Lymphocyte adhesion mediated by integrins

ADHESION

MOLECULES

Lymphocyte

IN LEUKOCYTE-ENDOTHELIUM

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709

adhesion mediated by integrins Y. van Kooyk and C.G. Figdor

Division

of Immunology, The Netherlands Cancer Institute, Antoni van Leeuwenhoek Plesmanlaan 121, 1066 CX Amsterdam (The Netherlands)

Introduction

In order to effectively defend the body against infectious organisms, cells of the immune system should be able to circulate as non-adherent cells in blood and lymph, and to migrate as adherent cells throughout the tissues. To arrive at sites of infection and to adhere to target cells bearing foreign antigen, cells need to cross endothelial and vessel walls. To control this rapid transition between adherent and non-adherent states, lymphocytes are equipped with different adhesion receptors. Integrins form a major group of adhesion receptors mediating the adhesion events of lymphocytes with antigen-bearing cells as well as between lymphocytes and endothelial cells (fig. 1). Lymphocytes express several integrin molecules at their cell surface which may all participate in the same intercellular interaction. Apart from integrins, lymphoid cells express other adhesion receptors which may regulate cell adhesion or contribute to the integrin-mediated cell-cell interaction. Cloning of the genes encoding these molecules and their counterreceptors revealed that they belong to distinct families of structurally related proteins : the selectins, immunoglobulin superfamily and the CD44 adhesion receptor (fig. 1). Here we focus on integrinmediated adhesion of lymphocytes; therefore, only integrins will be discussed in detail. Integrins

expressed

on lymphocytes

and their ligands

All integrins are heterodimers composed of an u-subunit non-covalently associated with a P-subunit (Hynes, 1987). Eight different P-subunits and fourteen different a-subunits have been identified thus far. Many distinct a-subunits can associate with only one P-subunit (Rouslahti, 1991; Hynes, 1992). In addition, certain u-subunits can associate with different P-subunits (Holzmann and Weissman, 1989; Hemler, 1990). Based on shared P-subunits, subfamilies can be distinguished within the integrin family. Only the molecules belonging to the pl, /32 and p7 subfamilies are expressed on lymphocytes (table I), and will be discussed below.

Huis,

The pl integrins are also called VLA (very late activation antigens). Thus far, eight members of this group are defined. They are expressed on a wide variety of cells, including non-haematopoietic cells (Hemler, 1990; Hemler et al., 1990; Hemler, 1988). Most VLA bind to extracellular matrix (ECM) components such as fibronectin (FN), laminin (LM) and collagen (Coll), and thus play a role in lymphocyte adhesion and migration into tissues (Shimizu et al., 1991; Rouslahti and Pierschbacher, 1987). VLA-4 (CD49d/CD29) is the only member of the pl integrins which recognizes, apart from the ECM component FN, a transmembrane molecule. VCAM-1 (vascular cell adhesion molecule-l) (Osborn et al., 1989 ; Elites et al., 1990) the ligand of VLA4, is expressed by activated endothelium (fig. 1). Therefore, VLA-4 is also involved in lymphocyte-endothelial cell interactions. The site on the ligands recognized by many VLA contains the sequence RGD or EILDV (Rouslahti and Pierschbacher, 1986; Wayner et al., 1989). The p2 integrins are also called the Leu-Cam family (leukocyte cell adhesion molecules), since their expression is restricted to leukocytes (T and B lymphocytes, monocytes, granulocytes), in contrast to all other integrin families. This group consist of three members : LFA-1, CR3 (or Mac-l) and pl50,95 (Sanchez-Madrid et al., 1982; Keizer et al., 1985). LFA-1 (leukocyte-function-associated antigen-l) consists of a 180-kDa u-subunit (CDlla) and a 95-kDa P-subunit (CD18) (Kurzinger et al., 1981), and is expressed on all leukocytes. It is involved in a broad range of leukocyte functions such as T-cellmediated killing, T helper cell and B-cell responses, natural killer cell activity, monocyte-mediated antibody-dependent cytotoxicity and leukocyte adhesion to endothelial cells (Springer, 1990; Martz, 1987). The involvement of LFA-1 in all these responses has been revealed by monoclonal antibodies directed against LFA-1 that inhibit these processes (Krensky et al., 1983). LFA-1 mediates cellcell interaction through binding of one of its three counterreceptors, ICAM(intercellular adhesion molecule-l) (Marlin and Springer, 1987), ICAM-

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B

ICAM-

VLA-4

Fig. 1. Multiple adhesion receptors can contribute to the interaction of lymphocytes with endothelial cells (A) or of lymphocytes with leukocytes (T, B lymphocytes, monocytes and dendritic cells) (B). Different families of adhesion molecules can be distinguished: blank = integrins. hatched = immunoglobulins, cross-hatched = selectins, stippled = unclassified molecules.

Table I. Integrins

Subunit

Names

p1 cil a2 u3 a4 a5

VLA-1 VLA-2 VLA-3 VLA-4 VLA-5 VLA-6 LFA-1 Mac-l, CR3 p150,95 -

82 :“, aM p7 2 aHML Abbreviations

used:

Coil

(collagen),

expressed

Distribution haemopoietically restricted

CD CD49a/CD29 CD49b/CD29 CD49c/CD29 CD49d/CD29 CD49eICD29 CD49f/CD29 CD1 la/CD18 CD1 lb/CD18 CD1 lc/CD18 CD49d/CDfc X (factor

X),

on lymphocytes.

