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ADVANCES IN IMMUNOLOGY, VOL. 54

CD44 and Its Interaction with Extracellular Matrix

JAYNE LESLEY,, ROBERT H Y M N , * A N D PAUL W. K l N C A D e

'Department of Cancer Biology, The Salk Institute, Son Diego, California 92186, and the tlmmunobiology and Cancer Program, Oklahoma Medical Research Foundation, Oklahoma City,

Oklohoma 73104

1. Introduction

CD44 is a broadly distributed family of cell surface glycoproteins that has been studied independently by many investigators in a variety of systems and under a variety of names. It is now generally believed to be a cell adhesion molecule with proposed functions in extracellular matrix (ECM) binding, cell migration, lymphopoiesis, and lymphocyte homing. Two reviews have sought to clarify the diverse historicaI nomenclature and present the evidence that has brought this assort- ment of molecules and their proposed functions together under the designation CD44 (Gallatin et al., 1991; Haynes et al., 1989). Most recently, study of the structure of the (probably) single gene that encodes CD44 has begun to reveal how the great variety of molecular forms of CD44 may be generated (see Section 11).

Our interest in CD44 was prompted by its involvement in lymphocyte development (see Section IV), during which it participates both in the earliest stages of T and B cell differentiation and in later stages of T and B cell activation in response to immunological stimuIi. In these and other contexts, CD44 seems to function by mediating cell-cell or cell-substrate interactions through recognition of elements of the extracellular matrix, intercellular milieu, and/or pericellular layer (Sec- tion 111). Perhaps all of the suggested functions of CD44 on motile cells, including metastatic cells (Section VI), can be explained as con- sequences of this recognition function. However, downstream events subsequent to ligand recognition by CD44 may vary greatly depending on cell type and on other stimuli in the cellular environment, perhaps accounting for the diversity of cellular responses observed to result after binding CD44 by ligand or antibody (Section IV).

Of particular interest to us is the finding that ligand recognition by CD44 is not constitutive in many CD44-expressing cells. Rather, like a number of other lymphocyte adhesion receptors (Dustin and Springer, 1991), CD44 receptor function is strictly regulated (Section V). Regu- lated, transient receptor activation may provide specificity for what would otherwise be an uncontrolled interaction between a broadly

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272 JAYNE LESLEY ET AL.

distributed cell surface molecule and common components of the extracellular environment. The association of elevated or altered CD44 expression with malignancy (Section VI) may be an indication of the importance of regulation of CD44 function to normal cell behavior.

II. Molecular lsoforms and Posttranslational Modifications

A. DISTRIBUTION AND HETEROGENEITY OF CD44 CD44 has been characterized in particular detail on lymphocytes,

macrophages, fibroblasts, epithelial cells and keratinocytes (Table I). However, it has also been detected on many other cell types, and several tissues previously considered to lack CD44 were subsequently shown to be positive under at least some circumstances. For example, a number of papers cite endothelial cells and erythrocytes as lacking this marker (Trowbridge et al., 1982; Kansas et al., 1989; Alho and Under- hill, 1989; Picker et al., 1989a). However, other reports have demonstrated expression on these cell types (Spring et al., 1988; Lucas et al.,

TABLE I SOME WELL-CHARACTERIZED FOHMS OF CD44

Tissue" Apparent M , ( X Protein isoformsb ~~

Lymphoid" 85-95 and 180-200 A, B, D, E, G M yeloidd 85-95 A, G Erythroide 82-92 A Fibroblastsf 80-85and180 A Epitheliala 177-250 Nervous systemh 74-86 A, C Endothelial' 116 ?

A, E, F, G, H, 1, J, K, L, M, N

Selected references (below) given for lymphoid and myeloid cells include leukemias; those for fibroblasts include fibrosarcomas; epithelial cells include carcinomas, keratinocytes, and melanoma; and the nervous system includes neuroblastomas, glioblastomas, and astrocytes. Also noteworthy is a thorough biochemic:d characterization of CD44 in placenta (St. Jacques et al., 1993).

" Protein isoforms correspond to Fig. 2. ' Jalkanen et al. (less), Jalkanen and Jalkanen (1992), Picker et al. (1989), Omary et al.

(1988). Trowbridge et al. (1982). Kalomiris and Bourguignon (1988), Flanagan et al. (1989), Gallatin et al. (1989).

Hughes et al. (1983), Camp et al. (1991). Lucas et al. (1989), Spring et al. (1988).

'Hughes and August (198l), Camp et al. (19!Jl), Kansas et al. (1989), Carter and Wayner (1988), Brown et al. (1991), Tarone et al. (1984), Culty et al . (1990).

Brown et al. (1991). Kansas et al. (1989), Haggerty et al. (1992). Asher and Bignami (1992). Vogel et al. (1998). Girgrah et 01. (1991b).

' Bourguignon et al. (1992).

CD44 AND ITS INTERACTION WITH ECM 273

1989; Bourguignon et al., 1992; Heider et al., 1993). These discrepan- cies may result from different experimental techniques, iise of cultured or transformed versus normal tissues, heterogeneity among endothelial cells, or genetic polymorphisms that dramatically influence levels of CD44 expression (see below). The few tissues that have consistently been described as CD44 negative include liver hepatocytes, kidney tubular epithelium, cardiac muscle, portions of the skin, and testis (Picker et al., 1989a; Flanagan et al., 1989; Wang et al., 1992; Heider et al., 1993). Expression in the nervous system of young normal individ- uals is restricted to the white matter, including astrocytes and glial cells, whereas its appearance in gray matter accompanies age and disease (Flanagan et al., 1989; Girgrah et al., 1991a,b; Vogel et al., 1992; Asher and Bignami, 1992). CD44 has been characterized in a number of species, which in addition to human and mouse, also includes hamster, baboon, rat, sheep, dog, and cow (Mackay et al., 1988; Idzerda et al., 1989; Aruffo et al., 1990; Sandmaier et aZ., 1990; Gun- thert et al., 1991; Gallatin et al., 1991; Bosworth et al., 1991).

The most abundant form of CD44 on lymphocytes has an apparent molecular mass on sodium dodecyl sulfate-polyacrylamide gel electro- phoresis (SDS-PAGE) of 85-95 kDa (Table 1). This is commonly termed the “standard” or “hematopoietic” form. The presence of intra- molecular disulfide bonds accounts for a slightly larger apparent molecular mass when run under reducing conditions. A similar, but not necessarily identical, type of CD44 has been characterized on macrophages, fibroblasts, fibrosarcomas, and astrocytes. The protein is acidic, with estimates of isoelectric point ranging from 4.2 to 5.8 (Jal- kanen et al., 1988; Kalomiris and Bourguignon, 1988; Picker et al., 1989,; Culty et al., 1990). Most of this charge is accounted for by the presence of sialic acid. CD44 can incorporate radiolabeled sulfate, which may be attached directly to the protein core (Jalkanen et al., 1988; Brown et al., 1991). The hematopoietic form is synthesized as a polypeptide of apparent molecular mass 42 kDa that undergoes subsequent N- and 0-linked glycosylation (Brown et al., 1991; Lokeshwar and Bourguignon, 1991). Also, depending on the tissue, CD44 is usually phosphorylated (Isacke et al., 1986; Kalomiris and Bourguignon, 1988; Carter and Wayner, 1988; Camp et al., 1991; Neame and Isacke, 1992).

It was apparent from biochemical studies that multiple forms of CD44 must exist (Omary et al., 1988; Kansas et al., 1989). A 180- to 200-kDa form of CD44 on lymphocytes was found to be sensitive to chondroitinase ABC, thus demonstrating that the molecule can exist as a proteoglycan (Jalkanen et al., 1988). This modification is not trivial


because it confers new ligand-binding ability on the molecule and unique functions to the cells that express it (Jalkanen and Jalkanen, 1992; Fassen et al., 1992) (see Section 111,D). Other size differences in CD44 have in some cases been accounted for by glycosylation (Quack- enbush et al., 1990; Camp et al., 1991). Indeed, half of the apparent molecular mass of the molecule is contributed by N-linked and 0- linked carbohydrate and, in addition to chondroitin sulfate, some species of CD44 bear heparan sulfate (Brown et al., 1991). Some CD44 molecules with a larger apparent molecular mass first noted in immu- nochemical studies (Omary et al., 1988; Kansas et al., 1989) were later shown to result from alternative exon utilization (see below). It is not clear whether glycosylation differences and/or alternative exon splicing confer tissue specific properties on CD44.

B. SOLUBLE CD44 Receptors of many kinds are frequently detectable in the circula-

tion in soluble form and their levels may reflect disease activity (Fernandez-Botran, 1991). Substantial quantities (5 pg/ml) of CD44 may be present in human serum and plasma (Dalchau et al., 1980; Telen et al., 1983; Lucas et al., 1989; Picker et al., 1989b; Baiil and HoiejSi, 1992). An inverse relationship was found between soluble CD44 levels in synovial fluid and numbers of immigrating blood cells in arthritic joints (Haynes et al., 1991). The cellular origin of soluble CD44 is not known and, depending on the individual, either one or two prominent species of CD44 were demonstrable (Baiil and HofejSi, 1992). Neutrophil granulocytes, but not lymphocytes, readily shed CD44 when stimulated in culture (Campanero et al., 1991; Baiil and HoiejSi, 1992). The size of the recovered material was somewhat smaller than that associated with the cells and there is evidence that this material is generated by an endogenous proteolytic mechanism. Crosslinkage of CD44 on lymphocytes and neutrophils, but not fibroblasts, resulted in loss of some surface antigen (Baiil and HoiejSi, 1992; Jacobson et al., 1984). Soluble CD44 has also been recovered from the culture medium of keratinocytes and carcinoma cells (Brown et al., 1991; Haggerty et al., 1992). It is not known if soluble CD44 has biological importance. However, at least one other cellular adhesion molecule (F3) is functionally competent when presented in soluble form (Durbec et al., 1992).

Cell surface CD44 is sensitive to a number of proteolytic enzymes. Trypsin releases a 65-kDa fragment from murine lymphocytes or macrophages (Trowbridge et al., 1982; Hughes et al., 1983), CD44 is cleaved in two steps by a bacterial glycoproteinase (Sutherland et al.,


1992), and bromelain releases all but 22 kDa of CD44 from T cells (Hale and Haynes, 1992). It has been noted that alternative splicing mechanisms can introduce arginine dipeptides into human CD44 iso- fornis (Dougherty et al., 1991). These sites in other receptors and proteoglycans correspond to sites involved in proteolytic release from the membrane. In mice, there is a similar site near the lipid bilayer in all known splice variants and an additional one in high molecular weight variants (He et al., 1992).

C. PROTEIN ISOFORMS The molecular cloning of CD44 from hematopoietic cells was imme-

diately informative with respect to structure and possible functions. The mature core protein is predicted to be 37-38 kDa and can be subdivided into several domains (Zhou et al., 1989; and see Fig. 1). There is one potential membrane-spanning region that shows 80-90% sequence homology among different species. A stretch of some 90 relatively hydrophobic residues comprises the amino terminus of the extracellular domain and also shows 80-90% sequence similarity among species. This domain has homology to cartilage link protein and other proteins known to interact with hyaluronan (HA) (Stamenkovic et al., 1989; Goldstein et al., 1989; Zhou et al., 1989; Nottenburg et al., 1989; Wolffe et al., 1990; Bosworth et al., 1991; Gunthert et al., 1991) (see Section III,B,l). However, CD44 lacks the conserved basic residues found in cartilage link and proteoglycan core proteins and thought to be involved in HA recognition (Goldstein et al., 1989; Wolffe et al., 1990). This region contains six cysteines that might be utilized to form a single globular domain (Goldstein et al., 1989). In the human, there are six potential sites for N-linked carbohydrate addition in this domain. The membrane-proximal domain is less well conserved, showing only approximately 50% sequence similarity among species. This domain contains three or four consensus sites for chron- droitin sulfate attachment and many potential sites of 0-glycosylation.

The cytoplasmic domain is highly conserved, showing 80-90% sequence similarity among species. Of the six serine residues in human CD44 cytoplasmic domain, five are conserved among the human, mouse, baboon, cow, and hamster (Stamenkovic et al., 1989; Zhou et al., 1989; Idzerda et al., 1989; Bosworth et al., 1991; Aruffo et al., 1990). Four of these residues are conserved in the rat; however, serine residue 317 is replaced by threonine (Gunthert et al., 1991). Of the conserved serine residues, Ser-296 (the first amino acid of the mature human protein is residue 1; for other species, residue numbers have been changed to correspond to the human sequence) i s not phosphorylated in intact epithelial cells (Neame and Isacke, 1992), although this

276 JAYNE LESLEY E T AL.

FIG. 1. Schematic representation of the human CD44 protein, “hematopoietic” or “standard” form. The solid filled area indicates the conserved region, amino acids 12 to 101, which shows sequence similarity to HA-binding domains of cartilage proteoglycan core protein and link protein. [Amino acid numbers correspond to the sequence given in Stamenkovic et al. (1989), with the first amino acid of the mature protein designated as residue 1.) The heavily stippled area indicates the membrane-proximal region, which shows more variability among species than other parts of the molecule, and which has been deleted in a mutant construct expressed in transfected cells (ANC in Table IV; He et al., 1992). The lightly stippled area indicate:; the transmembrane domain. 0-, Poten- tial N-linked glycosylation sites; 0--, possible 0-linked glycosylation sites; + -, potential sites for addition of chondroitin sulfate; -, site of insertion of alternately spliced exons (between amino acids 202/203); @-, four serines in the cytoplasmic domain that are conseved among all species and that are potential sites for phosphorylation; @-, serines 303 and 305, which have been shown to be required for phosphorylation in vivo (Neame and Isacke, 1992; Camp et al., 199.3a); S-S, probable, and S-+, possible, disulfide bonds between cysteine residues.


residue is a potential substrate for CAMP- and cGMP-dependent protein kinases (Wolffe et al., 1990). Both Ser-303 and -305 may be phosphorylated. Mutation of either abolishes the ability of the hematopoietic form of CD44 to be phosphoryIated in epithelial cells (Neame and Isacke, 1992). In other studies in which mutant constructs were transiently expressed in COS cells, mutation of residue 305 abolished phosphorylation, whereas mutation of residue 303 reduced it (Camp e t al., 1993a). As discussed by Neame and Isacke (1992), neither of these two residues appears to be a substrate for protein kinase C of CAMP- dependent protein kinases. Ser-271, which is located close to the transmembrane region, may be a substrate for protein kinase C (Wolffe et al., 1990), but it is not clear whether this residue is phosphorylated in intact cells. Mutation of this residue did not affect phosphorylation of CD44, nor did deletion of serine 317, when mutant constructs were expressed in COS cells (Camp et al., 1993a).

