European Journal of Clinical Investigation (1992) 22,443453

REVIEW

Cell adhesion/signalling: biology and clinical applications M. W. MAKGOBA*, A. BERNARD? & M. E. SANDERS$, *Reader/Head, Division of Molecular Endocrinology, Department of Chemical Pathology, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London, UK; ?Chef de Service, Laboratoire Central D’Immunologie Des HBpitaux De Nice, HBpital De L’Archet, Route de Saint Antoine de Ginestikre, B.P. 689-06012 Nice Cedex, France and $Director, Immunology/Rheumatology Clinical Research, Centocor, Inc., 200 Great Valley Parkway, Malvern, Pennsylvania 19355-1307, USA

Introduction

Cellular adhesion plays a central role in many biological processes [I] such as morphogenesis, cell migration, direct cell-cell co-operation with important clinical consequences involving, for instance, vascular thrombosis, inflammatory process, tumour metastasis, bacterial and parasite infections.The adhesion process is rigorously controlled and time-dependent. Adhesion molecules refer to those cell surface structures that directly play a decisive mechanical role in cell binding to its environments either to the extracellular matrix or to another cell. A central issue, when considering the events of cellular adhesion is that they are inherently linked to the events of cellular signalling. It is clear that the inherent coupling of adhesion to signalling is not only due to cell stabilization by the adhesion molecules permitting other surface molecules to bind their ligands, but that adhesion molecules per se are also directly involved in the transduction of powerful signals controlling cell activation/proliferation. Adhesion Pathways

The concept of leukocyte adhesion pathways was developed by Shaw et al. (1986) by the demonstration of two distinct molecular adhesion mechanisms utilised by cytotoxic T cell clones on antigen-negative targets [2]. This simple model has since evolved to encompass an array of other pathways as more receptor-ligand pairs become defined (Fig. I). Cellular adhesion in man, is mediated via multiple molecular pathways which involve specific intermolecular events, are made of multimolecular complexes and can display strong influences from one pathway on the affinity of Correspondence: M. W. Makgoba, Division of Molecular Endocrinology, Department of Chemical Pathology, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 ONN. UK.

another, and are likely to involve many regulatory mechanisms of cell avidity. The CD2 adhesion pathway CD2 was discovered as an important adhesion molecule since it was identified as the T cell surface molecule mediating spontaneous rosettes with erythrocytes [3]. It is restricted to T and NK cells, but its major ligand, CD58 (LFA-3) is present on most cell types [4] (Table I). The CD2 pathway is involved in T-cell adhesion to most other cells. The extracellular segment of CD2 is made of 2 Ig-like domains [5]. CD2 has a long intracellular segment, that is certainly involved in the transduction of activation signals [6]. Indeed, CD2 is the prototype of molecule displaying both adhesion and signalling activities. An important feature of CD2 is that its overall surface density, degree of sialylation and shape criticaly depend on T-cell differentiation and activation. This was demonstrated by the discovery of restricted epitopes (‘CD2R’) to thymocytes and activated T-cells, that can be induced to after binding of certain CD2 mAbs (‘epitope modulation’) [7,8]. Activated T-cells display high densities of CD2, poorly sialylated and displaying epitopes not present on resting T-cells The discovery that pairs of CD2 mAbs in appropriate combinations, can trigger T-cell activation as efficiently as--or even more efficiently than-mAb against the CD3-TCR, raised the question of whether T-cells could be activated independently from antigen recognition [8,9]. Yet, at least in most cases, engagement of the CD2 pathway would complement the primary signal transduced by the CD3-TCR complex [lo]. Another striking aspect of CD2 is that it appears to be able to transduce, within T-cells, quite different activation signals according to CD2 molecular engagement: binding a single mAb on CD2 blocks T-cell activation or T-cell executive functions. This effect was attributed to a simple mechanical effect of a single CD2 443

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M. W. MAKGOBA et al.

\

T cell

Target

I

Soluble CD 54

CAM-2

\

Figure 1. Schematic, simplified model of the multiple adhesion/signalling molecules involved in T cell/target interactions. HA is hyaluronic acid, CHO is carbohydrate and ? means uncertain site or molecule of interaction.

