Integrins, ICAMs, and Selectins: Role and Regulation of Adhesion Molecules in Neutrophil Recruitment to Inflammatory Sites

Integrins, ICAMs, and Selectins: Role and Readation of Adhesion Molecules in Nlutrophil Recruitment to Inflammatory Sites ~~

Takashi Kei Kishimoto and Robert Rothlein Immunology Department Boehringer-Ingelheim Pharmaceuticals, Inc. Ridgefeld, Connecticut 06877

1. Introduction The immune system is constantly being challenged by a myriad of microbial pathogens. Neutrophils are the front line of defense and are rapidly mobilized and recruited to sites of tissue injury or infection. This efficient response requires that neutrophils gain access to virtually any tissue. Thus, a key step in the inflammatory response is the ability of circulating neutrophils, which are in a nonadhesive state, to become adhesive and attach to the vascular endothelium at the appropriate place and time. It is clear that these adhesive interactions must be highly selective and transient in nature. Over the past decade research on cell adhesion molecules involved in this process has grown at a phenomenal pace. To date, three families of adhesion molecules have emerged as key players in neutrophil-endothelial cell interactions (Springer, 1990): (1) the CD 18 or p2 integrins, which include LFA-I (CDl la/CD18), Mac-1 (CD1 lb/CD18), and p150,95 (CD1lcKD18); (2) the intercellular adhesion molecules (ICAMs), which include ICAM-1 (CD54), ICAM-2, and ICAM-3; and (3) the selectins, which include L-selectin, E-selectin, and P-selectin (CD62). The study of the molecular basis of cell adhesion has been made possible Advances in Pharmaco/ogy, Volume 25 Copyright 6 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.

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by the development and use of molecular tools to isolate and analyze specific molecules-monoclonal antibodies which affect function and cDNA clones which can be mutated or engineered. This approach has yielded a wealth of information on structure-function and receptor-ligand relationships. Another central theme is the regulation of adhesive interactions. Regulation of both expression and function of adhesion molecules contributes to the specificity and reversible nature of cell-cell adhesion. It has also become increasingly clear that neutrophil localization is a multistep process. In I989 a simple two-step adhesion model was proposed based on the inverse regulation of Mac-1 and L-selectin expression on 1989~;Jutila neutrophils exposed to chemotactic agents (Kishimoto er d., e? a/.. 1989). Over the past several years new insights from many laboratories have greatly expanded this model to encompass other cell adhesion molecules. chemotactic factors, and cell types (Zimmerman et al., 1992; Schweighoffer and Shaw, 1992; Butcher, 1991).This generalized model of leukocyte-endothelial cell interactions has been referred to as an adhesion cascade-a precisely orchestrated cascade of events which ensures a rapid self-limiting response to isolate and destroy invading microbes while minimizing damage to healthy tissue. There are. however, circumstances under which these carefully regulated events go awry or are aberrantly initiated, resulting in neutrophil contribution to the pathogenesis of inflammatory diseases. The discovery that patients genetically deficient in CD18 integrin expression have defective neutrophil recruitment to inflammatory sites (Anderson and Springer, 1987) led to the speculation that adhesion antagonists may be of therapeutic benefit in treating inflammatory diseases. This review briefly introduces the key adhesion molecules involved in leukocyte trafficking to sites of inflammation, focuses on mechanisms to regulate adhesion molecule expression and function, defines some of the steps involved in neutrophil-endothelial cell interactions, and finally summarizes the progress made in testing antiadhesion molecule monoclonal antibodies (MAbs) in animal models of inflammatory diseases.

II. Structure, Function, and Distribution of Adhesion Molecules

A. CD18 (&) Integrins The CD18 integrin family includes three structurally related (YP heterodimers-LFA- 1, Mac- 1, and p 150.95 (Fig. I ) . The CDI 8 integrins were among the first leukocyte adhesion molecules discovered and have been

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p 150,95

LFA- 1

Mac- 1

Subunits

CDl la CD18

CD 1 Ib CD18

Ligands

ICAM- 1 ICAM-2 ICAM-3

ICAM- 1 C3bi fibrinogen factor X others

C3bi ? others ?

Distribution

All leukocytes

Granulocytes Monocytes Macrophages Some activated lymphocytes LGL

Granulocytes Monocytes Macrophages Some activated lymphocytes LGL

0

00

CDllc CD18

Fig. 1 The CD18 (pz) family of leukocyte integrins. LGL, Large granular lymphocyte,

the focus of several comprehensive reviews (Kishimoto et al., 1989a; Kishimoto and Anderson, 1992; Arnaout, 1990). The structure and function of the CD18 integrins are only briefly reviewed here. The CD18 integrins are a subset of a large and functionally diverse family of integrins. To date there are at least 12 a-subunits and seven 0subunits in the integrin family, whose combinations yield at least 19 distinct ap heterodimer receptors (Hynes, 1992; Kishimoto and Anderson, 1992). LFA-1, Mac-1, and p150,95 have distinct a-subunits which share in common the p2 (or CD18) subunit (Sanchez-Madrid et al., 1983), thus defining this as a subfamily. Most of the other integrin members are extracellular matrix receptors, which guide cell interactions with matrices such as fibronectin, vitronectin, laminin, fibrinogen, and collagen (Hynes, 1992; Ruoslahti and Pierschbacher, 1987). These integrins are involved in such fundamental processes as development, hemostasis, and wound healing. The CD18 integrins are primarily involved in cell-cell interactions of leukocytes, although Mac- 1 has been implicated in leukocyte-matrix interactions as well. These molecules not only help guide leukocyte migration, but are also involved in antigen presentation function and leukocyte effector functions. All integrins share common structural features. The a-subunits contain multiple divalent cation binding pockets (Corbi et al., 1987, 1988; Arnaout et ul., 1988; Larson et al., 1989). Not surprisingly, most integrin functions are Mg2+or Ca2+dependent. The a-subunits of LFA-1, Mac-1, and p150,95 are unusual since they have a large insertion (I) domain, which is homolo-

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gous to similar domains found in von Willebrand factor, complement components C2 and B, and the cartilage matrix protein. The only other integrin a-subunit known to share this feature is the a? subunit of VLA2 (Takada and Hemler, 1989). The CD18 integrin @-subunit, like other integrin P-subunits, contains 56 conserved cysteine residues in the extracellular domain (Kishimoto et al., 1987a; Law et al., 1987). Most of these cysteine residues are concentrated in four small repeats of an unusual motif. A heritable immunodeficiency disease, termed leukocyte adhesion deficiency (LAD),is due to mutations in the CD18 @-subunitwhich prevent surface expression of all three CD18 integrins (Kishimoto et al., 1987b). This disease has been described in humans (reviewed by Anderson and Springer, 1987; Todd and Freyer, 1988; Anderson et a / . , 1994), canines (Giger et al., 1987: Trowald-Wigh et al., 1992), and bovines (Kehrli et al., 1992). Interestingly, most of the mutations determined to date map to a region near the N terminus of the @subunit (Kishimoto et al., 1989b; Matsuura et al., 1992; Sligh et al., 1992; Corbi et al., 1992; Arnaout et al., 1990;Wardlaw era/., 1989;Nelsonetal., 1992;Backetal., 1992)which is highly conserved among all integrin @-subunits.This region appears to be critical for association of the a- and @-subunits. The corresponding region of the & integrin has also been implicated in ligand binding (D’Souza et al., 1988). While integrins in general are expressed on diverse cell types, the CD18 integrins are restricted in expression to leukocytes (reviewed by Kishimot0 et al., 1989a; Kishimoto and Anderson, 1992; Arnaout, 1990). LFA1 is expressed by all lineages of white blood cells, including lymphocytes, monocytes, and granulocytes. Mac-1 and p150,95 are restricted primarily to myeloid cells, although some activated lymphocytes and natural killer cells can be induced to express Mac-1 or p150,95. The CD18 integrins play at least a contributing role in most leukocyte adhesion-related functions. Early characterization of the CD18 integrins focused on their role on mononuclear leukocytes. LFA-1 was first described as an adhesion molecule involved in cytolytic T-cell binding to target cells (Davignon et al.. 1981) and later as an accessory molecule involved in other lymphocyte functions (reviewed by Springer et al., 1987). Mac-I was independently defined as a macrophage marker (Springer et al., 1979). Its first functional role was described as a receptor for the C3bi fragment of complement (Beller et af., 1982; Wright et al.. 1983). A role for the CD18 integrins in leukocyte localization-specifically, interactions between circulating leukocytes and vascular endothetial cells-was not really suspected until the revelation that the molecular basis of LAD disease was due to genetic deficiency in the expression of all three CD18 integrins.

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The hallmarks of LAD are striking: these patients suffer from recurrent life-threatening bacterial infections of soft tissues (reviewed by Anderson and Springer, 1987;Todd and Freyer, 1988;Anderson et al., 1994). Analysis of infected lesions revealed the lack of a neutrophil infiltrate, despite the fact that circulating levels of neutrophils in the blood were abnormally high. Significantly, normal leukocytes transfused into these patients are capable of reaching inflammatory sites (Bowen et al., 1982). Molecular studies revealed deficient expression of all three CD 18integrins (Anderson et al., 1984; Beatty et al., 1984; Dana et al., 1984; Springer et al., 1984), which was later shown to be due to defects in the @-subunitcommon to LFA-1, Mac-1, and pl50,95 (Kishimoto et al., 1987b). In uitro function analysis revealed that lymphocytes from LAD patients display the same range of adhesion-related defects as normal lymphocytes treated with antLCD18 MAbs. However, lymphocyte function seems only somewhat diminished in L 4 D patients; clearly, the most severe impact of CD18 deficiency is the apparent inability of circulating neutrophils to migrate from blood vessels into inflamed tissues. This observation triggered a wave of research on the role of CD18 integrins in mediating leukocyte-endothelial cell interactions (Pohlman et al., 1986; Zimmerman and McIntyre, 1988; Harlan et al., 1985; Harlan, 1985; Smith et al., 1988). Several groups demonstrated that neutrophil adhesion to cytokine-stimulated endothelial cells is partially inhibited by anti-CD18 MAbs. Also, an increase in the CD18-dependent adhesion component occurs upon neutrophil activation. Furthermore, neutrophil migration across the endothelial monolayer is totally blocked by anti-CD18 MAbs (Smith et al., 1988, 1989b). Studies with leukocytes from LAD patients confirmed these studies. Notably, LAD neutrophils are still capable of binding interleukin-1 (IL-1)stimulated endothelial cells, albeit less efficiently than normal leukocytes, but they are unable to undergo transendothelial migration (Smith et al., 1988, 1989b). Both LFA-1 and Mac-1 mediate adhesion through multiple ligands. LFA-1 binds to the ICAMs: ICAM-I (Rothlein et al., 1986), ICAM-2 (Staunton et al., 1989a), and ICAM-3 (de Fougerolles and Springer, 1992; see also below). Mac-1 also binds to ICAM-1 (Smith et al., 1989b), although at a site distinct from that of LFA-1 (Diamond et al., 1991). In addition, Mac- 1 mediates neutrophil homotypic aggregation and presumably binds to an undefined ligand on activated neutrophils. Mac-1 also recognizes a wide spectrum of unrelated molecules, including the C3bi fragment of complement (Beller et al., 1982;Wright et al., 1983),fibrinogen (Wright et al., 1988; Altieri et al., 1988), factor X (Altieri and Edgington, 1988), and microbial antigens (Bullock and Wright, 1987). The molecular basis for this promiscuous nature of ligand recognition is not well defined,

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although there appears to be distinct binding sites for C3bi binding versus homotypic cell aggregation (Anderson et nl., 1986; Dana et af., 1986).