Almost all studies have concluded that CD44 represents a single gene, located on the short arm of chromosome 11 in humans (Good- fellow et al., 1982; Franke et al., 1983) and on chromosome 2 in mice (Lesley and Trowbridge, 1982; Colombatti et al., 1982). There are at least 19 exons (Screaton et al., 1992), as indicated in Fig. 2, and unpublished studies with rodents suggest there is an additional exon between exons 5 and 6 (Screaton et d., 1992; U. Gunthert, personal communication, 1992). Variation in the cytoplasmic tails of CD44 results from differential utilization of one of two exons (exon 18 or 19) (Screaton et al., 1992). The first three amino acids common to both tails are coded by exon 17. Exon 18 encodes an A+T-rich untranslated region, which might confer instability on the mRNA. The additional amino acids present in the long cytoplasmic tail are encoded in exon 19. It is not certain whether mature CD44 molecules with the short tail are expressed; however, CD44 mRNA for a short-tailed form has been detected by the polymerase chain reaction (Goldstein and Butcher, 1990).

In the most simple pattern of expression, at least three major CD44 mRNA species are observed in Northern blots. These are approximately 1.6,2.2, and 4.8 kb in humans (Stamenkovic et al., 1989; Gold- stein et al., 1989; Quackenbush et al., 1990; Brown et al., 1991; Shtivelman and Bishop, 1991) and 1.6,3.3, and 4.6 kb in mice (Wolffe et al., 1990; Haegel and Ceredig, 1991). This size variation apparently results from the use of multiple polyadenylation signals (Shtivelman and Bishop, 1991; Ham et al., 1991).

At least 18 CD44 transcripts have been described to date (Fig. 2). This heterogeneity results from the facts that 12 of the 19 exons can undergo alternative splicing and that consensus splice donor/acceptor


MON I 2 3 4 5 1 6 7 8 9 10 I1 12 13 14 15 I6 I7 I8 19

lM

1 .....I ..... a .... 9 ..... n .................................................................... n..p ..... Jl.......... 1 0. .... 1 ..,,.I ..,. .n ..,. n ......................................................... ........... u ..,. P.J ...! .El U.1.J ................ 0 I ................ ........._......... ........................ B...? ..... El .......... fl .... .I ..... RJ ..... 0 .............. ........ *............*I .......................... ... a .... a .... .B .......... 1

0. ...@..,.I ..... Q .... 0.. ...................................................... ..... a,.a,p .,,. 4 .......... 0 U...,.~....~.,..Q....n........................................................ N.*Jt.R...J ....I.....*...J P

n. [email protected].,..4 .... n ................................................ n..OI.OP*O.~...J ..,. 1 .......... n .... I.,J .... a .... 1. ............................... ..........4.J..Jl., RO.U ..R..,.R........J u....i.,.l.%.n.,.n........................... ........ l..,lJ..ul.I.O.n..J ..... 4 .......... 1 n...i.,.i...n .... n ...................... ~,..E. ~~4..~..,~."~...U...R.,.~..........U 0 .... R..J .... n .... n .......)...... .R.~~.~.~,r . .~n. .~ . . ~RUn...R .... 4 .......... 0 n....l.... 1 .... .... .......-........ 1.J .... R.u~. .~n. .~ . .~~."~.O.U. . .R .... 1 .......... D 9,.1.*,. kQ .... ....... *. ....... 1 ............................. 4..P.R.4.J ..... a .......... 1 .... .IJ ..... QJ. .................. .... a..p.LB..J.. ........................... U..B ....._.... 0 0 .... l..J .... a .... a ......." ....... B.J..,a.,B..,s ..........._*........ " ........... ....._.... 1 0 .... 1.J .... a .... 0 ................................... I..) ................ 8,.n .*,2 *II, ...... I ....-... . D P

1 .... 4.J .... n....n .......)....... 1 ............................. m..*R ....-... nm......... .... .I..... a .... 9 ..... n ............. .................................... I..! ..... "..J...? .... 1 .....-.... 0

A

C ..........

E

c

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FIG. 2. Multiple transcription products of the CD44 gene. The genomic structure of human CD44 as described by Screaton et ol. (1992) is shown at the top with leader peptide (LP) and transmembrane domains (TM) indicated. Exons (2, 3) encode the putative HA-binding domain. An additional exon may exist between exons 5 and 6 [Screaton et al. (1992) and U. Gunthert, personal communication]. Also illustrated are splice variants in published cDNA sequences that may encode unique protein isoforms. The corresponding reference citations are as follows: A (Stamenkovic et al., 1989; Wolffe et ol., 1990; Zhou et al., 1989; Aruffo et al., 1990; Gallatin et ol., 1991; Idzerda et al., 1989; Bosworth e t al., 1991; Nottenburg et al., 1989; He et al., 1992, Harn e t ol., 1991); B (Goldstein and Butcher, 1990; Goldstein et al., 1989; Screaton e t al., 1992); C (Shtivel- man and Bishop, 1991); D (Arch et al., 1992); E (Jackson et al., 1992; Cooper et al., 1992);


sites occur within two exons (exons 5 and 7). Splicing within exons sometimes accounts for deletion of part of exon 5 in neuroblastoma cells (Fig. 2C) (Shtivelman and Bishop, 1991) and part of exon 7 in keratinocytes (Fig. 2L) (Hofmann et al., 1991). Furthermore, one example has been found in which a “constant” exon (exon 15) is deleted (Fig. 2N) (Gunthert et al., 1991). This is an impressive amount of diversity. However, in the case of another cell adhesion molecule, NCAM, which can alternatively splice 12 of its 27 exons, as many as 192 proteins are possible (Barthels et al., 1992; Screaton et al., 1992). A satisfactory nomenclature for CD44 transcripts and corresponding core proteins has not yet been introduced. Although the “hematopoietic” or “standard” isoform (Fig. 2A) is the most prominant on blood cells, it is also expressed by fibroblasts and melanoma cells (Stamenkovic et al., 1989; Idzerda et al., 1989; Zhou et al., 1989; Nottenburg et al., 1989; Wolffe et al., 1990; Aruffo et al., 1990; Gallatin et al., 1991; Bosworth et al., 1991; He et al., 1992). Similarly, transcripts corresponding to the “epithelial” form of CD44 (Fig. 2G) have been described in leukemias (Dougherty et al., 1991; Gunthertet al., 1991; Stamenkovic et al., 1991; Jackson et al., 1992).

Although it is not yet certain that every possible splice variant is translated into a mature protein, expression of a number of splice variants has been demonstrated. For example, long CD44 transcripts have been found in keratinocytes and carcinoma cells (Hofmann et al., 1991; Kugelman et al., 1992; Haggerty et al., 1992) and deglycosylated core proteins of a large size have been characterized in such cells (Brown et al., 1991; Haggerty et al., 1992). When compared with the “standard” CD44 transcript that predominates in hematopoietic cells (Fig. 2A), this long core protein contains an additional 338 amino acids in the extracellular domain. Besides doubling the length, three potential N-glycosylation sites, numerous 0-glycosylation sites, and two chondroitin sulfate attachment sequences are added to the molecule. Consensus attachment sequences for heparan sulfate side chains have not been described, but it is clear that they are present in this form of CD44 (Brown et al., 1991; Haggerty et al., 1992). As noted above, the

F (He et al., 1992; Jackson et al., 1992); G (Dougherty e t al., 1991; Gunthert et al., 1991; Stamenkovic et al., 1991; Hofmann et al., 1991; Jackson e t al., 1992; Brown et al., 1991; He et ul., 1992); H (Jackson et al., 1992); I (He et ul., 1992); J (He et al., 1992); K (Kugelman et id., 1992; Haggerty et al., 1992; Hofmann et al., 1991); L (Hofmann et al., 1991); M (Jackson et ul., 1992; Hofmann et al., 1991); N (Gunthert et al., 1991); 0, P, Q, and R (Hofmann et al., 1991). Additional splice variants have been reported by Herrlich et a1. (1993), but their sequences are not yet published.


inserted sequences also contain potential protease cleavage sites. Indi- vidual cells can simultaneously synthesize multiple isoforms of CD44 and our experiments with murine carcinoma cells revealed that the apparent molecular weights of the proteins corresponded to mRNA length (He et al., 1992).

It is uncertain whether aberrent splicing occurs in malignant cells, or whether the diversity of transcripts frequently observed in them is a consequence of elevated levels of CD44 transcripts that are normally only minor components and/or only present in activated cells. Activa- tion of normal lymphocytes correlated with the presence of a splice variant (Arch et al., 1992; Koopman et al., 1993). Also, a change in CD44 size, which may have been caused by posttranslational modification, occurred on stimulation of normal macrophages (Camp et ul., 1991). Transformed cells might therefore be representative of some normal differentiation stage or activation state. If splice variants are selectively expressed in tumor cells, this could provide a useful diag- nostic tool, because 1 tumor cell expressing a variant transcript may be detectable among lo6 blood cells, using the polymerase chain reaction (Matsumura and Tarin, 1992). In addition, particular CD44 isoforms correlate with metastasis in certain cases (Gunthert et al., 1991) (see Section VI).

Expression and alternative splicing of CD44 are clearly regulated in a way that is tissue and differentiation stage specific. In lymphocytes, these are also subject to change on activation (see Section V). Analysis of upstream regulatory sequences in the CD44 gene has been carried out in human neuroblastoma cells (Shtivelman and Bishop, 1991) and the human and mouse promoters have been analyzed by Herrlich and colleagues (1993). The neuroblastoma gene showed no TATA or CCAAT boxes, but contained a GC-rich region; however, the human and mouse genes cloned by Herrlich and colleagues (1993) show a TATA box at -31 to -35. In the neuroblastoma gene, RNA initiation sites were localized by primer extension experiments to 128 and 136 nucleotides upstream of the translation initiation codon, but the possibility of additional start sites in different tissues was not excluded. Functional promoter activity and the existence of multiple negative regulatory elements were demonstrated by transfection assays (Shtivelman and Bishop, 1991). Both the human and mouse genes show potential binding sites for the SP-1 transcription factor, although it is not certain whether these sites function in intact cells (Shtivelman and Bishop, 1991; Herrlich et al., 1993). An AP-1 binding site at - 108 to -114 in the human sequence and -109 to -115 in the mouse sequence appears to function, because mutation of this site reduces

CD44 AND ITS INTERACTION WITH ECM 28 1

transcription of an indicator gene in transient transfection assays, reduces ability to respond to induction by phorbol ester, and reduces the increased transcription seen after cotransfection of jun or ras constructs (Herrlich et al., 1993).

Several observations suggest that one or more genes act to influence the level of CD44 expression on the cell surface. A polymorphism in mice correlates with CD44 mRNA content (Haegel and Ceredig, 1991). The In" blood group antigen of humans is carried on the CD44 molecule and its expression on erythrocytes and monocytes, but not lymphocytes, is influenced by the dominant inhibitor gene Zn(Lu) (Telen et al., 1983; Spring et al., 1988). An inverse relationship may also exist between expression of CD44 and the multidrug resistance gene (Cian- friglia et al., 1991).

D. CYTOPLASMIC DOMAIN

Functional importance has been demonstrated for the cytoplasmic domains of many cell adhesion molecules and this is also the case for CD44 (Lesley et al., 1992; Thomas et al., 1992; see Sections II1,C and V,C,l). It has long been known that CD44 interacts at least indirectly with components of the cytoskeleton, including actin and ankyrin (Jacobson et al., 1984); Tarone et al., 1984; Lacy and Underhill, 1987; Carter and Wayner, 1988; Kalomiris and Bourguignon, 1988; Geppert and Lipsky, 1991; Bourguignon et al., 1992). Binding to erythrocyte ankyrin by purified CD44 has been demonstrated by in uitro assays (Kalomiris and Bourguignon, 1988); however, there is no sequence similarity between murine CD44 and the region of erythrocytic band 3 that is thought to be involved in ankyrin binding (Wolffe et al., 1990). The ability of CD44 to bind ankyrin in uitro is influenced by acylation (Bourguignon et al., 1991). Two conserved cysteines, located in the transmembrane domain, and at the interface of transmembrane and cytoplasmic domains, are potential sites for attachment of palmitic acid. Some similarity has been noted between sequences in the CD44 tail and those of members of the G-protein superfamily (Lokeshwar and Bourguignon, 1992). Purified CD44 bound GTP and displayed some GTPase activity in in uitro assays (Lokeshwar and Bourguignon, 1992).

Phosphorylation of the cytoplasmic tail of CD44 has also been thought to affect cytoskeletal association. In uitro phosphorylation of the molecule may enhance its affinity for ankyrin as measured in uitro assays (Lokeshwar and Bourguignon, 1992). As discussed in more detail below (Section V,C,l), however, it is not certain how these in uitro observations relate to observations in intact cells. In macro-


phages, only nonphosphorylated CD44 was present in the detergent- insoluble pool (Camp et al., 1991). Activation of T lymphocytes by phorbol ester and ionomycin reduced the apparent interaction of CD44 with the cytoskeleton (Geppert and Lipsky, 1991), as did phorbol ester treatment of macrophages (Camp et al., 1993a). However, it is not clear that phorbol ester treatment affects phosphorylation of CD44 in intact cells. No enhanced phosphorylation was seen in epithelial cells after phorbol ester treatment (Neame and Isacke, 1992), although transient increases in phosphorylation were seen in macrophages within minutes after phorbol ester treatment (Camp et al., 1993a).

Two closely spaced and well-conserved serines in the cytoplasmic tail (residues 303 and 305 in human CD44) may be phosphorylated (Isacke et al., 1986; Carter and Wayner, 1988; Neame and Isacke, 1992; Camp et al., 1993a). Mutation of either serine residue prevented (Neame and Isacke, 1992) or greatly reduced (Camp et al., 1993a) phosphorylation. Although deletion of the cytoplasmic tail of CD44 prevented localization of the molecule to the basolateral membrane of transfected polarized epithelial cells, mutation of either serine residue 303 or 305 such that phosphorylation was prevented did not influence localization and did not affect the association of CD44 with the cytoskeleton (Neame and Isacke, 1992). Enhanced phosphorylation induced by phorbol ester was seen in COS cells transfected with a mutant CD44 construct in which serine residues 271, 303, and 305 were changed to glycine or alanine. This observation implies that the transient phorbol ester-induced phosphorylation was not occurring on these residues (Camp et al., 1993a).

111. CD44 and Extracellular Matrix

A. GENERAL FEATURES OF EXTRACELLULAR MATRIX The extracellular matrix (ECM) fills the spaces between cells. Al-

though it has long been recognized that the components of the ECM perform an important structural role, it has been realized more recently that the ECM communicates with the cell interior and thus modulates cell adhesion, proliferation, and differentiation (Toole, 1991; Schu- bert, 1992). Major constituents of the ECM include collagenous proteins (Linsenmayer, 1991) and proteoglycans (Wight et al., 1991). The latter consist of one or more glycosaminoglycans-linear poly- mers of repeating disaccharides-covalently bound to a protein core.


Hyaluronan (HA) is a glycosaminoglycan consisting of a linear polymer of [u-glucuronic acid (1-p-3) N-acetyl-D-glucosamine (l-p-4)],* (Laurent, 1989; Wight et al., 1991; Laurent and Fraser, 1992), which differs from other glycosaminoglycans in not being covalently linked to a core protein. The HA polymer can have a molecular weight of up to several million. In solution, the polymer behaves as a random coil (Laurent, 1989). A large quantity of solvent is trapped within the coil and the molecule can be considered to be a highly hydrated sphere (Laurent and Fraser, 1992). There is evidence for secondary structure via hydrophobic bonding (Scott, 1992), and the possibility of aggregation of HA molecules leading to infinite meshworks has been raised (Scott, 1989, 1992).