Ab [l 11; yet evidence has emerged that a single CD2 mAb that induces a rise on CAMPlevels might induce a negative signal on T-cell proliferation activation [ 12,131. It was also observed that distinct CD2 mitogenic pairs of mAbs can induce distinct activation pathways in terms of the patterns of cytokines secreted and resulting T cell functions which are raised [ 14,151. This effect can be seen at the level of a single T cell, as shown by using a potentially bifunctional T-cell clone [ 161. This finding does not exclude that distinct subset of T-cells might also be recruited by these distinct CD2 pairs. Several arguments, including the requirement for 2 mabs to activate T-cells suggested the possibility that CD2 would bind another ligand in addition to CD58 [17]. Recent evidence has shown that CD59-a GPI linked molecule also involved in protecting cells against attack by homologous C’-binds CD2 [18,191. CD58 and CD59 molecules together, have a synergistic effect on T-cell activation, but other molecular species are involved that strongly influence the CD2 pathway, such as the E2 molecule [20], a product of the mic 2 pseudo-autosomal gene [21], and CD44 [22]. Thus, the CD2 pathway is a multimolecular pathway playing a central and subtle role in T-cell adhesion/activation, to ‘pilot’ T-cells either in an unresponsive or an activated state. Integrin-mediatedadhesion path ways

The integrin supergene family of molecules form the largest group of cell adhesion molecules in man (Table

I). They are transmembrane sialoglycoproteins consisting of a single alpha and a beta subunit noncovalently linked. They are classified into subfamilies according to the type of beta subunit [23]. There are presently 11 alpha subunits and 6 beta subunits so far characterised in terms of their primary sequences. The integrin molecules mediate adhesion by three mechanisms of interaction: cell-cell, cell-matrix and cellsoluble factor interactions. The characteristic of integrin-mediated adhesion is the absolute requirement for cations especially magnesium and temperature [2]. They are the major mediators of cell-matrix interaction and cell migration out of the vascular compartment. Homologues of human integrins are conserved in evolution to the level of Drosophila and amoeba [24,25, Adams et al. 1992, submitted]. An emerging feature of integrin interaction is the multiplicity of ligands recognised by a single receptor e.g. VLA-4 binding to VCAM-1, fibronectin and ICAM-2 [26,27] or the binding of LFA-1 to ICAM-1,2 [26] and the ability of certain alpha subunits to complex with several different beta subunits. The classic example is the alpha-v subunit combining with either beta-3, beta5 or beta-6 [28,29]. This multiplicity of interaction allows for diversity and specificity within this recognition system. Of great clinical interest is the observation that some of the ligands for integrins may occur in soluble forms in the circulation such as ICAM-1 [27,30]. Whether these soluble forms are distinct from the membrane-bound forms or are the products of cell shedding, enzymatic cleavage or death; or whether they play any regulatory role in adhesion and/or signalling process remains to be determined. Their

a b c

Table 1. Molecules and pathways involved in adhesionisignalling Other name

Family

TI I Sheep erythrocyte receptor LFA-2

Tissue expression

Ligand/s

Oth

T and NK cells

CD58 CD59

LF ME

ELAM- 1

Selectin

Endothelial cells

Sialylated Lewis X

GMP-I40

Selectin

Endothelial and platelets

Lewis x

Selectin

Lymphocytes Activated T cells Activated T cells Lymphocytes Lymphocytes Lymphocytes

Unknown Laminin collagen Collagen/laminin Laminin/fibronectin VCAM-1, ICAM-2, Fibronectin Fibronectin Laminin

All leukocytes Monocytes/nutrophils Neutrophils/T cell clones

CDS4, ICAM-2, Others CD54, 3Cbi, Fibrinogen Unknown

Platelets

Von Willebrand's Vitronectin Fibrinogen

Ubiquitous

Hyaluronic acid

LECAM-1 VLAal or VLA-I VLAa2 or VLA-2 VLAa3 or VLA-3 VLAa4 or VLA-4 VLAaS or VLA-5 VLAa6 or VLA-6