B. Intercellular Adhesion Molecules (ICAMs) ICAM-I (Rothlein et nl., 1986), ICAM-2 (Staunton et nl., 1989a), and ICAM-3 (De Fougerolles and Springer, 1992) were all originally defined functionally as ligands for LFA-I . Coincidentally, all three ICAMs are also structurally related members of the immunoglobulin (Ig) supergene family (Fig. 2 ) . They are most closely related to each other and more distantly to other Ig-like adhesion molecules, such as VCAM-I and NCAM. ICAM-1 has five Ig-like domains, with a short hinge region separating the third and fourth Ig-like domains (Staunton et ul., 1988; Simmons et al., 1988). ICAM-3 is structurally very similar to ICAM-1 (Fawcett et uf., 19!92:Vazeuxeral., 1992;deFougerolles e t n l . , 1993);it hasfiveIg-like domains. with each domain most similar in sequence to the corresponding domain of ICAM-1. The highest degree of identity between ICAM-I and ICAM-3 is in a region spanning domain 2 and part of domain 3. ICAM2, in contrast, has only two Ig-like domains, which are most closely related to domains 1 and 2 of ICAM-1 (Staunton et ul., 1989a). ICAM-I is a ligand for Mac-1 as well as for LFA-I (Smith et al., 1989b).

8

ICAM- I

[CAM-2

ICAM-3

Structure

Five Ig-like domains

Two Ig-like domains

Five lg-like domains

Ligandr

LFA-I LFA- 1 Mac- I CD43 Rhinovirus Malaria infected RBC

LFA- I

Dimbution

Widely inducible Lymphocytes Monocytes Endothelium Others

Leukocyte-specific Lymphocytes M onccytes Neutrophils

Constitutive Endothelium Lymphocytes

Fig. 2 The intercellular adhesion molecules (ICAMs). Ig, Immunoglobulin.

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Interestingly, the ICAM-1 binding sites for LFA-1 and Mac-1 are quite distinct: LFA-1 binds to domains 1 and 2 (Staunton et af., 1990), while Mac-1 binds to domain 3 (Diamond et al., 1991). ICAM-1 has also been reported as a ligand for leukosialin (CD43) (Rosenstein er al., 1991), although it is not clear whether ICAM-1 and CD43 interact as counterreceptors between two cells (Rosenstein et af., 1991) or on the same cell within the plane of the lipid bilayer (Ardman er af., 1992). Interestingly, expression of CD43 is deficient in Wiskott-Aldrich syndrome. Several pathogenic organisms have managed to subvert ICAM-1 for their own purposes: ICAM-1 is a receptor for rhinovirus (Staunton et al., 1989b) and for malaria-infected red blood cells (Berendt er al., 1992; Ockenhouse er al., 1992). The binding sites for both pathogens map to distinct sites of domain 1. It is not known whether ICAM-2 or ICAM-3 can bind these other ligands. The distribution and regulation of the three ICAM molecules are distinct. ICAM-1 is expressed basally only at low levels on vascular endothelial cells and lymphocytes, and is expressed at moderate levels on monocytes (Rothlein et al., 1986; Dustin et al., 1986). However, ICAM-1 expression can be induced to high levels on a wide range of cells by stimulation with inflammatory cytokines, such as IL-1, tumor necrosis factor (TNF), and y-interferon, or with lipopolysaccharides (Dustin et al., 1986). Induced or greatly increased expression of ICAM-1 has been reported on endothelial cells (Dustin et al., 1986), keratinocytes (Dustin et al., 1988), synovial cells (Mentzer et al., 1988), epithelial cells (Gundel er al., 1991), fibroblasts (Dustin er af., 1986), hepatocytes (Roos and Roossien, 1987; Adams et al., 1989), and myocytes (Entman et al., 1992). In addition, ICAM-1 expression is increased on activated lymphocytes (Dustin et af., 1986). This increased ICAM-1 expression requires mRNA transcription and de nouo protein synthesis. ICAM-2 expression, in contrast to that of ICAM-1, is constitutive and restricted to endothelial cells and mononuclear leukocytes (de Fougerolles er al., 1991; Nortamo et al., 1991). Inflammatory cytokines do not increase ICAM-2 expression on vascular endothelium. ICAM-3 is even more restricted in expression. It is expressed only on monocytes, lymphocytes, and granulocytes (de Fougerolles and Springer, 1992). Interestingly, ICAM-3 is the only ICAM molecule highly expressed by neutrophils. As a ligand for LFA-1, ICAM-1 has been implicated in guiding cellmediated cytolysis, antigen presentation, lymphocyte homotypic aggregation, and leukocyte-endothelial cell interactions. Additionally, ICAM-1, as a receptor for Mac-1, is involved in neutrophil-endothelial cell interactions (Smith er af., 1989b), transendothelial migration (Smith et al., 1988, 1989b), and adhesion-dependent respiratory burst (Entman et af., 1992).

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Functionally, it is not clear what roles ICAM-2 and ICAM-3 play in the inflammatory response. However, aggregation of lymphoid cell lines can involve LFA-1 interaction with one or more of the ICAMs (de Fougerolles and Springer, 1992). It is not known whether ICAM-3 can serve as a ligand for Mac- 1 in neutrophil homotypic aggregation.

C. Selectins L-selectin (Gallatin ef al., 1983), E-selectin (Bevilacqua et al., 1987), and P-selectin (McEver and Martin, 1984; Hsu-Lin et a l . , 1984) were discovered independent of each other by groups pursuing seemingly unrelated areas (reviewed by Lasky, 1992; Kishimoto, 1992). Only recent cDNA cloning of the selectins (Johnston et al., 1989; Lasky et al., 1989; Siegelman et al., 1989; Bevilacqua ef al., 1989; Siegelman and Weissman, 1989;Tedder et al., 1989;Bowen ef ul., 1989)revealed that these molecules are highly conserved members of the same gene family (Fig. 3). All three members of the selectin family share a common structural motif an Nterminal C-type lectin domain, an epidermal growth factor (EGF)-homologous domain, a variable number of short consensus repeats (SCRs) found in many complement regulatory proteins (e.g., factor H), a transmembrane, and a C-terminal cytoplasmic domain. Like other C-type lectins t Drickamer, 1988). the selectins feature Ca*+-dependent carbohydratebinding properties (Stoolman and Rosen, 1983; Yednock and Rosen, 1989; Lowe ef al., 1990; Phillips ef al., 1990; Walz et al., 1990). Extensive Lectin domain EGF domain

Short consensus repeats (SCR)

Transmembrane dqmain Cytoplasmic doimain

E-selectin

P-selectin

Distribution Neutrophils Monocytes Lymphocytes

Stimulated endothelium

Stimulated endothelium Activated platelets

Function

Inflammation

Inflammation Hemostasis (?)

L-select in

Lymphocyte homing Inflammation

Fig. 3 The selectin family of adhesion molecules. EGF, Epidermal growth factor.

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mutagenesis of the E-selectin lectin domain and computer modeling of these mutants based on the crystal structure of mannose-binding protein, a related C-type lectin, reveal a probable binding pocket in the E-selectin lectin domain for its carbohydrate ligand sialyl Lewis X (SLe') (Erbe et al., 1992). Short peptide fragments of the lectin domain have also been reported to block carbohydrate-binding function (Geng et al., 1992). The contribution of other domains of selectins is still not clear. MAbs directed against the EGF domain of L-selectin can block cell adhesion of leukocytes without blocking interactions with isolated polysaccharides (Siegelman et al., 1990; Spertini et al., 1991a). The selectins vary in the number of SCRs: L-selectin has two SCRs, E-selectin has six, and P-selectin has alternatively spliced forms of eight or nine SCRs. One unusual MAb, EL246 (Jutila el al., 1992),maps to a unique SCR epitope which is common to both E- and L-selectin. Interestingly, this MAb also blocks function without affecting carbohydrate-binding properties: The selectins have distinct tissue distributions. L-selectin is restricted in expression to white blood cells; about 60% of circulating lymphocytes and virtually all monocytes and granulocytes constitutively express Lselectin on their cell surfaces (Lewinsohn et al., 1987). L-selectin was first described by Gallatin et al. (1983) as a lymphocyte homing receptor, guiding tissue-specific migration of lymphocytes to peripheral lymph nodes (reviewed by Butcher, 1986; Yednock and Rosen, 1989). More recently, L-selectin has been demonstrated to mediate, in part, the adherence of neutrophils to cytokine-stimulated endothelial cells (Hallmann et al., 1991 ; Spertini et al., 1991c; Smith et al., 1991). E-selectin is restricted in expression to cytokine-stimulated endothelium (Bevilacqua ef al., 1987). Gimbrone, Bevilacqua, and colleagues (Bevilacqua et al., 1987; Luscinskas et al., 1989) pioneered the discovery and characterization of this molecule, which is involved primarily in neutrophil and monocyte adhesion to stimulated endothelial cells. E-selectin expression is prominent on endothelial cells in acute inflammatory lesions and correlates with the large influx of neutrophils (Cotran et at., 1986; Munro et al., 1989, 1991; Red1 et al., 1991; Leung et al., 1991). Recent studies indicate that E-selectin can also be expressed on endothelium at some chronically inflamed sites of skin (Koch et al., 1991; Munro et al., 1989) and synovialjoints (Picker et al., 1991b). Moreover, in uitro studies show that E-selectin can support adhesion of a small subset of memory T lymphocytes defined by expression of the HECA-452 antigen (Picker et al., 1991b). Interestingly, the HECA-452-positive lymphocytes have previously been associated with dermal T-cell infiltrates (Picker et al., 1990). P-selectin is restricted in cell surface expression to activated platelets (McEver and Martin, 1984; Hsu-Lin et al., 1984) and endothelial cells

