©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Individually Distinct Ig Homology Domains in PECAM-1 Regulate Homophilic Binding and Modulate Receptor Affinity (*)

(Received for publication, February 6, 1996)

Qi-Hong Sun (1) Horace M. DeLisser (2) Mark M. Zukowski (3) Cathy Paddock (1) Steven M. Albelda (2)(§) Peter J. Newman (1)(§) (4)(¶)

From the  (1)Blood Research Institute, The Blood Center of Southeastern Wisconsin, Milwaukee, Wisconsin 53233-2194, the (2)Department of Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104, (3)Amgen Boulder, Inc., Boulder, Colorado 80301, and the (4)Departments of Cellular Biology and Pharmacology, The Medical College of Wisconsin, Milwaukee, Wisconsin 53226

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

PECAM-1 (CD31) is a 130-kDa member of the immunoglobulin (Ig) gene superfamily that is constitutively expressed at high concentration at endothelial cell intercellular junctions and at moderate density on the surface of circulating leukocytes and platelets. Recent in vitro and in vivo studies have shown that PECAM-1 plays a central role in mediating the extravasation of leukocytes from the vessel wall in response to inflammatory mediators. To study the binding characteristics of PECAM-1, phospholipid vesicles were prepared and examined by flow cytometry and immunofluorescence microscopy for their ability to associate with each other and with cells. Proteoliposomes containing high concentrations of PECAM-1 interacted homophilically with each other, forming large self-aggregates. PECAM-1 proteoliposomes, as well as soluble bivalent PECAM-1 in the form of a PECAM-1/IgG immunoadhesin, associated homophilically with cells expressing human, but not murine, PECAM-1. This binding could be completely inhibited by monoclonal antibody Fab fragments specific for Ig homology Domain 1 or Domains 1 + 2. Binding studies using cells expressing human PECAM-1 deletion mutants and murine/human chimeras confirmed that both Ig Domains 1 and 2 were both necessary and sufficient for homophilic binding. In contrast, engagement of membrane-proximal Domain 6 with monoclonal antibody Fab fragments had the opposite effect and augmented the binding of PECAM-1 proteoliposomes to cells. Thus, PECAM-1, like certain integrins, appears to be capable of antibody-induced conformational changes that alter affinity for its ligand. Similar changes induced by physiologic stimuli could be important in regulating the function of PECAM-1 in vascular cells.


INTRODUCTION

PECAM-1 (CD31) is a 130-kDa member of the immunoglobulin (Ig) gene superfamily (1, 2, 3) that is constitutively expressed at high concentration at endothelial cell intercellular junctions (4) and at moderate density on the surface of circulating leukocytes and platelets (for a recent review, see (5) ). Recent in vitro(6) and in vivo(7, 8) studies have shown that PECAM-1 plays a central role in mediating the extravasation of leukocytes from the vessel wall in response to inflammatory mediators, as transendothelial migration is markedly inhibited in the presence of anti-PECAM-1 antibodies or soluble recombinant PECAM-1. Although PECAM-1 can become phosphorylated on cytoplasmic domain serine residues following cellular activation(9) , the precise way in which circulating leukocytes and the underlying endothelial cell barrier regulate the adhesive state of PECAM-1 to control cell-cell interactions remains poorly understood.

Previous studies suggest that PECAM-1 may be capable of interacting with multiple cellular targets. The early finding that PECAM-1 localizes to cell-cell borders of monolayer cells in culture only when a PECAM-1-transfected cell is in contact with another cell expressing PECAM-1 (3) suggested, but did not prove, that PECAM-1 may be capable of interacting homophilically with itself. Later studies (10) showed that PECAM-1-transfected L-cells were capable of interacting with nontransfected L-cells in a calcium-dependent manner, thus implicating a heterophilic adhesion mechanism for PECAM-1 as well. Cell surface glycosaminoglycans were later shown to serve as at least one cellular target for PECAM-1 mediated L-cell aggregation(11) . More recently, the integrin alpha(v)beta(3) has been proposed as a candidate counter-receptor for PECAM-1 heterophilic interactions(12) , but these studies await further confirmation.

In the present study, we have used fluorescently labeled proteoliposomes containing highly purified PECAM-1, and a recombinant PECAM-1/IgG chimeric protein (PECAM/Ig) containing the complete extracellular domain of PECAM-1, to further examine the molecular mechanisms involved in PECAM-1-mediated cellular adhesive events. Our results indicate that the primary cellular target for human PECAM-1 is PECAM-1 and that Ig homology Domains 1 and 2 mediate PECAM-1 homophilic adhesion. In addition, Ig Domain 6 may play a heretofore unexpected regulatory role in modulating the adhesive properties of PECAM-1.


EXPERIMENTAL PROCEDURES

Antibodies

The specificities of all antibodies used in this study are summarized in Table 1. Domain-specific murine anti-human PECAM-1 monoclonal antibodies (characterized in (13) ) used in this study include: PECAM-1.1 (specific for PECAM-1 Ig Domain 5), PECAM-1.2 (specific for PECAM-1 Ig Domain 6), PECAM-1.3 (specific for PECAM-1 Ig Domain 1), 4G6 (specific for PECAM-1 Ig Domain 6), and hec7.2 (specific for PECAM-1 Ig Domains 1 + 2) (kindly provided by Dr. William A. Muller, Rockefeller University). The rat anti-murine PECAM-1 monoclonal antibody, mAb 390(14) , was also employed in selected studies. In addition, the anti-integrin antibodies, AP3 (specific for amino acids 348-421 of the human beta3 integrin subunit (15) and LM609 (a blocking antibody specific for the human integrin alpha(v)beta(3) complex(16) , (kindly provided by Dr. David Cheresh, Scripps Research Institute) were used in selected studies. Fab fragments were generated using immobilized papain (Pierce) according to the manufacturer's instructions and dialyzed against phosphate-buffered saline at 4 °C overnight and analyzed on SDS-PAGE (^1)to confirm that no intact IgG remained in the preparations. Prior to their use, the reactivity of all anti-PECAM-1 Fab fragments was determined by enzyme-linked immunosorbent assay analysis using soluble recombinant human PECAM-1 as the target antigen.



