Osteopontin Receptor

Osteopontin Receptor Gerard J. Nau* Infectious Disease Unit, Massachusetts General Hospital, Boston, MA 02114, USA * corresponding author tel: 617-726-3812, fax: 617-726-7416, e-mail: [email protected] DOI: 10.1006/rwcy.2000.18003.

SUMMARY The two classes of osteopontin (OPN) receptors identified so far, CD44 and integrins, each have an extensive literature independent of OPN. Multiple activities have been attributed to these receptors, from development to leukocyte and lymphocyte homing and activation. Further investigation is required first to characterize better what biological activities OPN possesses, and then to determine which receptors are responsible for these activities. There are promising therapeutic agents under development for inhibiting integrin function. Some of these biological effects may be the result of inhibiting OPN activity. The opportunities for modulation of CD44 activity and for specific OPN antagonists have yet to be pursued.

BACKGROUND

Discovery Several receptors for OPN have been identified. Broadly, these include CD44 and various integrins (Uede et al., 1997). It would be impractical to summarize all of the data related to these receptor classes. For the purposes of this chapter, general references and specifics regarding the interaction of these receptors and OPN will be reviewed. CD44 was identified on the surface of human leukocytes, T cells, and thymocytes by a monoclonal antibody raised against human lymph node lymphocytes (Dalchau et al., 1980). The first indication that CD44 was relevant to OPN physiology came with the discovery of an interaction between these two molecules on cells transfected with CD44 (Weber et al., 1996).

Integrins have been studied for nearly two decades. Early work identified major cell surface antigens on several cell types, such as platelets, fibroblasts, and leukocytes, and the interaction of these receptors with extracellular matrix proteins (Phillips et al., 1980; Jennings and Phillips, 1982; Horwitz et al., 1985; Pytela et al., 1985a,b; Springer et al., 1985). Great interest in integrins was generated when the first OPN sequence became available. This sequence identified an RGD integrin-binding motif within the molecule (Oldberg et al., 1986).

Alternative names After the specific monoclonal antibody was isolated, CD44 was referred to as human leukocyte-common antigen (Dalchau et al., 1980). The receptor is also known as the polymorphic glycoprotein Pgp-1 antigen and the gp90 Hermes antigen (Goldstein et al., 1989; Stamenkovic et al., 1989). The term integrin was first proposed by Tamkun and colleagues after cloning a cDNA that encoded a protein believed to link the extracellular matrix and intracellular actin (Tamkun et al., 1986). Alternative names for this chicken integrin include 140K complex, CSAT antigen, and JG22 antigen (Hynes, 1987). The integrin appellation has been applied to the many  heterodimers that have been identified. Alternative names have been used to identify these receptors, particularly with regards to the extracellular matrix to which they are associated. For example, the v 3 integrin is known as the vitronectin receptor (Pytela et al., 1985b). Other alternative names reflect the cell from which the molecule was identified. For example, the L 2 molecule on lymphocytes was termed leukocyte function-associated (LFA-1) and the M 2 on macrophages was identified as Mac-1, Mo-1, p150,95 (Hynes, 1987). The IIb 3 structure on platelets is known as GPIIb-IIIa (Hynes, 1992).

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Structure Upon cloning the gene, Stamenkovic et al. found that CD44 is a transmembrane glycoprotein of 341 amino acids (Stamenkovic et al., 1989). Goldstein and colleagues cloned a cDNA encoding a transmembrane protein of 294 amino acids (Goldstein et al., 1989). It is now known that the protein exists in several isoforms that are generated by the selection of 10 out of 20 exons through alternative RNA splicing. These variant exons are called v1±v10. There is one standard isoform that is present on erythrocytes, leukocytes, and in the brain (Borland et al., 1998). The extracellular domain is large and is heavily glycosylated (Borland et al., 1998). Integrins are transmembrane, heterodimeric receptors composed of  and subunits. In mammals, there are 17  chains, 8 chains, and 22 distinct heterodimers that have been identified (Kumar, 1998). There is a large extracellular portion comprised of three repeats of a cysteine-rich domain in the chain. The short transmembrane domain is followed by an intracellular domain of variable length (Kumar, 1998).