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(McEver et al., 1989; Bonfanti et al., 1989). In unstimulated cells Pselectin is stored in the a-granules of platelets (Stenberg et al., 1985; Berman et al., 1986) and the Weibel-Palade bodies of endothelial cells (McEver et al., 1989; Bonfanti et al., 1989). The cytoplasmic domain of P-selectin mediates the transport of P-selectin to these intracellular compartments (Disdier et al., 1992). Cell activation with histamine or thrombin results in rapid recruitment of P-selectin to the cell surface. Pselectin has been implicated in the interactions of leukocytes with both platelets (Hamburger and McEver, 1990; Larsen et al., 1989, 1990) and endothelial cells (Geng et al., 1990; Patel et al., 1991; Lorant et al., 1991). Thus, P-selectin may bridge the early events of hemostasis and inflammation (Palabrica et al., 1992). The nature of selectin ligands has been an intense focus of research in many laboratories. Rosen, Stoolman, and colleagues demonstrated a role for L-selectin in carbohydrate binding many years before gene cloning revealed the lectin domain structure (Yednock and Rosen, 1989).Although great strides have been made in the past several years in identifying carbohydrate structures that interact with selectins, the precise nature of physiological selectin ligands remains controversial. This ambiguity reflects the subtle complexities of carbohydrate recognition-carbohydrates can be joined through different linkages and exist as branched structures-and the fact that carbohydrate epitopes may be displayed on a wide range of carrier lipids and proteins-perhaps not all equal in their ability to support selectin binding. A major breakthrough in 1990 was the discovery that E-selectin can bind SLeX (Phillips ef al., 1990; Walz et al., 1990; Lowe et al., 1990), a myeloid-specific sialylated fucosylated carbohydrate moiety (Fig. 4). Several complementary lines of evidence support this claim. Liposomes containing SLeX-glycolipids (Phillips et al., 1990) or soluble SLeXglycoproteins (Walz et al., 1990) are capable of blocking E-selectin-dependent adhesion of myeloid cells to stimulated endothelial cells or E-selectin

alpha 2.3

NeuAc + Gal

Fuc

-

alpha 1.3

1

NeuAc

beta 1.4

j. GIcNAc

Fuc

alpha 2.3 A

Gal

-

1

alpha 1.4

1

beta 1.3

GIcNAc

Fig. 4 Sialyl Lewis X and sialyl Lewis A.

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transfectants. Similarly, anti-SLeXMAbs can aiso block E-selectin-dependent adhesion (Phillips et al., 1990; Walz et al., 1990). Moreover, CHO cell line variants expressing SLeXbind to E-selectin, while other variants expressing only LeXdo not bind (Phillips p t af., 1990). Finally, transfection of an al,3-fucosyltransferase into CHO cells results in expression of SLeXdeterminants and correlates with the ability of these cells to bind E-selectin (Lowe ef al., 1990; Goelz et at., 1990). More recently. SLeA, a related sugar which differs from SLeXin only the linkages between the N-acetylglucosamine with galactose and fucose, has been shown to bind E-selectin (Berg et al., 1991a; Tyrrell et al., 1991). Computer modeling suggests that crucial residues on the sialic acid and fucose are presented in a similar plane in both SLeAand SLeX.Another sialylated fucosylated structure, vim-2, has also been proposed as a ligand for E-selectin (Tiemeyer et al., 1991), although this has not been confirmed (Walz ef al., 1990). Lewis X was first described as a specific ligand for P-selectin (Larsen et al., 1990), based on blocking of P-selectin-mediated adhesion with antiLeX MAbs and with Lex-containing oligosaccharides. However, other groups, using strategies similar to those used with E-selectin, have shown that sialic acid is an important component of the P-selectin ligand, and that SLeXis a much higher-affinity ligand than LeX (Polley et al., 1991; Moore ef al., 1991; Corral ef al., 1990; Tyrrell et al., 19911. Although Eand P-selectin share a similar carbohydrate specificity, it is becoming increasingly clear that there are distinct differences in ligand binding (Tyrre11 et al., 1991; Larsen ef al., 1992). Adding to this confusion is the observation that L-selectin can also bind SLeXand SLeA(Foxall et af., 1992; Berg et al., 1992; Green et al., 1992). Thus, selectins, in uitro, can interact with a variety of similar carbohydrate structures. It is clear that there are subtle differences in carbohydrate structure and among selectins which contribute to ligand specificity. Recently, there has been an emphasis on identifying specific carrier proteins and lipids which are physiologically important in selectinmediated interactions. SLeXis present at extremely high density on neutrophils, decorating many, but certainly not all, glycoproteins and glycolipids. While it is clear that SLeX-bearingstructures can support E- and P-selectinmediated adhesion in uitro, it is not clear whether E- and P-selectin bind with equal efficiency to all SLeX-bearingstructures. One possibility is that SLeXmay be added to a variety of proteins and lipids, but the presentation or accessibility of the SLeX may differ significantly between different glycoproteins and glycolipids, thus creating a hierarchy in which E-selectin preferentially binds to a subset of the total available SLeX.Alternatively, Siegelmann et al. (1990) have hypothesized that the lectin domain of the selections provides the carbohydrate specificity while the EGF domain

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may bind to protein determinants, thus creating an additional level of specificity. In addition, cell surface distribution may be an important factor in determining accessibility of the SLeXresidues: Some proteins, such as L-selectin, are localized to microvilli and pseudopods which are more likely to mediate cell-cell contact (Picker et af., 1991a). Based primarily on MAb blocking data, we have found evidence that L- and E-selectin appear to operate in the same CD184ndependent adhesion pathway (Kishimot0 et af., 1991; Abbassi et al., 1993). Anti-L- and anti-E-selectin MAbs have nonadditive blocking effects in a variety of assays, while both are additive in combination with anti-CD18 and anti-ICAM-1 MAbs. Neutrophi1 and lymphocyte L-selectin are identical in primary sequence (Ord et al., 1990); however, Picker et al. (1991a) have shown that L-selectin isolated from neutrophils but not from lymphocytes bears the SLeXcarbohydrate and can support E-selectin-dependent adhesion. Moreover, neutrophils treated with chyrnotrypsin, which cleaves L-selectin from the cell surface but does not significantly affect overall SLeXlevels, bind poorly to E-selectin (Picker et af., 1991a). Similarly, activated neutrophils, which have shed L-selectin but have significant SLeX on their surface, bind significantly less than unstimulated neutrophils to E-selectin (Kishimoto er al., 1991). The nature of the P-selectin ligand is equally controversial. Arrufo et af. (1991) have reported that sulfatides are a major ligand for P-selectin. In contrast, Moore et al. (1991) have found that protease treatment of neutrophils completely eliminates binding of Auid-phase P-selectin, even though significant SLeXremainson glycolipids. Picker er af. (1991a) have speculated that L-selectin may also serve as a ligand for P-selectin. More recently, McEver and colleagues have demonstrated that purified labeled P-selectin reacts by Western blot analysis to a specific band at 120 kDa from neutrophil lysates (Moore et al., 1992). These investigators demonstrated that this band does not correspond to L-selectin or CD43. This report is striking since P-selectin binds preferentially to this band, even though a wide range of neutrophil proteins contain the SLeXdeterminant. Thus, it may be possible to observe selectin-mediatedadhesion to carbohydrate alone in v i m , but optimal adhesion activity may require both protein and carbohydrate determinants. The greatest strides in identifying physiologically relevant carbohydrate camer structures have been made with an high endothelial venule (HEV) Iigand for L-selectin. Butcher and colleagues developed an MAb, MECA79, which stains peripheral node HEV and inhibits lymphocyte binding in ui?ro and trafficking in uiuo (Streeter et af., 1988). The MECA-79, MAb is an IgM and appears to recognize a carbohydrate determinant. Western blot analysis and immunoaffinity purification of reactive antigen shows

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several distinct bands: a major band of 105 kDa, with minor bands of 65, 90,150, and 200 kDa (Berg et al., 1991b). The purified MECA-79 antigen supports tissue-specific lymphocyte binding, which is blocked by both MECA-79 and anti-L-selectin MAbs (Berg et al., 1991b). In an independent line of investigation, Rosen, Lasky, and colleagues have recently utilized soluble L-selectin, in the form of a bivalent L-selectin-IgG chimeric molecule, to immunopurify a 50-kDa protein from lymph node tissue (Imai et al., 1991). This protein is heavily glycosylated and contains sulfated sugars. Interestingly, this major 50-kDa product isolated with the L-selectin-IgG chimeric molecule also immunoreacts with the MECA-79 MAb (Imai et al., 1991). Molecular cloning of this molecule, termed Glycam-1 (Lasky et al., 1992), reveals homology to mucins which are heavily 0-linked glycosylated proteins. This molecule lacks a classic transmembrane domain, but may be anchored to other proteins via an unusual amphipathic helix.

111. Regulation of Cell Adhesion Molecule Expression and Function

An important consideration in neutrophil adhesive interactions is that neutrophils in circulation are normally in a free-flowingnonadhesive state. Moreover, when neutrophils are recruited to an inflammatory site, its adhesive interactions must be highly specific and transient in nature. Thus, regulation of cell adhesion molecules is a central theme, and a variety of strategies are utilized by the immune system to orchestrate the expression and function of these adhesion molecules.

A. De Novo Synthesis Regulation of surface expression by de nouo synthyesis of adhesion molecules is a relatively slow means to modulate adhesion. However, most vascular adhesion molecules, including ICAM-1 (Dustin et al., 1986; Pober et al., 1986), E-selectin (Pober et al., 1986; Bevilacqua et al., 1987), VCAM-1 (Rice et al., 1990; Carlos et al., 1990), and at least one ligand for L-selectin (Hallmann et al., 1991; Smith et al., 1991; Spertini et al., 1991c), are induced de n o w by inflammatory cytokines such as IL-1 and TNF. Although induced expression can be detected in the first hours following stimulation, peak expression is not detected until 4 hours or more. This correlates with the large influx of neutrophils into inflammatory sites at 3-4 hours. Gene promoter regions for these adhesion molecules

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contain several distinct transcriptional regulatory elements. Regulation at the level of gene expression may be an important mechanism to ensure specificity and control the magnitude of the response. Controlling the magnitude of the response, to minimize tissue damage, may be as important as regulating the speed of the response. Similarly, the inflammatory response must be self-limiting. E-selectin is typically down-modulated rapidly after 8-24 hours, correlating with decreased neutrophil recruitment, while ICAM-I and VCAM-1 are more stably expressed. This differential regulation may reflect regulation at the level of mRNA transcription, RNA stability. or protein turnover (Wertheimer et d., 1992; Ghersa et d., 1992; Myers ef NI., 1992). However, there must also be some overriding mechanism to sustain E-selectin expression in selected chronic sites of inflammation. The time course of L-selectin ligand expression remains somewhat controversial. A ligand for neutrophil L-selectin appears to be down-modulated by 8-24 hours (Kishimoto et al., 1991; Spertini er al., 1 9 9 1 ~ ) like . E-selectin; however, L-selectin-dependent lymphocyte adhesion to stimulated HUVEC is stable over 2-3 days (Spertini er al., 1991~). This suggests the possibility of distinct counter-receptors for neutrophil and lymphocyte L-selectin-perhaps correlating with acute versus chronic inflammatory signals.