Within the ECM, large aggregates are formed by noncovalent interactions between proteoglycan molecules and HA (Morgelin et al., 1988). The basic unit is a ternary complex consisting of proteoglycan monomer, HA, and link protein, a molecule of approximately 40 kDa that interacts with both the proteoglycan monomer and HA to stabilize the proteoglycan-HA complex (Neame et al., 1986; Wight et al., 1991). Aggrecan, the aggregating chondroitin sulfate proteoglycan of cartilage, contains a protein core of 220 kDa to which numerous glycosaminoglycan side chains are covalently attached. Two globular domains, termed G1 and G2, are present at the amino terminus (Doege et al., 1987, 1991). The G1, but not the G2, domain interacts noncovalently with HA. The function of the G2 domain is uncertain and some proteoglycans, such as those found in aorta, lack a G2 domain (Morgelin et al., 1989). Proteoglycan and HA form complexes that appear in the elec- tron microscope as “necklace-like” structures, with proteoglycan monomers randomly attached along the HA chain (Morgelin et ul., 1988). Addition of link protein converts this structure to a much more densely packed structure with a continuous coat of proteoglycan ex- tending along the HA strand (Morgelin et al., 1988, 1989), reflecting stabilization of the proteoglycan-HA interaction by the link protein.

The G1 domain of aggrecan is composed of subdomains containing disulfide-bonded loop structures of about 100 amino acids (Doege et al., 1987, 1991). These domains show sequence similarity to tandem repeat regions in the C-terminal region of link protein (Neame et al., 1987; Doege et al., 1987, 1991) that have been demonstrated to be involved in the binding of link protein to HA (Perin et al., 1987; Goetinck et al., 1987). Other HA-binding proteoglycans also show regions of sequence homology to the tandem repeat regions found in aggrecan and link protein: versican, isolated from fibroblasts (Le Baron et al., 1992), and TSG-6, a protein inducible by tumor necrosis factor a

284 JAYNE LESLEY ET AL

and interleukin 1 (IL-1) in fibroblasts and inducible by mitogens in peripheral blood mononuclear cells (Lee et al., 1992).

B. CD44 AS A RECEPTOR FOR HYALURONAN 1. Sequence of CD44 Implies a Role in Hyaluronan Recognition A portion of the N-terminal domain of CD44 in humans and mouse-

residues 12-101 in humans (the first amino acid of the mature protein is residue 1; see Fig. 1)-shows approximately 30% sequence similarity to the second (B) subdomain of cartilage proteoglycan core and link proteins (Goldstein et al., 1989; Stamenkovic et al., 1989; Idzerda et al., 1989; Wolffe et al., 1990; Doege et al., 1991). This sequence similarity increases to about 50% if conservative amino acid substitutions are considered (Goldstein et al., 1989). There is also a lower, but significant, sequence similarity to the B1 subdomain of cartilage proteoglycan core and link proteins (Goldstein et al., 1989). Th' is sequence similarity does not necessarily imply that this region of CD44 is actually functionally involved in the binding of CD44 to HA (Wolffe et al., 1990; Doege et al., 1991). Both link protein and the two HA- binding proteoglycan core proteins aggregan and versican contain at least two disulfide-bonded subdomains that form tandem-repeated loop structures and it may be that binding of more than one subdomain to HA is required to provide sufficient affinity for a stable ternary complex between link protein, HA, and proteoglycan core protein (Goetinck et al., 1987). Also, the interaction of link protein with HA is thought to be largely of an ionic nature (Goetinck et nl., 1987). If so, then it should be noted that the arginine and lysine residues of link protein thought to be important in this interaction are not conserved in CD44 (Goldstein et al., 1989; Wolffe et al., 1990). There is an arginine residue at position 70 of the human sequence and position 72 of the mouse sequence (Goldstein et al., 1989; Zhou et al., 1989; Wolffe et al., 1990), but it is not known whether this residue serves the same function as the basic residues found in the homologous region of the link protein. Nevertheless, although there is only limited sequence similarity between the HA-binding domains of proteoglycan core and link proteins and CD44, CD44 does function as a receptor for HA.

2. Evidence that CD44 Is a Receptor f o r Hyaluronan

a. Inhibition of Hyaluronan Binding by

It has been known for many years that HA can bind to the surface of nonchondrogenic cells in culture (Underhill and Toole, 1979). Many cell types are surrounded by a chondrocyte-like pericellular matrix

CD44-Specific Antibodies


(Goldberg et al., 1984; Knudson and Knudson, 1991). Formation of this pericellular matrix occurs on addition of HA and cartilage proteoglycan monomer to cells, but matrix formation occurs only when the cells are able to bind HA through an HA-specific cell surface receptor (Knudson and Knudson, 1991). This receptor is present on a variety of cell lines. In t)it)o, it is expressed on epithelial cells, especially proliferating epithelial cells such as in the basal layers of stratified epithelium and on the basolateral surfaces of cells at the base of the crypts of Lieber- kuhn of the intestine, although it is not found on cells lining the mouth of the crypts or on the villi (Alho and Underhill, 1989). Because this receptor has been best characterized on fibroblast cell lines, it will be termed the “fibroblast HA receptor.” It is distinct from other HA receptors found on liver endothelial cells (Yannariello-Brown et al., 1992a,b) and ras-transformed tumor cells (Hardwick et al., 1992).

The fibroblast HA receptor is a glycoprotein of M, 85,000 that binds to six sugar residues (three repeating disaccharide units) on the HA polymer (Underhill et al., 1983). It is phosphorylated and a proportion is associated with cytoskeletal actin filaments (Underhill et al., 1985, 1987; Lacy and Underhill, 1987; Culty et al., 1990). These properties resemble those of CD44 (Lesley et al., 1990a; Culty et al., 1990). Binding of [3H]HA to mouse cells was blocked by the CD44-specific monoclonal antibody (mAb) KM201 and this antibody depleted detergent cell lysates of material able to bind [3H]HA. These latter experiments indicate the identity of the fibroblast HA receptor and CD44 (Culty et al., 1990).

A number of cell types, including fibroblasts and CD44-positive hematopoietic cells and cell lines, aggregate in the presence of exoge- nous HA (Green et al., 1988; Lesley et al., 1990a). This HA-dependent aggregation can be blocked if the cells are preincubated with antibody specific to the fibroblast HA receptor (Green et al., 1988) or with CD44-specific mAb (Lesley et al., 1990a). Binding of fluorescein- conjugated HA to lymphoid cell lines or adhesion of these cells to HA-coated plates is also specifically inhibited by CD44-specific antibodies (Lesley et al., 1990a; Miyake et al., 1990b). Adhesion of a B-lineage hybridoma to a bone marrow-derived stromal cell line can be blocked by CD44-specific antibody or by pretreatment of the stro- ma1 cell line with hyaluronidase (Miyake et al., 1990b), demonstrating that cell adhesion can be dependent on CD44 and HA.

Only a subset of CD44-specific mAbs inhibits HA-dependent binding, indicating that only particular epitopes on the CD44 molecule contribute to (or border) the HA-specific binding site. However, not all CD44-positive cells bind HA (Lesley et al., 1990a; Miyake and


Kincade, 1990). This result indicates that although CD44 is necessary for HA binding, there is not a one-to-one correspondence between the expression of CD44 on the cell surface and the ability to bind HA. This point is extremely important and its implications will be discussed in detail below (Section V).

b. Transfection and Expression of CD44 cDNA Constructs Several groups have used transfection and expression of CD44 con-

structs to demonstrate that HA is a ligand for CD44. Aruffo and colleagues (1990) expressed a soluble human CD44-immunoglobulin construct in COS cells. The gene product of this construct was a di- meric molecule consisting of the extracellular domain of CD44 and the hinge, cH2, and c H 3 domains of human IgG,. The soluble CD44 immunoglobulin fusion protein bound to lymph node high endothelial cells in primary culture. Binding was abolished by pretreatment of the target cells with hyaluronidase but not by enzymes that did not have activity against HA. Binding of the CD44-immunoglobulin fusion protein was inhibited in the presence of HA but not other glycosaminoglycans. In complementary experiments, St. Jacques and colleagues (1993) showed that radiolabeled CD44 purified from placenta by affinity chromatography could bind to immobilized HA. Binding was inhibited by preincubation of the CD44 with soluble HA and the bound material was sensitive to hyaluronidase. Much less binding was seen to collagen I, collagen VI, fibronectin, or heparin. These results strongly imply that HA is a ligand for CD44.

Aruffo and colleagues (1990) isolated a cDNA clone for the hamster CD44 molecule and transfected this clone into COS cells. The transfected cells reacted with antibody specific for the hamster fibroblast HA receptor, which, as discussed above (Section 11I7B,2,a), is CD44. In other experiments, the same group transfected the CD44-negative human Burkitt B cell lymphoma line Namalwa with the hematopoietic form of human CD44 and examined binding of the transfectants to primary cultures of lymph node high endothelial cells (Stamenkovic et al., 1991). B cells transfected with the hematopoietic form bound to high endothelial cells and binding was blocked if the assay was done in the presence of HA or a polyclonal CD44-specific antibody or if the high endothelial cells were pretreated with hyaluronidase.

Lesley and colleagues (1992) examined the HA-binding phenotype of the CD44.2-negative murine T cell lymphoma AKRl transfected with a murine CD44.1 cDNA. Transfection of CD44 conferred the ability to bind fluorescein-conjugated HA from solution and to bind to immobilized HA. Binding could be blocked by preincubation with


CD44-specific mAb and/or by competition with unconjugated HA. Transfection also conferred a CD44-dependent and hyaluronidase- sensitive increase in adhesion to a lymph node endothelial cell line.

These experiments provide conclusive evidence that CD44 functions as a receptor for HA. However, as noted above (Section III,B,2,a), not all cells that express the hematopoietic form of CD44 bind ligand. This observation implies that the cell is able to regulate the ability of the CD44 molecule to bind ligand. Thus it is reasonable to assume that some cells will not bind HA even if transfected with CD44 cDNA constructs whose expression leads to acquisition of HA-binding function when they are transfected into other cells. This, in fact, is the case (see Section V,A,4). The interesting questions are why CD44 is able to bind HA only in certain cellular environments and what factors are required to confer an HA-binding phenotype. These points will be discussed further in Section V.

C. FEATURES OF CD44 MOLECULE MEDIATING INTERACTION WITH HYALURONAN

The coding region of CD44 can be divided into four domains: an amino-terminal highly conserved domain, a less conserved membrane-proximal domain containing the alternatively spliced exons, the transmembrane domain, and the highly conserved cytoplasmic domain (Zhou et al., 1989; and see Fig. 1). To examine the role of these regions in mediating the ability of CD44 to bind HA, mouse CD44 constructs that code for a product lacking either the membrane- proximal domain or the cytoplasmic domain have been transfected into the mouse T cell lymphoma AKR1. Transfectants that do not express the membrane-proximal region (between Val-161 and Arg-244) bind HA (He et al., 1992). This result indicates that the amino-terminal two-thirds of the CD44 molecule is sufficient for HA recognition. That this amino-terminal region is involved in binding of HA has been inferred from the fact that the region of sequence similarity to proteoglycan core and link proteins is located in the amino-terminal domain between residues 12 and 101 (the first residue of the mature human protein is residue 1) (Goldstein et al., 1989; see Section III,B,l). St. Jacques et. al. (1993) suggested that within this region, only residues 18-30 and 88-112 are likely to be exposed at the surface. An antibody to a peptide comprising residues 18-30 did not inhibit binding of CD44 to HA, suggesting that these residues do not contribute to the HA-binding site.

AKRl transfectants expressing a mutant CD44 construct with a stop codon at Gly-276 and coding for only the first six amino acids of the


cytoplasmic domain do not bind HA, from solution, although AKRl transfectants expressing the wild-type molecule bind soluble HA well (Lesley et al., 1992). The amount of CD44 expressed on the surface of cells transfected with this mutant CD44 construct was roughly equal to the amount expressed on control transfectants expressing wild-type CD44 (Lesley et al., 1992). This is an important point, because the ability to bind HA from solution appears proportional to the amount of CD44 expressed on the cell surface (He et al., 1992). Thus, in experiments in which the HA-binding activity of two cell lines is to be compared, it may be necessary to use fluorescence-activated cell sorting or other methods to isolate cell lines expressing equal amounts of CD44 in order for valid comparisons to be made. Both sets of transfectants bound to HA immobilized on plates and showed hyaluronidase- sensitive binding to a lymph node endothelial cell line. However, the HA-specific binding of transfectants expressing the mutant construct lacking most of the cytoplasmic domain was lower than that of transfectants expressing a wild-type CD44 construct. Similar results were observed by Thomas and colleagues, who transfected melanoma cells with human CD44 constructs expressing only the first 6 or 16 amino acids of the cytoplasmic domain (Thomas et al., 1992). Transfectants not expressing most of the cytoplasmic domain bound to HA-coated plates less well than transfectants expressing the wild-type CD44 molecule. Also, melanoma transfectants lacking most of the cytoplasmic domain of CD44 did not migrate on HA-coated surfaces, even though the cells did attach. Therefore interaction of the cytoplasmic domain of CD44 with the cytoskeleton may be important in mediating postattach- ment events, such as cell motility.

Although the AKRl transfectants expressing a molecule lacking most of the cytoplasmic domain did not bind soluble HA, they could be induced to do so if the transfected cells were “activated” by pretreatment with the CD44-specific mAb IRAWB 14 (Lesley et al., 1992). This observation suggests that one function of the cytoplasmic domain is to allow preexisting CD44 molecules to enter a state whereby they are able to bind HA. Although these transfectants do not bind soluble HA unless “activated” by antibody, they do bind to immobilized HA, although less well than transfectants expressing wild-type CD44. These points will be discussed in more detail in Section V.

Although the membrane proximal domain is not required for HA recognition, insertion of exons in this region may modulate the ability to bind HA. Stamenkovic and colleagues found that the Burkitt lymphoma cell line Namalwa transfected with the human CD44 epithelial isoform [equivalent to the M2 isoforni of the mouse (He et al., 1992);


Fig. 2G] did not show HA-dependent binding to primary cultures of lymph node high endothelial cells (Stamenkovic et al., 1991) or to immobilized HA in microwells (Sy et al., 1991), whereas the same Burkitt lymphoma cell line transfected with the hematopoietic form did show HA-dependent binding. Flow cytometric analysis indicated that both sets of transfectants expressed approximately equal levels of CD44 (Stamenkovic et al., 1991; Sy et al., 1991). Melanoma cells transfected with a human CD44 epithelial isoform construct also bound poorly to immobilized HA (Thomas et al., 1992). These observations have led to speculation that the epithelial isoform and possibly other isoforms of CD44 containing exons inserted in the membrane proximal region may be unable to bind HA. The inserted sequences contain potential sites of glycosylation that might affect ability to bind HA. If so, then the particular cells into which the constructs have been transfected might determine the extent to which CD44-dependent binding to HA is observed. Higher molecular weight CD44 species purified from placenta (which is likely to comprise a mixture of isoforms) did not bind to immobilized HA (St. Jacques et al., 1993).