FG (fibronigen),

no no no no no no yes yes yes yes yes FN (fibronectin)

Counterstructure LM, Co11 LM, Co11 LM, Coil, FN FN, VCAM-1 FN LM ICAM-1, -2, -3 ICAM-1, FG, fc X, C3bi FG, C3bi FN, VCAM-1 ? and LM

(laminin).

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(Staunton et al., 1989a) or ICAM(de Fougerolles et al., 1991; de Fougerolles and Springer, 1992). All three ICAM are members of the immunoglobulin superfamily. ICAM-I has five Ig-like domains, ICAMhas two Ig-like domains, that are 35 070 identical to the two N-terminal domains of ICAM(Staunton et al., 1990). Recent cloning of ICAMrevealed that it consists of five Ig-like domains, 48 % identical to ICAMand 31 % identical to ICAM(Vazeux et al., 1992; Fawcett et al., 1992). p2 integrins which bind immunoglobulin superfamily counterreceptors recognize specific domains within the counterreceptor (Staunton et al., 1990; Diamond et al., 1991). Recently, it has been described that the LFA-I/ICAM-1 interaction can also be inhibited by peptides (Dustin et al., 1986). In contrast to LFA-1, ICAMis not exclusively expressed on leukocytes but on a wide variety of cells, including endothelial cells (Dustin et al., 1986). Inflammatory mediators strongly induce ICAMexpression on endothelium (Dustin el al., 1986; Pober et al., 1986). ICAMis found on haematopoietic cells and is highly expressed on endothelial cells. Its expression cannot be upregulated by inflammatory cytokines (Staunton et al., 1989a; de Fougerolles et al., 1991). Because ICAMexpression is high on endothelial venules in lymph nodes and vascular tissue, it has been hypothesized that it plays a role in leukocyte trafficking through the lymph. ICAM- has thus far only been found on leukocytes. Because of its high expression on resting leukocytes, it is thought to play an important role in the initiation of the immune response (de Fougerolles and Springer, 1992). CR3 (CDllb/CD18; Mac-l) consists of a 170-kDa a-subunit and a 95kDa P-subunit. It is expressed at low levels on a subset of T lymphocytes, whereas expression on monocytes and granulocytes is high (Miller et al., 1986; Keizer et al., 1987). In resting monocytes and granulocytes, CR3 is stored in peroxidase-negative granules (Bainton et al., 1987). Activation of these cells by inflammation mediators results in rapid degranulation and increased surface expression of CR3 (Bainton et al., 1987; Miller et al., 1987). CR3 functions as a receptor for the complement component C3bi and mediates binding of unopsonized microorganisms (Wright et al., 1983). Other ligands of CR3 are fibrinogen, Factor X (fc X) and ICAM(Loike et al., 1991; Diamond et al., 1990). LFA-1 and CR3 recognize distinct Ig domains on the ICAMmolecule (Diamond el al., 1991). p150,95 (CDllc/CD18) consists of a 150-kDa a-subunit and a 95-kDa P-subunit. Like CR3, it is expressed on a small subset of lymphocytes, whereas it is present at high levels on monocytes, macrophages, granulocytes and dendritic cells (Miller et al., 1986; Keizer el al., 1987; Lanier et al., 1985). The a-subunit of p150,95 shows 63 % homology with the a-subunit of CR3. Like CR3, p150,95 is stored in

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711

peroxidase-negative granules (Bainton et al., 1987). p150,95 binds to fibrinogen and C3bi (Loike et al., 1991; Myones et al., 1988). No transmembrane ligand of p150,95 has yet been identified. Both p150,95 and CR3 bind to denatured proteins (Davis, 1992), indicating that they are involved not only in cell-cell adhesion but also in cell-substrate adhesion. p7 integrins a4@7 (also called a4pp) and HML/P7 have recently been identified on lymphocytes (Erie et al., 1991; Holzmann et al., 1989). Similar to a4/pl, a4/p7 binds to fibronectin as well as to VCAM-1 (Ruegg et al., 1992). Structure

and function

of integrins

Both a- and P-subunits of integrins are type I transmembrane glycoproteins with a single hydrophobic transmembrane segment and a short cytoplasmic tail. The extracellular domains of both subunits associate to form the ap heterodimers (Hynes, 1992). The characteristics of each subunit will be discussed in detail below (fig. 2). The extracellular

domains

of the a- and P-subunits

The P-subunit of integrins contains a large extracellular domain consisting of 750 amino acids (p2 integrins). Characteristic of all P-subunits is a 4-fold repeat of a cysteine-rich segment in the extracelluiar part of the P-subunit (fig. 2) (Kishimoto et al., 1989b). The N-terminus may be tightly folded due to internal disulphide bonding (fig. 3). The extracellular domain of the P-subunit contains a region of 100-200 amino acids that is highly conserved between different P-subunits. Point mutations in this area revealed that this region is important for a/p association and ligand binding (Kishimoto et al., 1989; Wardlaw et al., 1990; Arnout et al., 1990). The a-subunits are also transmembrane glycoproteins and have a large extracellular part (LFA-1: 1,150 amino acids). In the N-terminal part of the protein, a 7-fold repeat of a homologous segment is located. Three (p2 integrins) or four (almost all pl integrins) of these repeats at the C-terminal part contain a sequence which shows homology with the calcium binding proteins troponin C, calmodulin and parvalbumin (Tuckwell et al., 1992; Kirchhofer et al., 1991). These repeats most likely contribute to the divalent cation-binding properties of the integrins (Kirchhofer et al., 1991 ; Martz, 1980). This EF-h and motif is made up of 13 residues, with a coordination typically supplied by residues 1, 3, 5, 7, 9 and 12 (Larson et al., 1989; Kretsinger, 1980). The a-subunits of the p2 integrins (CDlla-c) contain the metalbinding consensus nanopeptide DXDXDGXXD (D = aspartic acid, G = glycine, X = any amino acid)

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Fig. 2. Schematic

diagram

IN IMMUNOLOGY

of the structure

of p2 integrins.