B. Rapidly Mobilized Intracellular Compartments Neutrophils are not idle during the first hours required to induce E-selectin and ICAM-1 expression. P-selectin is stored in the Weibel-Palade bodies of endothelial cells (McEver et ul., 1989; Bonfanti et al., 1989) and the a-granules of platelets (Stenberg et al., 1985; Berman et NI., 1986).Stimulation by histamine and thrombin induces rapid mobilization of these compartments to the cell surface, resulting in cell surface expression within minutes of activation. Similarly, intravital microscopy studies suggest the possibility that a ligand for L-selectin may also be rapidly induced (von Andrian et al., I991; Ley et al., 1991). P-selectin may play a critical role in bridging the processes of hemostasis and inflammation in the earliest stages of vascular insult. P-selectin is also rapidly cleared (within 30 minutes) from the cell surface in v i m (Mcsver ef d.,1989; Bonfanti et al.. 1989). However, there is indication that P-selectin, like E-selectin. can be expressed at chronically inflamed synovial joints (Stoolman et ul., 1992). Low levels of oxygen radicals induce prolonged expression of Pselectin in v i m (Pate1 et al., 1991) and at least the mouse P-selectin gene appears to be responsive to induction by TNF (Weller et al., 1992). The Mac-1 integrin is also stored in intracellular granules of neutrophils, which are rapidly recruited upon neutrophil exposure to chemotactic fac-

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tors (Todd et al., 1984; Berger et al., 1984; Bainton et al., 1987). The most readily mobilizable store of Mac-1 is in a novel type of granule (Borregaard et al., 1987)-distinct from the classic primary and secondary granules. Thus, significant up-regulation of Mac-1 can occur without exocytosis of the destructive proteolytic enzymes found in primary and secondary granules. This pool of Mac-1 is likely to be important in neutrophil interactions with endothelium, while Mac- 1 stored in secondary granules may be more important for effector functions.

C. Receptor Activation An insightful but puzzling observation is that neutrophil aggregation and adhesion to endothelium induced by a single high dose of chemotactic agent is mediated by the constitutively expressed cell surface Mac-1 (Buyon et d.,1988; Vedder and Harlan, 1988). Under these experimental conditions the large pool of Mac-1 recruited from intracellular stores is not involved in these processes. However, resting neutrophils do not spontaneously aggregate. These observations suggest that neutrophil activation results in qualitative as well as quantitative changes in Mac-1 expression. Similarly, phorbol esters induce LFA-1-dependent lymphocyte aggregation without affecting the surface expression of LFA-1 or its ligand ICAM-1 (Rothlein and Springer, 1986; Patarroyo et al., 1985). Furthermore, stimulated lymphocytes bind to both purified LFA-1 and ICAM-1 immobilized onto plastic, while resting lymphocytes bind only to purified LFA-1 (Dustin, 1990). Interestingly, lymphocytes activated by crosslinking of the T-cell receptor complex show only a transient increase in LFA-1-mediated adhesion (Dustin and Springer, 1989; Van Kooyk et al., 1989), perhaps corresponding to the transient nature of adhesive events. These studies indicate that LFA- 1 functional activity can be modulated by activation. The CD18 integrin activation states can be identified and mimicked by MAbs which recognize activation-dependent epitopes. Figdor and colleagues first described the NKI-L16 MAb which recognizes a Ca2+dependent epitope on primed or activated lymphocytes but absent on resting lymphocytes (Keizer et al., 1988; Van Kooyk et al., 1991). This unusual antibody can induce lymphocyte aggregation which is inhibited by other anti-LFA-1 MAbs. Hogg and colleagues have described MAb24, which recognizes an Mg2+-dependent epitope common to all three asubunits and whose expression parallels receptor activity. MAb24 blocks leukocyte functions such as antigen presentation and neutrophil chemotaxis. However, inhibition by MAb24 does not inhibit ligand binding, but rather appears to lock the CD18 integrins into an active conformation and

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prevents de-adhesion (Dransfield and Hogg, 1989; Dransfield et al., 1992). This interesting observation underscores the necessity for the transient nature of adhesion events. A third MAb, Kim-127, recognizes a temperature-sensitive but divalent cation-insensitive epitope on the common p-subunit (Robinson et al., 1992). Kim-127 induces CD18-dependent adhesion. The precise relationship between the epitopes recognized by these three MAbs remains to be determined. Studies with these activation MAbs clearly indicate that the CD18 integrins undergo a conformational change which results in enhanced binding activity. However, the mechanisms by which cellular activation can influence the conformation of CD18 integrins on the outside of the cell remains unclear. Early studies indicated that phorbol esters induce phosphorylation of the P-subunit (Hara and Fu, 1986); however, more recent studies show that neutrophil activation with chemoattractants does not induce significant phosphorylation of the ,&subunit (Chatila et al., 1989; Merrill et af., 1990; Hibbs et al., 1991a). Moreover, staurosporine, an inhibitor of protein kinase C. inhibits neutrophil aggregation induced by phorbol ester but actually enhances aggregation induced with fMLP. Association of the p-subunit with cytoskeletal components has been proposed as a mechanism to alter CD18 function. In fact, the term "integrin" (Hynes, 1987) was originally chosen to define a class of membrane receptors which integrate the extracellular environment (matrix and other cells) with the intracellular cytoskeleton. For example, the fibronectin receptor colocalizes with fibronectin bundles on the outside of the cell and actin filaments on the inside. Similarly, LFA-1 colocalizes with talin, a cytoskeletal component which associates with the fibronectin receptor, on activated, but not on unactivated, lymphocytes (Burn et al., 1988; Kupfer and Singer, 1989).Moreover, LFA- I-dependent lymphocyte aggregation is inhibited by cytochalasin B (Rothlein and Springer, 1986).Finally, recent mutagenesis studies indicate that the cytoplasmic domain of the p-subunit is critical for CD18 function (Hibbs er al., 1991a,b). Interaction of CD18 with the cytoskeleton is an attractive model to explain regulation of receptor activity, since cytoskeletal interactions can be transient and easily modulated. It is notable that ICAM-I (Carpen et al., 1992) is also thought to associate with the cytoskeleton. L-selectin function is thought to be in a constitutively active state, since L-selectin-dependent adhesion of resting leukocytes can be readily observed at low temperatures (4°C) (Gallatin et al., 1983; Hallmann et al., 1991; Spertini et al., 199ic). However, Tedder and colleagues have shown that L-selectin can also be regulated by an activation-induced increase in ligand-binding activity (Spertini et al., 1991b). Both lymphocytes and neutrophils show increased L-selectin activity in binding carbo-

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hydrate ligands, such as the yeast phosphomannan (PPME), and in adhesion to HEV. This increased affinity is presumably transmitted through a conformational change in the L-selectin extracellular domain, although the precise mechanism is not known. Crommie and Rosen (1992) have found evidence for oligomerization of L-selectin, which could also affect receptor avidity.

D. Rapid Down-Regulation of Adhesion Molecule Expression It is clear that mechanisms to regulate rapid de-adhesion are as important as rapid adhesion mechanisms. L-selectin is constitutively expressed on resting neutrophils in a seemingly functional form. However, within minutes of neutrophil exposure to low levels of chemotactic factors, Lselectin is down-regulated from the cell surface (Kishimoto et al., 1989~). A broad range of neutrophil-activating agents, including CSa, fMLP, TNF, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-8, can induce this response (Kishimoto et al., 1989c;Jutila et al., 1989; Smith et al., 1991; Griffin et al., 1990). Interestingly, the rapid shedding of Lselectin follows the kinetics of Mac- 1 up-regulation from intracellular stores (Kishimoto et al., 1989c) (Fig. 5). Analysis of neutrophils which are recovered from the inflamed peritoneum in uiuo (Jutila et al., 1989) and immunohistological analysis of neutrophils in inflamed skin sites (Kishimoto et al., 1989c) suggest that this inverse regulation of adhesion molecules occurs in uiuo as well. These observations suggest that chemotactic factors provide a critical trigger which turns off one adhesion mecha-

0 min.

+

a

5 min.

Fig. 5 Inverse regulation of L-selectin and Mac-1 upon neutrophil activation. Chemotactic factors trigger a rapid transition in the surface expression of two major neutrophil adhesion proteins. L-selectin is down-regulated by apparent proteolytic cleavage from the cell surface, while Mac-1 is up-regulated by mobilizing intracellular granules containing Mac-1.These results suggest that Mac-1 and L-selectin mediate distinct but complementary adhesion pathways.

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nism (L-selectin)and simultaneously induces another (Mac-1) (see below). It seems likely that the rapid shedding of L-selectin involves an activation-dependent proteolytic event: however, the precise mechanism remains unresolved. A large fragment of L-selectin can be recovered from the supernatant of activated cells, suggesting that L-selectin is proteolytically clipped close to the transmembrane domain (Kishimoto et al., 1989~; Berg and James, 1990). We have recently identified a 6.2-kDa transmembrane peptide of L-selectin in activated lymphocytes (Kishimoto and Kahn, 1993).The nature of the putative protease is not known. Low levels of exogenous chymotrypsin removes L-selectin without affecting the surface expression of several other membrane proteins (Jutila er d..1991). However, a panel of chymotrypsin and other serine-protease inhibitors does not affect activation-dependent L-selectin down-regulation (Jutila et d.,1991: Kishimoto 1990). In a concentrated mixed population of neutrophils and lymphocytes, phorbol myristate acetate induces down-regulation of both neutrophil and lymphocyte L-selectin. In contrast, fMLP, a myeloid cell-specific activating agent, induces down-regulation of only the neutrophil L-selectin, even though lymphocytes are in close proximity (M. A. Jutila and T. K. Kishimoto, 1989). These results suggest that the protease acts in cis and is either membrane bound or rapidly inactivated. Another curious observation is that L-selectin transfected into a nonhematopoietic cell line (e.g., COS cells) is continuously shed into the medium (Lasky et a [ . , 1989). There are several possible explanations: The proteolytic mechanism is ubiquitous and serves as a general mechanism to regulate the expression of other cell surface proteins, the protease is ubiquitous but is involved in unrelated membrane events on other cell types, or Lselectin itself has protease activity and can cleave itself. Finally, Jutila and colleagues have found evidence that cross-linking of L-selectin, either nonspecifically with chemical cross-linkers or specifically with MAbs or polysaccharides, can induce L-selectin down-regulation in the absence of any overt activation (Palecanda et al., 1992).