/31 Integrin Integrin /31 Integrin /31 Integrin /31 Integrin /31 Integrin fll

LFA- 1 MaC-I p150/95

/32

gpl Ib/l1 la

/33

HERMES PgP- 1

Cartilage link protein

Integrin Integrin 8 2 Integrin

/32

Integrin

IC IC -

446

M. W. MAKGOBA et al.

greatest impact is likely to come from their diagnostic and predictive potential in inflammatory and neoplastic diseases [27,30]. The expression of some integrins is known to be regulated during development. It is generally postulated that temporal expression of some of these molecules during development may be necessary for organogenesis as homologous structures in Drosophila have been shown to be critical to this process [24]. Some integrins such as the leukocyte integrins CDl8/ CDlla-c (LFA-I, Mac-1 and p150/95), the platelet and megakaryocyte integrin gpl lb/l 1 la and the epithelial and epithelial tumour specific integrin alpha-6/ beta4 are expressed in a tissue specific pattern. A number of growth factors such as T G F beta may affect integrin expression [311. Under normal physiological state integrins largely exist in an inactive form. Cell activation by a number of substances such as PMA, LPS, tissue injury or inflammation leads to rapid activation of integrin binding. The ability to exist and rapidly change from an inactive, nonadherent state to an active and adherent/signalling state as a result of some stimulus is a feature of integrin adhesion. The mechanisms underlying this change are poorly understood, but are of immense interest in understanding the regulation of integrin-mediated adhesion/signalling. The simplest explanation would be that the inactive forms exist to facilitate blood flow as adherent, aggregated cells would be unsuitable for the circulation. Most integrin receptors recognise a tripeptide sequence RGD on their respective ligands with the exception of CD18/CD1 la (LFA-1) [32,26]. The precise mechanism of how this simple tripeptide is utilised by so many receptors for different adhesion and signalling processes is not known. Temporal and tissue specific expression, inducible expression, the ability to exist as either active or inactive moieties are all mechanisms by which integrin-mediated function is regulated. The leukocyte integrins. These are also known as the beta-2 integrins or CDl8/CD 11a-c. They are the main mediators of leukocyte diapedesis and homing by binding to high endothelial venules. CD18/CDl l a shows differential expression between naive and memory T lymphocytes [33]. CD18/CDlla is found mainly on lymphocyte, CD18/CDll b predominantly on monocytes and neutrophils and CD 18/CD1lc mainly on neutrophils. CD18/CDl l a binds to the molecules ICAM-1 (CD54), ICAM-2 [26] and possibly other undefined molecules. CD18/CDl l b binds to CD54, iC3B and fibrinogen [26]. The cellular ligand for pl50/95 remains undefined. The ligand ICAM- 1 has become a very interesting adhesion molecule: it is a highly inducible molecule by various cytokines, associates with the IL2 receptor, correlates with T-cell infiltrations into tumours and auto-immune thyroiditis [34], and exists in several forms in the circulation [27]. It is a receptor for the rhinoviruses and falciparum malaria and that soluble ICAM-1 inhibits rhinovirus

infection [34,26]. The diversity of interaction found in the integrin molecules is also found in their ligands. The VLA beta-I. This family of molecules is the major mediator of cell-extracellular matrix interactions and homing. T and B lymphocytes express different types of VLA molecules. B cells express large amounts of VLA-4, low amounts of VLA-2 and 3. They do not express VLA-1,5 and 6. T-cells on the contrary express a combination of VLA molecules depending on activation or differentiation state of the cells. Resting unactivated human T-cells express VLA3, 4, 5 and 6. They do not express VLA-1 or 2 [35]. Following activation the level of expressed VLA-3, 4 and 5 increases, that of VLA-6 decreases and there is induced expression of VLA-1 and 2 (36). VLA-4 binds to fibronectin, laminin and VCAM. VLA-6 binds to laminin on platelets and lymphocytes to mediate adhesion. Platelet gpl l b / l l la, a beta-a3 integrin, binds fibronectin and vitronectin to mediate adhesion. This receptor also interacts with soluble factors such as fibrinogen. The integrin alpha-v/beta-3 found on osteoclast binds to an RGD sequence found on osteopontin [37]. This mediates the adhesion of osteoclasts (the effector cell in osteoporosis) to bone. Selectin mediated adhesion pathways