In contrast, transfectants of the mouse T cell lymphoma AKRl expressing several higher molecular weight mouse CD44 isoforms (F, G, I, and J in Fig. 2), including the mouse equivalent of the human epithelial form, did bind HA (He et al., 1992). Binding of soluble HA was proportional to the level of CD44 expression, and for all isoforms, the level of HA binding was markedly increased if the transfectants were first “activated” by pretreatment with IRAWB 14 mAb. All of the isoforms mediated CD44-dependent adhesion to immobilized HA (He et al., 1992). This result indicates that CD44 isoforms containing in- serts in the membrane-proximal region are not intrinsically unable to bind HA, although it is not excluded that the presence of the insert may have quantitative effects on the ability of CD44 to bind HA when expressed in particular cellular environments (e.g., by affecting the affinity of the binding site or the overall avidity of a particular cell for HA). It is interesting to note that Namalwa cells transfected with the epithelial form of CD44 did bind to BHK cells (Stamenkovic et al., 1991). Although no evidence was presented that this binding was HA dependent, BHK cells do express high levels of cell surface HA (Un- derhill and Toole, 1982).

There is another possible explanation for these seemingly differing results. The last two amino acids derived from exon 5 (Fig. 2) are alanine and threonine. These are near the boundary of nearly all splice variants of CD44 and have been independently found in both genomic and cDNA sequences in many laboratories for human, rat, hamster,


and mouse CD44 (see, e.g., Aruffo et al., 1990; He et al., 1992; Screaton et al., 1992). A single exception is in the “epithelial” CD44 cDNA described by Stamenkovic et al. (1991), in which the nonpolar alanine is replaced by a basic arginine residue. It seems possible that this nonconservative change influences the HA recognition capability of this particular clone of the human epithelial form of CD44.

D. INTERACTION OF CD44 WITH OTHER EXTRACELLULAR MATRIX PROTEINS

There is evidence that CD44 can interact with other extracellular matrix proteins in addition to HA. The details, however, are somewhat contradictory, and it is not clear to what extent CD44 functions in intact cells as an adhesion receptor for ECM components other than HA. The class I11 extracellular matrix receptor (ECMR 111) was characterized as a phosphorylated glycoprotein of M, 90,000 that bound to type I and type VI collagen in affinity chromatography experiments or when ECMR I11 was incorporated into liposomes (Wayner and Carter, 1987; Carter and Wayner, 1988). One-dimensional peptide mapping and sequential immunoprecipitation experiments indicated that ECMR I11 and CD44 were closely related or identical entities (Gallatin et al., 1989). Although binding of CD44 to collagen was demonstrated in these experiments when CD44 was incorporated into liposomes, only low levels of binding of radiolabeled soluble CD44 isolated from placenta to collagen I or VI could be demonstrated (St. Jacques et al., 1993). It seems possible that the affinity of CD44 for collagen may be quite low, and it is not clear under what circumstances (or whether) CD44 mediates the adhesion of intact cells to collagen.

Fassen and colleagues (1992) have characterized a chondroitin sulfate-containing proteoglycan of mouse melanoma cells that binds to type I collagen. This molecule appears related to CD44, because it is recognized by CD44-specific antibodies on Western blots, although only after treatment with chondroitinase. The melanoma molecule does not seem to function as an adhesion receptor for collagen I, because treatment with P-D-xyloside, which inhibits synthesis of chondroitin sulfate proteoglycans, or chondroitinase does not inhibit attachment of melanoma cells to collagen I. Migration of the cells within collagen gels is, however, inhibited after treatment with P-D-xyloside, suggesting that this molecule functions in mediating cell movement after the cells have attached via other receptors and that the chondroitin sulfate modification is necessary for it to do so.

CD44 purified from lymphocytes that has been covalently modified by addition of chondroitin sulfate has been shown to bind fibronectin


and laminin as well as collagen I in in vitro binding assays (Jalkanen and Jalkanen, 1992). The presence of the chondroitin sulfate moiety is essential for binding activity, because only the M , 180,000-200,000 chondroitin sulfate-substituted form of CD44 has binding activity; the 90,000 form is inactive. Heparin, heparan sulfate, and chondroitin sulfate inhibit the binding of purified CD44 to fibronectin. These observations suggest that CD44 is interacting with fibronectin via its covalently attached chondroitin sulfate side chains. CD44 purified from placenta, however, binds fibronectin or collagen I only poorly (St. Jacques et al., 1993). Whether the interaction of chondroitin sulfate moieties on lymphocytes with fibronectin occurs in vivo and whether this interaction is important in mediating lymphocyte homing to specific sites is uncertain.

IV. CD44 and Lymphocyte Homing

A. EXPRESSION OF CD44 ON HEMATOPOIETIC CELLS 1. Distribution of CD44 during Hematopoietic Development in

Most hematopoietic cells of mouse and human express CD44 at some level, although the degree of expression can be quite heterogeneous, with some cells expressing close to background amounts whereas other cells express large amounts (Trowbridge et al., 1982; Kansas et al., 1989,1990; Horst et al., 1990a). In the mouse, it has been shown that pluripotent stem cells are CD44+ (Trowbridge et al., 1982; Spangrude et al., 1989), and CD44' cells are found in every hematopoietic lineage. In hematopoietic lineages that have been studied in detail, there are differentiation-related changes in the level of CD44 expression (see below). It has been proposed that these changes in CD44 expression during differentiation are related to differences in the adhesion requirements of sessile vs migratory lymphocytes (Horst et al., 1990a) and to bone marrow stromal cell-progenitor cell interactions in the early stages of hematopoiesis (Kansas et al., 1990; Miyake et al., 1990a).

In the mouse, the prothymocyte, a progenitor cell in bone marrow capable of homing to and populating the thymus, expresses CD44 (Trowbridge et al., 1982; Spangrude et al., 1989). It is likely, but not certain, that this progenitor cell is a multipotent stem cell, which becomes committed to the T cell lineage at some time after colonization of the thymus (Spangrude and Scollay, 1990; Wu et al., 1991a,b). Once in the thymus, this progenitor retains the CD44+ phenotype and

Mouse and Human


the ability to home to and repopulate the thymus for an unknown period of time as a “thymus-homing thymocyte progenitor” (Lesley et al., 1985a; Wu et aZ., 1991b). As differentiation of the bone marrow- derived progenitor proceeds in the thymus, the a chain of the interleukin 2 receptor (IL-2R) is expressed and CD44 is lost. The kinetics of appearance of populations of “double negative” (CD4-, CD8- ) thymocytes in irradiated thymuses during repopulation by bone marrow- derived progenitors indicates the following progression: CD44+/

lowed by CD4, CD8, and T cell receptor expression (Lesley et al., 1990b; Petrie et al., 1990; Scollay, 1991). CD44 reappears on some more mature thymocytes; however, the number of mature thymocytes expressing CD44 varies among mouse strains (Lesley et al., 1988; Lynch and Ceredig, 1989). Mouse strains expressing the CD44.1 allele, especially BALB/c mice, have relatively large numbers of CD44+ thymocytes, which include relatively mature “single-positive” cells, whereas CD44.2 strains, such as C57BL/6 and AKR/J mice, have few CD44+ thymocytes.

The appearance of CD44+ cells in the mouse fetal thymus supports the sequence of CD44 expression described above (Lesley et al., 1985b; Husmann et al., 1988; Penit and Vasseur, 1989). The first cells that can be phenotyped in the mouse fetal thymus are CD44+ (at 12-13 days). As these cells expand and differentiate, IL-2R is expressed and CD44 is lost. This occurs rapidly, over about 3 days, preceding expression of CD4, CD8, and T cell receptor. Reexpression of CD44 on more mature CD8+ and CD4+ single-positive thymocytes of fetal and new- born mice shows the strain variation described above (Lynch and Ceredig, 1989).

In peripheral T cells of the mouse, consistent with the differences in CD44 expression in the thymus, all BALB/c T cells are detectably CD44+ at some level, whereas AKR/J and C57BL/6 T cells contain a CD44-negative subpopulation (Lynch and Ceredig, 1989; Lee and Vitteta, 1991; Lesley and Hyman, 1992). Recent thymus emigrants in the periphery of AKR/J mice are heterogeneous in CD44 expression (our unpublished results, 1992) and the thymus is the source of CD44- negative peripheral T cells in C57BLi6 mice (Budd et al., 1987b); thus CD44 expression does not appear to be a factor in determining emigra- tion from the thymus.

In the human fetal thymus, a pattern of CD44 expression similar to that described in the mouse is observed (Horst et al., 1990b). The earliest lymphoid immigrants to the thymus express CD44 at relatively high levels. CD44 is then downregulated or lost during early differen-

IL-2R- + CD44+/IL-2R+ + CD44-/IL-2R+ + CD44-/IL-2R-, fol-


tiation and reexpressed later. Thus most cortical thymocytes are CD44-, whereas medullary thymocytes are CD44+ (Isacke et al., 1986; de 10s Toyos et al., 1989; Kansas et al., 1989; Horst et al., 1990b). The first lymphocytes to appear in fetal lymph nodes are CD44+ T lymphocytes. As lymph node (LN) organization is completed with delineation of B and T cell areas (around 19 weeks of gestation), all lymphocytes are CD44+ (Horst et al., 1990b). Later in LN development and in the adult, LN lymphocytes are strongly CD44+, except for germinal center B cells, which are CD44'" to negative (Kansas et al., 1989; Horst et al., 1990b). In human peripheral blood also, most lymphocytes are CD44+ (Isacke et al., 1986; de 10s Toyos et al., 1989; Kansas et al., 1989; Horst et al., 1990b).

B lineage expression of CD44 in human bone marrow cells was studied by using the expression of CDlO to identify immature pre-B cells and CD20 to identify mature B cells (Kansas and Dailey, 1989). CD44 expression was low on immature B cells and upregulated on mature B cells. B cell progenitors were not examined in this study, because they are a minor subpopulation of bone marrow cells, (but see Section IV,B,4). A large panel of non-Hodgkin's lymphomas and lymphoid leukemias thought to represent a complete spectrum of T and B cell development shows a pattern of expression similar to that seen in normal development, with the most immature stages expressing high CD44 levels, immature cells showing low or negative CD44 expression, and mature cells again expressing relatively high CD44 levels (Horst et al., 1990a).

From these studies in both mouse and human the general conclusion emerges that CD44 is expressed at relatively high levels during early stages of lymphoid development. At immature stages, the CD44 antigen is temporarily lost from the cell surface to be reacquired later in maturation.

CD44 expression in human bone marrow during myeloid and erythroid differentiation was studied by using a panel of cell surface markers to identify different stages of maturation in these lineages (Kansas et al., 1990). In agreement with studies in the mouse, lineage negative undifferentiated cells, presumed to be very early progenitors, were high in CD44 expression. Subsequent changes in CD44 expression showed different patterns in monocyte, granulocyte, and erythroid lineages. In another study, human bone marrow cells were separated by fluorescence-activated cell sorting into CD44hi, CD44m"d, and CD44l" subpopulations and assayed for myeloid and erythroid progenitor activity. Granulocyte-macrophage colony- forming unit and erythroid burst-forming unit precursors were pre-


dominantly in the CD44hi fraction (Lewinsohn et al., 1990). It has been suggested that CD44 is a general hematopoietic adhesion molecule, mediating interactions with bone marrow stromal cells (Kansas et al., 1990; Lewinsohn et al., 1990; Miyake et al., 1990a), which are thought to be important in the survival and differentiation of hematopoietic progenitors (Dexter et d., 1977; Whitlock and Witte, 1982).

2. CD44 Expression on Memory I’ Cells and Activated T Cells

In C57BL/6 mice, which express low numbers of CD44+ T cells in the thymus and periphery (Lynch and Ceredig, 1989), memory cytotoxic T cell precursors, elicited in response to several different antigens, have been shown to be almost exclusively in the CD44+, CD8+ population of peripheral T cells (Budd et al., 1987a,b). Before immunization, alloresponsive CTLp (cytotoxic T lymphocyte precursors) were found equally in both CD44+ and CD44- populations of CD8+ cells. Eight to 12 weeks after immunization, the frequency of allospecific CTLp increased only in the CD44’ population and, unlike the responding cells of unimmunized mice and responding CD44- cells of immunized mice, the cytotoxicity of these “memory” CD44’ cells was not inhibited by CD8-specific antibody, indicating a higher avidity for the target antigen (Budd et al., 1987a). For nonallogeneic antigens, to which no responding cells were detectable in unimmunized mice, 8- to 30-fold higher frequencies of antigen-specific CTLp were found in the CD44’ population than in the CD44- population of CD8+ cells of immunized mice (Budd et al., 1987a,b). These results indicate that elevated CD44 expression is stably acquired as a result of antigenic stimulation in viuo. It has also been shown that murine T cells expressing low levels of CD44 upregulate CD44 expression on stimulation in vitro (Budd et al., 1987b; Lynch et al., 1987). As pointed out by Lynch and Ceredig (1989), the usefulness of CD44 as marker for “memory” or previously activated T cells is limited to mouse strains that express low numbers of CD44+ cells in mature thymus and peripheral T cell populations.

Elevated CD44 expression also occurs in helper (CD4+ ) memory T cells of C57BL/6 mice, defined by functional responses to antigen and by coordinate expression of high CD44 levels with other markers for memory T cells such as low CD45RB and low Mel-14 expression (Butterfield et al., 1989; Swain et al., 1990; Lee and Vitteta, 1991). Furthermore, this “memory” population secretes a different array of lymphokines, characteristic Of Th2 helper cells, on in uitro stimulation as compared to naive helper cells (Swain et al., 1990,1991; Vella et al., 1992). When resting CD44hi and CD.44’” helper T cells (CD4’ ) were


compared for DNA, RNA, and protein content, Stout and Suttles (1992) concluded that CD44hi memory T cells (also defined by high levels of asialo-G, and low levels of CD45RB) are maintained in G1 (but not necessarily cycling) rather than resting out of cycle in Go, as were CD44’” CD4+ cells. This conclusion was based on the higher mean RNA content, higher mean total protein content, and stronger responses to in uitro stimulation for CD44hi cells as compared to CD44’” cells.

Both CD8+ and CD4+ peripheral T cells of the CD44’” or -negative phenotype are gradually depleted following adult thymectomy of C57BL/6 mice (Budd et al., 1987b; Swain et al., 1990), whereas CD44”’ T cells are depleted after a course of treatment with anti-mouse thymocyte serum given intraperitoneally, which preferentially affects circulating T cells (Swain et al., 1990). Furthermore, T lymphocytes expressing high levels of CD44 accumulate with age in mice (Lerner et al., 1989) and in the abnormal T cell populations of mouse strains that develop lymphoproliferative disease (Davidson et al., 1986; Budd et al., 1991). All these data support the proposal that the thymus is the source of CD44’” or CD44- T cells (depending on the mouse strain), and that stimulation in the periphery results in stably elevated CD44 expression. Differences in adhesion receptor expression between previously activated and naive T cells are thought to contribute to the different recirculation patterns (MacKay et al., 1990) and to the different endothelium-binding specificities (Pober and Cotran, 1991) of these two cell types.