The extracellular part of the a chain (a,.) contains an I domain and 7 tandem repeats, of which three contain cation binding motifs (hatched). The extracellular part of the /3 chain (p2) contains four cystein-rich repeats (cross-hatched) and a 100-200 amino acid long region (stippled) which is highly conserved among the p subunits of integrins. (*) represents sites of naturally occurring amino acid substitutions or deletions in the p2 subunit, which results in impaired up heterodimer formation, or the LAD syndrome. In the cytoplasmic tails of the c( and p chain, serine (S), tyrosine (Y) and threonines (T) residues are mentioned which can be phosphorylated ( p ) upon PKC activation and are important for functional activity of LFA-I (see text).

these repeats, but lack the glutamic acid at position 12, as is found in other typical EF-hand motifs (Tuckwell et al., 1992). It has been proposed that the glutamic acid residue, critical for cation binding, is provided by: 1) a segment in the u-subunit; 2) an oxygenated residue or segment within the P-subunit, or; 3) an aspartate residue present in the ligands recognized by these receptors (Dransfield, 1991; Ginsberg et a/., 1991). Binding of the divalent cations Cal+ and Mg2+ is necessary for receptor function (Martz, 1980; Staatz et a/., 1989; Elites et al., 1991 ; Rothlein and Springer, 1986). They can modulate u/p associations as well as ligand binding affinity (Kirchhofer et al., 1991). Recently, a novel site within the I domain of CR3 (CD1 lb/CD18) has been shown to function as a divalent cation binding site, which seems essential for ligand binding (Michishita et a/., 1993). The u-subunit of the p2 family contains an inserted region (I domain), which shows homology to cartilage matrix protein, von Willebrand factor and complement factor B and Cl.. The I domain is thought to be a flexible recogmtion element within the a-chain, involved in ligand binding (Diamond et al., 1993). Electron microscopic studies of the p3 integrins revealed that the a/p complex has a globular head with two tails. Similarities in structure are expected for the leukocyte-specific p2 integrins (fig. 3) (Carrel1 et al., 1985; Dana et al., 1991). Thus, the ligand binding site of the integrins seems to be formed by the cation domains and the I-domain within the u-subunit and the conserved region within the P-subunit (fig. 3). within

The intracellular

domains

of the u- and P-subunits

The cytoplasmic domains of the integrins are thought to be important for receptor function. Both phosphorylation of the cytoplasmic domain and its induced association with the cytoskeleton may direct receptor activation through induction of a conformational change. Interaction with cytoskeletal proteins, to contribute to focal adhesion, is important for integrin function, since disruption of the cytoskeleton abolishes receptor-mediated adhesion (Reszka et al., 1992; Pardi et al., 1992b). The cytoplasmic domain of the P-subunits is short (47, 46 up to 52 amino acids, pl, p2 and p7, respectively) and can interact with cytoskeletal proteins such as talin and u-actinin @l and p2 integrins) (Pardi et al., 1992b; Otey et al., 1990; Burn et al., 1988; Pavalko et al., 1991 ; Pavalko and Burridge, 1991). Moreover, the cytoplasmic domains may also interact with other cytoplasmic components (Brown et al., 1990). Deletion of the cytoplasmic domain of the pl-P2-subunit can inactivate receptor function (Hibbs et a/., 1991a,b; Buyon et a/., 1990). Three threonine residues, which are highly conserved within the pl, p2 and p7 integrin subfamilies, are most important for the p2 integrin receptor function (fig. 2) (Hibbs et al., 1991b). Upon stimulation with PMA or fMLP, serine756 present in the cytoplasmic domain of the p2 subunit is phosphorylated (Pardi et al., 1992; Chatila et al., 1989; Buyon et al., 1990). Whether this phosphorylation affects receptor function is not known.

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the regulation of distinct post-receptor binding events (Shimizu and Shaw, 1991).

Regulation

of lymphocyte

adhesion

Lymphocytes are equipped with several mechanisms to direct and regulate adhesion and migration to sites of inflammation (table II). First, upregulation or downregulation of surface expression, or the state of glycosylation of particular adhesion receptors is employed to control adhesive processes. Moreover, clustering of integrin receptors may increase the avidity of cell-cell interactions. Enhanced receptor expression and receptor clustering on the cell surface do not necessarily correlate with enhanced adhesiveness of cells. This requires activation of the adhesion receptor itself, resulting in’s higher affinity for its ligand. Activation of integrins can be achieved through “inside out” signalling, whereby signals are generated from within the cell (Hynes, 1992) (fig. 4). Activation of integrins involves conformational changes within the receptor, which lead to a higher affinity for its ligand, establishing cellcell interaction. Binding of the ligand by the integrin can result in “outside in” signalling, which may affect migration or proliferation and differentiation of the cell (Shimizu and Shaw, 1991; Pardi ef al., 1992a). The following sections will describe each of these processes in more detail. Fig. 3. Hypothetical three dimensional representation of

the LFA-1 molecule. The a- and P-chain form two stalks extending from the lipid bilayer, with both N-terminals tightly folded to form a globular head. The ligand binding site seemsto be formed by the cation binding domains and the I-domain within the a-subunit and the conserved region within the P-subunit.