Main activities and pathophysiological roles CD44 function has been the subject of much study (Borland et al., 1998). The receptor is known to be involved in lymphocyte homing, allowing binding to mucosal high endothelial venules (Duijvestijn and Hamann, 1989). Homing of prothymocytes is also dependent on CD44. CD44 binds hyaluronic acid (HA) in the extracellular matrix, fragments of HA, fibronectin, and collagen (Kincade et al., 1997). Small HA fragments, acting through CD44 (McKee et al., 1996), can induce expression of nitric oxide synthase 2 (NOS2; McKee et al., 1997). When coupled with IFN , the CD44±HA interaction can enhance the expression of some chemokines (Horton et al., 1998). CD44 has also been shown to present the chemokine MIP-1 (Tanaka et al., 1993). Finally, many tumors express altered isoforms and glycosylation of CD44 which presumably enhances metastatic potential (Borland et al., 1998). Integrins are known to mediate cell-to-cell and cellto-extracellular matrix (ECM) interactions. Many, though not all, integrins bind to ligands with an RGD motif; some show strict specificity to their ligands (Kumar, 1998). Multiple second messenger pathways are known to be activated upon integrin receptor ligation. These pathways include phospholipase activity, phosphatidylinositol turnover, increases in

intracellular calcium, and intracellular alkylanization. The cytoskeleton reorganizes after engagement of integrins and is believed to be related to cell adhesion, migration, and possibly phagocytosis (Hynes, 1987). In Glanzmann's thrombasthenia, there is a defect in platelet aggregation manifested by hemorrhagic symptoms. This is attributable to a deficiency of the IIb 3, GPIIb-IIIa integrin (George et al., 1990). Deficiencies in Mac-1, LFA-1, or p150,95, which share a common 2 subunit, lead to leukocyte adhesion deficiency syndrome characterized by granulocytosis, impaired leukocyte adhesion, and increased susceptibility to pyogenic infections (Anderson and Springer, 1987).

GENE

Accession numbers A search of the GenBank nucleic acid database for CD44 yields 221 listings. The cloning of human CD44 was published simultaneously by two groups and entered in M25078 and M24915 (Goldstein et al., 1989; Stamenkovic et al., 1989). Goldstein and colleagues used a gt11 expression library derived from a human B lymphoblastoid line that expressed high levels of gp90Hermes (Goldstein et al., 1989). Stamenkovic et al. used transient transfection of COS cells and antibody panning on dishes to select for positive clones (Stamenkovic et al., 1989). Pgp-1, the murine homolog of the human Hermes antigen, was cloned from a cDNA library derived from PU5-1.8 cells, accession number M30655 (Zhou et al., 1989) A search for integrins in GenBank yields an even larger number of entries: 2136. The first chain was cloned in 1986, accession number M14049, using a gt11 expression library constructed from chicken fibroblasts and a polyclonal antisera that recognized the chicken fibroblast 140 kDa complex (Tamkun et al., 1986). The human fibronectin receptor  chain was also cloned with a gt11 screen, accession number M13918 (Argraves et al., 1986). Particularly relevant for OPN binding, the human v chain was cloned by a similar approach, accession number M13918 (Suzuki et al., 1986).

PROTEIN

Accession numbers CD44 AAA36138 (Goldstein et al., 1989) AAA35674 (Stamenkovic et al., 1989) AAA39922 (Zhou et al., 1989)

Osteopontin Receptor 1861 Integrins AAA48926 (Tamkun et al., 1986) AAA52467 (Argraves et al., 1986) AAA52467 (Suzuki et al., 1986)

