Clustering of Ligand on the Surface of a Particle Enhances ... · Clustering of Ligand on the Surface of a Particle Enhances Adhesion ... Abteilung fur Immunologie, ... Here we ask

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

VOl. 263, No . 33, Issue of November 25, pp. 17822-17827,1988 Printed in U.S.A.

Clustering of Ligand on the Surface of a Particle Enhances Adhesion to Receptor-bearing Cells*

(Received for publication, April 8, 1988, and in revised form, June 17, 1988)

Anne Hermanowski-VosatkaS, Patricia A. Detmersp, Otto Gotzen, Samuel C. Silversteinll, and Samuel D. Wright** From the Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, New York 10021, 1Universitatsklinik Gottingen, Abteilung fur Immunologie, Kreuzbergring 57, 0-3400 Gottingen, Federal Republic of Germany, and the I1 Department of Physiology and Cellular Biophvsics, Columbia University College of Physicians and Surgeons, New York, New York 10032

” _ ”

Human leukocytes express a receptor that mediates the binding of cells and particles coated with C3bi, a fragment of the third component of complement. Pre- vious data indicate that the capacity of this receptor to mediate binding is regulated by changes in its aggregation state. Randomly distributed receptors bind ligand very inefficiently, but stimulation of polymorphonuclear leukocytes with phorbol esters causes a ligand-independent clustering of the receptors in the membrane, and the clustered receptors avidly bind C3bi-coated cells (1). We examined whether the aggregation state of surface-bound ligands also affects the efficiency of binding between receptors and ligands. We found that erythrocytes bearing C3bi in clusters were bound by both macrophages and polymorphonuclear leukocytes far more avidly than erythrocytes bearing the same number of ligands in random array. We made similar observations with erythrocytes coated with C3b, a ligand that is recognized by a separate receptor. Our observations show that the ability of a receptor-bearing cell to bind particles coated with the corresponding ligands is dramatically affected by the distribution of ligand on the surface of the particle. Cell-cell interactions may thus be regulated by alterations in the two-dimensional distribution of receptors and ligands on opposing cell surfaces.

Recent evidence suggests that the capacity of receptors to mediate adhesion between cells may be regulated by changes in the distribution of the receptors in the plane of the membrane (1). Human polymorphonuclear leukocytes (PMN)’

* This work was supported by United States Public Health Service Grants A122003 and A124775 (to S. D. W.) and A120516 (to S. C. S.), and by a grant in aid from the American Heart Association with funds contributed in part by the American Heart Association, Florida affiliate (to P. A. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Andre and Bella Meyer Fellow at The Rockefeller University. To whom correspondence should be addressed 1230 York Ave., Box 198, New York, NY 10021.

§ Arthritis Foundation Investigator. ** Established investigator of the American Heart Association.

The abbreviations used are: PMN, polymorphonuclear leukocytes; ECBb, erythrocytes bearing C3b; EC3bi, erythrocytes bearing C3bi; EC3bclustered, erythrocytes bearing C3b in clusters; EC3b,.,d0,, erythrocytes bearing C3b in random array; EC3bi,l,.te,d, erythrocytes bearing C3bi in clusters; EC3bi,,,do,, erythrocytes bearing C3bi in random array; HSA, human serum albumin; PMA, phorbol myristate acetate.

express a receptor that allows the binding of cells and particles coated with C3bi, a fragment of the third component of complement. Receptors for C3bi on resting human PMN are randomly distributed in the plane of the membrane when viewed by immunoelectron microscopy, and C3bi-coated erythrocytes are bound very poorly by these cells. Brief stimulation (15 min) of the PMN with the phorbol ester phorbol myristate acetate (PMA) dramatically enhances the efficiency with which the C3bi receptor binds ligand, and C3bi receptors on the cell surface show a coincident, ligand-independent aggregation into clusters. Continued stimulation (60 min) of PMN with PMA causes loss of binding activity, and the C3bi receptors return to a random distribution (1, 2). These and other data suggest that clustering of C3bi receptors is a prerequisite for efficient binding of ligand-coated erythrocytes.