The short cytoplasmic domain of the u-subunit (ranging from 24 to 58 amino acids) has several potential phosphorylation sites. The LFA-1 a-chain is constitutively phosphorylated (Pardi ef al., 1992b ; Chatila et al., 1989; Buyon et al., 1990). Hyperphosphorylation of the u-chain has been suggested to be associated with LFA-1 activation (Pardi et al., 1992). However, deletion of the cytoplasmic domain of the a-chain of the p2 integrins does not abrogate the capacity of LFA-1 to bind to ICAM(Hibbs et al., 1991a). This indicates that the cytoplasmic tail of the u-chain of the l32 integrins is not important for receptor activation. Characterization of the cytoplasmic domains of the u-chain of the l31 integrins revealed that these domains are important in

A) Regulation of lymphocyte of receptors and ligands

adhesion

by expression

Cell activation by cytokines or chemotactic factors can up- or down regulate cell surface expression. Expression of integrins on lymphocytes seems poorly sensitive to cytokine treatment. Expression of the pl integrins is not affected by cytokine treatment, while LFA-1 expression @2 integrin) is enhanced only 2-fold by IL2 or IFNy (Shimizu et al., 1990). On the other hand, expression of cellular ligands of integrin receptors is most sensitive to cytokines. Inflammatory mediators, such as IFNy, IL1 and TNFcr strongly induce ICAM(Dustin et al., 1986; Pober et al., 1986) or VCAM-1 expression (Osborn et al., 1989) in a wide variety of tissue, such as endothelium, resulting in enhanced binding of lymphocytes (Dustin and Springer, 1988). In contrast, other ligands of LFA-1 (ICAMand ICAM-3) seem less sensitive to inflammatory cytokines (Staunton et al., 1989; de Fougerolles et al., 1991; de Fougerolles and Springer, 1992). Differences in the state of glycosylation may also contribute to the capacity of adhesion receptors to bind to their ligand (Larson et al., 1989; Dahms and Hart, 1986; Shimizu et al., 1990a).

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Table

Regulation

II. Mechanisms

IN IA4A4UNOLOGY

that regulate P2-mediated

at the level of

A. Expression of receptors and their ligands B. Affinity/avidity modulation of adhesion receptors - “inside out” signalling - conformational changes - clustering of receptors - adhesion cascades C. Signalling through adhesion receptors - “outside in” signalling

Fig. 4. Activation and inactivation of integrins. When the receptor is in an inactive state (closed), “inside-out” signalling(I) can activate the receptorby inducing a conformational change (2) whereby the open receptor can bind its ligand. Ligand occupation of the receptorcan triggerintracellulareventsby “outside-in” sig-

nailing (3). When signalling has stopped, the activated receptor can convert back into the inactive state.

B) Affinity avidity adhesion receptors “Inside-out”

modulation

of

lymphocyte

signalling

A second mechanism to control cell adhesion is modulation of the affinity of the receptor for its ligand. Resting lymphocytes do not adhere spontaneously, but a variety of stimuli can induce PI-(VLA4, -5, -6) or PZ(LFA-1, CR3)-mediated cell-cell interactions. Exposure of lymphocytes to phorbol ester (PMA) strongly induces PI- and P2-dependent cell adhesion, suggesting that activation of protein kinase C (PKC) changes the affinity of these receptors for their ligands (Martz, 1980; Rothlein and

cell adhesion.

Influenced

by

Cytokines,

infectious agents, glycosylation

(De-)phosphorylation, second messengers Divalent cations, integrin mAb Divalent cations ? Second messengers, kinase activity

Springer, 1986; Shimizu et al., 1990). More recently, also, elevation of intracellular Ca2+ or cyclic AMP (CAMP) levels have been shown to activate the LFA-1 molecule (Haverstick and Gray, 1992a,b; van Kooyk el al., 1993). In addition, monoclonal antibodies directed against different cell surface structures expressed by T or B lymphocytes can stimulate LFA-l-mediated adhesion (table III). Triggering of these molecules activates intracellular signalling pathways leading to LFA-1 activation, and subsequent enhanced ligand (ICAM-1) binding. The finding that activation of the CD2 and CD3 molecules results in LFA-l-mediated adhesion (Dustin and Springer, 1989; van Kooyk et al., 1989) provides a link between TCR-mediated recognition of antigen and regulation of cell adhesion (Figdor et al., 1990). The observation that an increase in LFA-1 affinity induced by CD3 triggering is transient provides the cell with a mechanism to facilitate de-adhesion. Triggering of CD2 or CD3 has been shown to activate pl (VLA-4, -5, -6) (Shimizu et al., 1990b). LFA-1 activation induced through triggering of CD2 or CD3 depends on PKC activation, PTK (protein tyrosine kinase) activity and CAMP levels (Dustin and Springer, 1989; van Kooyk er a/., 1989). Also, increased intracellular Ca-+ levels induced by CD2 or CD3 triggering directly correlate with the activated state of the LFA-1 receptor (van Kooyk et al., 1989). Recently, other reports have shown that antibodies directed against MHC class I or II (Spertini et al., 1992), CD43 (Axelsson et al., 1988), CD44 (Koopman et al., 1990), CD28 (Shimizu et al., 1990b), CD7 (Shimizu et al., 1990b) and CD3 1 (Tanaka et al., 1992) expressed on T cells, and antibodies directed against CD19 (Smith ef al., 1991), CD20, CD40 (Kansas and Tedder, 1991; Barrett ef al., 1991) and MHC class II (Mourad et a/., 1990) expressed on B cells all induce LFA- 1-mediated adhesion (table III). Depending on the surface receptor triggered, different signal transduction pathways seem to be involved. These findings demonstrate that

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Table

Stimuli

IN LEUKOCYTE-ENDOTHELIUM

III.