Description of protein As outlined in the background section, both of the receptor classes for OPN, i.e. CD44 and integrins, are transmembrane glycoproteins. The standard form of CD44, CD44s (Borland et al., 1998), is composed of a single polypeptide with a 248 amino acid extracellular domain, a 21 amino acid transmembrane domain, and a 72 amino acid intracellular domain. CD44 isoforms are created by alternative splicing of 10 out of 20 exons during RNA processing. CD44s is extensively glycosylated, accounting for the molecular weight difference between the predicted polypeptide mass of 40 kDa and the 80±95 kDa observed on SDSPAGE (Borland et al., 1998). These glycosylation sites include N-linked (primarily in the N-terminus cartilage link domain) and O-linked moieties. The murine CD44 is similar in structure and has six possible N-linked and six possible O-linked sites (Zhou et al., 1989). In contrast to CD44, integrins are  heterodimers. The  subunits range between 120 and 180 kDa and the chains between 90 and 110 kDa (Hynes, 1992). The chains span the cellular membrane and have a cysteine-rich motif that is repeated four times in the extracellular domain. The  chains are also transmembrane proteins, some of which are posttranslationally cleaved, yielding subunits that are linked by disulfide bonds (Hynes, 1987).

Relevant homologies and species differences CD44 is highly conserved between the murine and human sequences (Zhou et al., 1989). The 155 amino acids on the N-terminus of the receptor have 89% identity between the two species and the 105 amino acids on the C-terminus have 86% identity (Zhou et al., 1989). Interestingly, there is an area of significant divergence in the extracellular domain near the plasma membrane; this area shares only 42% identity between the species, which is likely the result of the splice variations (Zhou et al., 1989). Integrins are homologous between Drosophila and vertebrates (Hynes, 1992). Hynes has proposed that these similarities allowed for the development of multicellular organisms and that the differences

permitted the development of variety and specificity of binding (Hynes, 1992; Borland et al., 1998).

Affinity for ligand(s) CD44 is known to bind several ligands, including HA, HA fragments, fibronectin, laminin, and collagen type I (Borland et al., 1998). OPN was shown to bind CD44 transfected into a murine embryonic cell line; however, a dissociation constant was not calculated (Weber et al., 1996). Integrins have a range of ligands where some receptors have single ligands and others have multiple ligands (Hynes, 1987, 1992). Of the 20 or more heterodimers identified, the v 3 complex appears to be the most promiscuous, binding at least seven ligands (Hynes, 1992). The Kd values of the fibronectin receptor for fibronectin and laminin are 10ÿ6 and 2  10ÿ6 M, respectively (Horwitz et al., 1985). OPN binding to integrins has largely been demonstrated by the use of blocking antibodies. Table 1 summarizes the specific integrin heterodimers that are believed to bind OPN. One report identified a binding constant of 2.35  10ÿ10 M of radiolabeled OPN on P388D1 cells (Nasu et al., 1995). However, it is unclear to which integrin OPN was binding.

Cell types and tissues expressing the receptor The tissue distribution of CD44 is broad. CD44 is expressed on T lymphocytes and B lymphocytes, thymocytes, granulocytes, and dendritic cells (Dalchau et al., 1980; Weiss et al., 1997). The murine homolog is also known to be present on macrophages and 3T3 cells (Hughes and August, 1982; Hughes et al., 1983). Immunostaining of frozen tissue showed thymic medulla, white pulp in spleen, and lymph node cortex express this molecule (Dalchau et al., 1980). Integrins also have broad cell and tissue distribution. Some integrins, however, are cell-specific. For example, the IIb 3 integrin is expressed only on platelets. The 2 subfamily is found on leukocytes (e.g. L 2 (LFA-1) on lymphoid and various myeloid cells and M 2 (Mac-1) on macrophages; Hynes, 1987).

Regulation of receptor expression CD44 is constitutively expressed. Modulation of receptor expression occurs when normal resting lymphocytes are activated, resulting in a transient shift

1862 Gerard J. Nau Table 1 Integrin heterodimers that bind OPN Integrin heterodimer

Cell type

Assay

Reference

v 3

Osteosarcoma cells

Adhesion

Oldberg et al., 1986

Smooth muscle

Adhesion/migration

Liaw et al., 1994, 1995; Yue et al., 1994

Fibroblasts

Adhesion

van Dijk et al., 1993a

Platelets

Adhesion

Bennett et al., 1997

v 5

Smooth muscle

Adhesion

Liaw et al., 1995

v 1

Smooth muscle

Adhesion

Liaw et al., 1995

9 1

Melanoma lines

Adhesion

Smith et al., 1996

4 1

HL-60 promyelocytic, Ramos lymphoblastoid

Adhesion

Bayless et al., 1998

8 1

K562 line

Adhesion

Denda et al., 1998

4ÿ, 5ÿ

P388D1 cells

Adhesion, haptotaxis

Nasu et al., 1995

a

Not dependent on the presence of an RGD sequence.