Here we ask whether the converse is true: is clustering of ligand (C3bi) a prerequisite for the binding of C3bi-coated erythrocytes to C3bi receptor-bearing cells? We have taken advantage of the finding that C3 binds covalently to cells and that its preferred target on erythrocytes is the integral membrane protein glycophorin-a, the major erythrocyte sialogly- coprotein (3). Due to extensive cross-links between glycophorin-cy and the spectrin-containing membrane skeleton (4), glycophorin-a is relatively immobile in the membrane. Thus, ligands bound to glycophorin-a should be relatively immobile on the cell surface and should retain the pattern of distribution in which these ligands were initially bound. We exploited these conditions to prepare erythrocytes bearing C3bi on their surfaces in clustered or random array. We found that both macrophages and PMN recognized the clustered ligands far more efficiently than the randomly distributed ligands.

We obtained similar results using another receptor-ligand pair. Macrophages and PMN express a second receptor which recognizes C3b, a cleavage product of the third component of complement distinct from C3bi. Erythrocytes coated with C3b distributed in clusters were bound much more efficiently by macrophages and PMN than erythrocytes coated with C3b distributed randomly. Our results indicate that clustering of ligands, as well as clustering of receptors, may enhance adhesion between cells. Changes in the aggregation state of ligands and receptors may constitute a cellular strategy for the control of cell-cell adhesion.

MATERIALS AND METHODS

Reagents and Buffers-PMA, diisopropyl fluorophosphate, and aprotinin were supplied by Sigma, human serum albumin (HSA) by Worthington, trypsin by Cooper Biomedical, Inc., and fibronectin by the Greater New York Blood Center (New York, NY).

Buffers used were: phosphate-buffered saline (137 mM NaCl, 2.7 mM KC1, 0.9 mM CaC12, 0.5 mM MgC12, and 8 mM phosphate, pH 7.4)

17822

Clustering of Surface-bound Ligand Enhances Adhesion 17823

(5); phosphate-buffered saline without CaCI2 or M$12 (PD); PD containing 1 mg/ml HSA (PD/HSA); 2.5 mM veronal buffer, pH 7.5, 75 mM NaCI, 2.5% dextrose, 0.05% gelatin, 0.15 mM CaCI2, and 0.5 mM MgC12 (DGVB++); and 5 mM veronal buffer, pH 7.5, 150 mM NaCI, 0.1% gelatin, 0.15 mM CaC12, 0.5 mM M$12, and 0.15 mM NiCl2 (GVBNi).

Fab fragments of mouse monoclonal anti-C3 IgG (Bethesda Re- search Laboratories) were prepared by the method of Porter (6) and were purified by affinity chromatography on C3-zymosan as described previously (7). Fab anti-C3 was biotinylated by standard procedures (8) for use in immunoelectron microscopy.

C3 was the generous gift of Dr. R. Paul Levine (Washington University, St. Louis, MO). C3 was iodinated by the Iodogen procedure (9) to specific activities of IO6 to 10' cpm/pg for use in quantitation of C3 on C3-coated erythrocytes. Factors B and D were purified by published procedures (10, 11). C3b inactivator (factor I) was purchased from Diamedix (Miami, FL) and was treated with diisopropyl fluorophosphate prior to use (12).

5 nm colloidal gold was prepared by the method of Slot and Geuze (13) and was conjugated to streptavidin by standard procedures (14). Colloidal gold was characterized by electron microscopy to determine the size distribution of the particles and the degree of aggregate formation. Only those preparations with uniform size and having >99% of gold particles present as individuals were used for labeling experiments.