Inside-out

signalling

INTERACTIONS

of 82 integrins.

Type of cell

2nd Messenger Activating

PMA (*’ Forskolin Ionomycin LPS CD2 (‘*’ CD3 CD7 CD19 CD28 CD31 CD39 CD40 CD43 CD44 MHC cl I MHC cl II

T, B

PKC CAMP, Ca’+, PKC, PKC, PKC PKC’ ? PKC 9

B T B T T T B T T B B J-9 B T T, B T

PKA PTK, PTK

Caz+i Ca*+i ’

(conformational

CAMP CAMP CAMP CAMP

9

?

PKC PTK PKC 9 PTK 9 Ca’+. I

activation of LFA-1 can be induced through triggering of different surface receptors, and illustrate that the LFA-I/ICAM-1 pathway is a common adhesion pathway that may be utilized by lymphocytes under quite different physiological conditions. alterations

Inactivating

PKA

(*) The addition of PMA, forskolin, ionomycin or LPS to B or T cells generates distinct 2nd messengers LFA-l-mediated adhesion. (**) Cross-linking of distinct cell surface molecules, by mAb generates different LFA-I activation. PKC (protein kinase C), PTK (protein tyrosine kinase). Ca’- i (intracellular Ca’* mobilization), mono-phosphate).

Intramolecular

715

changes)

An increasing amount of evidence indicates that activation of pl p2 is accompanied by a conformational change within the integrin molecule. 1) Unique monoclonal antibodies have been described that recognize integrins only in their activated state (active conformation). This is determined by the expression of neoepitopes on the integrin or the expression of a ligand-induced binding site (LIBS) on the integrin, which appears upon binding of their ligand (Dransfield et al., 1992a,b; van Kooyk et al., 1991). 2) Binding of Mg2+ to LFA-1 possibly induces a conformational change, as detected by enhanced reactivity with a certain mAb 24 (Dransfield and Hogg, 1989). Also binding of Ca2+ to LFA-1 can alter the configuration of LFA-1, as detected by enhanced reactivity with mAb NKI-L16 (van Kooyk el

activating messengers CAMP

or inactivating which regulate (cyclic adenosine

1991 ; and submitted). Extracellular binding of Mn2+ (Dransfield et al., 1992a; Altieri, 1991) and/or Co’+ (Dransfield et al., 1990) can induce a conformational change in both pl and p2 integrins, which leads to an increased affinity of the receptor for its ligand. 3) Phosphorylation or dephosphorylation of the cytoplasmic tails of the p2 or pl integrins (a- or P-subunit) or cytoskeletal components may induce a conformational change in the integrin (Pardi et al., 1992). 4) Specific antibodies directed against the @l and p2 integrins that enhance instead of inhibit adhesion have been described. Also, Fab fragments of these mAb enhance integrin-mediated adhesion which demonstrates that cross-linking of receptors is not required. Binding of these mAb most likely results in a conformational change in the adhesion receptor, thus increasing the affinity for its ligand. For example, mAb directed against a Ca’+-dependent epitope located on the LFA-1 a-subunit (van Kooyk et al., 1991; Keizer et al., 1988; van Kooyk, unpublished) and mAb directed against an epitope on the common P2-subunit (Robinson et al., 1992) enhance binding of LFA-1 to ICAM-1. Similarly, mAb directed against the VLA-4 a-chain can enhance

al.,

54th FOR 1/M IN IMMUNOLOG

VLA4-dependent cell aggregation (Campanero et al., 1990). However, unique anti-p1 mAb can also stimulate binding of VLA-4 to VCAM-1 as well as binding of leukocyte to ECM components (Kovach et a/., 1992 ; van de Wiel van Kemenade et al., 1992). This indicates that antibodies directed against the uor P-subunit can activate integrins by inducing a conformational change.

Clustering of receptors

Clustering of adhesion receptors has been shown to be important in facilitating integrin-ligand interaction. Clustering of CR3 receptors has been reported to be temporally correlated with its ability to bind their ligand (Detmers et a/., 1987). Moreover, binding of Cal+ to LFA-1 is associated with clustering of receptors on the cell membrane, which facilitates LFA-1 activation by increasing the avidity of LFA-1-ligand interaction (van Kooyk, unpublished).

Adhesion cascades

Several experiments indicate that adhesion molecules may operate successively in time. For example, leukocyte adhesion to endothelium at sites of inflammation is a multistep process initially involving unstable adhesion, followed by stable adhesion by p2 integrins and finally strong adhesion of Different I32 integrins to their counterreceptors. examples can be given of adhesion cascades leading to stable cell-cell binding. Recognition of antigen by lymphocytes is mediated by the T-cell receptor-antigen/MHC interaction, which precedes stable LFA-l/ICAM-1 interaction. Similarly, CD2/LFA-3 interaction may activate the LFA-l/ICAM-1 interaction (van de Wiel van Kemenade et al., 1992). Also CD44, which may play a role in lymphocyte homing, can stimulate the LFA-1 adhesion pathway (Koopman et al., 1990). Selectins or “rolling receptors” act very early in the adhesion cascades (Lawrence and Springer, 1991; Lawrence et al., 1990; Lo et al., 1991). They enable rolling of neutrophils along the endothelial cells, thereby reducing their speed. By bringing the neutrophil into close proximity of the endothelial cell, P2-mediated binding (CR3) to endothelium is facilitated (Lo et al., 1991). More recently, it has been described that cytokines can be immobilized by proteoglycans present on endothelial cells which trigger VLA-bmediated adhesion of specific lymphocyte subsets and thus regulate adhesion and migration of lymphocytes through endothelium (Tanaka et al., 1993).