Cells and Cell Lines

Human umbilical vein endothelial cells (HUVECs) were isolated and cultured in RPMI medium containing 15% horse serum and 30 µg/ml endothelial cell growth factor. Mouse L-cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and cultured in RPMI medium with 10% fetal bovine serum (FBS). Cells were harvested with 10 mM EGTA in Hanks' balanced salt solution (HBSS). PECAM-1-transfected L-cells expressing full-length human and murine forms of PECAM-1, as well as recombinant human/murine PECAM-1 chimeras, have been described previously(13) . L-cells were grown in RPMI medium containing 10% FBS and 0.5 mg/ml G418 (Life Technologies, Inc.) and harvested with 10 mM EGTA.

Purification of PECAM-1 and GPIIb-IIIa (Integrin alphabeta(3))

Human platelets were obtained from volunteer donors to The Blood Center of Southeastern Wisconsin, washed, and solubilized in Tris-buffered normal saline (TBS) containing 1% Triton X-100 and 400 µM phenylmethylsulfonyl fluoride, 10 mMN-ethylmaleimide, 0.1 mg/ml leupeptin, pH 7.4. Following clarification at 40,000 times g for 30 min at 4 °C, the detergent lysate was applied to an affinity matrix composed of the monoclonal antibody PECAM-1.3 coupled to Affi-Gel-10 (Bio-Rad). The unbound fraction was saved for affinity purification of GPIIb-IIIa (see below). The column was exchanged into a buffer comprised of TBS containing 30 mM beta-D-octyl glucoside (Calbiochem Corp.). Purified PECAM-1 was eluted in 100 mM glycine, pH 2.4, containing 30 mM octyl glucoside. GPIIb-IIIa was recovered from the PECAM-1-depleted detergent lysate by affinity chromatography of the unbound fraction from the PECAM-1.3-Sepharose column through KYGRGDS-Sepharose, as described previously(17) . Purified PECAM-1 and GPIIb-IIIa complex were examined by SDS-PAGE and immunoblot analysis (see ``Results''). Final protein concentrations were determined using the BCA protein assay (Pierce).

Preparation of PECAM-1 Phospholipid Vesicles

One mg of egg phosphatidylcholine (PC) (Avanti Polar Lipids, Alabaster, AL), supplied as a 25 mg/ml solution in chloroform and 50 µg of the fluorescent PC derivative, 7-nitrobenzo-2-oxa-1,3-diazole (NBD)-PC, were mixed with 10 mg of octyl glucoside in 200 µl of acetone, dried under a stream of filtered nitrogen, redissolved in 250 µl of diethyl ether, and finally dried under vacuum. The lipid/detergent residue was solubilized in 0.5 ml of 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 30 mM octyl glucoside, containing 0-250 µg of purified PECAM-1 or GPIIb-IIIa complex. After dispersion in a water bath sonicator (Laboratory Supplies, Hicksville, NY) at 80 watts for 5 min at 22 °C, proteoliposomes were formed by dialysis against TBS at 4 °C over 36 h. Proteoliposomes prepared in the presence of 100-250 µg of PECAM-1/mg of PC spontaneously aggregated during the later stages of dialysis (see ``Results''); however, GPIIb-IIIa vesicles, or PECAM-1 proteoliposomes formed in the presence of less than 40 µg protein/mg PC, remained largely monomeric and were size-selected and separated from unincorporated protein by gel filtration through Sepharose CL-4B. Fractions were monitored using a CytoFluor II fluorescence microplate reader (PerSeptive Biosystems, Bedford, MA) and the most highly fluorescent fractions pooled. The incorporation of PECAM-1 or GPIIb-GPIIIa complex into proteoliposomes was confirmed by SDS-PAGE and immunoblotting and the protein content quantitated using a monoclonal antibody-based immunoblot assay using purified PECAM-1 or GPIIb-IIIa as standards. Phospholipid concentration was estimated using the sub-micro quantitative phosphorous determination method of Böttcher et al.(18) . Vesicle preparations were examined by both fluorescence and phase contrast microscopy using a Zeiss Axioscope microscope. The size and fluorescence properties of proteoliposome preparations were also examined flow cytometrically using a FACScan (Becton Dickinson Corp., San Jose, CA). Vesicles were stored at 4 °C under nitrogen in the dark for up to 2 weeks before use.

Quantitative Evaluation of PECAM-1 Proteoliposome Binding to Cells

A total of 5 times 10^5 cells in 100 µl of HBSS containing 10% FBS were mixed with 125 µl of HBSS containing varying concentrations of monoclonal antibody Fab fragments, heparin (400 µg/ml), or heparan sulfate (400 µg/ml) (Sigma) and 25 µl of phospholipid vesicles. Following a 90-min incubation at 22 °C, the association of liposomes was determined by both immunofluorescence microscopy and flow cytometry. Protein-free NBD-labeled lipid vesicles were used to establish background levels of cell binding. In some experiments, cells were preincubated for 30 min at 22 °C with 40 µg/ml of intact monoclonal IgGs or 200 µg/ml of Fab fragments and then washed three times before addition of proteoliposomes. Selected binding assays were performed in the presence of 2.5 mM CaCl(2).