Coating Erythrocytes with C3b or C3bi-C3 is composed of a 127- kDa a chain and a 75-kDa 6 chain. Enzymatic activation by a protease (a "convertase" or trypsin) removes a 10-kDa fragment (C3a) from the a chain and alters the molecule in such a way as to expose an internal thioester group. The exposed thioester is the "labile binding site" of C3b. It has a very short half life (60 ps ; Ref. 15), either undergoing hydrolysis in the fluid phase or forming an ester or imidoester bond with a nearby reactive surface (16). When trypsin is added to a solution containing erythrocytes and C3, the C3 will be activated to C3b and the C3b will form a covalent bond, predomi- nantly to the hydroxyl groups of sugar molecules on glycophorin-a (3). Such trypsinization thus produces EC3b on which C3b has been attached singly and randomly (EC3b,.,do,).

lo9 sheep erythrocytes were mixed with C3 in concentrations ranging from 0.5 to 10 mg/ml and trypsin in DGVB++ at one-tenth the concentration of C3. After a 5-min incubation at 37 "C, the cells, now bearing a range of amounts of randomly bound C3b (EC3b,..d0,, Fig. U), were treated with 1 TIU/ml aprotinin to inhibit proteolysis, washed three times with ice-cold DGVB++, and resuspended to 10' cells/ml in DGVB++.

T o generate EC3b,lU.,d, each randomly placed C3b served as a nidus for the formation of a C3 cleaving enzyme of the alternative complement pathway. Cells with a random array of C3b were incubated with factors B and D. Factor B attaches to C3b, and factor D cleaves factor B to factor Bb; factor Bb, together with C3b, is known as C3 convertase. The convertase proteolytically activates C3 molecules, and the extreme lability of the binding site on C3b ensures that it will bind to the erythrocyte only in the immediate vicinity of the

A. E+ C3+ trypsin

8. E+ C3 + trypsin

C3, E, D, N i"

convertase. Generation of cell-bound C3 convertase and addition of more C3 allows for creation of clusters of C3b on the red cell surface (see Fig. 1).

A portion of the EC3b,.d0,,, decorated with C3 at the lowest concentration (0.5 mg/ml) was resuspended in GVBNi a t 4 "C. A nickel- stabilized C3 convertase was formed by modification of the procedure of Ross et al. (17). Factor B a t 100 pg/ml and factor D a t 400 ng/ml were added to EC3b,,do, and incubated for 2 min at 37 "C. After two washes in ice-cold GVBNi, C3 was added in concentrations ranging from 12.5 to 250 pg/ml, and cells were incubated for 20 min a t 37 "C to create erythrocytes with varying amounts of C3b clustered around each randomly placed convertase (EC3bcluSed, Fig. 1B). The cells were washed two times in ice-cold DGVB++ and resuspended to loR/ ml in DGVB++.

In order to cleave cell-bound C3b to C3bi, a portion of the EC3b were then treated with factor I at 83 units/ml at 37 "C for 3 and 6 h for EC3b,a,d,, and EC3bclustcd, respectively, in the presence of peni- cillin and streptomycin. Cells were washed three times in ice-cold DGVB++ and resuspended to 10"/ml.

The number of C3 bound per erythrocyte was determined in each preparation of erythrocytes by using "'I-C3 of known specific activity.

Characterization of Ligand-coated Erythrocytes-EC3b and EC3bi bearing '2'11-C3 were prepared as described above. After base-catalyzed scission of the ester linkage between C3 and the erythrocyte surface (16), the polypeptide structure of erythrocyte-bound C3 was determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (18). An autoradiogram of the gel revealed that the C3 eluted from both EC3b,.d., and EC3bclUsed exhibited the 127-kDa a chain and the 75-kDa 6 chain characteristic of intact C3b (Fig. 2, lanes a and c). Treatment of EC3b,,,,do,,, and EC3bc~uaed with factor I caused complete cleavage of the 127-kDa a chain to a 60-kDa species characteristic of C3bi (Fig. 2, lanes b and d). Thus, the C3b deposited by a cell-bound convertase has the same covalent structure as that deposited by trypsin, and neither preparation is contaminated to a measurable extent with C3bi. (Contamination with >5% C3bi would be detectable.) Native C3b binds factor B and acts as a cofactor for the cleavage of factor B by factor D. This activity of C3b has been observed for C3b deposited either by a cell-bound convertase or by trypsin (17) and is confirmed by our work; we find that the enhancement of cleavage of B by D is equivalent using C3b deposited by both methods (data not shown). Thus the conformation and availability of C3b appear identical whether deposited by a cell-bound convertase or by trypsin.

