Phagocytes express a family of structurally related receptors, LFA-1, CR3, and p150,95, that mediate adhesion of leukocytes to a variety of cells and surfaces. LFA-1 mediates the binding of killer T cells to targets, CR3 mediates binding of phagocytes to iC3b-coated surfaces and to endothelial cells, and LFA-1, CR3, and p150,95 each mediate the binding of bacterial lipopolysaccharide. Here we review the structure and function of each of these receptors and present evidence that they are related to a larger class of adhesion-promoting receptors called integrins. Of particular emphasis are observations that the capacity of these receptors to promote adhesion is strongly and reversibly modulated by both soluble and surface-bound stimuli. We review this form of regulation and present evidence that changes in the binding activity of adhesion-promoting receptors is accomplished by changes in the two-dimensional distribution of receptors in the plane of the membrane. Inactive receptors are randomly distributed in the membrane, and their ability to bind a ligand-coated surface is enabled by a ligand-independent movement into small clusters. The implications of these structural features are discussed.

Adhesion between phagocytes and other cells, particles, or the extracellular matrix is crucial for a variety of functions such as phagocytosis, diapedesis, and positioning of phagocytes in the liver, lungs, and other tissues. It has long been assumed that specific receptors mediate cellular adhesions, but only recently have such receptors been isolated and characterized. We describe here a newly recognized class of receptors that mediate cell-cell and cell-substratum adhesion, and we review the functions of these receptors on phagocytes.

The notion that a few receptors could be responsible for a wide range of adhesion events first arose with the discovery of a class of patients that exhibit recurrent lifethreatening infections with gram-positive, gram-negative and fungal pathogens (reviewed in Anderson & Springer, 1987; Todd & Freyer, 1988). These patients exhibit extreme leukocytosis but a failure to form pus at sites of infection. In vitro experiments showed that polymorphonuclear leukocytes (PMN) from these patients are defective in adhesion to iC3b-coated erythrocytes, to protein-coated glass or plastic surfaces (Anderson et al. 1984), and to endothelial cells (Harlan et al. 1985), and that failure to adhere results in the failure to display chemotactic responses. Thus, susceptibility to infection probably results both from an inability to bind and ingest opsonized pathogens and a failure to recruit cells to sites of infection. More importantly, these observations suggest that the leukocytes of LAD patients fail to extravasate and are retained in the vasculature by a failure to adhere to endothelial cells. Characterization of the proteins missing from the patients’ leukocytes have amply confirmed their role in adhesion to endothelium and in several other adhesion events.

The phenotype of LAD patients is caused by a failure of leukocytes to express three related proteins, LFA-1, CR3, and p150,95 (Springer et al. 1984). Each of these cellsurface glycoproteins consists of an α1ß1 dimer composed of a 150-190K (K = 103Mr) a chain and a 95K ß chain, schematically depicted in Fig. 1. The ß chain is identical in each of these three proteins (Sanchez-Madrid et al. 1983), and has been given the designation CD 18 by the International Leukocyte Antigen Workshop. The α chains, on the other hand, are structurally and antigenically distinct. Thus a monoclonal antibody against the α chain of CR3 (OKM1 for example) will bind and precipitate CR3, but not LFA-1 or p150,95, while a monoclonal against the common ß chain (IB4 for example) will precipitate all three of these dimeric proteins (Wright et al. 1983a). The α chains of LFA-1, CR3, and p150,95 are termed CD11a, CD11b, and CDllc, respectively, and CR3 may thus be referred to as CD11b/CD18.

Fig. 1.

Schematic representation of the LFA-1, CR3, p150,95 family of leukocyte antigens. Each protein is a dimer composed of a unique α chain in association with a ß chain that is identical in all three proteins.

Fig. 1.

Schematic representation of the LFA-1, CR3, p150,95 family of leukocyte antigens. Each protein is a dimer composed of a unique α chain in association with a ß chain that is identical in all three proteins.

LAD patients are deficient in all three of these proteins because of an inherited defect in the ß chain (Springer et al. 1984). Cells from LAD patients do synthesize normal precursors of the α chains, but these precursors are very rapidly degraded and do not appear on the cell surface. The α chains can, however, be ‘rescued’ in cells fused with a murine partner that expresses normal ß chain (Marlin eí al. 1986). Here the human α chain appears on the cell surface joined with a murine ß chain.

The cellular distribution of LFA-1, CR3, and p150,95 is outlined in Table 1. Lymphocytes express abundant LFA-1, but neither CR3 nor p150,95 can be detected. PMN express abundant CR3, and LFA-1 and p150,95 are minor but easily detectable components. Macrophages express large amounts of all three of these proteins. Expression of these antigens appears restricted to leukocytes, and they are not found on other mammalian cell types.

Table 1.

Adhesion-promating receptors of leukocytes

Adhesion-promating receptors of leukocytes
Adhesion-promating receptors of leukocytes

The role of the individual receptors has been dissected primarily with the use of monoclonal antibodies against the individual a chains. Some of the principal antibodies that have been used are listed in Table 1.