Construction and Purification of PECAM-1/Ig Chimeric Protein

A cDNA fragment encoding the entire extracellular domain of PECAM-1 (amino acids 1-574) was ligated with a cDNA encoding the hinge region, C(H)2, and C(H)3 domains of human IgG1 and the chimeric construct expressed in the mammalian expression vector, EMC3 (generously provided by Dr. Glenn Larsen, Genetics Institute, Cambridge, MA). Following stable transfection into Chinese hamster ovary (CHO) cells, the secreted IgG chimeric protein was purified by protein A-Sepharose chromatography using standard methods. The final purified product was examined by silver-stained SDS-PAGE gels and was verified to be greater than 95% pure. Endotoxin levels of the final purified protein were measured by the limulus amebocyte assay and ranged from 40 to 400 ng/ml.

Quantitative Evaluation of the Binding of PECAM-1/IgG Chimera to Cells

5 times 10^5 cells in 100 µl of HBSS containing 10% FBS were added to 125 µl of HBSS containing varying concentrations of monoclonal antibody Fab fragments, heparin, or heparan sulfate as described above. Five µg of PECAM-1/IgG in 25 µl of HBSS was then added and allowed to incubate for 90 min at 22 °C. The cells were washed by centrifugation with 1 ml of HBSS and then resuspended in 100 µl of HBSS containing 0.8 µl of FITC-labeled goat anti-human IgG chain (Jackson ImmunoResearch Laboratories, West Grove, PA). Following a 30-min incubation at 4 °C, the cells were washed once more in HBSS, resuspended in 0.6 ml of ice-cold HBSS, and subjected to flow cytometric analysis.


RESULTS

Adhesive Properties of PECAM-1 Proteoliposomes

We have previously found that soluble recombinant PECAM-1, comprised of only the extracellular domain (amino acids 1-574), binds negligibly to cells, presumably due the low affinity of monomeric forms of PECAM-1 for its cellular target. (^2)In its naturally occurring state, however, PECAM-1 is highly concentrated within the plane of the membrane(3) , where as many as 10^6 molecules of PECAM-1 are localized to endothelial cell intercellular junctions (19) . Therefore, we attempted to mimic the in vivo valency of PECAM-1 by reconstituting highly purified PECAM-1 derived from human platelets (Fig. 1A) into phospholipid vesicles comprised of 95% PC and 5% NBD-PC (a fluorescent derivative of PC used as a tracer). PECAM-1 proteoliposomes of varying protein/lipid ratios were prepared by detergent dialysis, and their ability to associate with each other and with cells was evaluated using flow cytometry and immunofluorescence microscopy. As shown in Fig. 1B, proteoliposomes prepared using 10 or 40 µg of PECAM-1/mg of PC remained evenly dispersed in solution; however, vesicles containing 100 or 250 µg PECAM-1/mg of PC interacted homophilically with each other, forming large self-aggregates. Control proteoliposomes containing GPIIb-IIIa (integrin alphabeta(3)) at similar protein/lipid ratios displayed no such tendency to self-associate. These data represent the first direct demonstration that purified PECAM-1 is capable of interacting homophilically with itself and is capable of promoting vesicle association only when sufficiently concentrated within the plane of a lipid bilayer, consistent with its subcellular distribution at cell-cell junctions.


Figure 1: Adhesive properties of PECAM-1 proteoliposomes. A, SDS-PAGE analysis of PECAM-1 and GPIIb-IIIa incorporated into phospholipid vesicles. PECAM-1 (lane 1) was purified from human platelet detergent lysates by immunoaffinity chromatography, while GPIIb-IIIa (lane 2) was prepared using KYGRGDS-Sepharose, as described under ``Experimental Procedures.'' Purified proteins were analyzed on a silver-stained 4-15% gradient polyacrylamide gel. The positions of molecular mass standards are shown at left in kilodaltons. Homophilic aggregation of PECAM-1-containing proteoliposomes. PECAM-1 (top) and GPIIb-IIIa (bottom) proteoliposomes of varying protein/lipid ratios were prepared by detergent dialysis and viewed by fluorescence microscopy at times 400 magnification. Note that NBD-labeled proteoliposomes having PECAM-1-to-lipid ratios of greater than 100 µg/mg PC spontaneously formed large aggregates, while vesicles containing the integrin GPIIb-IIIa remained evenly dispersed in solution. C, association of PECAM-1 proteoliposomes with HUVECs. NBD-labeled PECAM-1 proteoliposomes, prepared in the presence of 40 µg of PECAM-1/mg of PC, were incubated with HUVECs at 22 °C for 90 min and their association examined by fluorescence (right) and phase contrast (left) microscopy at times 400 magnification. GPIIb-IIIa proteoliposomes (top panels) were employed as a control. D, flow cytometric analysis of the association of PECAM-1 proteoliposomes with HUVECs. NBD-labeled liposomes containing either no protein (bottom left), GIIb-IIIa (top right), or PECAM-1 (bottom right) were mixed incubated with cells as described above and bound fluorescence quantitated by flow cytometry. Note that the binding of GPIIb-IIIa proteoliposomes was only slightly greater than that of the no-protein vesicles, while PECAM-1 proteoliposomes associated significantly with cells.