IV. A Model for Neutrophil-Endothelial Cell Interactions The question of how these multiple adhesion systems are orchestrated to allow efficient neutrophil emigration is a perplexing one. In 1989 a simple two-step adhesion model for neutrcphil interaction with stimulated endothelium was proposed (Kishimoto et al., 1989c: Jutila et al., 1989) based primarily on the following observations: ( 1 ) The CD18 integrins were known to show increased functional activity upon activation and to be

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involved in neutrophil aggregation (Buyon et al., 1988) and transendothelial migration (Smith et al., 1988, 1989b); (2) the CD18 integrins did not appear to be required for adhesion under shear stress in uiuo (Arfors et al., 1987) or in vitro (Lawrence et al., 1988)-neutrophils from LAD patients adhere under flow conditions; (3) antibodies against both Mac-1 and L-selectin inhibit neutrophil recruitment in uiuo (Jutila et af., 1989); and (4)Mac-1 and L-selectin expression are inversely regulated by chemotactic factors-L-selectin is rapidly down-regulated with a concurrent This latter observarapid up-regulation of Mac-1 (Kishimoto et al., 1989~). tion suggested that L-selectin and Mac-1 must mediate distinct but complementary adhesion events. The rapid down-regulation of L-selectin indicated that its participation must be confined to the earliest stages of extravasation, mediating the initial interaction of the circulating neutrophil with the activated endothelium (Fig. 6). This initial binding or rolling event would guide the unstimulated neutrophil to the appropriate site of inflammation, where it becomes exposed to low levels of chemotactic factors released on the endothelial surface at the site of inflammation. The chemoattractants provide the signal for the neutrophil to enter the inflamed tissue and concomitantly trigger the transition from L-selectinmediated to CD18-mediated adhesion. The CD18 integrins, LFA-1 and Mac-1, are largely responsible for subsequent adhesion strengthening, neutrophil aggregation, and the actual process of transendothelial migration. Over the last several years insights from many laboratories have supported and greatly expanded this simplified model to include additional adhesion molecules, chemotactic factors, and other cell types. The appeal of this generalized model is that it provides a mechanism to closely regulate

Selectin-dependent Rolling

Initial binding

Transition Activation

. ) CD18fiCAM-dependent Adhesion strengthening Transmigration Aggregation

Fig. 6 A model of neutrophil-endothelial cell interactions. In this simplified model the selectins mediate initial binding and rolling behavior of neutrophils, while CDINICAM-1 molecules are involved in transendothelial migration. Chemotactic factors trigger the transition between the two pathways. (Adapted by permission from Kishimoto, J . NZH Res., 1991, 3: 75-77.)

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neutrophil localization and neutrophil activation, thus ensuring minimal damage to surrounding healthy tissue.

A. Initial Interactions of Circulating Neutrophils with Endothelial Cells Neutrophils are subjected to tremendous shear forces generated by the flow of blood through the circulatory system. Classic intravital microscopy studies, dating back as far as a century ago (Cohnheim, 1889), described the curious phenomenon of leukocyte rolling. In these types of studies, the vast majority of cells, including the red blood cells, are an indistinguishable blur traveling at 300-1000 pm/sec (Atherton and Born, 1972). Within this flow neutrophils can be observed to distinctly roll along the surface of the vascular endothelium at a velocity about two orders of magnitude slower than the main flow of cells. This rolling phenomenon is thought to be an early inflammatory response to the tissue damage caused by the surgical manipulation needed to prepare the animal for intravital microscopy (Fiebig e l al., 1991). Neutrophil rolling is minimal or absent in intravital microscopy studies that require little or no surgical manipulation. Furthermore, at sites of inflammation leukocyte rolling precedes firm adherence at inflammatory sites. The molecular basis of this rolling behavior has been unknown until recently. Several lines of evidence now suggest a direct role for the selectin adhesion molecules in mediating neutrophil rolling. Using intravital microscopy, Arfors et al. (1987) demonstrated that antiCD18 MAbs inhibit leukocyte extravasation into sites of inflammation but have no effect on neutrophil rolling behavior. In fact, anti-CD18 MAbs appear to actually increase the velocity of neutrophil rolling (Perry and Granger, 1991). In an independent line of investigation, Smith and colleagues developed a parallel plate flow chamber to study the effects of shear stress on neutrophil interaction with cultured endothelial cells in uitro (Lawrence ef al., 1988). These investigators found that neutrophils exhibited rolling and sticking behavior on cytokine-stimulated endothelium but not on unstimulated endothelium. Moreover, the CD18-dependent component of neutrophil adhesion decreases with increasing shear stress and disappears by 2 dyn/cm2(Lawrence et al., 1988). An important insight was the observation that neutrophils from LAD patients deficient in CD18 expression roll and adhere under flow as well as normal neutrophils. These observations, together with the observation that L-selectin and Mac-1 are inversely regulated on stimulated neutrophils, led to the hypothesis that L-selectin may be involved in these early adhesion events under flow conditions (Kishimoto et al., 1989~;Jutila et al., 1989). To directly

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test this model, we developed MAbs against human L-selectin (Kishimoto et al., 1990) and examined their ability to block neutrophil adhesion to stimulated endothelium in the flow chamber. Anti-L-selectin MAbs dramatically reduced, but did not totally inhibit, neutrophil rolling and adhesion under flow conditions which are CD18 independent (1.85 dyn/cm2 (Smith et al., 1991). Activated neutrophils show increased adhesion to stimulated endothelium under static conditions, and this adhesion is almost entirely CD18 dependent. However, under conditions of flow, activated neutrophils, which have shed L-selectin but have increased Mac-1 expression, show substantially reduced neutrophil rolling and adhesion. AntiL-selectin MAbs do not further reduce adhesion of activated neutrophils. In a parallel line of investigation, two groups have used intravital microscopy to show a role for L-selectin in mediating neutrophil rolling in uiuo. Ley et al. (1991) demonstrated that a polyclonal serum against L-selectin reduces by 84% the number of rolling neutrophils in postcapillary venules of the rat mesentery. Significantly, a soluble bivalent L-selectin-IgG chimera also blocks neutrophil rolling by 89%. These investigators utilized a technique to inject the antagonists into venules directly upstream of the observation site. Thus, inhibition of rolling is readily reversible within seconds after administration of the antagonist is terminated, indicating that neutrophil blood levels are not grossly affected. Similarly, von Andrian et al. (1991) demonstrated that a single bolus of an anti-human L-selectin MAb (DREG-200), which cross-reacts with rabbit L-selectin, inhibits the number of neutrophils rolling in postcapillary venules of the rabbit mesentery. Furthermore, Fab fragments of this MAb were also effective and had no effect on circulating neutrophil counts. Anti-CD18 MAbs had no effect on neutrophil rolling but blocked firm adhesion of leukocytes. Together, these and other studies (von Andrian et al., 1992) indicate that L-selectin directly participates in neutrophil rolling. Since L-selectin is closely related to the vascular adhesion molecules P- and E-selectin, an obvious question is, Do these other selectins support neutrophil rolling from the endothelial cell side? Lawrence and Springer (1991) modified the parallel plate flow chamber to include lipid planar membranes containing purified adhesion molecules instead of endothelial cells. Purified P-selectin incorporated into the lipid planar membrane supports neutrophil rolling under flow conditions similar to those used with endothelial cells (2 dynlcm2). Significantly, neutrophils did not attach to artificial bilayers containing ICAM-1 under these conditions of flow. It is interesting that activation of the neutrophils greatly reduces their ability to roll on P-selectin. Similarly, Abbassi et al. (1993) have shown that mouse L-cell fibroblasts, transfected with the E-selectin gene, support neutrophil rolling and

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adhesion at shear forces of 1.85 dyn/cm2 to levels comparable to those seen with IL- I-stimulated endothelial cells. Neutrophils show no rolling or adhesion under these conditions on ICAM-I transfectants or the parent L-cell line. Neutrophil rolling on the E-selectin transfectants was completely blocked with anti-E-selectin MAbs. Interestingly, anti-L-selectin MAbs significantly inhibited. but did not completely block, neutrophil rolling on E-selectin (Abbassi er al., 1993). These results support the concept that L-selectin may partially contribute to E-selectin-mediated adhesion. The degree of adhesion under flow to E-selectin shows a high correlation with L-selectin surface density and only minimal correlation with SLeX density. However, neuraminidase treatment of neutrophils. which eliminates SLeXepitopes, blocks neutrophil rolling on E-selectin (Abbassi ef al., 1993). These data suggest that SLeXis necessary, but not all SLe' is utilized. Activation of neutrophils results in L-selectin downregulation without a significant effect on SLeXdensity, but activated neutrophils show diminished rolling on E-selectin monolayers. This small component of activated neutrophil binding to E-selectin transfectants under flow, however, is blocked by anti-E-selectin but not by anti-L-selectin. Anti-CD18 had no effect on the number of neutrophils rolling. but did increase the average rolling velocity (Abbassi er nl.. 1993). This suggests that CD18 integrins are not involved in rolling per se, but may act to slow the neutrophil down once the selectin-mediated rolling begins. Further, intravital microscopy studies are required to demonstrate E- and P-selectin involvement in neutrophil rolling in uiuo.