CR3

The first molecule shown to be deficient from LAD patients (Arnaout et al. 1982) had been previously identified as a receptor for a cell-bound fragment of complement (Beller et al. 1982; Wright et al. 1983a), and the name CR3 (complement receptor type three) derives from the nomenclature of complement receptors (see the chapter in this volume by S. K. A. Law for more detailed discussion of complement and other complement receptors). CR3 has also been referred to as Mac-1 and Mol because it is the first phagocyte-specific antigen identified by monoclonal antibodies in mouse and man respectively. CR3 functions as an opsonic receptor, and promotes the binding of iC3b-coated cells and particles by monocytes, macrophages, and PMN (Wright & Silverstein, 1982; Wright & Meyer, 1986). In appropriately stimulated cells, binding is followed by phagocytosis of the particle. The credentials of CR3 as an adhesion-promoting receptor are thus well established.

CR3 recognizes surface-bound iC3b but does not recognize either the precursor, C3b, or the product of further cleavage, C3dg (Carlo et al. 1979; Wright & Silverstein, 1982; Ross & Lambris, 1982). (C3b is recognized by a separate receptor, CR1, and this receptor is described by Law (1988).) Binding of monomeric or dimeric iC3b to CR3 has not been reported, presumably because of a low binding affinity, and the above statements on the specificity of the receptor rest primarily on studies using particles coated with defined complement fragments to measure binding to CR3-bearing cells. These particles, usually sheep erythrocytes, bear from 104 to 105 iC3b per red cell, and the resulting multivalent interaction with phagocytes is very avid. By analogy, it is likely that LFA-1 and p150,95 also bind their target ligands with low affinity and that adhesion events mediated by these receptors are driven by multivalent interactions. As described below, a functional consequence of this low affinity is a potential to reverse adhesion and promote detachment.

CR3 is composed of two non-covalently linked polypeptides, an α chain of 185K and a ß chain of 95K, both of which are exposed at the cell surface (Sanchez-Madrid et al. 1983; Wright et al. 1983a). Two lines of evidence suggest that the binding site for iC3b is located, at least in part, on the a polypeptide. First, monoclonal antibodies directed against the α polypeptide have been raised, and a subset of these block the binding of iC3b, while antibodies against the ß chain do not (Wright et al. 1983a). Further evidence for the role of the α chain in the binding of iC3b comes from observations of the ß chain in dimeric association with alternative α chains. Neither LFA-1 nor p150,95 recognizes iC3b (Wright & Jong, 1986) and since both of these receptors express ß chain, it is unlikely that the ß chain binds iC3b.

CR3 requires relatively high concentrations of divalent cations (about 0·5mM-Ca2+ and Mg2+) in order to interact effectively with ligand (Wright & Silverstein, 1982). This behaviour contrasts with that of other opsonic receptors (CR1, and receptors for the Fc domain of IgG (FcR)), which do not require divalent cations for binding activity. In addition, the binding capacity of CR3 is temperature dependent, and is absent in cells held at 0°C (Wright & Jong, 1986), again distinguishing CR3 from other opsonic receptors (CR1, FcR) which bind well at 0°C.

The region of the iC3b molecule that is recognized by CR3 has recently been defined by the use of synthetic peptides (Wright et al. 1987). CR3 binds particles coated with a 21 amino acid peptide that spans residues 1383-1403 of C3. This peptide contains the triplet Arg-Gly-Asp (RGD) in its midsection. As discussed below, RGD serves as a recognition structure for many receptors involved in cell adhesion events, and CR3 thus appears to be a member of a larger family of adhesionpromoting receptors.

Molecules other than iC3b may also serve as ligands for CR3. As noted above, CR3 is the principal adhesion-promoting receptor of PMN, and PMN from LAD patients fail to adhere to endothelial cells both in vitro (Harlan et al. 1985) and in vivo (reviewed in Anderson & Springer, 1987; Todd & Freyer, 1988). Moreover, antibodies against CR3 block the binding of phagocytes to endothelial cells in vivo (Arfors et al. 1987; Rosen & Gordon, 1987; K.-E. Arfors & S. D. Wright, unpublished observations) and in vitro (Wallis et al. 1986). Thus, CR3 is likely to recognize a structure on endothelial cells and to mediate diapedesis.

LFA-1

Monoclonal antibodies against LFA-1 were identified by screening clones for the ability to block T-cell-mediated cytolysis (Sanchez-Madrid et al. 1982). These studies identified three ‘lymphocyte function-associated antigens’, LFA-1, LFA-2, and LFA-3, which are required for efficient cytolysis. LFA-2 and LFA-3 are structurally and functionally distinct from the proteins under consideration in this review and will not be discussed further.

Several studies indicate that anti-LFA-1 antibodies block cytolysis by blocking adhesion of the killer cell to the target. The step that is blocked by binding of the antibody to LFA-1 on the killer cell had previously been described as a Mg2+-dependent, temperature-dependent adhesion event (reviewed in Martz, 1986). Anti-LFA-1 antibodies also block natural killer-mediated cytotoxicity (Mentzer et al. 1986a), macrophage-mediated tumour cytotoxicity (Strassman et al. 1986), and PMN-mediated antibody-dependent cytotoxicity (Miedema et al. 1984; Kohl et al. 1984). In all of these cases, the inhibition is caused by binding of the antibody to LFA-1 on the killer cell (not on the target), and thereby blocking adhesion to the target cells. Consistent with these data are results that show that T cells, NK cells, and PMN from LAD patients all show defects in cytotoxicity assays (Kohl et al. 1984; Mentzer et al. 1986b).