Since PECAM-1 vesicles containing only 40 µg of protein/mg of lipid remained largely monomeric in solution (Fig. 1B, middle panel) we used these in all subsequent cell association assays. Such PECAM-1 proteoliposomes bound HUVECS, as determined by both immunofluorescence microscopy (Fig. 1C) and by flow cytometric analysis (Fig. 1D).

To examine potential cellular targets for PECAM-1, murine L-cells and L-cells stably expressing PECAM-1 (Fig. 2A) were also examined for their relative ability to support binding of PECAM-1 proteoliposomes. PECAM-1 proteoliposomes bound to HUVECS and PECAM-1 transfected L-cells (Fig. 2B), but failed to associate with nontransfected L-cells. Additional studies (not shown) revealed that PECAM-1 proteoliposomes associated with other PECAM-1-positive cell lines, such as U937 cells, but not with cells lines (COS, CHO) that did not express PECAM-1. Thus, PECAM-1 proteoliposome binding appears to require cell surface expression of PECAM-1. These findings were confirmed in parallel studies that employed soluble bivalent PECAM-1 in the form of a PECAM-1/IgG construct. As shown in Fig. 2B, PECAM-1/IgG bound well to both HUVECS and PECAM-1-transfected L-cells, but failed to associate with nontransfected L-cells. Interestingly, the binding of both PECAM-1 phospholipid vesicles and PECAM-1/IgG was independent of the presence of divalent cations, as addition of EDTA had no effect on their association. Thus, the mechanism by which purified PECAM-1 associates with cells differs significantly from that implied by the aggregation of PECAM-1-transfected L-cells, in which PECAM-1-transfected L-cells were found to aggregate in a calcium-dependent manner and interacted as readily with nontransfected cells(3, 10) . Furthermore, addition of heparin or heparan sulfate had no effect on the binding of either PECAM-1 proteoliposomes or PECAM-1/IgG to HUVECS or PECAM-1-transfected L-cells (not shown), suggesting that cell surface glycosaminoglycans are incapable of supporting the binding of purified human PECAM-1.


Figure 2: Homophilic adhesion of PECAM-1. A, cell surface expression of PECAM-1. The indicated cells were removed from culture flasks with 10 mM EGTA, washed, stained with PECAM-1.3 monoclonal antibody (40 µg/ml) at 4 °C for 60 min, followed by FITC-conjugated goat anti-mouse IgG, and analyzed by flow cytometry. Shown are nontransfected murine L-cells that do not express PECAM-1, HUVECs, and PECAM-1-transfected L-cells. Normal mouse IgG (NMIgG, 40 µg/ml) was used as a control. B, homophilic association of PECAM-1-containing proteoliposomes and PECAM-1/IgG chimeric protein with cells expressing PECAM-1. For the vesicle binding experiments, cells were incubated with PECAM-1 proteoliposomes (prepared in the presence of 40 µg of PECAM-1/mg of PC) and analyzed by flow cytometry as described in the legend to Fig. 1. GPIIb-IIIa proteoliposomes (40 µg of GPIIb-IIIa/mg of PC) were used as a control. PECAM-1 proteoliposomes bound to HUVECs and PECAM-1-transfected L-cells, but not to nontransfected L-cells. Addition of 2.5 mM CaCl(2) had no beneficial effect on proteoliposome binding. For the PECAM-1/IgG experiments, cells were incubated with PECAM-1/IgG (20 µg/ml), followed by FITC-conjugated goat anti-human IgG, and analyzed by flow cytometry. Normal human IgG (NHIgG, 20 µg/ml) was used as the control. Consistent with the cell binding of PECAM-1 proteoliposomes, PECAM-1/IgG bound HUVECs and PECAM-1 transfected L-cells, but did not associate with nontransfected L-cells.



Ig Homology Domains 1 and 2 Mediate PECAM-1/PECAM-1 Interactions

To determine the region of PECAM-1 responsible for cell binding, we used a series of monovalent Fab fragments having specificity for different Ig domains of PECAM-1 (13) as inhibitors of PECAM-1 proteoliposome (Fig. 3A) or PECAM-1/IgG (Fig. 3B) interactions with PECAM-1 expressing cells. As shown in Fig. 3, murine monoclonal Fabs specific for Ig Domain 1 (PECAM-1.3) or Domains 1 + 2 (hec7.2) completely blocked the association of PECAM-1 proteoliposomes with HUVECS, either when added to the liposomes directly or when preincubated only with the cells. As a control, cells pretreated with an anti-PECAM-1 Fab (PECAM-1.1) having specificity for Ig Domain 5 were able to bind vesicles normally. Parallel studies using PECAM-1/IgG (Fig. 3B) produced identical results, as PECAM-1.3 and Hec7.2 completely inhibited binding, while PECAM-1.1 (Domain 5), PECAM-1.2 (Domain 6), and 4G6 (Domain 6) Fabs were without effect. Identical results were obtained using either HUVECS (Fig. 3B, left) or PECAM-1-transfected L-cells (Fig. 3B, right) as targets.