from CD44s to splice variants (Ponta et al., 1998). Langerhans cells and dendritic cells increase their expression of certain isoforms after activation (Weiss et al., 1997). The regulation of integrin expression appears to be involved in development (Hynes, 1987). More important in the regulation of integrin function, however, is the concept of inside-to-out signaling (Hynes, 1992). Both the IIb 3 and the 2 subfamily integrins exhibit this mechanism of control; activation of platelets and leukocytes is required to permit integrin-mediated signaling/events. This is believed to occur through a conformational change in the integrin because extracellular activating antibodies can achieve the same end (Hynes, 1992).

Release of soluble receptors The release of soluble CD44 (sCD44) has been welldocumented and a molecular basis for this phenomenon has been identified (Yu and Toole, 1996). sCD44 from non-Hodgkin's lymphoma cells blocks cell interaction with hyaluronate and inhibits lymphocyte binding to high endothelial venules (Ristamaki et al., 1997). This suggests that sCD44 could inhibit immune responses against the tumor cells. Naturally occurring soluble integrins have not been described. A soluble recombinant molecule has been produced in which the heterodimers were joined with fos- and jun-binding domains (Eble et al., 1998).

SIGNAL TRANSDUCTION Little is known about signal transduction from CD44 (Lesley and Hyman, 1998). In contrast, much information has accumulated on the signaling pathways associated with integrins; excellent reviews are available on this topic (Howe et al., 1998; Kumar, 1998; Dedhar, 1999).

Associated or intrinsic kinases One report indicates that monoclonal antibody ligation of CD44 on T cells leads to ZAP-70 tyrosine phosphorylation (Taher et al., 1996). This was attributed to increased p56lck activity. In addition, CD44 was found to be physically associated with p56lck (Taher et al., 1996). However, CD44 lacks a CXCP sequence necessary for p56lck binding (Lesley and Hyman, 1998). An adapter molecule may be required for this signaling pathway. Crosslinking or clustering of integrins leads to the formation of focal adhesions that activate focal adhesion kinase (FAK), a Rho-GTPase phenomenon (Kumar, 1998; Dedhar, 1999). Integrin ligation appears to inhibit the serine-threonine kinase that is associated with the chain, integrin-linked kinase (ILK) (Hannigan et al., 1996). Importantly, integrin engagement that leads to FAK activation can initiate the MAP kinase cascade (Howe et al., 1998). The adapter protein Shc can associate with the  chain via

Osteopontin Receptor 1863 caveolin, which can also activate the MAPK (Howe et al., 1998). There may also be a Ras-independent mechanism for integrin activation of the MAPK (Howe et al., 1998). Regarding integrins that bind OPN, phosphatidylinositol 3-kinase (PI-3 kinase) and Src kinase, in addition to FAK, are found associated with v 3 (Hruska et al., 1995). OPN signaling increases FAK activity (Hruska et al., 1995). OPN also appears to activate Src kinase, at least in melanoma cells (Chellaiah et al., 1996). Not surprisingly, OPN treatment of cells has been found to increase tyrosine phosphorylation (Lopez et al., 1995).

Cytoplasmic signaling cascades Ligation of CD44 by HA leads to a rise in intracellular Ca2‡ in mouse T lymphoma cells (BW5147) (Bourguignon et al., 1993). Increased intracellular Ca2‡ by either HA or calcium ionophore leads to patching and capping of CD44 in these same cells (Bourguignon et al., 1993). This effect has not been demonstrated with OPN. Several papers have evaluated potential signaling cascades induced by receptor±OPN ligation. OPN binding to v 3 results in immediate signals in osteoclasts (Miyauchi et al., 1991). These include a reduction of cytosolic Ca2‡ through a calmodulindependent activation of a Ca2‡-ATPase (Miyauchi et al., 1991). Because peptides had a similar effect as whole protein, the authors argue that immobilization is not required (Miyauchi et al., 1991). OPN binding to v 3 also initiates phophatidylinositol turnover, including the production of phosphatidylinositol triphosphate (Hruska et al., 1995). This is due to increased activity of PI-3 kinase (Hruska et al., 1995). An important consequence of this kinase's activity is to inhibit apoptosis (Kumar, 1998), though this has yet to be demonstrated with OPN as the integrin ligand. Other intracellular signals generated by integrin ligation have been identified in other systems. These include tyrosine phosphorylation, cytoplasmic alkalinization, and increases in intracellular Ca2‡ (Hynes, 1992).