It is important to point out in this context that LFA-1 does not provide the specificity for the cytotoxic cells. Rather, targets are selected by separate recognition molecules (T3Ti for example) on the killer, which bind to structures on the target. The bond created by these recognition molecules, however, is weak and LFA-1 functions to strengthen the adhesion (Martz, 1986).

LFA-1 also participates in adhesion events that are unrelated to cytotoxicity. Anti-LFA-1 blocks both the spontaneous (Mentzer et al. 1985) and the PMA-stimulated aggregation of B lymphocyte cell lines (Rothlein et al. 1986), and very recent data indicate that LFA-1 is involved in strengthening the adhesion between T cells and dendritic cells (Inaba & Steinman, 1987). Effective adhesion with a dendritic cell appears to be a prerequisite for stimulation of resting T cells (Steinman et al. 1986).

The ligand that LFA-1 recognizes on target cells has long eluded investigators, but recent studies have identified a molecule, intercellular adhesion molecule-1 (ICAM-1), that serves as a ligand in at least some instances. ICAM-1 is recognized by a monoclonal antibody that blocks LFA-1-dependent aggregation of lymphoblastoid cell lines (Rothlein et al. 1986). This inhibition is additive with that caused by anti-LFA-1. While the aggregation of some cell lines is strongly inhibited by anti-ICAM-1 antibody, the LFA-1-dependent aggregation of other lines cannot be inhibited. Thus, it appears that ICAM-1 is not the only ligand for LFA-1.

ICAM-1 is a membrane-bound polypeptide of 90K (Rothlein et al. 1986) found on epithelial cells, endothelial cells, B cells, and macrophages (Dustin et al. 1986). By homology one would postulate that ICAM-1 contains an RGD sequence similar to that recognized by CR3 in C3bi, but sequence data on ICAM-1 are not yet available.

p150,95

A unique function for p150,95 has not yet been described and its name reflects only the molecular weight of its constituent polypeptide chains. Nevertheless, it does appear to function in adhesion events. p150,95 participates in binding of unopsonized bacteria (Wright & Jong, 1986) and fungi (Bullock & Wright, 1987), a function shared with LFA-1 and CR3 (see below). Further, certain cytotoxic T cell clones express appreciable levels of p150,95, and the killing mediated by these cell lines can be blocked by anti-p150,95 antibodies (Keizer et al. 1987). As observed with anti-LFA-1 antibodies, the anti-p150,95 antibodies block formation of conjugates of killer and target. A unique ligand for p150,95 has not yet been identified.

Antibodies against p150,95 were first obtained by immunizing mice with cells from patients with hairy cell leukaemia (Schwarting et al. 1985). Hairy cells express very abundant p150,95 and very little CR3 or LFA-1 (S. D. Wright, unpublished observations), but the significance of this observation is not clear. p150,95 is also expressed in high abundance on tissue macrophages (Hogg et al. 1986), and may thus be expected to serve a. major function of these cells.

Several workers have noted that antibodies against CR3 not only inhibited the binding of iC3b-coated particles but also partially inhibited binding of particles such as zymosan (Ezekowitz et al. 1984; Ross et al. 1985a), Leishmania (Blackwell et al. 1985; Mosser & Edelson, 1985), and Staphylococcus (Rosset al. 1985b). Since these binding experiments were performed in the absence of a source of complement, it was proposed that CR3 interacts not with C3bi but with the particles directly. Further support for this idea came from the observation that cells from LAD patients exhibit defective recognition of zymosan (Ross et al. 1985b) and Escherichia coli (S. D. Wright, unpublished observations), again in the absence of serum opsonins.

More detailed experiments have indicated that CR3, LFA-1, and p150,95 each share the capacity to bind directly the microbes Histoplasma capsulatum (Bullock & Wright, 1987) and E. coli (Wright & Jong, 1986). In these experiments, individual receptors were cleared from the apical surface of the macrophage membrane by allowing the cells to spread on surfaces derivatized with specific anti-receptor monoclonal antibodies. The receptors diffuse in the plane of the membrane and are trapped by antibody at the basal surface of the macrophage (Fig. 2). By this means it was shown that clearing all three receptors, CR3, LFA-1, and p150,95, inhibits the binding of microbes, but binding is still observed if any one of these receptors is present. Thus, each of these homologous receptors is individually capable of binding to El. capsulatum and E. coli.

Fig. 2.

Clearing receptors from the apical portion of the plasma membrane by surfacebound anti-receptor antibodies. Macrophages spread on serum albumin-coated surfaces exhibit cell surface receptors that are distributed around the entire cell perimeter (lefthand side). In contrast, macrophages spread on surfaces coated with anti-receptor monoclonal antibodies exhibit depletion of receptors from the apical membrane (righthand side). This clearing of receptors from the apical surface occurs by diffusion of receptors in the plane of the membrane to the basal surface where they are trapped by interaction with a specific antibody. A single monoclonal antibody only clears its target antigen, not other receptors on the cell surface.

Fig. 2.