Figure 3: PECAM-1 homophilic adhesion is mediated by Ig Domains 1 and 2. The interaction of either PECAM-1 proteoliposomes (A) or PECAM-1/IgG (B) with cells was measured in the presence of the indicated monoclonal antibody fragments (Fabs). Fabs specific for Ig homology Domain 1 (PECAM-1.3) or Domains 1 + 2 (hec7.2) were inhibitory down to background levels, whereas Fabs specific for Ig Domain 5 had no effect. Inhibition by PECAM-1.3 and hec7.2 occurred whether Fabs were added to the liposomes (A, left) or preincubated with cells (A, right). Antibodies bound to Ig Domains 1 or 1 + 2 effectively blocked adhesion of PECAM-1/IgG whether HUVECs (B, left) or PECAM-1-transfected L-cells (B, right) were used as target cells.



To further investigate the involvement of the amino terminus in mediating PECAM-1 homophilic interactions, we examined the binding of human PECAM-1/IgG to L-cells expressing an isoform of PECAM-1 lacking Ig Domain 1 (Delta1 cells). As shown in Fig. 4, L-cells expressing wild-type full-length PECAM-1 bound the murine monoclonal antibodies PECAM-1.2 (Domain 6) and PECAM-1.3 (Domain 1), while the PECAM-1 Delta1 L-cells bound PECAM-1.2, but failed to react with PECAM-1.3, as would be predicted. PECAM-1 proteoliposomes bound normally to L-cells expressing full-length PECAM-1, but did not associate with surface-expressed PECAM-1 that lacked Ig Domain 1 (Fig. 4B). Moreover, PECAM-1/IgG failed to interact with isoforms of PECAM-1 that lacked either Ig Domains 1 or 2 (Table 2), suggesting that both NH(2)-terminal Ig homology domains might participate in the formation of the homophilic contact site.


Figure 4: PECAM-1 homophilic adhesion requires Ig Domain 1. A, expression of PECAM-1 on the surface of transfected L-cell lines. PECAM-1-transfected L-cells expressing either full-length PECAM-1, or a recombinant isoform missing NH(2)-terminal Ig homology Domain 1 (PECAM-1 Delta1), were harvested with 10 mM EGTA in HBSS, washed, and incubated with either PECAM-1.3 (specific for Ig Domain 1) or PECAM-1.2 (specific for Ig Domain 6). Normal mouse IgG (NMIgG) was used as the control. The cells were then washed, stained with FITC-conjugated goat anti-mouse IgG, and analyzed by flow cytometry. Note the PECAM-1 (Delta1) L-cells bound PECAM-1.2 normally, but failed to react with PECAM-1.3. B, PECAM-1 proteoliposomes and PECAM-1 IgG each bound normally to L-cells expressing full-length PECAM-1, but failed to bind to to the PECAM-1 isoform lacking Ig Domain 1, demonstrating that the amino terminus of the molecule is required for PECAM-1 homophilic interactions.





To rule out the possibility that deletion of an entire Ig homology domain inadvertently resulted in conformational changes that led to ``loss of function,'' we constructed and expressed in murine L-cells a series of human/murine chimeric proteins that much more closely resembled wild-type PECAM and examined their ability to interact homophilically with human PECAM-1/IgG. As shown in Table 2, human PECAM-1/IgG failed to bind murine PECAM-1 expressed on the surface of L-cells, demonstrating species specificity for PECAM-1 homophilic interactions and permitting us to splice selected regions of human PECAM-1 onto the unreactive murine backbone to search for ``gain of function'' adhesive phenotypes. Substitution of murine Ig Domains 1 + 2, or murine Domain 2 alone, into a human PECAM-1 backbone resulted in loss of human PECAM-1/IgG binding, consistent with a role for both of these domains in mediating homophilic adhesion. Finally, a chimeric construct consisting of human PECAM-1 Ig Domains 1 + 2 fused to a murine PECAM-1 backbone (Domains 3-6) restored homophilic binding. Together with the domain-specific antibody blocking studies shown in Fig. 3, our data strongly implicate Ig homology Domains 1 and 2 as both necessary and sufficient for the formation of the primary homophilic contact site of PECAM-1.

The Endothelial Cell Vitronectin Receptor, alpha(v)beta(3), Is Incapable of Supporting the Binding of Purified PECAM-1

HUVECS express approximately 100,000 copies of the integrin alpha(v)beta(3)(20) and approximately 10^6 molecules of PECAM-1 (19) on the cell surface. Recently, Piali et al.(12) have reported that both polyclonal and monoclonal antibodies to murine alpha(v)beta(3) are able to inhibit the binding of murine lymphokine-activated killer (LAK) cells to an immobilized truncated, recombinant murine PECAM-1 construct consisting of Ig Domains 1-3. To examine whether human alpha(v)beta(3) might be able to serve as a heterophilic counter-receptor for a naturally occurring, full-length form of PECAM-1, we pretreated HUVECS with antibodies specific for the alpha(v)beta(3) complex (LM609)(16) , beta(3) alone (AP3)(21) , or PECAM-1.3. As shown in Fig. 5, LM609 and AP3 each failed to inhibit the binding of PECAM-1 proteoliposomes to HUVECS. RGD peptides were also without inhibitory effects (not shown). In contrast, preincubation of HUVECS with Fab fragments of the PECAM-1 Ig Domain 1-specific antibody, PECAM-1.3, completely blocked the association of PECAM-1 proteoliposomes with the cell surface. Similar results were obtained using PECAM-1/IgG in place of PECAM-1 proteoliposomes (not shown). These data are most consistent with cell surface PECAM-1 serving as the primary cellular target for PECAM-1-mediated cellular interactions and would appear to rule out a direct role for alpha(v)beta(3) in supporting the binding of native human PECAM-1 to cells.