Transcription factors activated It is known that integrin receptor ligation activates several different transcription factors. For example, NFB is activated in the monocytic line THP-1 when the cells are cultured on fibronectin (Rosales and Juliano, 1996). Shear stress on endothelial cells triggers MAP kinase activity, culminating in the activation of AP-1, SP-1, Elk-C, and NFB (Shyy and Chien, 1997). Integrins also function as costimulatory receptors in T lymphocytes. For example, fibronectin binding to 5 1, the very late activation antigen-5 (VLA-5) receptor, induces AP-1 activity in human peripheral blood T cells (Yamada et al., 1991). T cells treated with immobilized anti-CD3 antibody and fibronectin have increased IL-2 production, IL-2 receptor expression, and proliferation compared to cells without exposure to fibronectin (Yamada et al., 1991).

Genes induced Several studies have examined the gene expression changes after integrin ligation. In one analysis, crosslinking of the fibronectin receptor increased the expression of collagenase and stromelysin (Werb et al., 1989). Granulocyte±macrophage colony-stimulating factor (GM-CSF) mRNA increases after macrophages adhere to fibronectin (Thorens et al., 1987). Monocyte adherence to fibronectin increases the expression of TNF and CSF-1 (Eierman et al., 1989). Another report demonstrated that macrophages cultured on collagen or fibronectin-coated plastic increase their expression of IL-8 (Standiford et al., 1991). Using two-dimensional gel electrophoresis and a subtractive cloning strategy, Danen et al. identified 12 novel genes whose expression is increased in human salivary gland cells cultured on fibronectin or collagen (Danen et al., 1998). Presumably, these gene expression changes are the result of signaling through integrins, though it is unknown if OPN is capable of eliciting any of these responses.

DOWNSTREAM GENE ACTIVATION

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY

To date, studies of gene activation induced by OPN± receptor interactions have not been done. Gene expression changes after receptor activation have been studied for integrins, but not for CD44.

The details of OPN's biological effects have been summarized in the OPN chapter. What follows here is an overview of some biological effects of integrins and CD44.

1864 Gerard J. Nau

Unique biological effects of activating the receptors CD44 is intimately involved in immune cell physiology. CD44 is an important receptor for lymphocyte homing to high endothelial venules (HEV) of lymphoid tissue (Duijvestijn and Hamann, 1989). Langerhans and dendritic cell homing to T cell zones of lymph nodes also relies on variant CD44 isoforms (Weiss et al., 1997). Structures similar to HEV are associated with chronic inflammation and are likely to participate in lymphocyte recruitment to these areas (Duijvestijn and Hamann, 1989). In at least one instance, CD44 was shown to immobilize a chemokine, MIP-1 , thereby augmenting adhesion in vitro (Tanaka et al., 1993). Several studies have shown that anti-CD44 monoclonal antibodies enhance T cell activation in vitro. For example, the proliferation of human peripheral blood T cells in response to antiCD2 or anti-CD3 antibodies increases when antiCD44 antibodies are included in the culture (Denning et al., 1990). In contrast, Rothman and colleagues have generated an anti-CD44 monoclonal that inhibits activation induced by anti-CD3 antibodies (Rothman et al., 1991). CD44 can mediate redirected lysis by NK cells (Sconocchia et al., 1994, 1997). CD44 ligation participates in the activation of monocytes (Webb et al., 1990), which may account for the monocyte-dependence of T cell proliferation augmented by anti-CD44 antibodies (Denning et al., 1990). Related to macrophage activation, HA fragments induce the expression of NOS2 after binding CD44 (McKee et al., 1997). Integrins have significant effects on cellular adhesion, cell viability, and cell proliferation (for example, see reviews by Howe et al., 1998, and Hynes, 1992). Endothelial cells (Meredith et al., 1993) and epithelial cells (Frisch and Francis, 1994) separated from their extracellular matrix undergo apoptosis; in this context, it has specifically been named anoikis (Frisch and Francis, 1994; Frisch and Ruoslahti, 1997). The inhibition of anoikis depends on integrin activation of phosphoinositide 3-OH kinase and activation of protein kinase B/Akt (Khwaja et al., 1997). Failure to undergo anoikis after detachment is characteristic of some tumors, particularly those with activated Src or Ras (Kumar, 1998). Integrin±ligand binding also appears to potentiate the effects of growth factors, presumably through activation of the growth factor receptor (Schneller et al., 1997; Howe et al., 1998). Schneller et al. found that v 3 integrins were associated with PDGF receptors and that v 3 ligation by vitronectin increased the mitogenic and chemotactic effects of PDGF (Schneller et al., 1997).