Clearing receptors from the apical portion of the plasma membrane by surfacebound anti-receptor antibodies. Macrophages spread on serum albumin-coated surfaces exhibit cell surface receptors that are distributed around the entire cell perimeter (lefthand side). In contrast, macrophages spread on surfaces coated with anti-receptor monoclonal antibodies exhibit depletion of receptors from the apical membrane (righthand side). This clearing of receptors from the apical surface occurs by diffusion of receptors in the plane of the membrane to the basal surface where they are trapped by interaction with a specific antibody. A single monoclonal antibody only clears its target antigen, not other receptors on the cell surface.

The capacity of CR3, LFA-1, and p150,95 to bind directly to microbes without the intervention of antibody or complement represents a mechanism by which macrophages may recognize potential pathogens before the onset of adaptive immunity. Consistent with this notion is the observation that LAD patients suffer frequent lifethreatening infections primarily in the first 2 years of life. Those patients that survive the first years, however, may live to adulthood in relatively good health.

What is the nature of the ligand on the surface of microbes that is recognized by adhesion-promoting receptors? Recent work indicates that the chemical structure of the ligands on E. coli is very different from the proteins, iC3b or ICAM-1. Macrophages bind E. coli by recognizing lipopolysaccharide (LPS), the most prevalent molecule on the surface of the bacterium (Wright & Jong, 1986). The portion of the LPS molecule that is recognized is the Lipid A region, which consists of a fatty-acylated diglucosamine bisphosphate. Since the fatty acids of Lipid A are buried in the outer membrane of the bacterium, it is likely that the diglucosamine phosphate provides the recognition structure bound by CR3, LFA-1, and p150,95. It is also likely that sugar phosphates on the surface of H. capsulatum, zymosan, and Leishmania may account for their recognition by these receptors.

Bacterial LPS (endotoxin) causes profound physiological effects in man and animals. These include fever, shock, and the induction of the acute phase response (Morrison & Ulevitch, 1978). The cell type primarily responsible for these effects is the macrophage, which synthesizes large amounts of interleukin-1 and cachectin (TNFα) in response to LPS (Beutler et al. 1985; Durum et al. 1985). The studies described above indicate that CR3, LFA-1, and p150,95 can bind LPS, but whether these receptors directly mediate the many biological effects of LPS is not yet certain.

How can adhesion-promoting receptors bind ligands as different as iC3b and LPS? Ross et al. (1985a) originally suggested that CR3 has two distinct binding sites, one site for iC3b and another for microbes, and recent data support this claim. The binding site for iC3b can be blocked by certain monoclonal anti-ar chain antibodies (OKM10 for example), which do not block binding of LPS (S. D. Wright, unpublished results) or the CR3-dependent spreading of PMN on protein-coated plastic (Dana et al. 1986). Conversely, binding of LPS (S. D. Wright, unpublished results) and spreading on plastic (Danaet al. 1986) can be blocked by an anti-a chain antibody (mAb 904) that does not block the binding of iC3b. These data suggest that CR3 has two binding sites, one for iC3b and another for LPS. It is of interest to note that LPS is a common contaminant of all laboratory solutions, binds avidly to tissue culture plastic (Wright & Jong, 1986), and may thus contribute to the avid adhesion of phagocytes to artificial substrata in vitro.

Work from several laboratories indicates that CR3, LFA-1, and p150,95 are members of a large, broadly distributed family of proteins that promote cell adhesion (Ruoslahti & Pierschbacher, 1986; Hynes, 1987). This class of receptors, recently termed ‘integrins’ by Hynes (1987), includes the fibronectin receptor, vitronectin receptor, gp Ilb/IIIa of platelets (the fibrinogen receptor), the VLA antigen series, and several others. Sequence data show extensive homology among a chains and among ß chains of all the integrins sequenced. A detailed description of the structural properties of integrins and their relation to one another is presented by Law (1988) and will not be covered here. Rather, we will point out several functional similarities among integrins, which allow a unified view of their actions.

A summary of the properties of several integrins is shown in Table 2. While not all properties have been documented for all receptors, the consensus emerges that each integrin is a large dimeric surface protein that promotes relatively weak adhesion either to ligand-coated cells or substrata. Effective adhesion requires warm temperatures and divalent cations. The requirement for divalent cations is explained by the presence of tandem repeats of a cation-binding domain in the a chain of all integrins thus far sequenced. The requirement for warm temperatures remains to be explained.

Table 2.

Integrins, a family of adhesion-promoting receptors

Integrins, a family of adhesion-promoting receptors
Integrins, a family of adhesion-promoting receptors

Integrins appear capable of binding to two types of ligands. The first and best characterized are protein in nature. These ligands show far less sequence homology than the receptors, but nearly all contain the amino acid sequence, Arg-Gly-Asp (RGD), at the recognition site (Ruoslahti & Pierschbacher, 1986). For example, the fibronectin receptor recognizes the sequence GRGDS in fibronectin, the vitronectin receptor recognizes TRGDV in vitronectin, and CR3 recognizes YRGDQ in C3bi. Nevertheless, integrins do not cross react with each others’ ligands. For example, CR3 does not bind fibronectin, nor does the fibronectin receptor bind iC3b (Wright & Meyer, 1985). A likely explanation for this is that the amino acids flanking RGD exhibit only weak similarity, thus providing a possible basis for discrimination (Wright etal. 1987).