To determine whether our procedures succeeded in producing clustered and random distributions of C3 on the erythrocyte surface, we used immunoelectron microscopy to localize c3 on EC3b,.d,, and EC3bclUaed (19). EC3b a t 10' cells/ml were incubated with an equal volume of biotinylated Fab anti-C3 antibody a t 1 pg/ml in DGVB++ for 50 min on ice. Cells were washed three times with ice-cold DGVB++ and the cell pellet resuspended into 5 nm streptavidin colloidal gold diluted to an As20 of 0.5 in PD/HSA. Cells were then incubated for 50 min on ice, washed once in PD/HSA, and twice in PD buffer. Pelleted cells were resuspended in 5 pl of PD, and cells were lysed by pipetting 1 pl of concentrated cells onto a 2 0 4 drop of 2.2 mM phosphate, pH 7.4. Membranes floating on the surface of the drop were picked up on 300-mesh formvar- and C-coated grids (E. F. Fullam, Inc., Latham, NY) and washed briefly in phosphate buffer.

FIG. 1. A diagrammatic representation of the generation of EC3br..d,, and EC3b,l.,d. When erythrocytes are mixed with trypsin and C3, C3b is deposited on the cell surface in random array ( A ) . A portion of EC3b,.d,,,, with a low amount of attached C3b is further treated with factors B and D and C3 in the presence of nickel, which generates a C3 cleaving enzyme at the site of the original C3b. Addition of more C3 results in deposition of the C3b in the immediate vicinity of the original C3 ( B ) .

a b c d FIG. 2. SDS-polyacrylamide gel electrophoresis of eryth-

rocyte-bound C3. Erythrocytes were coated with I2'1-C3 and prepared for gel analysis of the bound C3 as described under "Materials and Methods." An autoradiogram of the resulting gel is shown here. Lane a, EC3b,.do,; lane b, EC3bi,.d.,; lane c, EC3bCl.,d; and lane d, EC3bLlusere+

17824 Clustering of Surface-bound Ligand Enhances Adhesion Grids were air-dried and examined on a JEOL 100 CX microscope a t 80 kV, and cells were photographed at random. The specificity of labeling was verified by the observation that there was no binding of streptavidin gold to EC3b when biotinylated Fab anti-C3 was omitted from the labeling protocol (data not shown). The efficiency of labeling was verified by the observation that the number of gold particles counted per erythrocyte was 73% as great as the number of C3b counted by radiolabeling methods (data not shown).

The distribution of C3b on the erythrocyte membrane was determined by counting the number of streptavidin-labeled gold particles (bound to biotinylated Fab anti-C3) found singly or in clusters on the membranes of Cab-coated erythrocytes. T o warrant inclusion in a cluster, gold particles had to be no more than 20 nm apart. For each experimental condition, a t least 4000 gold particles were counted, and the percent of the total gold particles in each size cluster was determined and graphed as a histogram (Fig. 3). The data presented are

30

0 0 20

a

- 01 ; I O &

c 0 0 s I 2 3 4 ?5

Gold particles per cluster

FIG. 3. Quantitation of C3 distribution on erythrocytes by immunolocalization. Erythrocytes bearing approximately 3000 clustered (0) or randomly distributed (0) C3b were prepared and incubated with biotinylated Fab anti-C3 and with 5 nm streptavidin- conjugated gold particles, and the number of gold particles per cluster was quantitated as described under “Materials and Methods.”

representative of three labeling experiments. Assay for Attachment of Erythrocytes-Human monocytes were

isolated from blood and cultured 4-8 days in Teflon beakers as described previously (7). During culture, the monocytes differentiate to macrophages. PMN were isolated from fresh whole blood on Ficoll- Hypaque gradients (20).