Like integrins, selectins form a family of molecules which are structurally related and designed to mediate adhesion (see Table I): they have an NH2 terminal mammalian lectin-like domain and they are issued from evolutionary highly related genes [38]. They also display common important functional features: they mediate adhesion of blood cells to vascular endothelium in privileged sites and their expression is strongly modulated upon cell activation. ELAM-1, a selectin induced on endothelial cells from post-capillary venules upon inflammation by IL-1, T N F and bacterial LPS [39] mediates the adhesion of neutrophils and monocytes by binding the sialyl-Lewis X determinant (a sialylated, fucosylated polyactosamines) [26]. ELAM-1 also mediates the adhesion of memory T-cells, but not virgin T cells, to ‘activated’ endothelial cells [40]. These observations on privileged expression of an adhesion pathway on defined tissues and T cell subsets were emphasized as an example of site specific migration [41]. The LECAM-I, or LAM-I molecule. This was first described in mice as the MEL-14 Ag, allowing the homing of lymphocytes to lymph nodes by mediating their adhesion to high endothelial venule cells. LAM-1 binding to its ligand is specially inhibited by polysaccharides rich in mannose 6-phosphate, such as polyphosphomonoester core polysaccharide derived from the yeast Hansenula (PPME) [42]. The affinity for PPME is increased when lymphocytes or neutrophils are activated [43]. Yet, LAM- 1 expression disappears after lymphocytes have been activated and are no

CELL ADHESION SIGNALLING longer present on most memory T-cells [44]. However, LAM-1 is present on cells that d o not normally migrate into peripheral lymph nodes, namely haemapoietic progenitors suggesting that its role encompasses homing of lymphocytes/monocytes into the periphery [45]. CD62, also called PADGEM or GMP-140 Ag. This is an inducible granule membrane protein of platelets and endothelial cells [46,47]. In resting platelets it is located within the alpha granules and is translocated on to the surface upon activation induced by thrombin, histamine and the C5b-9 complex. In endothelial cells it is present in Weibel-Palade bodies and mobilized to their plasma membrane upon their activation. CD62 mediates adhesion of myelomonocytes to platelets or endothelial cells. CD62 binds to CD15 (Fucosyl N-acetyllactosamine, Lewis X) [48] and also to sulfatides (heterogeneous 3-sulfated galactosyl ceramides). CD44 mediated adhesion The CD44 molecule. This has a wide tissue distribution. It is a single sialoglycoprotein of 80-90 kDa. The higher molecular forms are expressed differentially on cells [22]. Unlike the integrins, selectins or the immunoglobulin-like adhesion receptors CD44 have homology with chick and rat cartilage link proteins. Differential expression in mouse lymphocytes distinguishes the naive from memory T lymphocyte subsets [49]. CD44 mAbs have been shown to inhibit T cell rosetting and B cell adherence to stroma. Also CD44 mAbs have been shown to enhance T cell proliferative responses. The ability of some CD44 mAbs to inhibit lymphocyte adhesion to high endothelial venules has suggested the role of this molecule in the migration and homing of lymphoid cells from the vascular compartment into the lymph nodes [50]. The above studies have shown the role of CD44 in adhesion interactions and signalling. Recent studies have shown that the ligand for CD44 is hyaluronic acid [51]. This binding explains some, but not all of the CD44 interactions suggesting that other ligands may be involved. Types of potential anti-adhesion therapies

The recent molecular characterization of adhesion pathways has led to exploration of their role in the pathogenesis of a variety of diseases and has opened the door for the development of a new class of pharmaceutics which would inhibit adhesion. In order for an anti-adhesion therapy to be clinically useful, it must have several characteristics including: the ability to inhibit an adhesion interaction that is of sufficiently critical importance to the target disease pathogenesis that its disruption will lead to clinical benefit; sufficient specificity of anti-adhesion effect to permit other critical adhesion processes to continue, giving the therapy a suitable margin of safety; pharmacokinetic and pharmacodynamic characteristics which permit a