A second type of ligand for integrins is glycolipid. Ganglioside GD2 co-localizes with the vitronectin receptor in adhesion plaques and co-purifies with the vitronectin receptor in detergent solutions, indicating the presence of a binding site on this integrin for glycolipid (Cheresh et al. 1987). As detailed above, CR3, LFA-1, and p150,95 each recognize LPS, another glycolipid (Wright & Jong, 1986). It is not clear at present whether the binding site for LPS on the CD 18 complex is homologous with the binding site for GD2 on the vitronectin receptor, since, in the former case, the receptors bind LPS that is anchored in an adjacent bilayer, and in the latter, receptors bind GD2 that is anchored in the same bilayer. It is also not known if other integrins bind glycolipids, but it appears likely since cell types that do not express CR3, LFA-1 or p150,95, such as endothelial cells, do show marked physiological responses to LPS (Schleimer & Rutledge, 1986).

Integrins appear not to be involved in the strong, long-term adhesions found between cells in tissues. Thus, integrins appear unrelated to proteins of gap junctions, tight junctions, or desmosomes. Rather, integrins mediate relatively weak adhesions that may be readily broken.

The reversible nature of adhesions caused by integrins is illustrated by several findings. (1) Cells bearing fibronectin receptors can migrate rapidly across surfaces coated with fibronectin (Rovasio et al. 1983), indicating that sequential, transient attachments are made between fibronectin receptors and ligand. (2) The LFA-1-mediated binding of killer T cells to targets can be as short as 10 min, ending when the killer detaches and moves to another target (Sanderson, 1976). (3) The iC3b receptor mediates the binding of polymorphonuclear leukocytes (PMN) to endothelial cells, which must be short-lived to enable diapedesis to occur. (4) High concentrations of synthetic peptides bearing the RGD sequence, which competitively block the action of several integrins (Ruoslahti & Pierschbacher, 1986), prevent normal migration of cells in avian, amphibian (Boucaut et al. 1984), and insect embryos (Naidet et al. 1987), a process for which transient adhesions are obviously required.

One can imagine two models of receptor behaviour that could be used to generate transient adhesions. In the first model, cells would deliver adhesion-promoting receptors to the cell surface only at the place and time required, and after they had functioned, either the receptor or the ligand would be proteolytically inactivated to break the adhesion. In a second model, adhesions are made or broken by allosteric alteration of the receptors, which effectively turn them on or off. An important prediction of this second model is that receptors may be left on the cell surface in the ‘off’ state such that they can be turned on when and where needed. We present evidence below that supports the second of these two hypotheses, and demonstrates that CR3 is expressed on the cell surface in a form that is unable to bind ligand. We believe that this property of on/off regulation will prove to be a general feature of integrins.

Several observations suggest that integrins might exist on the cell surface in an inactive state. Transformed fibroblasts fail to bind fibronectin despite apparently normal levels of fibronectin receptors (Hirst et al. 1986; Chen et al. 1986). LFA-1 may also exist in an inactive form, since the LFA-1-dependent aggregation of B cells or lymphoblastoid cell lines is not observed unless the stimulant phorbol myristate acetate (PMA) is added (Rothlein & Springer, 1986). The most detailed studies of on/off behaviour, however, are available for CR3 on macrophages and PMN. We review here the evidence for regulation of binding and phagocytosis by CR3 in phagocytes and discuss the agents that mediate the regulation. We will first discuss regulation of the binding capacity of CR3.

CR3 on resting human monocytes and macrophages avidly mediates binding of iC3b-coated erythrocytes with half-maximal binding at about 5000 iC3b per erythrocyte (Wright & Silverstein, 1982). Culture of the phagocytes for 48 h with interferon (IFN)y (but not IFNa) causes a striking decrease in the binding capacity of C3 receptors: half-maximal binding of erythrocytes is not obtained even with 120 000 iC3b per erythrocyte (Wright et al. 1986). This result contrasts with the behaviour of Fc receptors in that IFNγ causes dramatically enhanced expression of FcγRp72 (Guyre et al. 1983) and enhanced binding of IgG-coated erythrocytes (Wright et al. 1986).

The reduced binding activity of CR3 in IFNγ-treated cells is not associated with changes in the number of cell surface receptor molecules, nor is it associated with proteolytic inactivation of the receptors (Wright et al. 1986). Rather, CR3 appears to be reversibly disabled. This point is emphasized by the observation that the binding capacity of CR3 can be fully restored in minutes by allowing the phagocytes to interact with surfaces coated with the extracellular matrix protein, fibronectin (see below).

The physiological significance of this ‘deactivation’ of CR3 by IFNγ is not currently clear, but IFNγ can be expected to diminish complement receptor activity on all macrophages except those in contact with the appropriate extracellular matrix components. Since IFNγ-treated macrophages possess extremely potent cytolytic activity, lowered capacity to adhere may control inappropriate cytolysis.

The binding activity of CR3 on granulocytes is also regulated but in a manner different from that seen in mononuclear cells (Wright & Meyer, 1986). Resting PMN have a low capacity to bind C3-coated particles. This binding activity is very rapidly up-regulated by several agents including PMA and C5a. Though PMA is not a physiological stimulus, its activities are the best characterized and are described in detail below.