Attachment of ligand-coated erythrocytes to monolayers of macrophages or PMN was measured as described (7). Briefly, 50 erythrocytes were added per cell and the preparation was incubated a t 37 “C for 45 min. Unbound erythrocytes were removed by washing and attachment was scored by phase contrast microscopy. The number of attached erythrocytes per 100 phagocytes is termed the attachment index. Due to inherent variation in the C3b and C3bi binding capacity of each set of phagocytes, averages between experiments using different phagocyte preparations do not provide useful data. However, differences in EC3b and EC3bi attachment to a single set of phagocytes were reproducible and do provide useful data. Each experiment cited in this study is representative of at least three experiments.

RESULTS

Distribution of C3 on the Surface of Erythrocytes-Quanti- tative immunoelectron microscopy was used to determine the distribution of C3 on EC3b,,,dom and EC3bcIUswd bearing similar amounts of ligand (see Fig. 1 and “Materials and Methods”). On EC3b,.d0, labeled with monoclonal Fab anti- C3 and 5 nm colloidal gold, 53% of the gold particles were present as individuals and 90% of the particles were present in clusters of three or fewer. Clusters of four or more particles were rarely observed (3% of total gold particles) (Fig. 3). In contrast, on EC3bc,used only 30% of the particles were present as individuals and the amount of gold in clusters of five or more particles had increased to 18% of total. Thus, the principal difference between EC3b,,dom and EC3bc1uswd is in the frequency of large clusters. Examples of labeled EC3b,..do,

FIG. 4. Electron micrographs of the surface of EC3b. Immunoelectron microscopy using biotinylated monoclonal Fab anti-C3 bound to streptavidin-conjugated gold particles reveals the arrangement of C3b bound to the membrane. Arrowheads point to single molecules of C3b on EC3b,.d0, (a) and typical clusters of C3b on EC3bc~us,e4 ( b ) . Bar indicates 0.1 pm.


' " O L I zoo / L /

1 E C3b/E " 0

0 800 I600 2400

C3b i /E

FIG. 5. Clustered ligand is more avidly bound by the C3b and C3bi receptors on macrophages than randomly distributed ligand. Erythrocytes bearing C3 in clustered (closed circles) or random array (open circles) were prepared using different amounts of C3. The number of C3 per erythrocyte shown here represents the average of three separate erythrocyte preparations generated in an identical fashion and using identical reagents (see "Materials and Methods"). The binding of the coated erythrocytes to monolayers of macrophages plated on fibronectin was then measured. In three experiments, the binding of erythrocytes with clustered C3 was 6.2- fold greater (range 4.5-7.8) for EC3b or 3.1-fold greater (range 2.3- 6.65) for EC3bi than binding of erythrocytes with random C3. These binding comparisons are for erythrocytes bearing 1250 C3 molecules. A, binding of EC3b. b, binding of EC3bi.

and EC3bC~,,*,d are shown in Fig. 4. Observations on the distribution of C3b on erythrocytes

also apply to ECSbi, since EC3bi are derived from EC3b by treating with factor I to cleave the bound C3b to C3bi in situ.

Clustered C3 Binds More Efficiently Than Random C3 to Receptors on Macrophages-Monolayers of macrophages were incubated with EC3b,.,do, or EC3b,1,,~,d bearing a range of C3b per erythrocyte. Binding of EC3bCl,,*,d was at least 5- fold greater than binding of EC3b,,,d,, throughout the entire spectrum of ligand densities tested (Fig. 5A) .

A similar difference in the efficiency of binding of EC3bi,,,d0, and EC3biclused was observed. Throughout the range of amounts of C3bi per erythrocyte, binding of EC3bi,l,,~red was between 2- and %fold greater than binding of EC3bi,.,d0, (Fig. 5B).

Similar results were obtained for both EC3b and EC3bi using freshly isolated monocytes or cultured monocytes spread on surfaces coated with human serum albumin or fibronectin (data not shown).