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convenient dosing schedule suitable for the target disease; and, drug delivery characteristics suitable for devising a convenient and effective dosing route. Therapies to be used for acute disease indications can be appropriately administered via intravenous infusion, and therefore are less restricted by considerations of route and schedule of administration. A greater challenge is presented by the development of antiadhesion therapies for chronic diseases, since these therapies must have characteristics permitting their administration over long periods of time by convenient dosing routes and schedules. Research in the development of anti-adhesion therapies has identified at least four types of potentially adhesion-inhibiting molecules which may demonstrate utility in the treatment of human diseases (Table 2). Each of these types of potential therapies has different advantages and disadvantages. Monoclonal antibodies are currently the best characterized of the various types of potential anti-adhesion therapies. Their advantages include the potential for a relatively long pharmacokinetic half-life and a prolonged biologic effect due to their binding with high affinity to effector cells, such as leukocytes or platelets, or to endothelial cell adhesion receptors. Their disadvantages include a high potential for evoking an immune response to themselves, as well as their requirement for parenteral administration. It is likely that the treatment of chronic diseases with whole monoclonal antibodies specific for adhesion receptors will prove to be non-feasible due to these restrictions on chronic administration and route of administration. However, immunogenicity may be decreased by chimerization or humanization of murine monoclonal antibodies, so that they consist of predominantly human protein sequences, and through the use of antibody fragments containing the antigen-binding region. Another class of potential anti-adhesion therapies are soluble adhesion receptor proteins, such as soluble ICAM-1, or soluble GMP-140 [27,30,52]. The occurrence of soluble ICAM- 1 in vivo is well established, and it is likely that soluble ICAM-1 and GMP-140 function in vivo as natural inhibitors of adhesion events. A potential advantage of soluble adhesion receptor molecules as therapies is that they are unlikely to be highly immunogenic, making them suitable for the treatment of chronic diseases. However, the molecular weight of soluble adhesion receptors will likely require that they be parentally administered. The frequency with which a soluble adhesion receptor would have to be administered would be dependent upon its in vivo pharmacokinetic and pharmacodynamic characteristics. If the soluble receptor has sufficient affinity for its ligand, and if it can be formulated for slow-release subcutaneous administration, it is likely that intermittent administration which would be acceptable to patients with chronic diseases would be possible. Small peptides which inhibit adhesion interactions are a third type of potential anti-adhesion therapy [53,54]. Such peptides may be based upon the known

448

M. W. MAKGOBA et al. Table 2. Potential types of anti-adhesion therapies Type of Therapy

References

Monoclonal antibodies Soluble adhesion receptors Peptides based on adhesion receptors Adhesion recognition carbohydrates

60,61,63,66,67,73-76,78-81,83-86 27, 30, 52,68 53-55 56.57

sequences of adhesion molecules, or may be based upon aminoacid sequences obtained from the complementarity determining regions of monoclonal antibodies which bind to adhesion molecules [55]. The advantages of small peptides are that they are unlikely to be immunogenic, and have the potential for oral delivery. Small peptide adhesion inhibitors may be useful for the treatment of chronic diseases, particularly if oral delivery systems, such as liposome formulations, can be developed which will protect them from digestion and permit absorption from the gastrointestinal system. A fourth class of potential anti-adhesion therapy may be based on complex carbohydrates which are critical for certain adhesion recognition events. For instance, sialyl-Lewis X, a complex carbohydrate found on the neutrophil selectin molecule LECAM-1, is critical for adhesion of neutrophils to ELAM-1 and GMP-140 [56,57]. Small molecular weight synthetic carbohydrates which could inhibit the naturally occuring carbohydrate-dependent adhesion interactions, may be suitable for the treatment of chronic diseases since they are unlikely to be immunogenic. However, they must have affinity and specificity to inhibit the naturally occurring carbohydrate recognition events. In addition, an ideal carbohydrate based anti-adhesion therapy would be orally available, resistant to digestion in the gastrointestinal tract, resistant to rapid metabolism in circulation, and have pharmacokinetics which permit a therapeutic concentration of the molecule to be maintained with a convenient dose and schedule. Animal models of anti-adhesion therapy