Two studies directly demonstrate the participation of stimulated cells expressing high levels of CD44 in immunological responses in uiuo. Mobley and Dailey (1992) detected the appearance of a minor Mel-14-negative, CD44hi population of CD8’ cells in LN draining an allograft. This population contained all detectable cytolytic activity. A similar Mel-14-negative, CD44hi population represented the majority of CD8+ cells in a grafted sponge matrix containing allogeneic cells, and the adhesion molecules LFA-1 and ICAM-1, were also elevated on this population. Rodrigues et al. (1992) employed adoptive transfer of antigen-specific cloned cytolytic cell lines to study the requirements for a protective response against malaria infection. Two sets of clones, which did not differ in epitope fine specificity or ability to lyse target cells in vitro, were either “protective” or “nonprotective” in naive mice injected with malaria sporozoites. High expression of CD44 and VLA-4 correlated with in uiuo protective function, and the importance of CD44 expression was further substantiated by sorting CD44hi and CD44’” subpopulations from a partially protective clone. Sorted


CD44hi cells were protective, whereas CD44'" cells were not. Histo- logical studies of infected liver revealed that injected radiolabeled protective cells were found closely associated with parasitized hepatocytes, whereas nonprotective cells, although present in equal numbers in the liver, were not in close contact with infected hepatocytes. This is the most direct demonstration that CD44 participates in the effector stage of an immunological response.

Studies of human lymphocytes also suggest that elevation of CD44 expression is a consequence of activation. "Memory" T cells, recognized by their high expression of LFA-3, LFA-1, and CD2 and by their absence in fetal umbilical cord blood, showed a two-fold elevation in CD44 expression compared to naive cells, and this population gave enhanced responses to soluble antigen in uitro relative to the adhesion receptor-low population (Sanders et nl., 1988). In vitro stimulation of human T cells induced a two-fold increase in CD44 expression and induced increased adhesion to and migration on lymphokine-activated HUVEC (human umbilical vein endothelial cells), which was partially inhibited by mAb (Hermes-3) against CD44 (de 10s Toyos et al., 1989; Oppenheimer-Marks et al., 1990). Haynes et al. (1991) found that CD44 expression was high on lymphocytes and macrophages in synovial tissue of rheumatoid arthritis patients. Two studies have presented evidence that higher molecular weight isoforms of CD44 are transiently expressed in response to antigenic stimulation in uiuo in rats (Arch et al., 1992) and in human T cells in response to phorbol ester or anti-CD3 plus IL-2 stimulation in vitro (Koopman et al., 1993).

In studies of macaque lymphocytes, Willerford et al. (1989) concluded that activated lymphocytes were CD44hi, expressing 5- to 10- fold more CD44 than CD44l" cells, and that CD44'" lymphocytes upregulated CD44 during in vitro activation. They suggest that CD44hi and CD44l" lymphocytes follow different migratory pathways in the circulation, because splenic and peripheral blood cells were mostly CD44hi, whereas recent emigrants from lymph node and Peyer's patches, collected from the thoracic duct, were predominantly CD44'".

B. PHYSIOLOGICAL EXPERIMENTS IMPLYING A ROLE FOR CD44 IN

1 . Relationship to Hermes Antigen Mediating Peripheral Lymph

Lymphocyte migration into secondary lymphoid tissues occurs via adhesion to a specialized endotheliurn identified histologically as high endothelial venules (HEV) in lymph nodes and Peyer's patches (Woodruff et aZ., 1987). Stamper and Woodruff (1976) developed an in

LYMPHOCYTE HOMING AND HEM.4TOPOIESIS

Node Adhesion


uitro assay to measure the capability of circulating lymphocytes to specifically recognize and bind to HEV. In this assay, lymphocyte populations are incubated with frozen sections of lymphoid tissue. After washing off unbound cells, cells adhering to the high endothelium of blood vessels are quantitated microscopically. Using this assay, Mel-14 mAb, which recognizes murine L-selectin (Lasky et al., 1989), was identified on the basis of its ability to inhibit murine lymphocyte and lymphoid cell line adhesion to HEV (Gallatin et al., 1983). The Hermes antigen of human lymphocytes was identified by cross- reactivity with Mel-14 mAb and given the designation “lymphocyte homing receptor” (Jalkanen et al., 1986), but antibodies raised against Hermes antigen were later found to recognize human CD44 (Gallatin et al., 1989; Picker et al., 1989b; Stefanovd et al., 1989; St. John et al., 1990). One of the Hermes-specific antibodies, Hermes-3, and a polyclonal antiserum produced against the isolated antigen blocked adhesion of human lymphocytes to frozen sections of HEV (Jalkanen et al., 1987), as Mel-14 does in the murine system. Pals et al. (1989a) found that two other mAbs specific for human CD44 caused partial inhibition of lymphocyte HEV binding. It is surprising that different cell adhesion molecules appear to mediate this activity in different species, yet this seems to be the case: Culty et al. (1990) showed that binding of murine cells to HEV is not sensitive to hyaluronidase or to mAb known to block HA binding by CD44. In the mouse, carbohydrate ligands other than HA have been shown to mediate adhesion via Mel-14 (Imai et al., 1990). Thus there is no evidence that CD44-HA interactions participate in lymphocyte adhesion to HEV in the mouse, although they can mediate adhesion to cultured endothelial cell lines (Lesley et al., 1992; Uhlig et al., 1993).

Hermes-3 mAb, which blocks human lymphocyte adhesion to frozen sections of HEV, does not block HA-dependent adhesion of a B cell line transfected with human CD44 to cultured rat endothelium (Stamenkovic et al., 1991) or [ 3H]HA binding by detergent extracts of human CD44’ cells (Culty et al., 1990). This is consistent with the report that the determinant recognized by the Hermes-3 mAb maps to the membrane-proximal portion of the external domain of CD44 (Gold- stein et al., 1989), whereas the predicted HA-binding sequences are near the amino-terminal, membrane-distal portion of the molecule (see Section 111). These results imply that Hermes-3-sensitive binding of human lymphocytes to HEV comprises a CD44-ligand interaction distinct from the CD44-HA interaction described in Section 111. However, because the role of HA in CD44-mediated binding of human lymphocytes to HEV in the frozen section assay has not been tested,


other explanations are possible. Hermes-3 mAb could indirectly affect CD44-mediated HA binding in normal lymphocytes, for example, by interfering with CD44 interaction with other molecules on the cell surface involved in regulating its HA-binding function (see Section V). Such regulatory interactions may not occur in the transfected B cell system studied by Aruffo et at. (1990) or in the solubilized receptor- [ 3H]HA-binding assay of Culty et al. (1990).

In conclusion, it has not been shown, in either murine or human systems, that CD44-mediated binding to HA is involved in the proposed “homing receptor” activity of CD44, defined experimentally by CD44-dependent lymphocyte binding to HEV in the frozen section assay (Jalkanen et al., 1987). However, CD44-mediated adhesion of lymphocytes to cultured endothelial cells and cell lines, and to stromal cell lines, has been shown to involve HA binding (Aruffo et at., 1990; Stamenkovic et al., 1991; Miyake et al., 1990b; Lesley et al., 1992), indicating that CD44 binding of HA may be involved in lymphocyte- endothelial interactions under certain circumstances.

2. In Vivo Role of CD44 in Lymphocyte Migration

The adhesion of lymphocytes to HEV in frozen sections of lymphoid tissue in uitro is thought to reflect in uiuo interactions of circulating blood cells with specific endothelial structures that determine selective entry of cells into distinct organ:;. In mice, extravasation of lymphocytes has been studied in uiuo by intravenous injection of radiolabeled lymphocytes and determination of the distribution of injected cells by counting radioactivity in various tissues. Uhlig and colleagues (1993) found no effect of Fab fragments of CD44-specific mAb on gross localization of labeled mature lymphocytes one hour after injection into mice. In similar experiments, LFA-l-specific mAb and Mel-14 mAb specific for L-selectin, which inhibits murine lymphocyte adhesion to HEV in frozen section assays, did inhibit localization in LN (Gallatin et al., 1983; Hamann et al., 1988, 1991). Injection of CD44- specific antibody in uiuo causes loss ofCD44 from the lymphocyte cell surface (Camp et al., 1993b; see Section V,C,4). Camp et al. (1993b) found that peripheral L N lymphocytes, stripped of cell surface CD44 by in uiuo exposure to CD44-specific mAb, entered lymphoid organs normally. These studies argue against a role for CD44 in the extravasation of lymphocytes into lymphoid organs during normal trafficking in the mouse.

Removal of cell surface CD44 by exposure to CD44-specific mAb, however, did result in inhibition of edema and leukocyte infiltration at a site of cutaneous delayed-type hypersensitivity 24 hours after chal-


lenge (Camp et al., 1993b). Subsequently, a normal inflammatory response developed with the infiltrating cells reexpressing CD44. Even in these mice, which were responding to immunological challenge, there was no effect of CD44 mAb treatment on the number of lymphocytes in draining LN, although the majority of these lymphocytes remained CD44 negative at 72 hours postchallenge. The authors suggest that CD44 is required for extravasation into an inflammatory le- sion involving nonlymphoid tissue, but not for normal leukocyte recirculation through lymphoid organs.

3. Inhibition of Migration of Thymocyte Progenitors

CD44 was shown to be present on pluripotent bone marrow stem cells that give rise to multilineage 10-day spleen colonies and on the bone marrow prothymocytes that populate the thymus (Trowbridge et al., 1982). Cytotoxic depletion of bone marrow, using CD44-specific mAb and complement, dramatically reduced these activities in irradiated, bone marrow-repopulated mice. The possibility that CD44 might be involved in homing of these two progenitors was suggested by the fact that incubation with mAb alone was as effective at inhibiting repopulation as incubation with mAb and complement. Similarly, repopulation by a thymus homing progenitor resident in the thymus whose progeny can be detected in the thymus at 12-15 days postirradi- ation was prevented by pretreatment of progenitor cell enriched populations with CD44-specific mAb alone (Lesley et al., 1985a). It is possible, however, that antibody acted to remove these progenitors from the circulation via opsonization and phagocytosis in the liver rather than by blockade of a homing receptor, because the experiments used intact antibody (IgG2, and IgGZb) rather than Fab antibody fragments.

4 . Inhibition of Hematopoiesis in Vitro by CD44-Spec$c Antibodies

Monoclonal antibodies to CD44 were found to inhibit B cell lym- phohematopoiesis completely in long-term bone marrow cultures (Mi- yake et al., 1990a). The antibodies used in these experiments were initially prepared and selected on the basis of their ability to inhibit adhesion of a B lineage hybridoma to a cloned stromal cell line. Fur- ther definition of that model revealed that CD44 on the lymphoid cells was recognizing HA on the surface of stromal cells (Miyake et al., 1990b). However, the actual mechanism of inhibition of long-term cultures remains uncertain. Addition of CD44-specific antibodies to cultures that were already well established had no effect, whereas antibodies to either VCAM-1 or VLA-4 dislodged B lymphocyte pre-


cursors from the stroma at that time (Miyake et al., 1991). This indicates that CD44 and its ligands are important for an early step in culture initiation, but not for later interactions of relatively abundant lympho- hematopoietic progenitor cells with the microenvironment. Because of the ubiquitous nature of CD44 and the complexity of in vivo experiments, the role of this molecule and its ligands in normal lymphohe- matopoiesis remains an open issue.

5. Signaling through CD44-Ligand Znteractions

A number of studies have shown that treatment of human T cells with antibodies specific for CD44 in conjunction with other activation signals can result in either enhancement or inhibition of lymphocyte responses (see Table 11). These experiments are believed to mimic possible effects of engagement of CD44 with its ligand on stimulation through other receptor-ligand interactions, such as T cell receptor binding to antigen. On the basis of observed stimulatory effects, it is concluded that interaction of CD44 with an unknown ligand on endothelial cells or antigen-presenting cells may enhance the ability of circulating T cells to respond to antigen-specific signals. This conclusion is indirect, and a few points should be noted about these experiments: CD44-specific mAbs alone usually do not elicit any activity; not all CD44-specific mAbs have the described activities; the primary stimuli being influenced by CD44-specific mAbs are often suboptimal; some of the effects seen are dependent on the presence of monocytes in peripheral blood lymphocyte (PBL) cultures, so different results are obtained with purified T cells.

Denning et al. (1990) suggested that CD44 may enhance T cell activation by increasing T cell-monocyte interactions through stimulation of LFA-l/ICAM-mediated or CD2/LFA-S-mediated adhesion. It has been shown that CD44-specific mAb treatment of human T lymphocytes can induce LFA-l-mediated cell aggregation (Pals et al., 1989a; Koopman et al., 1990). Also, overexpression of baboon CD44 in murine fibroblasts resulted in increased spontaneous cell aggregation that probably did not involve LFA-1 (St. John et al., 1990).

Only a few studies, involving myeloid lineage cells, have examined responses of hematopoietic cells to HA. Hiro et al. (1986) found that soluble HA stimulated IL-1 production by cultured human monocytes and rabbit macrophages. Although CD44 was not directly implicated in this study, the specificity of the reaction for HA, but not other glycosaminoglycans, suggests that CD44 may be the receptor. More recently, Noble et al. (1993) showed that murine bone marrow-derived macrophages are stimulated by HA to synthesize mRNA for IL-lp,

TABLE I1 In Vitro EFFECTS OF CD~~-SPECIFIC MONOCLONAL ANTIBODIES ON HUMAN PERIPHERAL BLOOD LYMPHOCYTE RESPONSES

CD44-specific mAb Costimulation Effect (result) Ref.

H90 and Fab of H90

NIH-44.1

A3D8

A3D8

Fab of A3D8 212.3

Fab of 212.3 9F3

Suboptimal, CD2-mAb pairs Immobilized CD&mAb Suboptimal, CD2-mAb pair Suboptimal, immobilized CD3-mAb None None

Suboptimal, CD3-mAb Suboptimal, CD2-mAb pair High levels, CD2-mAb pair Suboptimal, CD2-mAb pair Optimal, immobilized CD3-mAb

Suboptimal, CD2-mAb pair PHA, PMA" Optimal, immobilized CD3-mAb None

F(ab')e of 9F3 None NIH-44 Immobilized, CD3-mAb

CD4-mAb + tumor cells s5 None FabofS5 None 8B2.5 Submitogenic PMA

Immobilized CD3-mAb CDZ-mAb

Immobilized None 8B2.5 None

Increased T cell proliferation Increased T cell proliferation, induced ILPR expression Increased T cell proliferation Increased T cell proliferation Partial inhibition of E rosettes Inhibition of T cell E rosettes

Increased T cell proliferation Increased T cell proliferation (monocyte dependent) Increased T cell proliferation (monocyte independent) Increased T cell proliferation (monocyte dependent) Inhibited T cell proliferation, IL-2 production, IL-2R expression, CaZ+

flux Increased T cell proliferation No effect Inhibited T cell proliferation Redirected CTL-mediated lysis to antigen-negative, Fc-receptor+ target Inhibited CTL-mediated lysis of antigen+ target Stimulated lymphokine production by IL-2-cultured T cells (LAK-T cells) Inhibited lymphokine production by LAK-T cells Enhanced NK lysis of NK targets Enhanced NK lysis of NK targets Increased T cell proliferation Increased T cell proliferation Increased T cell proliferation Induced monocytedependent T cell proliferation Induced monocyte-independent T cell proliferation

Huet et al. (1989)

Shimizu et al. (1989)

Hale and Haynes

Denning et al. (1990) (1989)

Rothman et al. (1991)

Seth et al(l991)

Chong et al. (1992)

Tan et al. (1993)

Pierres et 01. (1992)

a PHA, Phytohemagglutinin; PMA, phorbol myristate acetate.


tumor necrosis factor a (TNF-a) and insulin-like growth factor 1 (IGF-1) and to secrete IGF-1. This activity was inhibited by CD44- specific mAb, suggesting that a CD44-HA interaction on the macrophage cell surface might initiate a cytokine cascade. Stimulation of macrophages with CD44-specific mAb can result in release of cy- tokines: TNF, IL-1, and macrophage colony-stimulating factor (M-CSF) (Webb et al., 1990; Gruber et al., 1992). Hyaluronan has also been shown to stimulate neutrophil migration in Boyden chamber assays, in conjunction with fibronectin (Hakansson and Venge, 1985), and this could be mediated by CD44, which is abundant on neutrophils.