Stimulation of PMN with PMA produces a biphasic response (Wright & Meyer, 1986). During the first 20 min, the capacity of CR3 to bind and phagocytose iC3b-coated erythrocytes is dramatically enhanced. During the following 30 min, the capacity to bind and phagocytose is depressed to levels below those shown by resting cells. Treatment of PMN with PMA causes a rapid rise in the expression of CR3 on the cell surface (Berger et al. 1984; O’Shea et al. 1985; Wright & Meyer 1986). Specific granules (Todd et al. 1984; O’Shea et al. 1985), granules identified by the presence of gelatinase (Petrequin et al. 1987), and granules identified by the presence of alkaline phosphatase (Borregaard et al. 1987) have all been implicated as a source of the newly expressed CR3. Though increased expression of CR3 is temporally associated with the enhanced capacity to bind and ingest C3-coated particles caused by PMA, an increased number of receptors is unlikely to be responsible for either the increased binding or phagocytosis. During activation of adherent PMN by PMA, the attachment of iC3b-coated erythrocytes increases 8-to 10-fold and phagocytosis increases 30-to 40-fold while the number of CR3 per cell increases only threefold (Wright & Meyer, 1986). More strikingly, the capacity of CR3 to bind ligand and signal phagocytosis is eliminated during incubation with PMA from 20 to 65 min, but the expression of CR3 does not change in this time. Increasing the expression of CR3 on the cell surface is clearly not sufficient to induce ligand binding, since treating PMN with the chemotactic peptide N-formyl-methionyl—leucyl—phenylalanine (fMLP) causes a twofold increase in the amount of CR3 on the cell surface (Berger et al. 1984) but does not affect the binding activity of this receptor (Detmers et al. 1987). The capacity of CR3 to bind ligand must therefore be controlled in another way.

What biochemical events could explain the transient changes in CR3 activity? Several observations suggest that phosphorylation may provide an answer. (1) The time course and reversibility of C3 receptor activation are consistent with the hypothesis that phosphorylation controls receptor activity. (2) PMA is a potent activator of a ubiquitous Ca2+-activated, phospholipid-dependent protein kinase (Castagna et al. 1982). (3) Loading PMN with inorganic thiophosphate (thioP) allows irreversible activation of C3 receptors (Wright & Meyer, 1986). ThioP resembles phosphate and is incorporated into nucleotides and phosphoproteins, but the resulting thiophosphoproteins are resistant to phosphatases. One would thus expect that in a cell loaded with thioP, phosphorylation caused by stimulation of a kinase would result in a pool of thiophosphorylated proteins, which are resistant to dephosphorylation. It is thus likely that the irreversible activation of receptors observed in loaded cells is a consequence of irreversible thiophosphorylation. (4) Very recent data show that CR3 is a phosphoprotein, and its state of phosphorylation is regulated by PMA (S. D. Wright, unpublished results).

How could phosphorylation of CR3 alter its capacity to bind ligand? Our recent work indicates that CR3 aggregates in the plane of the membrane in response to PMA, and such aggregation appears to be a prerequisite for ligand binding (Detmers et al. 1987). When the surfaces of resting PMN are labelled with monoclonal anti-CR3 (OKM1) and colloidal gold and viewed by transmission electron microscopy, the gold particles depicting CR3 are present in a random distribution, with many as individuals (Fig. 3A). After PMN are treated with PMA for 25 min, which dramatically enhances binding activity, clusters of receptors are apparent (Fig. 3B). The time course of aggregation corresponds precisely with the time course of enhancement of binding. Aggregation is high at 25 min in PMA, and there is disaggregation by 50 min in PMA, by which time binding declines to levels comparable to those observed in resting cells (Fig. 4). Other surface antigens on PMN (FcR, HLA) do not exhibit changes in their aggregation state in response to PMA, indicating that this is not a general property of membrane proteins. As mentioned above, treating PMN with the chemotactic peptide fMLP causes increased expression of CR3 on the cell surface (Berger et al. 1984) but does not enhance the binding activity of the receptor (Detmers et al. 1987). fMLP also does not cause aggregation of CR3, again supporting the idea that clustering of CR3 is required for binding ligand.

Fig. 3.

Clustering of CR3 on the surface of PMN in response to PMA. A, Resting PMN and B, PMN treated for 25 min at 37°C with 33 ng ml-1 PMA were labelled with biotin-OKM1 and 10-nm streptavidin-conjugated colloidal gold as previously described (Detmers et al. 1987). Whereas most gold particles are present on resting cells as individual particles, distinct clusters of receptors are apparent after treatment with PMA (see text for details). Bar, 0·2 μm.

Fig. 3.

Clustering of CR3 on the surface of PMN in response to PMA. A, Resting PMN and B, PMN treated for 25 min at 37°C with 33 ng ml-1 PMA were labelled with biotin-OKM1 and 10-nm streptavidin-conjugated colloidal gold as previously described (Detmers et al. 1987). Whereas most gold particles are present on resting cells as individual particles, distinct clusters of receptors are apparent after treatment with PMA (see text for details). Bar, 0·2 μm.

Fig. 4.

Transient aggregation of CR3 on the surface of PMN treated with PMA. PMN were incubated for 5Omin at 37°C with 33 ng m-1 PMA added for 0 (A), 10 (B), 25 (C) or 50 (D) min. Cells were labelled with biotin-OKMl and 10-nm streptavidin gold, and the gold particles in each size of cluster were quantitated as previously described (Detmers et al. 1987). Aggregation increases from 0 to 25 min, and disaggregation occurs from 25 to 50 min (see text for details).