In recent years a large amount of research has demonstrated the potential utility of anti-adhesion therapies through in vitro investigation, and in vivo studies using various animal models of human diseases. A representative, though incomplete list of animal studies is contained in Table 3. It should not be surprising that anti-adhesion therapies hold potential in such a wide variety of diseases, since adhesion is critical in a variety of biologic processes including leukocyte trafficking, platelet aggregation, viral infectivity, and tumour cell metastasis. Furthermore, it is apparent that in vivo experimentation with antiadhesion therapies in animal models will experience a dramatic expansion over the next few years as additional candidate therapeutic molecules directed at an

Table 3. Animal models of diseases in which anti-adhesion therapies have demonstrated efficacy Condition

Species

Therapy

Reference

Asthma

Rat Rabbit

Anti-ICAM- I Anti-ICAM-1 AntLCD18 Anti-ICAM-1 Anti-ELAM-I

60 61 61 62 63

Monkey Arthritis

Rat Rabbit

Anti-ICAM-l AntLCD18

67 66

Cerebral malaria

Mouse

CDlla

73.74

Meningitis

Rabbit

AntLCD18

75,76

Myocardial ischaemia

Cat Dog

Anti-CDl8 Anti-CD1 lb

79 78

Shock

Rabbit

Anti-CD18

80

Spinal cord ischaemia

Rabbit

Anti-ICAM-1

81

Thrombosis

Dog

Anti-GPIIb/llla

83, 84

Transplant rejection

Monkey

Anti-ICAM-1

85,86

Tumour metastasis

Mice

RGD peptides

54

increasing number of potential molecular targets reach the stage of preclinical evaluation. The following overview will outline the current state of research in anti-adhesion therapies in a variety of disease categories. Asthma

Asthma is a chronic inflammatory disease characterized by the accumulation of neutrophils and eosinophils in the bronchial airways. The participation of ICAM-1 and ELAM-1 in the adhesion of neutrophils and eosinophils to endothelial cells stimulated with inflammatory cytokines has been established in virro, and the expression of these two adhesion molecules has been shown to be increased in biopsies of late phase allergic cutaneous reactions in humans [58,59]. The combination of ELAM- 1 monoclonal antibody plus either ICAM-1 or CD18 monoclonal antibodies com-

CELL ADHESION SIGNALLING pletely inhibits adhesion of neutrophils and eosinophils to activated endothelium, indicating that the adhesion mediated by the combination of these two receptors mediates most or all of the strong adhesion by neutrophils and eosinophils to activated endothelium [58]. These findings, however, do not exclude the potential participation of other adhesion molecules such as GMP-I40 or VCAM-I in the process of neutrophil or eosinophil accumulation in inflamed airways, perhaps by serving as ligands for initial weak adhesion of neutrophils and eosinophils under flow conditions. Treatment of a rat model of airways inflammation, induced by immune complexes, with anti-ELAM-1 led to a marked diminution of recruitment of neutrophils and vascular injury [60]. In a rabbit model of lung inflammation induced by administration of phorbol ester, administration of ICAM-1 or CD18 antibodies also led to a marked diminution of accumulation of neutrophils in the lungs [61]. These two animal models demonstrate the importance of both the ELAM-1 and ICAM-I adhesion ligands in the recruitment of neutrophils in non-specific models of lung inflammation. Similar findings have been obtained in primate models of antigen-specific airways inflammation. In a monkey model of airways hyperactivity, induced by intratracheal aerosolization of ascaris antigen to ascaris-sensitized animals, administration of anti-ICAM- 1 resulted in a decrease of infiltration of the eosinophils into the airways as assessed by bronchoalveolar lavage, and improvement in airways responsiveness as assessed by methacholine challenge [62]. However, administration of ICAM-I antibody did not inhibit the acute influx of neutrophils into the airways of ascarissensitive monkeys induced to have a late phase response in their airways following inhalation of ascaris antigens. Administration of monoclonal antibody to ELAM- 1 did block the influx of neutrophils in this model, and improved airway obstruction (63). The sum of these data indicate that both ICAM-1 and ELAM- 1 are critical to the influx of inflammatory cells in airways inflammation, and suggest that a combination of therapies which inhibit the adhesion of neutrophils and eosinophils mediated by these adhesion receptors would be beneficial in the treatment of human asthma. Arthritis