V. Regulation of Interaction of CD44 with Extracellular Matrix

A. EVIDENCE THAT HYALURONAN BINDING Is REGULATED 1 . Cell Lines

Evidence that the ECM receptor function of CD44 is regulated first came from studies of HA binding by murine hematopoietic cell lines expressing CD44. Although some CD44-positive cell lines bind HA in a CD44-dependent manner, as demonstrated by the inhibition of HA binding with certain CD44-specific mAb, many CD44-expressing cell lines do not bind HA (Table 111; and Lesley et al., 1990a; Miyake and Kincade, 1990). All of these cell lines express the hematopoietic isoform of CD44. Hyaluronan receptor activity is not masked by endogenous HA in these cell lines, because treatment with hyaluronidase or chondroitinase ABC does not reveal HA-binding function (Lesley et al., 1990a). Some T cell lines could be induced to bind HA after culture in phorbol ester. Maximal induction required 16 hours of incubation and was accompanied by increased CD44 expression but no change in the isoform expressed (Lesley et al., 1990a; Hyman et al., 1991). It does not appear, however, that differences in the HA-binding capacity of CD44-positive cell lines can be accounted for solely by quantitative differences in the level of CD44 expressed on the cell surface: a CD44hi variant selected by fluorescence-activated cell sorting for high levels of CD44 expression did not bind HA, whereas an HA-binding variant selected from the same parental line expressed less CD44 than the CD44hi variant (Hyman et al., 1991). Several B cell lines could not be induced to bind HA by culture in phorbol ester or lipopolysaccharide. Among CD44-positive B cell lines, HA-binding activity was observed only in the most mature cell types, those secreting immunoglobulin (Lesley et al., 1990a; Miyake and Kincade, 1990, and our unpublished results, 1992).


TABLE I11 CD44 EXPRESSION AND HYALURONAN BINDING BY CELL LINES^

Induced Adhesion to CD44 HA HA immobilized

Cell type Cell line expressionb binding‘ bindine HAe

T cell AKR 1 s49 SAKRTLS 12 CD44hi variant of

SAKRTLS Ha+ variant of

SAKRTLS EL4 BW5147

C1.18 (myeloma) BM2 (hybridoma) 70Z/3 (pre-B) RAW 253 (pre-B) Sp2/0 (myeloma) XS63 (myeloma)

B cell S 194 (myeloma)

Fibroblast L cell

- + +

+

+ + + + + + +

nd nd +

- +

+ + + + + + + +

-

- + -

- -

nd nd nd nd - -

-

ndr + + +

nd nd nd nd + - + + nd

” References: Lesley et al. (1990a. 1992). Miyake and Kincade (1990), Hyman et al. (1991); and our unpublished results (1992).

CD44 expression was determined by flow cytometery with fluorescein-conjugated mAb, IM7; + indicates fluorescein staining greater than threefold over background of unstained cells.

HA binding was determined by flow cytometry with fluorescein-conjugated HA; + indicates fluorescein staining greater than threefold over background of unstained cells.

Induced HA binding was determined by flow cytornetry with fluorescein-conjugated HA and the inducing rnAb, IRAWB14; + indicates an increase in binding at least threefold over that seen with fluoresceinconjugated HA alone.

Adhesion was determined by counting radiolabeled cells bound to HA immobilized on plastic culture wells.

’nd, Not determined.

Certain CD44-specific mAbs, such as IRAWB 14 (Lesley et al., 1992), “activated” HA binding by CD44 in some T cell lines that expressed CD44 but did not bind HA constitutively (see Table 111). The term “activate” is used to signify the rapid conversion of preexisting CD44 molecules on the cell surface from a state that does not bind soluble HA to a state that does bind HA. The rapid activation of CD44 HA-binding function by mAb was clearly distinguishable from induction of HA binding observed after exposure of cell lines to phorbol ester. Phorbol ester induction occurred over several hours in culture at 37°C and was accompanied by large increases in CD44 expression, indicating cell differentiation (Lesley et al., 1990a; Hyman et al.,


1991). Activation by CD44-specific mAb occurred at 0°C and in the absence of divalent cations (Lesley and Hyman, 1992). It did not involve intracellular signaling, because it occurred immediately on binding antibody at 0°C and in cells that had been prefixed. These results suggest a direct effect of the antibody on CD44 molecules on the cell surface. Monovalent Fab fragments were inactive, indicating that crosslinking of CD44 molecules is required and suggesting that HA-binding function may involve clustering of CD44 into a multivalent structure (Lesley et al., 1993). Not all cell lines that express CD44 and fail to bind HA were activated by mAb. Pre-B lines, such as RAW 253, could not be induced to bind HA by IRAWB 14, nor could L cells (Table 111).

Some CD44-positive cell lines that did not bind soluble HA did, however, bind HA immobilized on plastic surfaces. This was true of the T cell lines that could be induced by CD44-specific mAb to bind soluble HA (see Table 111), and of a transfected T cell line expressing a mutant CD44 molecule with a truncated cytoplasmic domain (Lesley et aZ., 1992). This suggests that either (1) the immobilized ligand represents a different structure that can be recognized by CD44 on these cells whereas soluble ligand cannot, or (2) the immobilized ligand itself activates the HA-binding function of CD44, perhaps by stabilizing low-affinity interactions through clustering of CD44 molecules into multivalent receptors. The CD44-positive pre-B cell line, RAW 253, which is not inducible by IRAWB 14 mAb, does not bind immobilized HA in the presence or absence of the mAb.

Data on HA binding by human hematopoietic cell lines or cells of other species are lacking. However, several brain-derived human cell lines were able to bind HA via CD44 only if they were pretreated with hyaluronidase (Asher and Bignami, 1992). In these studies also, not all CD44' lines bound HA, although all expressed the same hematopoietic CD44 isoform. A human melanoma line sorted for high and low CD44 expression bound immobilized HA, and the extent of binding correlated with the level of CD44 expression (Birch et al., 1991).

2. Normal Hematopoietic Cells

In view of the broad tissue distribution of both CD44 and its proposed ligands, it is not surprising that receptor-ligand interactions are restricted, especially among circulating cells. Indeed, in the mouse, normal, resting hematopoietic cells that express CD44 could not be shown to bind either soluble or immobilized HA (Lesley et al., 1990a, 1992; Murakami et al., 1990, 1991; Lesley and Hyman, 1992). Hyal-


uronidase treatment of bone marrow cells, among which myeloid precursors express especially high CD44 levels, did not expose HA binding function. In uitro activation of T cells with phorbol ester, ionomycin, concanavalin A (ConA), CD3-specific antibody, and several lymphokines after both short term (20 min at 37°C) and longer term (overnight culture) treatment, and of B cells with lipopolysaccharide (overnight culture), did not induce detectable HA binding (Lesley et al., 1990a; Lesley and Hyman, 1992). A population of HA-binding B cells could, however, be induced by culture for several days in IL-5 or by a chronic graft-versus-host reaction in uiuo (Murakami et al., 1990, 1991). The HA-binding cells induced under these conditions were secreting IgM antibody. The conditions and the time required to induce these HA-binding B cells were indicative of selection and differentiation processes.

Although in uitro stimulation of normal T cells did not induce HA binding, it has been possible to demonstrate that mouse T cells expressing CD44 can bind both soluble and immobilized HA after exposure to certain CD44-specific mAbs. The extent of HA binding induced by the CD44-specific mAb IRAWB 14 in splenic T cells was proportional to the level of CD44 expressed (Lesley and Hyman, 1992), which varies among mouse strains (Lynch and Ceredig, 1989; Lesley et al., 1988) and with cell activation (see Section IV,A,2). The same antibody, however, did not induce bone marrow cells or splenic B cells to bind HA. As with T cell lines, the mAb IRAWB 14 activated HA binding on normal T cells immediately on binding to preexisting cell surface CD44 molecules in a temperature- and cation-independent manner.

These demonstrations of CD44-mediated HA binding by normal T and B cells under specific conditions suggest that CD44-dependent HA binding may occur naturally in uiuo. Yet, if this is true, the absence of binding activity among hematopoietic cells taken from normal mice indicates that CD44-mediated binding of HA must be tightly regulated. The circumstances under which the HA receptor function of CD44 may be called on in uiuo, and the mechanisms by which receptor function is activated at the appropriate time and place, are unknown. Indeed, the role of CD44-mediated ECM- binding activity in hematopoietic cell function is unknown. But the association of elevated levels of adhesion receptors, including CD44, with effector cells engaged in immunological reactions and with memory cell phenotype and the effects of CD44-specific mAb on T cell function (see Section IV) suggest that CD44 does participate in lymphocyte function.


3. Three Activation States of Hematopoietic CD44

The evidence from both normal murine lymphocytes and lymphoid cell lines suggests that there are three possible states of HA receptor function for CD44 (Lesley and Hyman, 1992, He et al., 1992): (1) nonactivatable, corresponding to resting B cells, some pre-B lymphomas, and bone marrow cells, (2) activatable (able to rapidly con- vert preexisting CD44 to function to bind HA, e.g., by IRAWB 14 monoclonal antibody), represented by resting CD44+ T cells and some T lymphomas (Lesley et al., 1992), and (3 ) constitutively active, or able to bind HA without activation, represented by some B and T cell lines (Table 111) and IgM-secreting B cells induced by IL-5 in vitro (Murakami et aZ., 1990) or graft-versus-host reaction in uiuo (Murakami et al., 1991). The conversion of CD44-expressing cells from one HA receptor state to another may be a relatively slow process involving maturation/differentiation, or it may be rapid, as in the conversion of activatable cells to HA-binding function by CD44- specific mAb. A survey of B lineage cell lines “frozen” in different stages of differentiation (Coffman, 1982) indicates a progression from state 1, to state 2, to state 3 during differentiation from pre-B to immunoglobulin-secreting cells (our unpublished results, 1992). Among normal B cells, only nonactivatable (state 1; Lesley and Hyman, 1992) and constitutively active (state 3; Murakami et al., 1990, 1991) states have been observed. Resting T cells that express CD44, on the other hand, were activatable (state 2), although their ability to bind HA once activated was also a function of the level of CD44 expression (Lesley et al., 1992; Lesley and Hyman, 1992). The activatable state could allow rapid and reversible engagement of CD44-HA interactions under specific circumstances. As noted above (see Section V,A,2), HA binding in normal T cells was not activated by a number of reagents that induce or mimic stimulation through the T cell receptor and that have been shown to increase ligand binding by other adhesion molecules (see below, Section V,B). Thus the physiological inducers of CD44 activation in T cells are unknown. Possible mechanisms are discussed below (Section V,C).

4 . Control of Regulation of CD44 Function by Cellular Environment

Differences among cells in the HA-binding function of CD44 imply that this function is closely regulated by the cell. The best evidence that the cellular environment influences the ECM-binding function of CD44 comes from the transfection of identical CD44 constructs into different cell types (see Table IV). When the CD44-


TABLE IV POSITIVE AND NEGATIVE REGULATION OF HYALURONAN BINDING IN TRANSFECTED

CELL LINESO

CD44 expression

HA InducedHA Parental cell line Transfection CD44.1 CD44.2b binding bindingd

AKRl (CD44.2-) None Wild-type

CD44.1e ACYf ANCg MI isoform

(variant F)” M2 isoform

(variant G) M3 isoform

(variant I) M4 isoform

(variant J) L cells (CD44.2+ ) None

Wild-type CD44.1

Wild-type CD44.2

Wild-type CD44.2

S49 (CD44.1-) None

RAW 253 (CD44.1+) None

- + + + +

+ + + - + - -

+ +

References: Lesley et al. (1992); He et al. (1992); and our unpublished results (1992). ’ Expression of CD44 alleles was determined by flow cytometry with fluorescein-conjugated allele-specific mAb, RAMB44 for CD44.1 and C71/26 for CD44.2; + indicates fluorescein staining greater than threefold over background of unstained cells.

Hyaluronin binding was determined by flow cytometry with fluorescein-conjugated HA; + indicates fluorescein staining greater than threefold over background of unstained cells.

Induced HA binding was determined by flow cytometry with fluorescein-conjugated HAand the inducing mAb, IRAWB14; + indicates an increase in binding ofat least threefold over that seen with fluorescein-conjugated HA alone.

wt, wild-type CD44, “hematopoietic” or “standard’ form. JACY is a transfectant expressing a mutant CD44.1 molecule lacking all but the first six amino

acids of the cytoplasmic domain (Lesley et ol., 1992). 8 ANC is a transfectant expressing a mutant CD44.1 molecule lacking amino acids 162 to 244 of the

membrane-proximal region of the external domain (He et al., 1992). M1-M4 isoforms are transfectants expressing CD44.1 isoforms with inserted sequences in the

membrane-proximal region of the external domain, corresponding to splice variants F, G, I, and J illustrated in Fig. 2 (He et al., 1992).


negative murine T cell lymphoma AKRl (genotype CD44.2) was transfected with the hematopoietic form of CD44.1 cDNA, the result- ing transfectants were able to bind HA as measured in several different assay systems (Lesley et al., 1992). This cell line was also permis- sive for HA binding when it expressed higher molecular weight CD44 isoforms (He et al., 1992). We have also transfected a construct coding for the hematopoietic form of CD44.1 into L cells. Although L cells express endogenous CD44.2, they do not bind HA and cannot be induced to bind by mAb (Table 111). The transfected CD44.1 construct was expressed at levels equivalent to the endogenous CD44.2, but it did not confer HA-binding function on L cells (Table IV). Simi- larly, a construct coding for the CD44.2 hematopoietic form was transfected into a CD44-negative (CD44.1 genotype) T lymphoma, S49, and into the CD44.l-positive pre-B lymphoma RAW 253, which does not bind HA in either the absence or presence of the inducing mAb IRAWB 14. The S49 transfectants acquired CD44-dependent HA-binding function, but the RAW 253 transfectants remained unable to bind HA (Table IV). It is noteworthy that the transfected CD44 molecules expressed in L cells and RAW 253 cells, which were identified and immunoprecipitated with allele-specific mAb (Lesley and Trowbridge, 1982), were of the same molecular weight as the endogenous CD44 molecules, indicating that they were subject to the same posttranslational modifications. The molecular weight of the CD44.2 molecule expressed in RAW 253 was different than that of the same construct expressed in S49 cells, indicating differences in the posttranslational modification of CD44 in the two cell types (our unpublished results, 1992).