Clustered C3 Binds More Efficiently Than Random C3 to Receptors on PMN-On PMN, the capacity of the C3bi receptor to bind ligand is regulated by alterations in receptor distribution. Treatment of PMN with PMA causes C3bi receptors to aggregate and, on continued treatment, to disag- gregate, with a concomitant rise and fall in the binding capacity for C3bi-coated erythrocytes (1, 2). To determine if the clustered receptor binds more efficiently to clustered or randomly distributed ligand, we tested binding of EC3birand0, and EC3bi,1,,~.,,d to PMN exposed to PMA (30 ng/ml) for various lengths of time. Stimulation of cells with PMA for 20 min caused nearly a &fold increase in binding of EC3bi,l,,,,d. In this time, the surface of the PMN shows both an increase in expression of the C3bi receptor (2-3-fold) and a movement

I200

800

400

x U a,

.- = o c

Q)

r E 8 400 c

3 300

200

I O 0

G 0 20 40 60 80

Minutes in PMA FIG. 6. C3b and C3bi receptors on polymorphonuclear leu-

kocytes bind clustered ligand more efficiently than randomly arrayed ligand. Monolayers of PMN were exposed to PMA at 30 ng/ml for various lengths of time. Erythrocytes bearing approximately 3000 C3/erythrocyte in clusters (closed circles) or in random array (open circles) were then added, and after 30 min of incubation at 37 "C attachment index was measured. After 20 min exposure to PMA, the binding of erythrocytes with clustered C3 was 6.0-fold greater (range 4.1-7.8) for EC3b or 4.0-fold greater (range 2.0-5.9)

of EC3b. B, binding of EC3bi. for EC3bi than binding of erythrocytes with random C3. A, binding

"- FIG. 7. Deadhesion of cells held together by randomly

placed or clustered bridges. See text for description. 0, ligand p , receptor.

of these receptors into clusters (2). Enhanced binding is thus a product of both enhanced expression and altered distribution of receptors. During the subsequent 40 min of PMA treatment, binding gradually declined, reaching control levels at 60 min (Fig. 6B). In this time, C3bi receptor expression remains constant (2). The disaggregation of receptors that occurs in this time thus appears to be the principal cause of decreased binding activity. In contrast to the results with EC3bLlU,*,d, binding of EC3bi,,do, barely rose above baseline during treatment with PMA. Thus, attachment of particles to PMN is most efficient when both receptor and ligand are clustered.

The C3b receptor on PMN also exhibits a transient increase in binding capacity upon stimulation of cells with PMA (2). We measured the binding of EC3b,,,d0, or EC3b,lUsered to

17826 Clustering of Surface-bound Ligand Enhances Adhesion

PMN treated with PMA to determine if the C3b receptor binds more efficiently to clustered or randomly distributed ligand. We observed that binding of EC3b,l,,t,,,d also increased nearly 5-fold by 20 min in PMA, declining gradually to control levels by 60 min (Fig. 6A). Binding of EC3b,,,dOm barely rose above base line during treatment with PMA. Although the aggregation state of the C3b receptor has not been measured, the similarity of these results to those obtained with the C3bi receptor suggest that the C3b receptor may also form transient clusters in the plane of the membrane after exposure of cells to PMA.

DISCUSSION

Our observations indicate that adhesion between cells can be greatly affected by the distribution of the molecules in- volved in recognition. The binding of C3b-coated erythrocytes to macrophages exhibited at least a &fold increase when ligand was clustered on the surface of the red cell (Fig. 5A). Ligand clustering caused a similar enhancement in binding mediated by the structurally distinct C3bi receptor (Fig. 5B) . Binding of IgG-coated particles to Fc receptors on mouse peritoneal macrophages also is strongly enhanced by clustering of ligand.2

Our data suggest that relatively large clusters of C3b and C3bi (25) are the principal mediators of binding to macrophages or PMN. Small clusters of ligand (2-4 CS/cluster) are clearly insufficient for binding, since EC3b,,,d,, bear similar numbers of such clusters as EC3b,l,,~Ed (Fig. 3) yet are not recognized by phagocytes (Figs. 5 and 6). On the other hand, the presence of larger clusters (5 or more) is well correlated with binding. EC3b,l,,te,.~ bear 6-fold more of these clusters than EC3b,,,dOm (Fig. 3) and are recognized nearly 5-fold more avidly by macrophages and PMN (Figs. 5 and 6).