B. Chemotactic Factors as a Triggering Mechanism to Regulate Adhesion Pathways

Chemotactic factors play a multifaceted role in inflammation. In uitro studies with chemotactic factors can seem confusing and contradictory. Two factors are critical: the concentration of the chemotactic factor and whether or not there is a gradient. A gradient of chemoattractants can, of course, provide the signal for directed migration. At low concentrations chemoattractants also promote neutrophil adhesiveness, while at higher concentrations some chemoattractants have an antiadhesive effect (Gimbrone et al., 1989). At high concentrations chemotactic factors can also be costimulatory signals for the neutrophil oxidative burst and phagocytosis (Shappell et af., 1990). In addition, many chemotactic factors appear to be specific for subclasses of leukocytes, which may add another level of specificity to leukocyte recruitment to inflammatory sites (Schweighoffer and Shaw, 1992; Butcher, 1991). It seems clear that adhesion molecules can account for

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only part of the cell and tissue specificity of leukocyte migration. Leukocytes share many of the same adhesion molecules, yet some inflammatory infiltrates are dominated by neutrophils, while others may involve monocytes, eosinophils, or subsets of lymphocytes. Expression of an L-selectin ligand and ICAM-1 by endothelial cells is not sufficient to explain the specificity of neutrophil recruitment to acute inflammatory sites, since most leukocytes express L-selectin and CD18 integrins. However, a neutrophil-specific chemoattractant, such as IL-8, may help account for cell type specificity. In leukocyte recruitment chemotactic factors may provide a crucial trigger or a “go or no-go’’ signal to enter the inflamed tissue. In this model the selectin-mediated rolling of neutrophils along endothelium is readily reversible unless followed by an appropriate chemotactic signal. Neutrophil chemotactic factors trigger what is likely to be an irreversible commitment of the neutrophil to attempt to migrate into the inflamed tissue. This chemotactic factor trigger results in neutrophil shape change, indicating reorganization of the cytoskeleton, and the rapid inverse regulation of L-selectin and Mac-1. Thus, chemotactic factors trigger a transition from L-selectin-dependent adhesion to Mac-1-dependent adhesion. Lselectin is required for neutrophil localization to the endothelium adjacent to the inflamed tissue, while the CD18 integrins move the neutrophils across the vascular endothelium. It is not clear why L-selectin is downregulated, although it may be a necessary event for transendothelial migration to continue or it may be a protective mechanism which ensures that stimulated neutrophils which are freed by shear forces in the blood cannot gain access to other tissues. This transition in neutrophil adhesiveness can be observed in v i m . Under static conditions, in which no shear stress is applied, resting neutrophil adhesion is partially selectin dependent and partially CD18/ICAM- 1 dependent (Kishimoto et al., 1991; Smith et al., 1991; Spertini et al., 1991c; Luscinskas et al., 1989). Upon exposure of the neutrophil to fMLP, a chemotactic agent, the adhesion becomes almost entirely CD18/ICAM1 dependent (Dobrina et al., 1990; Smith et al., 1989a). Lawrence and Springer (1991) found that in conditions under flow, neutrophils remain round during their rolling interactions on a mixture of purified P-selectin and ICAM-1. If fMLP is infused into the chamber, the average rolling velocity decreases drastically and within 5 minutes the neutrophils become adherent and spread on the bilayer. Interestingly, if only P-selectin is present in the bilayer, neutrophils do not become arrested, even with infusion of fMLP. Thus, even in this artificial system, all three components-selectins, chemotactic factors, and CD WICAM-1-are required for this transition from rolling to firm adherence.

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The physiologically relevant chemotactic factor that triggers this transition in uiuo is unknown. There are probably multiple mediators, perhaps utilized in different types of inflammatory events. It would be most efficient if the stimulated endothelial cells themselves could produce the appropriate chemoattractant. Furthermore, it would be more efficient if the chemotactic factor is physically associated with the endothelial surface, rather than only in a free and soluble form which would be quickly carried downstream by the blood flow. This would ensure appropriate localization of the neutrophil to the inflammatory site. Endothelial cells are capable of producing several known neutrophil chemoattractants, including IL-8, GM-CSF, and platelet-activating factor (PAF). Neutrophils exposed to unstimulated endothelium remain spherical, while those exposed to IL-1-stimulated endothelial cells undergo a shape change response. IL-1 alone does not induce the neutrophil shape change. Smith et af. (1991) further demonstrated that coculture of freshly isolated neutrophils with IL- l-stimulated endothelial cells for 30 minutes induced a dramatic down-regulation of L-selectin and an up-regulation of Mac-1. Moreover, CD18-deficient neutrophils attached to IL-l-stimulated endothelial cells undergo shape change and detach from the endothelial monolayer. Furthermore, the conditioned medium from IL-l-stimulated endothelial cells can directly induce this transition in neutrophils (Smith et al., 1991). Huber et al. (1991) recently reported that an anti-IL-8 serum significantly blocked neutrophil transmigration across stimulated endothelial cells. Immunolocalization of IL-8 demonstrates association with both the endothelial cells and the underlying collagen gel matrix. These results suggest that in 3- to 4-hour stimulated endothelial cultures, IL-8 is the major chemotactic factor involved. Another interesting possibility is that neutrophil adhesion to E-selectin is sufficient to trigger activation of the CD18 integrins (Lo et al., 1991; Kuijpers et al., 1991). However, the process of neutrophil adhesion or rolling on P-selectin (Lawrence and Springer, 1991) or E-selectin (Abbassi et al., 1993) does not cause a shape change response. Zimmerman, Mclntryre, and colleagues have studied the role of endothelial cell-derived PAF in neutrophil adhesion and activation (Prescott et af., 1984; Zimmerman et al., 1990). In contrast to the soluble form of PAF produced by several cell types, endothelial cell PAF is expressed as a membrane-associated factor on the cell surface. Although PAF is synthesized de n o w , both PAF and P-selectin are coexpressed within minutes after thrombin or histamine stimulation of endothelium, correlating with increased neutrophil adhesiveness (Zimmerman and McIntyre, 1988; Zimmerman et al., 1985). Neutrophils bind directly to endothelial PAF and become primed for Mac-1 up-regulation and enhanced responses

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to chemotactic factors. These results suggest that in the earliest events following vascular insult, PAF and P-selectin may function cooperatively to mediate neutrophil localization. These investigators proposed that Pselectin acts as a tether to stop the circulating neutrophil, allowing PAF to trigger the transition to CD18-dependent transmigration (Lorant et al., 1991).

C. Neutrophil Adhesion Strengthening and Aggregation The transition from selectin-mediated adhesion to CD18-mediated adhesion is not an all-or-none phenomenon, but rather a gradual transition. As mentioned above, CD18 integrins do not directly mediate neutrophil rolling, but to contribute to the velocity of rolling (Perry and Granger, 1991;Abbassi et al., 1993).LAD neutrophils and anti-CD18normal neutrophils roll faster than untreated normal neutrophils (Abbassi et al., 1993). As neutrophils are exposed to chemotactic factors, L-selectin and Mac1 must work cooperatively to ensure that the primed neutrophil is not released by shear forces. Both L-selectin (Spertini et al., 1991b) and the CD18 integrins (Buyon et al., 1988; Vedder and Harlan, 1988) undergo a conformational change, resulting in a transient increase in affinity for ligand. This increased adhesiveness strengthens the interaction of neutrophils with the endothelium. Presumably, as L-selectin is shed, the recruitment of additional CD18 integrins contributes to neutrophil adhesiveness. Lawrence and Springer (1991) showed that purified P-selectin but not ICAM-I can support neutrophil attachment under shear stress. However, if neutrophils are first allowed to attach to these purified proteins before shear stress is applied, cells attached to ICAM-1 are much more resistant to detachment than those bound to P-selectin. In addition to becoming more adhesive for the endothelium, neutrophils exposed to chemotactic factors show increased adhesiveness for each other, resulting in neutrophil aggregation (Dana et al., 1986; Anderson et al., 1986; Schwartz et al., 1985). Neutrophil aggregation may amplify the recruitment process, by causing more cells to bind to that region in the blood vessel. During severe inflammation, some vessels become occluded with neutrophil aggregates, which helps to slow blood flow and may allow for even greater neutrophil accumulation. Neutrophil aggregation is a Mac-l-dependent event (Dana et al., 1986;Anderson et al., 1986;Schwartz et al., 1985). Interestingly, neutrophil-endothelial cell interactions and neutrophil-neutrophil aggregation utilize distinct Mac-1 ligands: ICAM1 is involved in the former but not the latter. Recent data suggest that Lselectin may serve as a counter-receptor for Mac-1 in neutrophil aggregation (Simon et al., 1992). It is noteworthy that the rate of L-selectin

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down-regulation coincides with the kinetics of neutrophil disaggregation: Typically, neutrophil aggregation induced by chemotactic factors peaks within several minutes and diaaggregation is evident by 5-10 minutes. However, phorbol ester stimulation of neutrophils results in L-selectin down-regulation but stable neutrophil aggregates, suggesting other potential ligands. The transient nature of neutrophil aggregation is more physiologically relevant, since the ultimate goal of the neutrophil is to migrate into the inflammatory site.

D. Neutrophil Transendothelial Migration A critical step in the recruitment of neutrophils is the ability of neutrophils to penetrate the vascular endothelium and the basement membrane (reviewed by Smith, 1992). Classic studies show that neutrophils first migrate between endothelial cell junctions, flatten out, and then proceed through the basement membrane. The development of primary endothelial cell cultures (Gimbrone, 1976)enabled the study of neutrophil transendothelial migration in uirro (Smith, 1992). By phase-contrast microscopy, neutrophils on the endothelial monolayer appear spherical and phase bright, while those cells which had migrated beneath the monolayer appeared flattened and phase dark. Neutrophils which come into contact with IL1-stimulated HUVEC rapidly undergo a shape change and migrate across the monolayer within minutes (Smith et al., 1988, 1989b). In contrast, migration across unstimulated endothelial cells is inefficient: Neutrophils remain spherical and stay on the apical surface of the monolayer. However, addition of exogenous chemoattractants to a Boyden-type chamber beneath the monolayer results in directed migration of neutrophils. Interestingly, direct addition of chemotactic factors to the neutrophils results in no migration, regardless of whether the endothelial monolayer was stimulated with IL-1 (Smith, 1992). These studies suggest that a chemotactic gradient is critical for efficient transendothelial migration of neutrophiis, and that IL-1 stimulation of endothelial cells induces production of an endogenous chemotactic gradient which can be detected by neutrophils. As mentioned above, stimulated endothelial cells can produce several known neutrophil chemoattractants, including IL-8, PAF, and GM-CSF. Transendothelial migration is a CD18 integrin-dependent event (Smith et al., 1988, 1989b; Luscinskas et af., 1989, 1991; Hakkert et al., 1991; Furie et ul., 1992; Smith er af., 1991). Neutrophils isolated from CD18deficient patients adhere to IL-1-stimulated endothelium but fail to transmigrate in uirro (Smith et al., 1988). This inability of neutrophils to transmigrate is probably the most severe functional defect in these patients. Both LFA-1 and Mac-1 appear to be involved, since MAbs against either