Y

C) Signalling through adhesion receptors “Outside-in”

signalling

In addition to regulation of the receptor affinity by signals generated inside the cell, an increasing amount of evidence indicates that adhesion receptors themselves can transmit signals into the cell (“outside-in” signalling). The use of monoclonal antibodies or purified immobilized ligands to cross-link specific integrin receptors, has identified cell adhesion molecules as signal transducer molecules. Clustering of p2 integrins by antibodies directed against the LFA-1 or CR3 u-subunit results in release of intracellular Ca*+ as well as an increase in intracellular pH (Ng Sikorski et al., 1991 ; Schwartz et a/., 1991b; Pardi et al., 1989; Richter et a/., 1990). Clustering of pl receptors by mAb directed against the pl-subunit can cause tyrosine phosphorylation of a 115-kDa molecular complex (Kornberg et al., 1991). Moreover, ligation of VLA-4 on T cells stimulates tyrosine phosphorylation of a 105-kDa protein, indicating that engagement of VLA-4 on T cells activates protein-tyrosine kinase (PTK) activity (Nojima et al., 1992). Indeed-specific protein-tyrosine kinases have been identified, which are concentrated in focal adhesions (FAK, or focal adhesion kinases), and which are phosphorylated in response to cell attachment to ECM components (Hanks et al., 1992). Binding of /31 integrins to FN induces clustering of the integrins and activates the Na + /H + antiporter (Schwartz et al., 1991a). In addition, ligation of LFA-1 on B cells improves the ability to present antigen to the T cells (Moy and Brian, 1992). Adherence of neutrophils via p2 integrins together with cytokines induces a respiratory burst and cell motility (Jaconi et al., 1991). Furthermore, monocyte adhesion seems to be associated with PLC activation, whereas intracellular Cal+ mobilization plays a role in cell spreading (Lefkowith et al., 1992). Several reports have indicated that anti-LFA-l-u antibodies (van Seventer et al., 1991~; van Noesel et a/., 1988), anti-ICAM(van Seventer et a/., 1991a; Cerdan et al., 1989) or anti-ICAMantibodies (Damle et al., 1992) enhance lymphocyte proliferation, when immobilized together with anti-CD3 antibodies (costimulation) (van Seventer et al., 1990). AntiP2-subunit antibodies were ineffective, suggesting that the a- and P-subunits of LFA-1 may have distinct signalling properties. VLA-4 as well as its cellular ligand VCAM-1, can also provide costimulatory signals (van Seventer et al., 1991a; Burkly et a/., 1991; Damle and Aruffo, 1991). Cross-linking of integrins by antibodies mimics the engagement of receptor with its ligand and affects cytoskeletal organization. “Outside in” signalling through adhesion receptors is mediated by different intracellular messengers and/or cytoskele-

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tal components, and may contribute directly to cell maturation, differentiation and proliferation (Pardi et al., 1992; van Seventer et a/., 1991b).

Role of lymphocyte

adhesion

receptors

in vivo

The importance of adhesion receptors in vivo is best illustrated in patients suffering from the leukocyte adhesion deficiency (LAD) syndrome (Staatz et al., 1989; Springer et al., 1984; Anderson and Springer, 1987; Kishimoto et al., 1987 ; Todd and Freyer, 1988 ; Fischer et a/., 1985), a genetic deficiency of the p2 integrin receptor caused by mutations in the common p2 subunit (fig. 2) (Kishimoto et al., 1989; Wardlaw et al., 1990; Arnaout et al., 1990). The LAD syndrome results in the absence of LFA-1, CR3 and p150,95 on the cell surface of leukocytes. Patients have recurring, life-threatening bacterial infections, and severe defects in adhesion-dependent leukocyte functions, which are often fatal in childhood, unless they can be corrected by bone marrow transplantation (Fischer et al., 1988). The potential of mAb directed against integrins or their counterreceptors to reduce vascular and tissue damage in a variety of clinical disorders, has been examined (acute and chronic allograft rejection, rheumatoid arthritis, inflammatory skin disease, asthma and ischaemia-reperfusion syndromes). Inhibition of leukocyte adhesion processes by “anti-adhesion” therapy represents a novel approach to treat disorders in which leukocytes contribute significantly to vascular and tissue damage (Carlos and Harlan, 1990). In vivo treatment with anti-VLA-4 antibodies inhibits migration of lymphocytes to cutaneous inflammatory sites (Issekutz, 1991). Anti-ICAMantibodies have been shown to reduce airway inflammation, hyperresponsiveness and asthma symptoms (Wegner et al., 1990). Moreover, a soluble form of ICAM-1, as well as anti-ICAMantibodies inhibit rhinovirus infections, which cause 50 % of common colds (Marlin et al., 1990). ICAMhas been identified as the cellular receptor for a subgroup of rhinoviruses (Staunton et al., 1989b; Greve et al., 1989) and is one of the receptors for Plasmodium falciparum(malaria)-infected erythrocytes (Ockenhouse et al., 1992; Berendt et al., 1992). It has been demonstrated that anti-CD18 antibodies prevent ischaemia-reperfusion injury (Vedder et al., 1988), whereas anti-CD1 lb antibodies reduce infarct size in a canine model (Simpson et al., 1988). Administration of anti-LFA-1 antibodies enhances different tissue transplant survival (Heaggy and Waltenbaugh, 1983; van Dijken et al., 1990; Harning et al., 1991), whereas anti-ICAMantibodies have been demonstrated to extend kidney and heart transplant survival in monkeys in the absence of tolerance induction (Cosimi et al., 1990; Flavin et al., 1991).