Infiltrates containing granulocytes and mononuclear leukocytes are a hallmark of synovial histology in various forms of inflammatory arthritis. Presumably, leukocytes in the synovium first adhere to and diapedese through the vascular endothelium, and are then retained in the synovial tissue by adhesion to synovial fibroblasts. A number of adhesion molecules are likely to be involved in this process. It has been reported that VLA4/VCAM- 1 -mediated adhesion is critical for the adhesion of T-lymphocytes to rheumatoid synovial endothelial cells, while ICAM- 1, and CD 11a/CD 18

449

interactions are less critical for T-cell adhesion to synovial endothelium [64]. In contrast, ICAM-1 and CDl 1a/CD18 interactions play a prominent role in the adhesion of T-cells to rheumatoid synovial fibroblast, although other adhesion receptors are involved in this process, since inhibition of T-cell adhesion by antibodies to these adhesion molecules is incomplete [65]. Treatment of a rabbit model of chronic antigeninduced arthritis with monoclonal antibody to C D 18 resulted in decreased numbers of inflammatory cells in synovial fluid, and improvement in synovial histology compared to control animals [66]. In a rat model of adjuvant-induced arthritis, administration of ICAM- 1 antibody resulted in a marked decrease in paw swelling, reduction of acute phase reactants, and improvement of radiographic appearance [67]. Thus, the use of anti-adhesion therapies for the treatment of human inflammatory arthritides such as rheumatoid arthritis may be of therapeutic utility, provided non-immunogenic adhesion-blocking therapeutics suitable for chronic administration can be developed. Infectious Diseases

Adhesion molecules may play a critical role in the pathogenesis of a variety of infectious diseases, either by serving as receptor molecules for the adherence or infectivity of infectious agents, or by their role in the recruitment of inflammatory cells, which may in turn contribute to tissue injury through the release of inflammatory mediators. ICAM- 1 has been shown to be a receptor for rhinovirus [68]. Soluble ICAM-1 molecule has been shown to inhibit rhinovirus infection of cultured human epithelial cells. CD18 and ICAM- 1 can participate in the formation of syncytium by T-cells infected with the human immunodeficiency virus [69,70] In addition, CD18 and ICAM-1 expression are increased on HIV-infected T-cells. This may augment their ability to disseminate into tissue and potentially increase their interaction with other CDCexpressing leukocytes, increasing the potential for infection of neighbouring cells [7 I]. To date, in uiuo studies with adhesion-inhibiting therapies in viral diseases have not been published, although in uitro data indicate this approach may be of utility for some viral diseases. ICAM-1 and CD36 have been shown to be receptors for cytoadherence of erythrocytes infected with malaria organism, plasmodium falciparum [72]. Adherence of plasmodium-infected erythrocytes to vascular endothelium within the brain is thought to be critical to the immunopathogenesis of the frequently fatal central nervous system manifestations of this type of malaria. In uitro studies with anti-ICAM- 1 antibody and soluble ICAM-1 have shown that they fail to inhibit the death of mice infected with malarial organisms [73,74]. However, in these models, treatment with antibody to C D l l a was effective at preventing mortality, even when administered late in the course of infection. The effect of anti-CDIla is

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thought to be due to inhibition of mononuclear leukocyte adhesion in the cerebral vasculature. Adhesion molecules are critical for the accumulation of inflammatory leukocytes at sites of bacterial infection. In some bacterial infections, such as meningitis, the accumulation of leukocytes contributes to irreversible tissue injury. Studies with anti-CD 18 monoclonal antibody treatment in rabbit models of gram positive and gram negative meningitis have shown a reduction of inflammatory cell infiltration into the cerebrospinal fluid, and decreased brain edema [75,76]. In the studies of gram positive meningitis, an effect on mortality was also observed. A critical question in the use of anti-adhesion therapies for bacterial sepsis is whether mortality would be increased due to the inability of an infected host to mount an adequate defence against the bacterial infection. In a study of abdominal sepsis in rabbits treated with antLCD18 antibody and antibiotics, antibody-treated animals demonstrated no difference in infectious complications, or mortality compared to control animals treated with antibiotics alone [77]. Thus, anti-adhesion therapy may be of use in some forms of bacterial infections in humans, such as meningitis, or sepsis-associated acute respiratory distress syndrome, in which inflammatory cell accumulation in tissues is thought to contribute to tissue injury.