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partially block transendothelial migration and together have an additive effect. Anti-CD 18 MAbs block neutrophil transendothelial migration almost completely. ICAM-1 appears to be a major endothelial ligand for both LFA-1 and Mac-1, as MAbs against ICAM-1 are effective inhibitors of neutrophil transendothelial migration (Smith et al., 1989b). Interestingly, anti-CD18 is consistently more effective than anti-ICAM-1, suggesting the possible involvement of other ligands (e.g., ICAM-2). Thus, Mac-1 plays a crucial role for both neutrophil aggregation and directed migration. However, the mechanics of how molecules involved in static adhesion events can also participate in directed migration has been unknown. Neutrophil aggregation requires only the constitutively expressed Mac-1, and Mac-1 mobilized from intracellular pools is not utilized (Buyon et al., 1988), yet the vast majority of the neutrophil Mac1 molecules reside within intracellular granules. Recently, elegant studies by Smith and colleagues (Hughes et al., 1992) indicated that exposure of neutrophils to minute stepwise increases in chemoattractants results in corresponding small stepwise increases in Mac-1 surface expression from mobilized pools. Importantly, each wave of newly recruited Mac-1 is localized to the leading edge of the migrating neutrophil. As the neutrophil migrates in response to small increases in chemoattractant levels, the wave of Mac-1 continues down the surface of the neutrophil until it reaches the trailing uropod and a new wave of Mac-1 is recruited to the leading edge. Migration continues only if there is another small increase in chemoattractant levels. Although it is widely accepted that CD18 integrins are necessary for neutrophil transendothelial migration (Smith et al., 1988, 1989b, 1991; Luscinskas et al., 1989, 1991; Hakkert et al., 1991; Furie et al., 1992), it is not clear whether other adhesion molecules contribute to this process. Antibodies against CD18, ICAM-1, L-selectin, and E-selectin all reduce the total number of neutrophils which can attach to the surface of stimulated endothelial cells; therefore, they also reduce the total number of cells that undergo transendothelial migration. However, Smith and colleagues distinguished inhibition of attachment from inhibition of transendothelial migration by showing that neutrophils that are adherent in the presence of anti-L- or anti-E-selectin MAbs are not affected in their ability to migrate across the endothelial monolayer (Smith et al., 1991; Kishimoto et al., 1991). Similar results with anti-E-selectin are reported by other groups studying neutrophil migration across cytokine-stimulated endothelium (Hakkert et al., 1991) and across unstimulated endothelium in response to an applied chemotactic gradient (Furie et al., 1992). In contrast, Luscinskas and colleagues found profound inhibition of transmigration with anti-E-selectin (Luscinskas et al., 1991) and, to a lesser extent, with

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anti-L-selectinMAbs (Spertini et al., 1991~).The basis for this discrepancy is not clear, but may ultimately reflect differences in the culture and assay systems.

V. Therapeutic Potential of Adhesion Molecule Antagonists

Despite the great wealth of information gained from elegant studies on the molecular basis of cell adhesion interactions, the ultimate test of concepts and models generated from in uitro studies is physiological relevance in uiuo.The earliest in uiuo studies with a specific adhesion molecule antagonist demonstrated the role of the L-selectin MAb in tissue-specific homing of lymphocytes to peripheral lymph nodes (Gallatin et al., 1983; Butcher, 1986). The therapeutic potential for adhesion molecule antagonists was largely unexplored until the discovery that LAD patients were deficient in expression of the CD18 integrins. The lesson learned from the study of LAD patients-that CD18 integrins are part of the critical path for neutrophil recruitment-led to the speculation that CD18 antagonists may be therapeutically useful for a broad range of inflammatory disease states in which neutrophils wreak havoc on healthy tissues. More recently, patients deficient in all fucose-containing carbohydrates, including carbohydrate components of selectin ligands, have been identified (Etzioni et al., 1992).These patients also suffer from severe and recurrent infections, and in uitru studies indicate defective selectin-dependent functions. Since the two-step adhesion model predicts that selectins and integrins work in series, rather than in parallel, these results suggest that antagonists of either selectin- or integrin-mediated adhesion may be therapeutically useful in inflammatory diseases (Fig. 7). This concept has been tested in a variety of clinically relevant animal models of inflammatory diseases (reviewed by Harlan et al., 1992). Only a brief overview of some of the progress made in this area is presented here.

A. In Vivo Neutrophil Recruitment The first in uiuo study, by Arfors et al. (1987), demonstrated that antiCD18 MAbs could block neutrophil emigration to sites of inflammation. These investigators showed by intravital microscopy that exogenous chemotactic factor-induced leukocyte recruitment to microvessels could be blocked by anti-CD18 MAbs. Subsequent studies demonstrated that antiCD18 MAbs reduced neutrophil emigration to inflammatory sites in the skin (Arfors et al., 1987; Lindbom et al., 1990; Nourshargh and Williams,

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Selectin-dependent Rolling

Initial binding

Transition b CDl8fiCAM-dependent Activation

Adhesion strengthening Transmigration Aggregation

Anti-L-Selectin MAb

Anti-CD 18 MAb

Fig. 7 Therapeutic potential of antiadhesion molecules. The two-step adhesion model predicts that selectins and CD18/ICAM-l work in series, rather than in parallel. Thus, antagonists of either pathway may be therapeutically beneficial in a variety of diseases involving neutrophil-mediated tissue and vascular damage. For example, anti-L-selectin MAbs are predicted to block initial binding events, including neutrophil rolling, thus preventing exposure to chemotactic factors and subsequent transendothelial migration. In contrast, anti-CD18 MAbs may allow initial events to occur, but result in the eventual detachment of neutrophils, thus blocking recruitment.

1990; Price et al., 1987),peritoneum (Mileski et al., 1990),lung (Doerschuk et al., 1990; Horgan et al., 1990; Barton et al., 1989), heart (Winquist et al., 1990; Dreyer et al., 1991), intestine (Kurtel et al., 1992), synovium (Jasin et al., 1992), and central nervous system (Tuomanen et al., 1989; Clark et al., 1991a). Of the three integrins, Mac-1 appears to be of central

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importance for neutrophil recruitment (Rosen and Gordon, 1987; Rosen et al., 1989b; Jutila et al., 1989; Simpson et al., 1988). There are circumstances when neutrophil recruitment is not affected by anti-CD18 MAbs. Doerschuk et al. (1990)demonstrated that neutrophil recruitment in response to Streptococcus pneumoniae, but not to Escherichia coli, in the lung is CD18 independent. Curiously, neutrophil response to S. pneumoniae in the abdominal wall is CD18 dependent. Thus, both the stimulus and the anatomical site may contribute to differences in adhesion molecule usage in neutrophil recruitment. Since LFA-1 and Mac-1 both interact with multiple ligands, it is possible that more than one ligand is utilized in neutrophil recruitment. However, ICAM-1, as an inducible ligand for both LFA-1 and Mac-1, has all the desired characteristics of an inflammation-specific endothelial adhesion molecule. One anti-ICAM-1 MAb, R6.5,is unique in blocking ICAM-1 binding to both LFA-1 and Mac-1 (Smith et al., 1989b; Diamond et al., 1991). Anti-ICAM- I inhibits neutrophil emigration into the inflamed lung (Barton et al., 1989), kidney (Cosimi er al., 1990), central nervous system (Clark et al., 1991a), and eosinophil emigration into the lung following antigen challenge (Wegner et al., 1990). In uiuo evaluation of anti-selectin MAbs is more limited, although this situation is changing rapidly. Early studies demonstrated that the anti-Lselectin MAb, MEL- 14, blocks neutrophil recruitment to the inflamed skin (Lewinsohn er at., 1987) and peritoneum (Jutila et a / . , 1989). Jutila er al. (1989) demonstrated that the anti-L-selectin MAb was as effective as an anti-Mac-] MAb. More recently, Watson et al. (1991) utilized an L-selectin-Ig chimeric molecule to block neutrophil emigration into the inflamed peritoneum. Blood levels of this chimeric molecule as low as 3 pg/ml were effective in reducing neutrophil emigration. anti-E-selectin MAbs have been shown to reduce neutrophil emigration into the inflamed peritoneum and lung (Gundel et al., 1991; Mulligan et al., 1991).

B. Animal Models of Leukocyte-Mediated Tissue and Vascular Damage Neutrophil recruitment to sites of tissue injury is a vital part of the normal inflammatory response. However. neutrophils are a potent destructive force capable of generating oxygen radicals, releasing proteases, and producing proinflammatory factors. A neutrophil component in vascular and tissue injury has been implicated in the pathogenesis of a wide variety of inflammatory diseases, including ischemia-reperfusion injury, adult respiratory distress syndrome, rheumatoid arthritis, and inflammatory skin disease (Rotrosen and Gallin, 1987; Harlan, 1985). A misdirected neutro-

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phi1 can cause extensive damage to healthy tissue and create a vicious cycle by inducing recruitment of additional waves of leukocytes. Given the crucial role of adhesion molecules in neutrophil localization, it has been speculated that blocking adhesion may disrupt the cycle of additional leukocyte recruitment.

1. Ischemia-Reperfusion Injury Ischemia-reperfusion injury encompasses a broad range of tissue and organ trauma. Prolonged ischemia causes tissue damage and eventually leads to irreversible loss of function unless the ischemic organ or limb is reoxygenated. Ironically, reperfusion can cause further damage, even though reperfusion is critical for survival of the tissue. Previous studies have shown by neutrophil depletion that neutrophils contribute significantly to the tissue damage following reperfusion of oxygen-starved tissue. Neutrophils which enter the reperfused organ become activated, perhaps in response to proinflammatory factors released by the injured tissue. The speculation is that this tissue damage due to reperfusion may be prevented if one could block neutrophil recruitment at these sites. An early study demonstrated efficacy in dogs of an anti-CDllb MAb in reducing reperfusion injury to the myocardium following occlusion of the left circumflex coronary artery (Simpson et al., 1988). Other groups have extended these studies with the anti-CD18 MAb in primate (Winquist et al., 1990), canine (Dreyer et al., 1991), and feline (Ma et al., 1991) models of myocardial reperfusion injury. Anti-ICAM-1 (Ma et al., 1992) and, most recently, anti-P-selectin (Weyrich et af., 1992; Ma et al., 1993) MAbs have also been shown to reduce myocardial damage in similar models. Thus, adhesion antagonists in conjunction with tissue plasminogen factor or streptokinase, which dissolves clots and allows reperfusion to affected areas of the heart, may provide an effective combination treatment for myocardial infarction. Anti-CD18 MAbs were further tested in hemorrhagic shock and resuscitation, a model which is clinically relevant for multiple organ failure syndrome. Rabbits treated with antLCD18 MAbs showed greatly diminished liver and gastrointestinal damage. All anti-CD18 MAb-treated animals survived the 5-day study, compared to only 29% survival among the control group (Vedder er af., 1988). Similar results were obtained in a primate model of hemorrhagic shock (Mileski er al., 1990). Significantly, anti-CD18 MAb-treated animals required substantially less fluid to sustain cardiac output. Vedder et af. (1990) provided a dramatic demonstration of the potent effects of anti-CD18 MAbs in preventing tissue reperfusion injury. A rabbit