Fig. 4.

Transient aggregation of CR3 on the surface of PMN treated with PMA. PMN were incubated for 5Omin at 37°C with 33 ng m-1 PMA added for 0 (A), 10 (B), 25 (C) or 50 (D) min. Cells were labelled with biotin-OKMl and 10-nm streptavidin gold, and the gold particles in each size of cluster were quantitated as previously described (Detmers et al. 1987). Aggregation increases from 0 to 25 min, and disaggregation occurs from 25 to 50 min (see text for details).

A possible mechanism by which clustering of receptors may endow them with enhanced binding activity is suggested by studies on the spatial distribution of the ligand, iC3b, on the surface of ligand-coated particles (A. Hermanowski-Vasotka, P. A. Detmers, D. Goetze, S. C. S. Iverstein & S. D. Wright, unpublished results). When C3 is deposited as random monomers, the resulting C3bi-coated erythrocytes are not bound by macrophages. However, if an equivalent number of C3bi molecules are deposited in clusters, binding to macrophages is avid. Similar results are observed with PMN. Even stimulated cells, in which CR3 is clustered on the surface of the phagocyte, are incapable of binding erythrocytes with randomly deposited iC3b, and avid binding is only observed when both the iC3b on the erythrocyte and the CR3 on the PMN are both clustered. Since the size of the clusters of iC3b (>5) is similar to the size of the clusters of CR3 (6—10), it is likely that clusters of ligand interact with clusters of receptors to mediate effective binding between cells.

Why does clustering of receptors and ligand promote the interaction of cells? Though we cannot rule out the possibility that clustering causes conformational changes in the proteins, we prefer the hypothesis that the multivalent binding between clusters stabilizes cell-cell interaction. To disassociate a cluster of ligands from a cluster of receptors, each of the individual receptor—ligand interactions would need to be simultaneously broken. The unlikelihood of this event could prevent the detachment of a iC3b-coated cell.

The observation that clustering of CR3 is required to mediate adhesion suggests a mechanism by which cells could detach from a ligand-coated surface or cell. Receptors that are actively mediating adhesion are held in position by two types of bonds, those among components of a receptor cluster and those between receptor and the opposing ligand. To reverse adhesion, these two bonds may be broken sequentially. The bond tethering a receptor to other members of a cluster appears to involve phosphorylation of the receptor and may thus be broken by the action of a phosphatase. Since the binding affinity of the second bond (between an individual receptor and an individual ligand) is very low, the ‘off rate’ will be high and receptors will release ligand often. Upon releasing ligand, untethered receptors would be free to diffuse in the plane of the membrane away from the cluster and away from ligand, thus reducing the chance of rebinding to ligand. Effective adhesion of a cluster of receptors with a cluster of ligands may thus be broken by sequentially breaking the individual receptor-ligand bonds and removing the receptors from the cluster.

Secretion

The behaviour of CR3 differs from that of other opsonic receptors in two respects. During Fc-receptor-mediated phagocytosis, phagocytes release large amounts of oxygen metabolites (superoxide and hydrogen peroxide) and arachidonic acid metabolites (prostaglandins and leukotrienes). These substances act to kill the coated target and to promote inflammation. In contrast, CR3 triggers neither release of hydrogen peroxide (Wright & Silverstein, 1983; Yamamoto & Johnston, 1984) nor arachidonic acid metabolites (Aderem et al. 1985). The observation that C3 receptors do not promote secretion of these inflammatory compounds suggests that C3 receptor-mediated phagocytosis may provide a means of clearing opsonized particles without initiating or perpetuating an inflammatory response. They also emphasize how well suited CR3 is for its role in cell migration, which is not associated with killing of cells en route.

Adhesion-promoting receptors may, however, play a role in potentiating secretion events mediated by other receptors. The agonists fMLP and TNFα cause PMN to secrete enormous amounts of hydrogen peroxide, but secretion only occurs if the cells are allowed to adhere to surfaces (Nathan, 1987). Preliminary experiments suggest that the actions of CR3, LFA-1, and p150,95 are needed for this adhesiondependent potentiation of secretion (C. F. Nathan & S. D. Wright, unpublished observations). Such an activity of adhesion-promoting receptors is ideally suited for use by cytotoxic cells such as T cells or NK cells. These cells do not secrete toxins constitutively, rather, secretion only occurs when the killers are attached to targets in an LFA-l-dependent manner.

Movement

The best appreciated function of adhesion-promoting receptors is to initiate elaboration of pseudopodia. Cells employ pseudopods in a similar way to accomplish spreading, phagocytosis, and locomotion, and we presume similar intracellular signals are used in all of these processes. While the nature of the intracellular signals used to cause movement of pseudopodia is unknown, recent experiments indicate that, like the binding activity, the signalling function of CR3 is regulated in phagocytic leukocytes. These studies have used phagocytosis of C3bi-coated erythrocytes as an assay, but the results are likely to apply to integrin-mediated migration as well.