B. PARALLELS WITH REGULATORY MECHANISMS OF OTHER ADHESION MOLECULES

The failure of many CD44-expressing cells to bind HA, and the ability to rapidly activate HA binding under specific conditions, suggests that CD44 is a member of a growing list of cell adhesion molecules that require activation to bind ligand most efficiently. Indeed, the ability to rapidly and transiently increase ligand-binding affinity may be a general property of leukocyte adhesion molecules (Dustin and Springer, 1991; Spertini et al., 1991). This list includes many of the integrins (see Dustin and Springer, 1991; Hynes, 1991) and L- selectin (Spertini et al., 1991). These adhesion systems exhibit regulatory features that are similar to the CD44-HA interactions we have described: they exhibit transitions in ligand-binding function on cell


differentiation and activation, and some show rapid activation by certain receptor-specific mAb.

1. Platelet gpIIb-IIIa (all&) lntegrin

Best studied is the platelet integrin gpIIb-IIIa, which on resting circulating platelets does not bind soluble ligands, although it can bind immobilized fibrinogen. Soluble fibrinogen could be bound after platelet cell activation by phorbol ester or physiological agonists, and this activation involved intracellular signaling (Shattil and Brass, 1987). Thus the activated platelet is able to modulate the affinity of the integrin receptor for its ligand from within. Receptor activity could also be induced by certain mAbs that bound the integrin (O’Toole et al., 1990) and by binding of ligands (Du et al., 1991). Solubilized integrin could be activated to bind soluble ligand by mAb as a monovalent Fab fragment (O’Toole et al., 1990), and the activated integrin could be specifically recognized by certain mAbs that did not bind integrin on resting cells (Shattil et al., 1985). On the basis of these and other observations, platelet integrin activation is thought to involve a conformational change in the molecule. The cytoplasmic domain of gpIIb-IIIa has been implicated in controlling transitions between activation states (O’Toole et al., 1991).

2. LFA-1

The a ~ & integrin (LFA-1) on T lymphocytes participates in the interaction between T lymphocytes and antigen-presenting cells and between T lymphoctyes and target cells, only if activated through stimulation of the T cell receptor or CD2 (Dustin and Springer, 1989; van Kooyk et al., 1989). As in the case of CD44 activation, an LFA-1- specific mAb, NK1-L16, could activate LFA-1-mediated adhesion in some T cells (Keizer et al., 1988); however, unlike the CD44 case, in which phorbol esters failed to activate HA binding in normal T cells, phorbol ester treatment did induce rapid LFA-1 activation. Figdor et al. (1990) suggested three activation states of LFA-1 on human PBLs: (1) inactive LFA-1, on resting PBLs that express little or none of the LFA-1 epitope recognized by NK1-L16, (2) intermediate or activatable LFA-1, expressing the NKl-L16 epitope but not LFA-1 adhesive activity, and (3) active LFA-1, which mediates adhesion. The authors proposed that progression from the resting to the activatable state involves maturation and perhaps Ca2+-dependent clustering of receptors (Figdor et al., 1990). As with the platelet receptor gpIIb- IIIa, it is thought that cells regulate the activity of LFA-1 through


intracellular interactions with the cytoplasmic domain of the integrin (Hibbs et al., 1991).

3. Mac-1 /CR3

The a& integrin on monocytes and neutrophils, also referred to as CR3 (complement receptor 3) or Mac-1, was the first integrin found to be activatable. On monocytes and neutrophils, the complement receptor function could be rapidly activated by phorbol esters (Wright and Silverstein, 1982) and this was thought to involve receptor clustering (Detmers et al., 1987). A change in receptor conformation upon activation to bind fibrinogen could be detected by a mAb specific for the activated state (Altieri and Edgington, 1988) and was dependent on extracellular calcium (Graham and Brown, 1991). Although cations are thought to play a role in activation of most of the integrins, there is no evidence for any such requirement for CD44 function.

4 . L-Selectin L-Selectin, a member of the family of adhesion receptors contain-

ing a lectin-like domain, expressed on lymphocytes and neutrophils, was activated by lineage-specific stimuli to increase its affinity for a carbohydrate ligand (Spertini et al., 1991). Lymphocyte L-Selectin was activated by crosslinking of T cell receptor or CD2, whereas L-Selectin activity on neutrophils was stimulated by G-CSF, GM- CSF, and TNF-a. These transitions occurred in minutes and without changes in the level of receptor expression. L-Selectin-mediated binding of murine lymphocytes to HEV was also increased by these treatments.

C. POSSIBLE MECHANISMS FOR REGULATING CD44

So far, we have presented evidence that CD44 on hematopoietic cells can exhibit three states of HA-receptor funtion. The functional state of CD44 in a particular cell type is related to the cell type and its state of differentiation (Tables I11 and IV, and Section V,A). The conversion between functional states may take place through differentiation and thus involve the synthesis of an array of new molecules that could influence CD44 function, or it might take place by rapid activation of preexisting CD44 molecules, as is seen on exposure to CD44-specific mAb. Below, we discuss several possible nonexclu- sive mechanisms by which cells might regulate the ECM receptor function of CD44: (1) interaction of the cytoplasmic domain with in-

RECEPTOR FUNCTION


tracellular proteins, such as elements of the cytoskeleton, (2) modification of the cytoplasmic domain by phosphorylation, (3) posttranslational modification of the extracellular domain by carbohydrate addition, (4) expression of alternate CD44 isoforms, (5) interaction with other cell surface molecules, (6) interaction with other extracellular ligands, and (7) masking or shedding of cell surface CD44.

1 . Regulation through the Cytoplasmic Domain and the Possible Role of Phosphorylation

Evidence for a role for the cytoplasmic domain in regulating HA binding comes from transfection experiments with CD44 constructs coding for CD44 molecules with truncated cytoplasmic domains (Lesley et al., 1992; Thomas et al., 1992). Although the wild-type hematopoietic form of CD44 binds HA when transfected into the T lymphoma AKR1, a “tailless” CD44 mutant molecule lacking all but the first six amino acids of the cytoplasmic domain does not bind HA in solution and cells transfected with this construct adhere poorly to immobilized HA (Lesley et al., 1992). Thomas et al. (1992) also found that melanoma cells transfected with mutant CD44 constructs coding for deletions in the cytoplasmic domain bound poorly to immobilized HA and did not migrate on an HA substrate, whereas transfectants expressing a wild-type CD44 construct did (see Section 111). These studies suggest two possible roles for the cytoplasmic domain of CD44: (1) an inside-out control of CD44-ligand-binding function, possibly by influencing the cell surface distribution of CD44 and/or conformation of the external domain {as has also been suggested for some integrins [see Dustin and Springer (1991) and Hynes (1992)l and for L-selectin (Spertini et u Z . 1991)]}, and (2) an outside-in signaling function, whereby cell motility or other cell functions could be activated in response to CD44 binding to ligand, possibly by engagement of the cytoskeleton or by interaction with intracellular signaling mechanisms.

The tailless CD44 construct expressed in AKRl cells does not bind soluble HA but can be activated to bind by exposure to the CD44- specific mAb IRAWB 14 (Lesley et al., 1992). As described above (Section V,A, l) , this antibody-induced activation does not involve intracellular signaling and cannot be mediated by monovalent Fab fragments (Lesley et al., 1993). This suggests that clustering of CD44 molecules into a multimeric configuration may be required for HA binding, and that the cytoplasmic domain of CD44 may influence HA binding by mediating the formation of a multimeric receptor. CD44 aggregation on the cell surface could be influenced by interaction of

3 12 JAYNE LESLEY ET AL.

the cytoplasmic domain with cytoskeletal elements or with other intracellular molecules, and this in turn might involve changes in phosphorylation of the cytoplasmic domain. It is also possible that alterations in the cytoplasmic domain could mediate a conformational change in the external domain of CD44.

Interactions I of CD44 with cytoskeletal elements, through its cytoplasmic domain, have been reported in several cell systems. In fibroblasts, Jacobson et al. (1984) related the distribution of actin stress fibers to that of CD44 crosslinked into patches by primary plus secondary antibodies. About 50% of cell surface gp85 (CD44) in BHK cells described by Tarone et al. (1984) was found to be Triton X-100 insoluble and distributed with actin filaments. Lacey and Underhill (1987) showed that a portion of the HA receptor of fibroblasts (CD44) was associated with cytoskeletal actin filaments in the Triton X-100- insoluble fraction of Swiss 3T3 cells. Bourguignon et nl . (1986) found an association between lymphoma gp85 and an ankyrin-like protein that also binds to actin and fodrin in lymphoma cells whose surface molecules were crosslinked with lectin or antibody. Kalomiris and Bourguignon (1988) later showed that purified gp85 (CD44) bound to purified erythrocyte ankyrin, and that phosphorylation of CD44 by exogenously added brain protein kinase C increased this interaction (Kalomiris and Bourguignon, 1989). Camp et al. (1991), however, found a negative correlation between phosphorylation of CD44 and its association with the cytoskeleton in macrophages isolated from the peritoneal cavity of mice. In resident macrophages, the fraction (about half) of CD44 that was associated with the cytoskeleton [1% Nonidet P-40 (NP-40) insoluble] was not phosphorylated, whereas detergent-soluble CD44 in these cells was phosphorylated. Cell activation induced a change in CD44 association with the cytoskeleton: elicited macrophages showed no association of CD44 with the cytoskeleton and, again, detergent-soluble CD44 was phosphorylated. Geppert and Lipsky (1991) also saw a reduction in cytoskeletal association of CD44 after phorbol myristate acetate (PMA) activation of human PBLs. Neame and Isacke (1992) examined the localization of CD44 in the basolateral membrane of polarized epithelial (MDCK) cells. This distribution was dependent on the cytoplasmic domain of CD44 because cells transfected with human CD44 constructs coding for molecules lacking most of cytoplasmic domain showed a diffuse distribution of human CD44, whereas cells transfected with wild- type constructs showed normal localization of the human CD44 to the basolateral membrane. This targeting presumably involves the interaction of the cytoplasmic domain of CD44 with other, intracellu-


lar molecules. Phosphorylation does not play a role in localization, however, because cells transfected with a mutant construct, in which the two serines responsible for in vivo phosphorylation in these cells were mutated to alanine or glycine, showed normal basolateral localization of the human CD44 molecules. The absence ofthe phosphorylation sites also did not affect cytoskeletal association of the mutant CD44 molecules expressed in 3T3 fibroblasts (Neame and Isacke, 1992).

Among the studies of CD44 interactions with the cytoskeleton and correlations with phosphorylation of the cytoplasmic domain, none has looked at binding of ECM components by CD44 in relation to these activities. Thus, although the association of CD44 with cytoskeletal elements seems a possible mechanism of regulating its receptor function, the data relating cytoskeletal interactions, phosphorylation, and ECM-receptor function are incomplete and, in some cases, contradictory. The apparent contradictions, could, however, be the result of the use of different experimental systems (i.e., different cell types and cell-free versus intact cell systems).

Two reports have defined intracellular molecules that influence the function of integrins, revealing additional mechanisms by which adhesion receptor-ligand interactions might be regulated from inside the cell. Hermanowski-Vosatka et al. (1992) isolated a small lipid from stimulated polymorphonuclear leukocytes that can activate ligand binding by CR3 integrin in unstimulated polymorphonyclear leukocytes and by the purified integrin. Levels of expression of this lipid in polymorphonuclear leukocytes paralleled the state of activation of CR3 function. In another study, Pullman and Bodmer (1992) used adhesion to collagen to select transfectants expressing a cDNA clone coding for a molecule that increased integrin-mediated adhesion to components of the ECM. The encoded protein of 142 amino acids had an N-terminal myristylation motif and a consensus tyrosine- phosphorylation site at the C terminus, which was required for the enhancement of adhesion. The authors suggested that this is a signal transduction molecule that contributes to integrin function. These studies indicate potential means by which the function of cell adhesion molecules might be regulated by the intracellular environment. These, or other unknown mechanisms, could modulate the HA- binding function of CD44.

2. Modijication of Extracellular Domain

Among human cell lines, a wide variety of glycosylation patterns of CD44 has been described (see Section 11; e.g., Omary et al., 1988; Brown et al., 1991; Jalkanen et al., 1988), with each cell type seem-


ing to have “decorated” CD44 with a unique array of carbohydrate structures. There is no doubt that these modifications may influence CD44 function and its interactions with the ECM.

In considering a possible role for cell type-specific glycosylation patterns in regulating CD44 interactions with the ECM, one must take into account potential effects of the use of alternate CD44 isoforms. In most of the studies of differences in glycosylation patterns, it is not certain which core proteins underlie the diversity of molecular weight patterns observed. The inserted amino acid sequences in the nonstandard (nonhematopoietic) isoforms of CD44 all contain numerous potential glycosylation sites, as do the predicted HA-binding amino-terminal region and the membrane-proximal region of the extracellular domain of “standard” CD44 (see Section 11). To address the functional role of the variant CD44 isoforms, it will be important to determine their glycosylation status in uiuo, which requires know- ing the in uiuo cellular distribution. The development of mAbs specific to the inserted domains of the different isoforms (Matzku et d . , 1989; Koopman et aZ., 1993) should soon make this problem amena- ble to analysis. There is evidence for the transient expression of alternate (nonstandard) CD44 isoforms in hematopoietic cells in response to in uiuo immunological challenge (Arch et al., 1992) and in uitro activation (Koopman et aZ., 1993). Thus, switching isoforms provides another mechanism for regulating CD44-mediated interactions with ECM. Below, we discuss several studies bearing directly on the question of alternate CD44 isoforms and the role of posttranslational carbohydrate additions in CD44 interactions with the ECM.

Fassen et u Z . (1992) found that migration of a melanoma cell line into a collagen matrix was dependent on the presence of a chondroitin sulfate-modified cell surface glycoprotein. The approximately 110-kDa core protein, remaining after chondroitinase digestion, reacted with CD44-specific mAbs, suggesting that it is probably a higher molecular mass isoform of CD44. Removal of chondroitin sulfate prevented cell migration into collagen and prevented binding of collagen by the isolated glycoprotein. Jalkanen and Jalkanen (1992) found that only a relatively minor form of chondroitin sulfate- modified standard form CD44 purified from human PBL was able to bind fibronectin. Chondroitinase treatment did not affect PBL binding to HEV in a frozen section assay, but did reduce, slightly, the binding of a B cell line to fibronectin. Thus, although interaction of chondroitin sulfate-modified CD44 with fibronectin may contribute to cell adhesion, it does not appear to be involved in CD44- dependent adhesion to HEV.