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ear was transected, leaving only the central artery and vein. Ischemia was induced by clamping the artery for 10 hours, followed by reperfusion. AntLCD18 reduced swelling and allowed the tissue to survive. Significantly, Winn et al. (1992) have recently reported that anti-P-selectin was also effective in this model. These results indicate that targeting either the selectin-mediated adhesion events or the CD18-mediated adhesion events can be of therapeutic benefit. The acute response to transplanted organs can also be considered, in part, an ischemia-reperfusion injury. Once the organ is removed from the donor, it is subjected to ischemia until it is transplanted into the recipient, where it becomes reperfused. Anti-ICAM-1 MAbs given over a course of 12 days, starting 2 days prior to transplantation, extended survival of renal (Cosimi et al., 1990) and cardiac (Flavin et al., 1991) allografts to more than twice that of allografts in control-treated cynomolgus monkeys. Anti-ICAM- 1 also reversed acute kidney allograft rejection episodes in monkeys given suboptimal doses of cyclosporine A (Cosimi et al., 1990). However, it is important to remember that anti-CD18 and anti-ICAM-1 not only affect leukocyte trafficking but also have profound effects on leukocyte effector functions and antigen presentation. One report suggested that a combination of anti-LFA-1 and anti-ICAM-1 therapy results in long-term specific tolerance to the transplanted organ well after MAb administration was ceased. Skin grafts from the same donor major histocompatibility complex (MHC) were accepted with no further treatment, while grafts from other MHC-disparate donors were rapidly rejected. These studies are provocative but must be confirmed. The use of anti-CD18 and anti-ICAM-1 MAbs has been expanded to show efficacy in preventing reperfusion injury to the lung (Horgan et al., 1990, 1991; Bishop et al., 1992), liver (Jaeschke et al., 1990), muscle (Carden et al., 1990), intestine (Hernandez et al., 1987; Kurtel et al., 1992), and central nervous system (Clark et af., 1991a,b). Lower torso (Welbourn et al., 1992) and intestinal (Hill et al., 1992) ischemia and reperfusion not only cause local tissue injury, but also result in neutrophil sequestration and activation in the lung. Anti-CD18 protects against both the local muscle damage and the remote lung damage. Severe bum causes irreversible tissue damage, but the surroundingtissue experiences reduced blood flow and subsequent inflammation, resulting in a progression of tissue injury in this marginal zone. The reduced blood flow, perhaps due in part to neutrophil plugging of vessels, may cause ischemia-reperfusiontype injury. Anti-CD18 and anti-ICAM-1 were both effective in restoring blood flow to the marginal zone and preventing extension of tissue injury (Mileski et al., 1992; Lipsky et al., 1992). Significantly, anti-adhesion molecules showed protective effects if administered as late as 3 hours

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after the bum injury. These and other models show potential clinical indications for use of antiadhesion molecules in myocardial infarction, trauma, bum, frostbite, limb reattachment, and organ transplantation.

2. Inflammatory and Immunological Diseases Antiadhesion molecules have also been shown to be therapeutically effective in attenuating a variety of inflammatory and immunological disease models (Harlan et al., 1992). MAbs against the leukocyte integrins have been shown to be protective in animal models of endotoxic shock (Thomas et al., 1992; Jaeschke et al., 1991), intimal thickening in arteries (Kling et al., 1992),bacterial meningitis (Tuomanen et al., 1989), aspiration pneumonitis (Goldman et al., 1992)) autoimmune diabetes (Hutchings et af., 1990), oxygen toxicity (Wegner et al., 1992), and edema (Arfors et al., 1987; Lindbom et al., 1990; Kaslovsky et al., 1990; Lo et al., 1992). Jasin and colleagues have tested the effects of antLCD18 in an antigeninduced arthritis model in rabbits (Jasin et af., 1990; Lipsky et af., 1992). Intraarticular injection of a soluble antigen into a previously sensitized rabbit induces an acute form of joint inflammation which is marked by a severe neutrophil infiltrate, followed by a chronic synovitis similar to that seen in rheumatoid arthritis. Anti-CD18 MAbs given immediately before and after the intraarticular challenge inhibit the acute infiltrate. Significantly, this protection extended to the chronic phase, without requirement of additional MAbs: Leukocyte accumulation in the joint was substantially reduced 2 and 4 weeks after antigen challenge, with corresponding decreases in lining layer hyperplasia. These results indicate that blocking the recruitment and activation of neutrophils in the acute phase may prevent progression to a chronic inflammatory condition. Anti-CD18 and anti-ICAM-1 have been found to inhibit the classic Shwartzman reaction in rabbits (Argenbright and Barton, 1991, 1992). Rabbits are injected intradermally with endotoxin and 18 hours later challenged systemically with zymosan or fMLP, resulting in hemorrhaging at the intradermal injection sites. This hemorrhaging is greatly reduced in the presence of anti-CD18 or anti-ICAM-1 MAbs. These results suggest that the primary endotoxin stimulus, which can be replaced with IL-1 or TNF, induces local up-regulation of ICAM-1, causing accumulation of leukocytes. The secondary systemic challenge induces neutrophil activation, resulting in the local thrombohemorrhagic response. The role of adhesion molecules in the pathogenesis of asthma has been investigated by Wegner, Gundel, and colleagues (Wegner et al., 1990; Gundel et al., 1991, 1992). Repeated antigen inhalation challenges in primates causes chronic airway eosinophilia and hyperresponsiveness (Weg-

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ner et al., 1990). Pretreatment with anti-ICAM-1, but not anti-E-selectin, inhibits the eosinophil influx and the onset of airway inflammation and hyperresponsiveness. However, anti-ICAM- 1 did not reverse an existing condition of airway inflammation or hyperresponsiveness. These studies indicate a role of ICAM-1 in the migration of newly recruited eosinophils. In contrast, neutrophil recruitment and the late-phase airway obstruction associated with acute airway inflammation after a single antigen inhalation challenge is not affected by anti-ICAM-1, but is significantly attenuated by anti-E-selectin MAbs (Gundel et al., 1991). These results indicate distinct adhesion molecule usage in acute versus chronic airway inflammation. Mulligan and colleagues have shown CD18 (Mulligan et al., 1992a), E-selectin (Mulligan et a f . , 1991), and P-selectin (Mulligan et a f . , 1992b) involvement in a rat model of immune complex injury in the lung.

C. Safety of Antiadhesion Molecule Therapy Antiadhesion therapy is potentially a two-edged sword (Harlan et a f . , 1992). The study of the LAD patients indicated that interrupting the CD18 adhesion process may be therapeutically useful in inflammatory diseases, yet it is also important to remember that these LAD patients also suffer from severe bacterial infections. It is possible that even limited anti-CD18 therapy may cause increased risk in the susceptibility and severity of bacterial infections. It is notable that LAD patients with a moderate phenotype (3-10% of normal cell surface CD18 integrin expression) suffer less severe infections, especially past infancy, and greatly improved survival; severely deficient patients (
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D. Clinical Trials The BIRRl anti-MAb (R6.5)is in phase I clinical trials to evaluate safety and dosage requirements of BIRRl for two indications: kidney transplantation (Cosimi et al., 1992) and rheumatoid arthritis (Kavanaugh et al., 1992). The phase I renal allograft study targeted cadaver donor renal allografts into patients at high risk for delayed graft function. All 18patients tolerated the antibody well, without the obvious “first-dose” reactions observed frequently with OKT3 MAb therapy. One patient died of Aspergillus pneumonitis and another patient developed a lymphoma and died; both patients had received a course of BIRRl followed by a course of OKT3 therapy. The target dosage of BIRRl was defined as trough blood levels sufficient to inhibit the in uitro LFA- l/ICAM-l-dependent lymphocyte aggregation response (>10 pg/ml). Early dosing studies indicated that target levels of BIRRl in the blood were not achieved. Later, patients received increased doses of BIRRl. Significantly, six of the seven patients not receiving a therapeutic dose of BIRRl had delayed graft function and all seven patients suffered acute rejection episodes; in contrast, eight of 11 patients with therapeutic doses had early graft function and only three had an acute rejection episode. Many of the contralateral kidneys from these cadaver donors were transplanted into other patients at other hospitals. Three of these contralateral kidneys were lost to primary nonfunction, in contrast to no primary nonfunction in the 18 patients in this study. These results suggest the efficacy of anti-ICAM-1 in promoting early function and survival of renal allografts. This is extremely encouraging, since these recipients were selected for high-risk factors.

VI. Future Directions The role of adhesion molecules in leukocyte interactions with endothelium has become a topic of intense study. Each year our understanding of the structure, function, and regulation of adhesion molecules increases dramatically. However, each year brings the discovery of new molecules, new functions, and new puzzles. New approaches in the coming years should include the use of transgenic animal models, gene-targeted knockouts, and crystal structures of isolated receptors and receptor ligands. Since this review was completed, there have been significant advances in this rapidly growing field. The authors apologize for any omissions. It is also not surprising that numerous biotechnology and pharmaceutical companies are intensely focused on adhesion molecule research. Nonsteroidal anti-inflammatory drugs have a huge potential market. The use of existing MAbs in a variety of animal models has already shown the efficacy

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of targeting adhesion molecules to prevent the inflammatory response. The in uivo roles of many adhesion molecules, including p150,95, ICAM2, ICAM-3, and P-selectin, and largely unknown. Each adhesion molecule has a distinct profile of function, regulation, and distribution. The selectins are involved primarily in leukocyte traflic, while the CD18 integrins and ICAM-1 have broader roles in antigen presentation and leukocyte effector functions. For some indications it may be desirable to affect multiple leukocyte functions, while in other indications it may be unnecessary or detrimental. Different indications may differ as to which molecule will be the most promising target for efficacy and safety. Murine MAbs have been of invaluable use in demonstrating efficacy, yet for safety reasons in chronic indications other alternative antagonists must be developed. Human anti-mouse antibody responses neutralize efficacy of the murine MAb and pose a risk of anaphylactic shock in response to further administration of the MAb. Currently, there is an active effort to “humanize” murine MAbs by grafting the complementary determining regions of the mouse MAb onto a human Ig framework, thus reducing, but not eliminating, immunogenicity. Ultimately, novel antiadhesion drugs might include small molecule antagonists which are nonimmunogenic and highly specific. In addition to traditional random screening of large chemical libraries, it may be possible to rationally design drugs based on known ligand structures, such as small carbohydrates for selectins, or peptide-based antagonists. Finally, in addition to direct receptor antagonists, drugs which modulate adhesion molecule regulation or expression may also be effective.

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