Figure 5: Endothelial cell alpha(v)beta(3) is incapable of supporting the binding of PECAM-1. A, expression of the integrin alpha(v)beta(3) on HUVECs. HUVECs were incubated with the alpha(v)beta(3) complex-specific antibody, LM609, the beta(3)-integrin subunit-specific antibody, AP3, or PECAM-1.3 at 4 °C for 60 min, followed by FITC-conjugated goat anti-mouse IgG, and analyzed by flow cytometry. NMIgG at 40 µg/ml was used as the control. LM609 is a blocking antibody which inhibits the interaction of alpha(v)beta(3) with vitronectin receptor as described previously(16) . B, anti-alpha(v)beta(3) antibodies do not inhibit the association of PECAM-1 proteoliposomes with HUVECs. HUVECs were preincubated with 40 µg/ml of the indicated monoclonal antibodies at 22 °C for 30 min, washed, and used as the target cells for PECAM-1 proteoliposome binding, which was performed as described under ``Experimental Procedures.'' NMIgG at 40 µg/ml was used as a negative control. Note that PECAM-1.3 (specific for Ig homology Domain 1) completely blocked the association of PECAM-1 proteoliposomes with HUVECs to background levels, suggesting that cell surface PECAM-1 serves as the sole target for homophilic PECAM-1 interactions.



Engagement of Domain 6 Modulates the Adhesive Properties of PECAM-1

There is considerable evidence that, upon cellular activation, adhesion molecules of the integrin family are capable of undergoing conformational changes leading to transition from a resting state to one that is ligand binding-competent(22, 23) . Physiologically, this transition likely occurs as a result of the interaction of cytosolic components with integrin cytoplasmic domains (24, 25, 26) , although certain murine monoclonal antibodies are able to mimic this event by binding to the extracellular domain and selectively recognizing, or inducing, the active conformation(27, 28) .

Like many integrins, PECAM-1 is constitutively expressed on the surface of vascular cells that, under normal conditions, do not interact with each other. To determine whether PECAM-1 might be capable of undergoing conformational changes that result in increased adhesiveness, we attempted to modulate the affinity of PECAM-1 proteoliposomes for cells using murine monoclonal antibodies that bind to domains of PECAM-1 not directly involved in homophilic adhesion. Fab fragments were used to avoid potential activation of Fc receptors. As shown in Fig. 6A, when Fab fragments of PECAM-1.2 and 4G6, each specific for Ig Domain 6, were prebound to PECAM-1 proteoliposomes, binding to HUVECS increased 2-4-fold. The increased association of PECAM-1 proteoliposomes with the cell surface was dependent upon the concentration of ``activating'' Fab used (Fig. 6B) and could be completely inhibited by preincubation of the cells with the Ig Domain 1-specific antibody, PECAM-1.3 (not shown). Not all anti-PECAM-1 antibodies induced enhanced binding, as increasing concentrations of Domain 1-specific Fabs (PECAM-1.3, hec7.2) alone resulted in inhibition of proteoliposome binding down to background fluorescence levels. Thus, PECAM-1, like certain integrins, appears to be capable of undergoing antibody-induced conformational changes that alter affinity for its ligand. For full-length, naturally occurring human PECAM-1 presented in the context of a phospholipid bilayer, that ligand is PECAM-1.


Figure 6: Engagement of Ig homology Domain 6 modulates PECAM-1 adhesive interactions. A, monoclonal antibodies specific for Ig Domain 6 increase the association of PECAM-1 proteoliposomes with HUVECs. PECAM-1 proteoliposome binding to HUVECs was performed as described under ``Experimental Procedures,'' with 200 µg/ml of the indicated monoclonal Fabs added to cell/liposome mixture. Protein-free fluorescent PC vesicles, as well as GPIIb-IIIa proteoliposomes, were used as controls for nonspecific binding. 100% binding was taken as the median fluorescence intensity obtained as a result of the association of PECAM-1 proteoliposome with HUVECs in the absence of anti-PECAM-1. Note that the binding of two different monovalent Fabs specific for Ig homology Domain 6 (PECAM-1.2 and 4G6) resulted in increased PECAM-1 proteoliposome binding to HUVECs. B, dose-dependent modulation of PECAM-1 adhesive interactions. HUVECs were incubated with PECAM-1 proteoliposomes in the presence of various concentrations of anti-PECAM-1 monoclonal Fabs. NMIgG Fab fragments were employed as a control. Note that Ig Domain 6 Fabs enhanced binding in a dose-dependent manner, while Ig Domain 1 Fabs inhibited binding down to background fluorescence levels.




DISCUSSION

In the present study, we have employed a well defined system using isolated adhesion molecules in the form of proteoliposomes and IgG chimeric constructs to investigate mechanisms by which human PECAM-1 mediates cellular interactions. Each of these multivalent forms offer the potential to achieve affinities hundreds of times greater than monovalent soluble recombinant receptor molecules(29, 30) . Our major findings may be summarized as follows. First, full-length PECAM-1, when present at sufficient concentration within the plane of the membrane, is capable of interacting directly with itself in a divalent cation-independent, homophilic manner (Fig. 1). Second, both phospholipid vesicles expressing isolated, purified human platelet PECAM-1, and a chimeric bivalent form of PECAM-1 expressed as an immunoadhesin molecule, bind specifically to cells that express PECAM-1 (Fig. 2). Third, these homophilic PECAM-1 interactions are mediated by the first two NH(2)-terminal Ig homology domains, Ig Domains 1 and 2 ( Fig. 3and Fig. 4). Fourth, other cellular targets, including cell-surface glycosaminoglycans and surface-expressed integrins (Fig. 5), are insufficient to support the direct binding of purified human PECAM-1, even when it is presented in a high-affinity, multivalent form. Finally, PECAM-1 appears to be the first homophilic adhesion molecule capable of undergoing affinity modulation, as engagement of membrane-proximal Ig Domain 6 results in increased Domains 1 + 2-mediated homophilic binding (Fig. 6).