Clusters of C3bi may be required for efficient adhesion to clusters of C3bi receptors. Previous data indicate that the C3bi receptor of stimulated PMN is clustered in a ligand- independent fashion into aggregates of 6-10 receptors and that the clustered state is a prerequisite for efficient binding to C3bi-coated erythrocytes (1). Those studies employed erythrocytes opsonized with C3bi by a method which would result in the deposition of clusters of ligand. We demonstrate here that clustered ligand is a prerequisite for binding to stimulated PMN, and efficient interaction of ligand-coated erythrocytes is only obtained when both C3bi and the C3bi receptor are present in clusters (Fig. 6B). Given the similar size of the clusters of ligand (25) and the clusters of receptors (6-10 receptors), we presume that these clusters interact.

Why does clustering of receptors and ligand promote the interaction of cells? Although we cannot rule out the possi- bility that clustering is accompanied by conformational changes in the proteins, we prefer the hypothesis that the multivalent binding between clusters stabilizes cell-cell interaction. Fig. 7 illustrates cell-cell adhesion mediated by either clustered or randomly placed bridges. A weak force may readily disrupt binding that is mediated by randomly placed links (top panel) because the force resisting each step of deadhesion is a single receptor-ligand bond of low affinity and fast dissociation rate. In contrast, deadhesion may be halted when a cluster of ligand and receptor is encountered (bottom panel). To dissociate a cluster of ligands from a cluster of receptors, each of the individual receptor-ligand interactions would need to be broken simultaneously. In addition, if a subset of the receptor-ligand pairs in a cluster were to dissociate, their confinement to a cluster would increase the like-

’ 0. Gotze and S. C. Silverstein, unpublished observations.

lihood of relocating each other. The extent to which deadhesion might be slowed by clustering can be appreciated from observations on the dissociation of oligomers of antibody from an antigen-coated surface (21). Dimerization causes a 5-10- fold decrease in dissociation rate but essentially no change in association rate. We would like to hypothesize that the aggregation of receptors and ligands in the two-dimensional plane of the membrane is comparable to the oligomerization of soluble ligand in three dimensions. Aggregation of the binding sites of immunoglobulin, or aggregation of ligand and of receptors into clusters, will in both cases make the binding reaction more favorable.

A possible functional role for ligand clustering is suggested by recent experiments which show that the C3bi receptor functions not only in binding of C3bi-coated cells but also in the movement of PMN out of the vasculature. PMN from patients with a genetic deficiency in the C3bi receptor fail to bind to endothelium both i n vivo (22) and i n vitro (23). The binding to endothelium is presumably mediated by a ligand on the endothelial cell, and thus PMN might be recruited from the circulation by aggregation of this ligand on the endothelial cell surface.

The C3bi receptor is structurally related to a class of broadly distributed receptors that mediate transient adhesion events (24). This class of receptors, termed “integrins,” includes the fibronectin receptor, vitronectin receptor, C3bi receptor, gly- coproteins IIb/IIIa of platelets (the fibrinogen receptor), lym- phocyte function-associated antigen-1 of killer T cells (a receptor for structures on target cells), and several others. Because of the high degree of structural homology between the C3bi receptor and other members of the integrin family (25-27), it is likely that our observations on the regulation of adhesion between the C3bi receptor and its ligand will be relevant to a large number of cellular adhesion events.

Acknowledgment-We thank Dr. Zanvil A. Cohn for critical read- ing o f this manuscript.

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27. Kishimoto, T. K., O’Connor, K., Lee, A., Roberts, T. M., and

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