in leukocyte traffic and recirculation (4,5).