Cell-cell contacts between adjacent endothelial cells are mediated by adhesion molecules belonging to several different gene families, including the integrins alpha(2)beta(1) and alpha(5)beta(1)(31) , at least two members of the cadherin family(32) , and one member of the immunoglobulin gene superfamily, PECAM-1(1, 4, 33) . Previous studies have shown that PECAM-1 is expressed diffusely on the surface of isolated endothelial cells or PECAM-1-transfected fibroblasts and that upon contact with another PECAM-1-expressing cell, the subcellular distribution of PECAM-1 changes dramatically, with a majority of cell surface PECAM-1 becoming localized to the intercellular junction(3) . In contrast, PECAM-1 is not present at the intercellular junctions formed by PECAM-1-transfected cells in contact with PECAM-1-negative cells, leading to the hypothesis (3) that PECAM-1 interacts homophilically with PECAM-1 on an adjacent cell to effect its concentration at cell-cell borders. The possibility, however, that an intermediary ligand might link PECAM-1 receptors on neighboring cells, much the same as activated GPIIb-IIIa receptors on adjacent platelets are bridged by the symmetrical ligand, fibrinogen(34, 35) , has never been addressed. Our finding that purified human PECAM-1 in a phospholipid bilayer interacts 1) directly itself in the absence of added proteins (Fig. 1) and 2) with PECAM-1 expressed on a cell surface ( Fig. 2and Fig. 3) would appear to rule out a requirement for intermediary co-factors in homophilic PECAM-1 interactions.

In seeming contrast to the homophilic interactions demonstrated in Fig. 1Fig. 2Fig. 3Fig. 4, a number of studies have suggested that PECAM-1 may also be capable of interacting heterophilically with cell surface glycosaminoglycans(11, 36) . These data are derived from experiments in which PECAM-1-transfected murine L-cells were shown to bind, in a divalent cation-dependent manner, as readily to nontransfected L-cells as they do to each other(10, 11) . Aggregation of PECAM-1-transfected cells with nontransfected L-cells was inhibited in the presence of added heparin and other selected glycosaminoglycans, suggesting that glycosaminoglycans might serve as a cellular target for PECAM-1 heterophilic interactions. As shown in the present study, however, when presented in purified form, both human PECAM-1 proteoliposomes and human PECAM-1/IgG interacted with cells efficiently only when PECAM-1 was expressed on the cell surface and did not associate at all with nontransfected murine L-cell fibroblasts (Fig. 2B) or with other PECAM-1-negative cell lines. Binding to cell surface PECAM-1 was unaffected by heparin or RGD peptides and was divalent-cation-independent, providing compelling evidence that the major ligand for purified human PECAM-1 is PECAM-1.

Our findings do not rule out a role for cellular PECAM-1 in mediating heterophilic cellular interactions, especially if one invokes a downstream signaling event that results from either cellular activation or PECAM-1/PECAM-1 engagement. There are several lines of evidence that suggest that PECAM-1-mediated signaling, rather than adhesion per se, could broker heterophilic interactions. First, a number of laboratories have independently shown that engagement of PECAM-1 leads to up-regulation of integrin function(37, 38, 39) , providing evidence for secondary adhesion that, while PECAM-1-dependent, does not involve PECAM-1 adhesion per se. Second, we (9) and others (40) have shown previously that PECAM-1 becomes phosphorylated on its cytoplasmic domain following cellular activation. Either of these two events, shown schematically in Fig. 7, could possibly lead to a change in the adhesive phenotype of the cell, enabling heterophilic adhesion mediated by non-PECAM-1 receptors. The recent findings of DeLisser and colleagues (36, 41) that deletion of portions of the PECAM-1 cytoplasmic domain results in a loss of heterotypic adhesion capability are consistent with a role for the PECAM-1 cytoplasmic domain, particularly exon 14(36, 42) , in mediating this signaling event. Studies are currently in progress to characterize the downstream signaling events that emanate from cell surface engagement of PECAM-1.


Figure 7: Schematic representation of the roles of PECAM-1 in mediating cellular interactions. Two distinct roles for PECAM-1 are depicted, PECAM-1 homophilic adhesion per se, and PECAM-1-dependent signaling leading to heterophilic adhesion mediated by non-PECAM-1 receptors. According to this model, both homophilic as well as heterophilic adhesion are PECAM-1-dependent. Anti-PECAM-1 Fabs binding at or near Ig Domain 1 interfere with homophilic PECAM-1/PECAM-1 interactions, while antibodies bound to Ig Domain 6 propagate long range conformational changes at the amino terminus of the molecule, leading to affinity modulation and tight adhesion. Binding of Domain 6 antibodies may also affect the conformation of the cytoplasmic domain and influence assembly of signaling complexes such that downstream events necessary to effect secondary heterophilic adhesion do not occur.