coronary thrombosis have shown that it is highly effective in preventing reocclusion by thrombosis following thrombolytic therapy [83,84]. The ability of F(ab’)2 fragments specific for gpl l b / l l l a to inhibit thrombosis in viuo indicates their potential utility for the treatment of human diseases involving thrombosis, such as myocardial infarction, unstable angina, pulmonary embolism, transient central nervous system ischaemic attacks, and stroke in evolution. Transplant rejection

Infiltration of transplanted organs by T-lymphocytes is critical to the pathogenesis of chronic transplant rejection. Studies with anti-ICAM-1 antibody in monkeys have shown that it inhibits rejection of renal and cardiac allografts [85,86]. Biopsies of transplanted kidneys following prophylactic administration of antiICAM-1 antibody showed decreased T-cell infiltration and decreased vascular inflammation. Biopsies of rejecting transplanted kidneys, from monkeys treated with anti-ICAM-1 antibody following onset of rejection, did not show a decrease in cellular infiltration, although there was a decrease in tissue oedema and haemorrhage, and a functional reversal of rejection in the majority of animals. Thus, treatment with adhesion-blocking therapies may be useful both in a preventative and therapeutic mode for human transplant rejection.

Ischaemic diseases

The accumulation of neutrophils in tissues following ischaemia and reperfusion is thought to be critical to the process of ischemic tissue injury. CDI l b monoclonal antibody has shown the ability to decrease myocardial necrosis in a dog model of myocardial ischaemia and reperfusion [78]. Similarly, CDI 8 monoclonal antibody has shown the ability to decrease myocardial necrosis in a cat model of myocardial ischaemia and reperfusion [79]. Ischaemic injury in a cat model of hemorrhagic shock and resuscitation was decreased in the lungs, liver and gastrointestinal mucosa of CDI 8 antibody-treated animals compared to control animals. In addition, mortality, metabolic acidosis, and gastrointestinal bleeding was decreased in the CD18 antibody-treated animals [80]. Ischaemic injury to the spinal cord was reduced by administration of anti-ICAM-l antibody to rabbits, although anti-ICAM-l failed to inhibit ischaemic injury in a rabbit model of brain ischaemia [81]. These models indicate the potential utility of adhesion-blocking therapies for human diseases such as myocardial infarction, traumatic shock, and spinal cord injury. Thrombotic diseases

Aggregation of platelets is critical in the process of thrombosis, and is mediated by the adhesion receptor gpl 1 b/l 1 l a [82]. Studies using F(ab)2 fragments of monoclonal antibody to gp 1 1b/ 1 1 1a in dog models of

Tumour metastasis

Adhesion molecules may contribute to tumour metastasis, either through upregulation of their expression on malignant cells, thus enhancing their ability to invade tissues; or through downregulation of expression on malignant cells, leading to protection of the malignant cells from destruction by cells involved in immune surveillance. Decreased expression of cell adhesion molecules appears to be an early event in the development of colorectal tumours, and overexpression of cell adhesion molecules correlates with a high risk of metastasis of colorectal tumours [87]. The depth of cutaneous invasion, and thus, the risk of metastasis of malignant melanoma correlates with the expression of ICAM-1 on the melanoma cells, although the mechanism by which ICAM-I expression on melanoma cells could contribute to metastasis is undefined [881. Metastasis of murine melanoma cells in an in uivo model is inhibitable by adhesion-blocking peptides containing the Arg-Gly-Asp-Ser (RGDS) sequence. These peptides are capable of inhibiting cellular adhesion to extra-cellular matrix components, such as fibronectin [54]. It is conceivable that administration of adhesion inhibiting therapies at the time of cancer surgery could decrease the incidence of metastasis, or prolong the time to onset of clinically evident metastasis, by inhibiting the ability of micrometastatic foci of malignant cells to colonize distant tissues.

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