Antibodies that block the v 3 integrin inhibit angiogenesis induced by FGF in the chicken chorioallantoic membrane (Brooks et al., 1994). These findings are of particular interest because v 3 is a receptor for OPN. A substantial literature has accumulated regarding integrins and leukocyte function. Leukocyte recruitment to sites of inflammation relies on the interplay between selectins on endothelial cells and their respective ligands (Springer, 1994). However, the model of leukocyte rolling, tight adhesion, and transendothelial migration also involves the interaction of leukocytes with integrin ligands: intercellular adhesion molecule (ICAM), vascular cell adhesion molecule 1 (VCAM-1), and mucosal addressin cell adhesion molecule 1 (MAdCAM-1) (Springer, 1994). Moreover, there are critical intercellular interactions between leukocytes involving ICAMs and the leukointegrins of the 2 family: CD11a (L), CD11b (M), CD11c (x2), CD11d (d2), and CD18 ( 2) (Gahmberg, 1997). These interactions are important for T cell activation, cytolysis, neutrophil function, and NK cell function (Gahmberg, 1997). Divalent cations are required and specialized outside-to-in and inside-to-out activation occurs (Gahmberg, 1997; Hynes, 1992). As mentioned previously, inside-toout activation refers to enhanced ligand binding after the activation of the integrin (Hynes, 1992); chemokines play an important role in this process (Gahmberg, 1997; Springer, 1994). The 4 family of integrins are also expressed by T cells, B cells, and monocytes. An example of this group is very late antigen 4 (VLA-4), 4 1, a receptor for VCAM-1 (Springer, 1994).

Phenotypes of receptor knockouts and receptor overexpression mice In spite of the multiple functions attributed to CD44, the phenotype of the null mouse was relatively modest (Schmits et al., 1997). The null animals generated fewer hematopoietic precursors, measured as colonyforming units in blood and spleen, after systemic administration of G-CSF. T and B cell function, CTL activity, and delayed-type hypersensitivity reactions appeared unchanged. Relevant to OPN physiology, CD44-null mice had enhanced granuloma burdens after i.v. injection of heat-killed Corynebacterium parvum. Transfection of CD44 to fibroblasts from these animals increased binding to HA. Finally, CD44ÿ fibroblasts transformed with SV40 large T antigen generated large tumors in syngeneic (CD44‡) animals compared to lines derived from CD44‡ fibroblasts (Schmits et al., 1997).