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A combination of anti-LFA-1 and anti-ICAMmAb leads to tolerance in heart transplantation in mice (Isobe et al., 1992) ; however tolerance is not observed in skin transplants (van Kooyk et al., unpublished). Pilot studies in humans revealed that anti-LFA-1 mAb prevented acute rejection of kidney transplants without induction of tolerance (Le Mauff, unpublished). Conclusions

These findings demonstrate that adhesion receptors play an important role in different immunologic reactions. Leukocyte-specific adhesion receptors such as LFA-I show a dynamic nature by the presence of distinct conformational changes, which can be regulated extracellularly by divalent cations or ligand binding, and intracellularly by the generation of distinct signals. The usage of mAb directed against these receptors or their ligands in vivo by an anti-adhesion therapy demonstrates a promising reduction in tissue destruction, caused by disease or by inflammatory responses. Future experiments will determine if combinations of antibodies directed against distinct adhesion receptors will give optimal acceptance of tissue transplants. Acknowledgements This work is supported by a grant from the Dutch Kidney Foundation “Niersticting” grant number C87.724. References Altieri, D.C. (1991), Occypancy of CD1 lb/CD18 (Mac-l) divalent ion binding site(s) induces leukocyte adhesion. J. Immunol., 147, 1891-1898. Anderson, D.C. &Springer, T.A. (1987), Leukocyte adhesion deficiency: an inherited defect in the Mac-l, LFA- 1, and p 150,95 glycoproteins. Ann. Rev. Med., 38, 175-194. Arnaout, M.A., Dana, N., Gupta, SK., Tenen, D.G. & Fathallah, D.M. (1990), Point mutations impairing cell surface expression of the common beta subunit (CD18) in a patient with leukocyte adhesion molecule (Leu-CAM) deficiency. J. Clin. Invest., 85, 977-981. Axelsson, B., Youseffi-Etemad, R., Hammerstrom, S. & Perlmann, P. (1988), Induction of aggregation and enhancement of proliferation and IL-2 secretion in human T cells by antibodies to CD43. J. lmmunol., 141, 2912-2917k. Bainton, D.F., Miller, L.J., Kishimoto, T.K. & Springer, T.A. (1987), Leukocyte adhesion receptors are stored in peroxidase-negative granules of human neutrophils. J. Exp. Med., 166, 1641-1653. Barrett, T.B., Shu, G. & Clark, E.A. (1991), CD40 sig-

nailing activates CD1 la/CD18

(LFA-1)-mediated

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son, T. (1991). Calcium signalling capacity of the CD1 I b/CD18 integrin on human neutrophils. Exp. Ceil Res., 195. 504-508. Nojima, Y., Rothstein, D.M., Sugita, K., Schlossman, F. & Morimoto, C. (1992), Ligation of VLA4 on T cells stimulates tyrosine phosphorylation of a l05-kD protein. J. Exp. Med., 175, 1045-1053. Ockenhouse, C.F., Betageri, R., Springer, T.A. & Staunton, D.E. (1992), Plasmodium falciparum-infected erythrocytes bind ICAM-I at a site distinct from LFA-I, Mac-l, and human rhinovirus. Cell, 68,63-69. Osborn, L., Hession, C., Tizard, R., Vassallo, C., Luhowskyj, S., Chi Rosso, G. & Lobb, R. (1989), Direct expression cloning of vascular cell adhesion molecule I, a cytokine-induced endothelial protein that binds to lymphocytes. Ce//, 59, 1203-1211. Otey, C.A., Pavalko, F.M. & Burridge, K. (1990), An interaction between alpha-actinin and the beta I integrin subunit in vitro. J. Cell Biol., I I I, 721-729. Pardi, R., Bender, J.R., Dettori, C., Giannazza, E. & Engleman, E.G. (1989). Heterogeneous distribution and transmembrane signalling properties of lymphocyte function-associated antigen (LFA-I) in human lymphocyte subsets. J. Imtnunol., 143, 3157-3166. Pardi, R., Inverardi, L. & Bender, J.R. (1992a), Regulatory mechanisms in leukocyte adhesion : flexible receptors for sophisticated travelers. Itntnunol. Today, 13, 224-230. Pardi, R., Inverardi, L., Rugarli, C. & Bender, J.R. (1992b), Antigen-receptor complex stimulation triggers protein kinase C-dependent CDI la/CDl8-cytoskeleton association in T lymphocytes. J. Cell Biol., 116, I21 I-1220. Pavalko, F.M. & Burridge, K. (1991), Disruption of the actin cytoskeleton after microinjection of proteolytic fragments of alpha-actinin. J. Cell Biol., 114, 481-491. Pavalko, F.M., Otey, C.A., Simon, K.O. & Burridge, K. (1991), Alpha-actinin : a direct link between actin and integrins. Biochem. Sot. Trans., 19, 1065-1069. Pober, J.S., Bevilacqua, M.P., Mendrick, D.L., Lapierre, L.A., Fiers, W. & Gimbrone, M.A. Jr. (1986). Two distinct monokines, interleukin I and tumor necrosis factor, each independently induce biosynthesis and transient expression of the same antigen on the surface of cultured human vascular endothelial cells. J. Immunol., 136, 1680-1687. Reszka, A.A., Hayashi, Y. & Horwitz, A.F. (1992), Identification of amino acid sequences in the integriri beta I cytoplasmic domain implicated in cytoskeletal association. J. Cell Biol., 117, 1321-1330. Richter, J., Ng Sikorski, J., Olsson, I. & Andersson, T. (1990), Tumor necrosis factor-induced degranulation in adherent human neutrophils is dependent on CDI I b/CDI8-integrin-triggered oscillations of cytosolic free Ca2 + . Proc. Nafl. Acad. Sci. (Wash.), 87, 9472-9476. Robinson, M.K., Andrew, D., Rosen, H., Brown, D., Ortlepp, S., Stephens, P. & Butcher, E.C. (1992), Antibody against the Leu-CAM beta-chain (CDl8) promotes both LFA-I and CR3-dependent adhesion events. J. Immunol., 148, 1080-1085. Rothlein, R. &Springer, T.A. (1986), The requirement for lymphocyte function-associated antigen I in homotypic leukocyte adhesion simulated by phorbol ester. J. Exp. Med., 163, 1132-1149.