Recently, Piali et al. have reported that murine lymphokine-activated killer (LAK) cells, which express PECAM-1 on their surface(43) , were able to bind truncated recombinant forms of murine PECAM-1 that had been immobilized on plastic microtiter wells(12) . Surprisingly, binding of LAK cells to a PECAM-1 construct consisting only of Ig Domains 1-3 could be inhibited by preincubating the cells with either RGD peptides or with intact, bivalent antibodies specific for the integrin alpha(v)beta(3). While only 2-3% of cells added actually bound their recombinant PECAM-1 construct, even in the absence of added antibodies, their results nevertheless differ significantly from our own observations in which not only did anti-alpha(v)beta(3) antibodies have no effect on full-length human PECAM-1 adhesion, but the binding of both PECAM-1 proteoliposomes or PECAM-1/IgG was completely blocked by pretreating HUVECS or PECAM-1 transfectants with PECAM-1.3 Fab fragments alone (Fig. 5). Since this Fab fragment is only 50 kDa, it is unlikely that it would interfere with accessibility of the 100,000 alpha(v)beta(3) complexes normally found on the surface of HUVECS(20) . While these discrepancies might be attributed to species differences (their cells and constructs were of murine origin), we speculate that the small amount of alpha(v)beta(3)-mediated binding observed by Piali and colleagues may have resulted from secondary signaling events that may follow PECAM-1/PECAM-1 interactions (Fig. 7). The fact that neither murine nor human PECAM-1 contains the highly conserved integrin-binding motif, I(L)-D(E)-S(T)-P (or S)-L, that is present in all integrin-binding Ig superfamily (IgSF) members described to date, including Ig domains 1 and 4 of VCAM-1 (44, 45, 46, 47) and in Ig Domain 1 of ICAM-1, ICAM-2, ICAM-3, and MadCAM(47, 48) , argues further that PECAM-1 is not a typical integrin-binding IgSF member.

Our findings that engagement of PECAM-1 Ig Domain 6 may regulate the adhesive properties of the amino terminus represent the first example of affinity modulation of an adhesion molecule outside of the integrin family. Clearly, Domain 6-specific Fab fragments are functioning as a surrogate modulator of PECAM-1 affinity in our system and serve only to demonstrate that PECAM-1 affinity modulation is possible. The actual physiological regulator(s) of PECAM-1 affinity states remain to be identified. However, in light of the fact that long range conformational changes can result in changes in the adhesive phenotype of PECAM-1, the opportunity presents itself to re-examine previous studies of PECAM-1-mediated adhesion in which Ig Domain 6 antibodies were employed and offer alternative interpretations. For example, the recent findings of Liao et al.(49) that antibodies prebound to PECAM-1 Ig Domain 6 prevented release of monocytes from recently transversed endothelium may reflect an antibody-induced increase in the affinity of Ig Domain 1 for its endothelial cell target (i.e. junctional PECAM-1). Likewise, Fawcett et al.(50) found that recombinant PECAM-1 constructs lacking Ig Domain 6, when plated onto microtiter wells, were unable to bind to and capture PECAM-1-positive cells and concluded from their studies that Domain 6 therefore represented a homophilic contact site. In light of the potential ability of Domain 6 to modulate the adhesive phenotype of PECAM-1 (Fig. 6), it is possible that artificial truncated constructs lacking this domain have reduced affinity for cell surface PECAM-1. The fact that truncated constructs extend less far out from the bottom of the microtiter well may also have contributed to their relative inability to bind cells.

In summary, we have used a series of well defined monoclonal antibodies and recombinant PECAM-1 constructs to map the homophilic contact sites involved in PECAM-1 cellular interactions. Both loss of function as well as gain of function mutations were employed and together strongly suggest that Ig homology Domains 1 and 2 are both necessary and sufficient to mediate PECAM-1/PECAM-1 associations. Finally, simulated engagement of Ig Domain 6 resulted in long range propagation of a conformational change at the amino terminus of the molecule, as manifested by a dramatic increase in binding of PECAM-1 proteoliposomes to cells. Future studies aimed at identifying the factors that regulate the adhesive phenotype of PECAM-1, and its subsequent ability to mediate the interactions of leukocytes and platelets with the vessel wall, should have important implications for our understanding of inflammation, angiogenesis, and the immune response.


FOOTNOTES

*
This work was supported by Grants HL-40926 (to P. J. N.), HL-43611 (to S. M. A.), and HL-03382 (to H. M. D.) from the National Institutes of Health and by a Robert Wood Johnson Foundation Minority Faculty grant (to H. M. D.). 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.

§
Established Investigators of the American Heart Association.

To whom correspondence should be addressed: Blood Research Institute, The Blood Center of Southeastern Wisconsin, 638 N. 18th St., Milwaukee, WI 53233-2194. Tel.: 414-937-6237; Fax: 414-937-6284.

(^1)
The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; HUVEC, human umbilical vein endothelial cell; FBS, fetal bovine serum; HBSS, Hanks' balanced salt solution; GP, glycoprotein; TBS, Tris-buffered normal saline; PC, phosphatidylcholine; NBD, 7-nitrobenzo-2-oxa-1,3-diazole; CHO, Chinese hamster ovary; LAK, lymphokine-activated killer; FITC, fluorescein isothiocyanate.

(^2)
C. Paddock and P. J. Newman, unpublished observations.


ACKNOWLEDGEMENTS

We are grateful to Drs. William A. Muller and David A. Cheresh for their gifts of monoclonal antibodies, to Barbara Karan-Tamir for assistance in constructing and producing Ig-chimeric constructs, and to Drs. Richard Gumina and Denise Jackson for their helpful advice and comments.


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