Osteopontin Receptor 1865 Several studies of integrin-null animals have demonstrated profound phenotypes. Attesting to the importance of integrins in development, numerous integrin knockout strains have embryonic lethal phenotypes with morphogenic and placental defects (Yang et al., 1993, 1995; Stephens et al., 1995; Fassler and Meyer, 1995; Kreidberg et al., 1996; Bader et al., 1998). Some integrin-deficient animals do not have lethal phenotypes. These strains have demonstrated the function of integrins in adhesion and in normal tissue architecture (Dowling et al., 1996; Gardner et al., 1996; van der Neut et al., 1996). In contrast to the anti-angiogenic effects of antiantibodies, embryos lacking v chains have normal vessel growth (Bader et al., 1998). In an effort to reconcile this discrepancy, Bader and colleagues point out there may be functional redundancy among integrins during development. Alternatively, model systems where v 3 antibodies are effective at blocking angiogenesis may be overly dependent on this integrin (Bader et al., 1998). This difference, though unexplained, is not moot because anti-v 3 reagents may be used clinically (see Therapeutic utility). Several groups have generated mouse strains deficient in the leukointegrins. Studies of these animals have reiterated the importance of these integrins in acute inflammation and immune function. Neutrophils from CD11b-null mice have diminished adhesion to endothelium and, unexpectedly, a reduced rate of apoptosis induced by phagocytosis (Coxon et al., 1996). In addition, these animals have impaired mast cell development and mast cell function in a model of acute peritonitis (Rosenkranz et al., 1998). Another CD11b-null mouse shows reduced neutrophil adherence to fibrinogen-coated disks but normal neutrophil accumulation (Lu et al., 1997). A neutrophil migration defect was expected, but CD11a appears to compensate for the CD11b deficiency (Lu et al., 1997). Mice expressing a low amount of 2, the common chain of the leukointegrins, have fewer neutrophils accumulate in the peritoneum after administration of thioglycollate and delayed rejection of cardiac tissue transplants (Wilson et al., 1993). Mice deficient in ICAM-1, an important ligand for CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1), have leukocytosis with diminished T cell function in mixed lymphocyte reactions, reduced contact hypersensitivity, and a reduced sensitivity to LPS (Xu et al., 1994).

CD44 was exclusively absent from red blood cells in a patient with anemia; lymphocytes, granulocytes, and monocytes had normal levels of expression (Parsons et al., 1994). Bone marrow sampling revealed abnormal erythropoiesis with erythroblast accumulation in G1 and G2 phases of the cell cycle (Wickramasinghe et al., 1991). This implicates at least one CD44 isoform in hematopoiesis. There are two well-described clinical syndromes that are the direct result of integrin defects. The case of a boy with recurrent infections and diminished neutrophil adhesion (Crowley et al., 1980) led to similar observations in other patients (Arnaout et al., 1982; Bowen et al., 1982; Dana et al., 1984; Kohl et al., 1984). Clinically, these patients had recurrent bacterial infections, granulocytosis, aggressive periodontitis, poor wound healing and delayed umbilical cord separation. Phagocytosis and neutrophil recruitment, grossly observed as the inability to form pus, was also impaired (Anderson and Springer, 1987). This constellation of findings was labeled leukocyte adhesion deficiency syndrome. Analysis of these patients revealed the absence of some plasma membrane integrins and a defect in 2 expression, though  chains were present within the cell (Springer et al., 1984). This observation led to the understanding that coexpression of both the  and the chains are required for the surface expression of integrins (Springer et al., 1984). Ultimately, the defect was characterized on the molecular level: mutant mRNAs were identified and the protein products failed to associate with the  subunits (Kishimoto et al., 1987). The second integrin defect is manifested as Glanzmann's thrombasthenia. This hemorrhagic disorder is characterized by cutaneous purpura and mucosal hemorrhage with a prolonged bleeding time and normal platelet numbers. However, the platelets fail to aggregate and form a platelet plug (George et al., 1990). A defect was found on platelet membrane glycoproteins (Nurden and Caen, 1974), later identified to be GPIIb/IIIa (Phillips and Agin, 1977). Ultimately this was found to be the IIb 3 integrin (George et al., 1990). The absence of this integrin reduces platelet adhesion to fibrinogen, fibronectin, and von Willebrand factor (Ruggeri et al., 1982).

THERAPEUTIC UTILITY

Human abnormalities

Effect of treatment with soluble receptor domain

There has been one report of a human who was deficient in some form of CD44 (Parsons et al., 1994).

Although soluble CD44 molecules have been defined (Yu and Toole, 1996) and are capable of blocking

1866 Gerard J. Nau lymphocyte adhesion to HEV (Ristamaki et al., 1997), this has not been exploited clinically. Recombinant 3 1 dimers have also been described (Eble et al., 1998), but these have not been used therapeutically.