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Ruegg, C., Postigo, A.A., Sikorski, E.E., Butcher, E.C., Pytela, R. & Erle, D.J. (1992), Role of integrin alpha 4 beta 7/alpha 4 beta P in lymphocyte adherence to fibronectin and VCAM-I and in homotypic cell clustering. J. Cell Biol., 117, 179-189. Ruoslahti, E. (1991), Integrins. J. C/in. Invest., 87, l-5. Ruoslahti, E. & Pierschbacher, M.D. (1986), Arg-Gly-Asp: a versatile cell recognition signal. Cell, 44, 517-518. Ruoslahti, E. & Pierschbacher, M.D. (1987), New perspectives in cell adhesion: RGD and integrins. Science, 238, 491-497. Sanchez-Madrid, F., Krensky, A.M., Ware, C.F., Robbins, E., Strominger, J.L., Burakoff, S.J. & Springer, T.A. (1982), Three distinct antigens associated with human T-lymphocyte-mediated cytolysis: LFA-I, LFA-2, and LFA-3. Proc. Null. Acad. Sci. (Wash.), 79, 7489-7493. Schwartz, M.A., Lechene, C. & Ingber, D.E. (199la), Insoluble fibronectin activates the Na/H antiporter by clustering and immobilizing integriri alpha 5 beta I, independent of cell shape. Proc. Narl. Acad. Sci. (Wash.), 88, 7849-7853. Schwartz, M.A., Ingber, D.E., Lawrence, M., Springer, T.A. & Lechene, C. (1991b), Multiple integrins share the ability to induce elevation of intracellular pH. Exp. Ceil Res., 195, 533-535. Shimizu, U., van Seventer, G.A., Horgan, K.J. & Shaw, S. (1990a). Regulated expression and binding of three VLA (beta I) integrin receptors on T cells. Nature (Lond.), 345, 250-253. Shimizu, Y. & Shaw, S. (1991), Lymphocyte interactions with extracellular matrix. FASEB J., 5, 2292-2299. Shimizu, Y., van Seventer, G.A., Horgan, K.J. & Shaw, S. (1990b). Roles of adhesion molecules in T-cell recognition : fundamental similarities between four integrins on resting human T cells (LFA-1, VLA-4, VLA-5, VLA-6) in expression, binding, and costimulation. Immunol. Rev., 114, 109-143. Shimizu, Y., Newman, W., Gopal, T.V., Horgan, K.J., Graber, N., Beall, L.D., van Seventer, G.A. & Shaw, S. (1991), Four molecular pathways of T cell adhesion to endothelial cells : roles of LFA- I, VCAM-I, and ELAM-I and changes in pathway hierarchy under different activation conditions. J. Cell Biol., 113, 1203-1212. Simpson, P.J., Todd, R.F., Fantone, J.C., Mickelson, J.K., Griffin, J.D. & Lucchesi, B.R. (1988), Reduction of experimental canine myocardial reperfusion injury by a monoclonal antibody (anti-Mol, antiCDI lb) that inhibits leukocyte adhesion. J. C/in. Invest., 8 I, 624-629. Smith, S.H., Rigley, K.P. & Callard, R.E. (l991), Activation of human B cells through the CD19 surface antigen results in homotypic adhesion by LFA-l-dependent and -independent mechanisms. Immunology, 73, 293-297. Spertini, F., Chatila, T. & Geha, R.S. (1992), Engagement of MHC class I molecules induces cell adhesion via both LFA-l-dependent and LFA-l-independent pathways. J. Immunol., 148, 2045-2049. Springer, T.A. (1990), Adhesion receptors of the immune system. Nature (Lond.), 346, 425-434. Springer, T.A., Thompson, W.S., Miller, L.J., Schmalstieg, F.C. &Anderson, D.C. (1984), Inherited deficiency of the Mac-l, LFA-I, pl50,95 glycoprotein

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61-64.

Staunton, D.E., Dustin, M.L., Erickson, H.P. & Springer, T.A. (1990). The arrangement of the imrnunoglobulin-like domains of ICAM-I and the binding sites for LFA-I and rhinovirus [published erratum appears in Cell 1990 Jun 15, 61(2), 11571. Cell, 61, 243-254.

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Van Seventer, G.A., Newman, W., Shimizu, Y., Nutman, T.B., Tanaka, Y., Horgan, K.J., Gopal, T.V., Ennis, E., O’Sullivan, D., Grey, H. & Shaw. S. (l99la), Analysis of T cell stimulation by superantigen plus major histocompatibility complex class II molecules or by CD3 monoclonal antibody: costimulation by purified adhesion ligands VCAM-I, ICAM-I, but not ELAM-1. J. Exp. Med., 174, 901-913. Van Seventer, G.A., Shimizu, Y. & Shaw, S. (199lb), Roles of multiple accessory molecules in T-ell activation. Curr.

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Itlvesr.,

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