Macrophages that are spread on human serum albumin or collagen exhibit low resting levels of CR3-mediated phagocytosis. However, when they spread on surfaces coated with fibronectin (Wright et al. 1983b; Pommier et al. 1983), or laminin (Bohnsack et al. 1985), their receptors are ‘activated’ and they promote phagocytosis. Several aspects of this type of activation of CR3 are worthy of note. First, fibronectin that is covalently bound to the substratum can activate the phagocytosis-promoting capacity of CR3 located on the apical portion of the phagocyte (Wright et al. 1983b). Thus, it is clear that fibronectin is not acting as an opsonin. Rather, it acts to regulate the activity of receptors for opsonins. Second, in order for fibronectin to activate C3 receptors, it must be bound to a substratum: soluble fibronectin is not capable of activating C3 receptors (Wright et al. 1983b; Wright & Meyer, 1985).

Fibronectin exerts its effect on CR3 by interacting with a receptor on macrophages which is also an integrin. Surface-bound synthetic peptides of fibronectin that contain the sequence RGD readily activate CR3 on macrophages, and soluble, monomeric peptides competitively inhibit activation by surface-bound fibronectin (Wright & Meyer, 1985). These experiments indicate that the activating effect of fibronectin is mediated by a receptor that recognizes the sequence RGD, and that fibronectin receptors must be crosslinked or immobilized in order to activate CR3. These data demonstrate that ligation of one member of the integrin family (the fibronectin receptor) may alter the behaviour of another integrin (CR3). It is worth noting that CR3 is not the only receptor that is regulated in this way. Macrophages express a structurally distinct C3 receptor, CRI, and the binding and signalling activity of CRI is regulated by fibronectin exactly as is that of CR3 (Wright & Silverstein, 1982; Wright etal. 1983b, 1984, 1986).

The phagocytosis-promoting activity of CR3 on human macrophages can be strongly enhanced by the pleiotropic compound, PMA. Within minutes of being exposed to PMA, macrophages become capable of rapidly ingesting iC3b-coated red cells (Wright & Silverstein, 1982). PMA affects only the ability of CR3 to signal phagocytosis, not the ability to bind ligand.

Complement receptors on macrophages may also be activated by soluble factors that are generated by stimulated T lymphocytes. Griffin & Griffin (1979, 1980) have described a novel lymphokine of approximately 10K, which activates the phagocytosis-promoting capacity of C3 receptors on murine peritoneal macrophages. A unique sequence of cellular interactions initiates release of this lymphokine. Upon ingestion of IgG-coated particles, macrophages elaborate a factor, which in turn causes T lymphocytes to secrete the lymphokine. The lymphokine appears to be different from other lymphokines such as IFNγ and interleukin-2, and cannot be elicited by classical mechanisms of lymphokine production such as antigenic stimulation of appropriate T cell clones (Griffin & Griffin, 1979). The receptor for the lymphokine has not been characterized.

How does ligation of one type of receptor (the receptor for lymphokine, fibronectin, or PMA) alter the capacity of a second type of receptor (CR1 or CR3) to generate an intracellular signal? The answer to this question is not in hand, but several possibilities can be ruled out. Activation of C3 receptors for phagocytosis does not require protein synthesis since activation by either lymphokine or PMA occurs in the presence of inhibitors of protein synthesis (Griffin & Griffin, 1979; Wright & Silverstein, 1982). Thus, the manufacture of new receptors does not explain activation. Activation is not accompanied by changes in the number of cell surface receptors since neither PMA nor fibronectin causes changes in the expression of C3 receptors on the cell surface (Wright et al. 1984). This observation suggests that activation is caused by a structural change in existing C3 receptors. Activation of C3 receptors is reversible. Observations with lymphokine, PMA, and fibronectin all show that receptors can be switched from inactive to active and back in the course of an hour (Griffin & Griffin, 1979; Wright et al. 1984). Thus, activation is not the result of irreversible modifications such as proteolysis. We speculate that, as with binding activity, the signalling activity of receptors may also be regulated through reversible phosphorylation events, perhaps at sites separate from those that control binding.

Students of embryology have long suspected that interactions of cells with matrix material or certain other cell types may influence the course of differentiation of that cell. Indeed, interaction of cells with fibronectin appears capable of altering their course of differentiation (West et al. 1979; Sieber-Blum et al. 1981; Loring et al. 1982). Since CR3, LFA-1, and p150,95 are structurally related to fibronectin receptors, one might expect that they would also direct differentiation events in leukocytes. Recent experiments by Dinget al. (1987) have shown that macrophages incubated for 2 days with a monoclonal antibody against murine CR3 exhibited the differentiative changes normally observed in response to IFNγ. Control experiments indicated that this was not caused by contaminating T cells or through autocrine production of IFNγ. These observations suggest a new facet of the function of adhesion-promoting receptors, which may have broad significance in understanding the interactions of cells.

We describe here one family of receptors that mediates a variety of adhesion events in leukocytes. These receptors are part of a larger group of receptors, integrins, which function in a great variety of adhesion events in many cell types. We wish to point out, however, that the search for receptors that mediate cell-cell adhesion is far from complete. We can expect that in the coming years many additional receptors will be discovered that deserve to be called ‘adhesion-promoting.’

Supported by USPHS grant AI-22003 and a Grant-in-Aid from the American Heart Association with funds contributed in part by the American Heart Association Florida Affiliate. S.D.W. is an Established Investigator of the American Heart Association. P.A.D is an Arthritis Foundation Investigator.

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