Effects of inhibitors (antibodies) to receptors Integrins make a critical contribution to inflammation, which makes them possible therapeutic targets. Antibodies, peptides, and peptidomimetics to block VLA-4 are being developed as anti-inflammatory drugs (Lin and Castro, 1998). A CD11b/CD18 (Mac-1) antagonist reduces inflammation in an animal model of colitis (Meenan et al., 1996). Because of the importance of the IIb 3 integrin in platelet aggregation, the therapeutic potential, and the financial possibilities, this platelet integrin has been a prime target for inhibition during acute coronary syndromes. Several glycoprotein inhibitors, namely tirofiban, lamifiban, and eptifibatide, have been studied in clinical trials with favorable results (Alexander and Harrington, 1998). Similar results of blocking platelet aggregation have been observed with antibody inhibitors and peptide inhibitors (Domanovits et al., 1998; Mousa et al., 1998). Several potential products are being developed as antagonists of v 3. A synthetic peptide antagonist inhibits bone resorption and osteoporosis in oopherectomized rats (Engleman et al., 1997). Presumably this results from disrupting osteoclast v 3 adhesion to bone OPN. A cyclic peptidomimetic antagonist of v 3 reduces restenosis after stent placement in a coronary artery (Srivatsa et al., 1997). The OPN±v 3 interaction is believed to be critical in the pathophysiology of restenosis (Hirota et al., 1993; Panda et al., 1997). A Fab product under development, abciximab, should be particularly enticing for interventional cardiologists because it inhibits both the platelet IIb 3 integrin and v 3 (Tam et al., 1998). Antibodies and a cyclic peptide antagonist of v 3 reduce neovascularization, attesting to a role for v 3 in vessel formation (Brooks et al., 1994; Drake et al., 1995; Hammes et al., 1996). Finally, the study of snake venom led to the discovery of an entirely new class of integrin antagonists, the disintegrins (Gould et al., 1990). These are short peptides that contain an RGD integrin-binding motif with an extremely high affinity for integrins (Gould et al., 1990). Further analysis revealed that these molecules are truncated from larger proteins (Paine et al., 1992). Full-length proteins contain a disintegrin

domain and a zinc metalloprotease domain, the hemorrhagin domain (Paine et al., 1992). Ultimately similar proteins that are named a disintegrin and metalloprotease (ADAM) were disovered in many species, including humans (Black and White, 1998). These are physiologically important molecules; for example, an ADAM controls the release of TNF (Black et al., 1997; Moss et al., 1997). Venom proteins are known for their inhibition of platelet function (Gould et al., 1990) and their activation of the coagulation cascade (Paine et al., 1992). The integrin-binding function is responsible for the block of platelet function (Huang et al., 1989); this integrin-binding property is being harnessed to study other cell biological and clinical questions. The disintegrin contortrostatin can bind v 3 and inhibit osteoclast attachment (Mercer et al., 1998). Echistatin also binds v 3 and inhibits M-CSFdirected cell migration and the formation of multinucleated osteoclasts (Nakamura et al., 1998). The disintegrin accutin is an v 3 antagonist that blocks angiogenesis by inducing endothelial apoptosis (Yeh et al., 1998). There are numerous reports using disintegrins to block platelet aggregation. For example, bitistatin has been used to block IIb 3, thereby preventing platelet loss during extracorporeal membrane oxygenation (Shigeta et al., 1992). Disintegrins may be useful anti-inflammatory agents (Schluesener, 1998). Thus, there is great promise for innovative applications of integrin antagonists. These applications include reducing inflammation, blocking platelet function, particularly during acute cardiac thromboses, blocking restenosis after angioplasty, and inhibiting vessel growth. The latter may be applicable to conditions such as diabetic retinopathy (Hammes et al., 1996) and neovascularization associated with cancer (Yeh et al., 1998).

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LICENSED PRODUCTS Multiple vendors of immunologic reagents have products related to CD44 and integrins. For example, Pharmingen (San Diego, CA) and Harlan Bioproducts (Indianapolis, IN) have multiple antibodies directed to these antigens. Sigma-Aldrich (St Louis, MO) sells extracellular matrix proteins, RGD-containing peptides, and several disintegrins.