The ability of different CD44 isoforms to bind HA has been studied by transfection of constructs coding for different isoforms into cell lines (see Section 111,C). Human hematopoietic and epithelial forms of CD44 expressed in a CD44-negative Burkitt lymphoma line dif- fered in their ability to bind HA, with the epithelial form of CD44 failing to mediate adhesion to immobilized HA (Sy et al., 1991) or HA-dependent adhesion to cultured endothelial cells (Stamenkovic et al., 1991). The epithelial form of CD44 expressed in melanoma cells also bound poorly to HA, whereas the hematopoietic form conferred HA-binding function (Thomas et al., 1992). But several high molecular weight isoforms of murine CD44, including that which is homologous to human epithelial CD44, bound HA when expressed in the CD44-negative lymphoma AKRl (He et al., 1992). These clearly conflicting results could result from differences in posttranslational modifications to the inserted isoform sequences mediated by different cellular environments. Another possibility, discussed further below, is that other cell surface molecules, or even soluble cell products, may interact with the external domain of CD44 to modulate its affinity for HA. Such external factors may have specificity for different CD44 isoforms and may differ for different cell types.

3. Regulation by Interactions of the Extracellular Domain of CD44 with Other Molecules

The discovery of multiple CD44 isoforms with inserted sequences in the membrane-proximal portion of the extracellular domain (Sec- tion I1,C) gives new inpetus to the suggestion that the HA-receptor function of CD44 might be regulated by interaction with other molecules at the cell surface (Lesley et al., 1990a). Alternate sequences in different isoforms could allow interaction of CD44 with different regulatory molecules in different cell types and tissue environments, adding to the possibilities of specific control of an otherwise simple interaction between a single amino-terminal, HA-binding region and a ubiquitous ligand HA.

There is, to date, no direct evidence for such heteromolecular interactions involving CD44 and other cell surface molecules. A number of studies have examined the effects of crosslinking cell surface molecules with mAbs. Antibody cross-linking is presumed to mimic the effect of binding of a receptor to its ligand. Thus these types of studies have been used to imply possible influences of the engagement of one receptor-ligand pair on the function of other receptor- ligand interactions. Rosenman et al. (1993) have observed cocapping of CD2 and of L-selectin with CD44 capped by antibody crosslinking


in human T cells from peripheral blood. Although the observed cocapping is thought to be mediated by the engagement of the cytoskeleton, the proximity of different adhesion receptors induced by cocapping could result in interactions of their external domains. Costimulation of CD44 with cell surface molecules such as CD2 and T cell receptor of human T cells, using combinations of antibodies to crosslink these cell surface molecules, can result in enhancement or inhibition of activation signals (see Table 11). It is not known whether interactions take place between the external domains of these molecules. Nevertheless, the observations indicate that CD44 interactions with other cell surface molecules may be dynamic and may change depending on the array of ligands available. This is consistent with the activatable state of normal T cells in the mouse. In other cell types, however, such as resting B cells, CD44 might be maintained in an inactive state by more stable interactions with a different set of cell surface molecules, which would be altered only by differentiation events.

Yet another possibility is that cell surface CD44 function might be influenced by direct interaction of its external domain with soluble extracellular factors. Factors that are secreted in restricted tissue environments and/or in response to specific stimuli, such as inflammation, could be specific ligands for the alternative sequences of different CD44 isoforms. Activation of CD44 HA-binding function by extracellular soluble factors would allow rapid and flexible engagement of CD44-HA-mediated adhesion and mobility, and expression of additional isoforms by stimulated cells could increase the reper- toire of factors that might influence CD44 function.

CD44 has been shown to contribute to cell mobility on ECM sub- strates (Fassen et al., 1992; Thomas et al., 1992). A number of motility factors have been shown to influence cell migration of specific leukocyte populations. For example, members of the IL-8 chemotactic factor family induce migration in different subpopulations of neutrophils, monocytes, and lymphocytes, but the receptors for these factors are unknown (Larsen et al., 1989; Schall, 1991). One family member, RANTES, is specific for monocytes and for memory T cells (Schall et al., 1990). Another family member, MIP-1P, was shown to be present on LN endothelium and to influence adhesiveness of specific T cell subpopulations (Tanaka et d., 1993). Although there is no evidence that these factors influence CD44 function, factors of this type might be capable of directly and specifically activating CD44 by binding to sequences in its external domain (Kincade, 1992).


4 . Regulation by Shedding, Masking, and Soluble CD44

Downregulation of CD44 by shedding of the external domain is a potentially rapid mechanism of regulating CD44 expression and, hence, ECM-binding function. Loss of cell surface CD44 by shedding of a protease-cleaved fragment of the cell surface molecule has been reported for human neutrophils and lymphocytes. Campanero et al. (1991) observed downregulation of cell surface CD44 and CD43 in human neutrophils after a 30-minute exposure to TNF, PMA, calcium ionophore, and fMLP (formyl-Met-Leu-Phe). This downregulation was believed to result from proteolytic cleavage, as it was inhibited by protease inhibitors. Baiil and HoiejSi (1992) also observed downregulation of cell surface CD44 (after 12 hours) in granulocytes in response to stimulation with PMA and, to a lesser degree, ionomycin, and in both granulocytes and lymphocytes in response to immobilized or soluble antibody specific for CD44. Again, shedding was believed responsible for the loss of cell surface antigen, in this case because an '251-labeled CD44-specific antibody- reactive species of reduced molecular mass (compared to cell surface CD44) could be isolated from supernatants of '251-surface labeled stimulated cells. A number of other hematopoietic cell surface molecules have been found to be downregulated by shedding in response to external stimuli: TNF receptor (Porteau and Nathan, 1990, CD6 (Huizinga et al., 1988), CD14 (Baiil and Strominger, 1991), L- selectin (Griffin et al., 1990; Kishimoto et al., 1989), ICAM-1 (Becker et al., 1991), CD23 (Guy and Gordon, 1987), and CD32 (Sarmay et al., 1991).

A soluble form of CD44 has been found in human serum. Lucas et al. (1989) reported an In-Lu antigen-related species in serum, and Baiil and HoiejSi (1992) found two species, isolated by immunoaffi- nity chromatography, of approximately 60 to 80 kDa and 100 to 150 kDa. These two species contained different peptides when analyzed after digestion with V8 protease, suggesting that they may be products of different CD44 isoforms. The presence of soluble CD44 in serum suggests that shedding of CD44 may take place in uiuo Baiil and HoiejSi, 1992). Experiments of Camp and colleagues (1993b) demonstrated that shedding of CD44 from the surface of murine lymphocytes was induced by CD44-specific mAb administered in uiuo and resulted in a 1.5- to twofold increase in soluble CD44 in serum. Soluble CD44 may also represent another mechanism of mod- ulating the function of cell surface CD44, through competition for


ligand between soluble and cell-associated forms (Haynes et al., 1989, 1991).

The ability of CD44 to bind HA could be modified by blocking of its HA-binding sites with either cell-associated molecules or soluble HA. Asher and Bignami (1992) were able to detect CD44-mediated HA-binding in brain-derived cell lines and primary cells only after treatment with hyaluronidase, indicating that the HA-binding domain of CD44 was occupied by endogenously produced HA. Knudson and Knudson (1991) showed that accumulation of a pericellular coat around some cell types in culture was dependent on the expression of a cell surface receptor for HA. Thus, in some cell types, CD44 may function in the assembly of a protective coat and perhaps to anchor a cell in a particular location, rather than to promote migration. In hematopoietic cells, however, HA-receptor function was not exposed by treatment of cell lines or bone marrow cells with hyaluronidase or chondroitinase ABC (Lesley et al., 1990a).

VI. CD44 and Tumor Cell Migration: Possible Role of CD44 in Metastasis

A. METASTASIS AND EXTRACELLULAR MATRIX Tumor metastasis is the end result of a multistep process (Sch-

irrmacher, 1985; Fidler, 1990). As discussed in Fidler (1990) and Blood and Zetter (1990), the primary malignant neoplasm must be- come vascularized if it is to grow beyond a minimum size (several cubic millimeters). Following vascularization, the tumor cell mass penetrates the basement membrane and invades lymphatics, venules, or capillaries. Cells from this invasive tumor cell mass must then detach and enter the circulation [often forming emboli of small tumor cell aggregates, lymphocytes and platelets (Fidler, 1990; Blood and Zetter, 1990)], be transported through the circulation, and arrest in and adhere to the vessels of the target organ. The adhering tumor cells must then exit from the vasculature, penetrate the subendothe- lial basement membrane, and enter the extracellular space (Blood and Zetter, 1990). For successful metastasis to occur, the tumor cells must be able to proliferate in the new location-a process that is highly dependent on both the local microenvironment and the ability of the invading tumor cells to respond to this microenvironment (Sch- irrmacher, 1985; Fidler, 1990).

Given the complexity of the metastatic process, there has been con- siderable debate as to whether successful metastasis is the end result of the selection of a “metastatic phenotype” or whether it is a consequence of the chance survival of a tumor cell through a series of


random traumatic events (Schirrmacher, 1985; Kerbel, 1990; Aslak- son and Miller, 1992). There is good evidence that tumor cell populations show heterogeneity in metastatic potential and that tumor cells that show an enhanced ability to metastasize to specific sites preexist in the tumor cell population and can be selected, at least experimentally (Schirrmacher, 1985; Fidler, 1990; Kerbel, 1990). It is not reasonable to conclude from this fact, however, that there is a single “metastatic molecule” whose presence (or absence) can account for metastatic behavior. Independent subpopulations derived from a single tumor may fail to disseminate at different points of the metastatic process, suggesting a marked heterogeneity even within a single tumor (Aslakson and Miller, 1992). Multiple changes in tumor cell phenotype that each lead to an only moderate increase in tumor cell survival at one or another step of the metastatic process may be the difference between success or failure in terms of the ability of a given tumor to metastasize. Successful tumor metastasis might best be thought of as the end result of a sequential process containing both random and selective elements (Schirrmacher, 1985).

This complexity means that it is difficult to make overall global generalizations from experimental models. As discussed by Aslakson and Miller (1992), properties measured in uitro that appear to correlate with metastasis may not be the same properties responsible for metastatic behavior in uiuo (for which in uitro assays may not exist). Conversely, properties that are expressed in both metastatic and nonmetastatic cells may be crucially important for metastasis, but the particular nonmetastatic cells examined may all have downstream de- fects that do not allow them to complete later steps in the metastatic process and thus the importance of the property might not be appre- ciated. Also, the metastatic behavior of a particular tumor is a reflec- tion both of the properties of the cells of the tumor itself and the organ environment in which these tumor cells are placed (Fidler, 1990). Thus, the behavior of a tumor injected subcutaneously or intra- avenously by the experimenter may be different than the behavior of the same tumor in its true in uiuo site.

The necessity for metastasizing tumor cells to move through the extracellular spaces and penetrate basement membranes implies that recognition of extracellular matrix components is an essential factor mediating tumor metastasis. Many factors contibute to tumor cell adhesion and motility in the extracellular matrix. Attachment to components of the matrix via a variety of cell surface receptors (Van Roy and Marcel, 1992) may be followed by localized degradation of the extracellular matrix (Liotta, 1986; Blood and Zetter, 1990; Liotta et


al., 1991). The tumor cells then can migrate into the region of localized degradation (Liotta, 1986), using the same or a different set of adhesion receptors as those used for the initial attachment.

Hyaluronan has been implicated in tumor cell migration, and some of these migratory events may be relevant to metastasis. Tumor cells may directly secrete HA or may stimulate neighboring fibroblasts to secrete HA (Knudson et al., 1989). This secreted HA is incorporated into the extracellular matrix (Yoneda et al., 1988; Knudson and Knudson, 1991). It may function to separate tissues, affect collagen fibril formation and packing, or affect adhesive forces between the cells and their substrate (Docherty et al., 1989). In model systems in uitro, HA has been reported to enhance fibroblast mobility in mono- layers (Turley et al., 1985) and in three-dimensional collagen matrices (Docherty et al., 1989). Hyaluronan is thus likely to be involved in tumor cell migration within extracellular spaces. Presumably, increased HA synthesis could enhance migration and in this way contribute to metastasis. Hyaluronan is also present around most, but not all, basement membranes (Underhill, 1989) and thus may contribute to the movement of tumor cells into and out of the vasculature. There are some data consistent with these ideas. Hyaluronan accumulation measured in collagen matrices has been correlated with tumor progression of preneoplastic mouse mammary cells (Hitzeman et d., 1992), and the peripheral invasive areas of human breast carcinomas showed a higher content of HA than the central noninvasive areas or adjacent normal tissue (Bertrand et al., 1992). Factors produced by human mesothelioma cells stimulate HA production by normal me- sothelial cells and fibroblasts (Asplund et al., 1993). It is conceivable that enhanced HA production could act in a positive feedback loop to facilitate tumor cell growth and migration. Similarly, increased expression of CD44 could contribute to adhesion to and mobility on an HA substrate. Thomas et al. (1992) showed in an in uitro assay that transfectants expressing high levels of the hematopoietic form, but not the epithelial form, of CD44 showed mobility on HA-coated surfaces.

Degradation of extracellular matrix components (Blood and Zetter, 1990) and of HA in particular (West and Kumar, 1989) has been implicated in angiogenesis. Thus, HA might be important in the estab- lishment of the initial tumor and of metastases at distant sites by inducing the vascularization of tumor cell foci. Degradation products of the HA polymer containing approximately 4-25 disaccharides induce the formation of blood vessels on the chick chorioallantoic membrane (West et al., 1985) and induce the synthesis of new


proteins in cultures of bovine aortic endothelial cells (Kumar et al., 1987). Degradation products of extracellular matrix proteins have also been thought to be chemotactic for tumor cells (Blood and Zetter, 1990). Although there seems to be no evidence directly implicating HA as a causal agent of tumor cell chemotaxis, HA has been shown to enhance the chemotactic response of granulocytes for fibronectin (Hakansson and Venge, 1985).

CD44 has been reported to participate in the internalization and degradation of HA (Culty et al., 1992; Sampson et ul., 1992), although the rate of internalization noted in these studies is slow. CD44 has a long half-time on the cell surface (Jacobson et al., 1984), and it is not certain whether CD44 is on the endocytic pathway. CD44 does have a sequence similar to the internalization recognition motifs of constitutively recycling receptors (Trowbridge, 1991). A calcium- independent HA receptor that is thought to be distinct from CD44 and is on the endocytic pathway has been reported in rat liver sinu- soidal endothelial cells (Yannariello-Brown et al., 1992a,b). The relative contribution of this receptor, of the HA receptor studied by Tur- ley and colleagues (Hardwick et d., 1992), and of CD44 to HA degradation in tissues that are major sites of HA uptake in uitio (e.g., lymph node; Fraser et al., 1988) or in the vicinity of proliferating tumor cells remains to be determined.

As noted above (Section III,D), chondroitin sulfate-substituted forms of CD44 have been reported to bind fibronectin, collagen I, and laminin (Jalkanen and Jalkanen, 1992). Tumor cells expressing this form of CD44 might have the ability to bind to these ECM components, and it is possible that acquisition of this ability may act to enhance tumor cell migration at several steps of the metastatic process. Perhaps most interesting in this regard is the demonstration that melanoma cells bearing a CD44-like chondroitin sulfate proteoglycan show enhanced mobility in collagen gels (Fassen et al., 1992). It is not unreasonable to think that this property could contribute to the success of tumor cell invasion.

In certain in tiitro experimental situations, CD44 has been shown to confer an adhesive phenotype on cells (St. John et al., 1990; Belit- sos et al., 1990) distinct from the aggregation that occurs when CD44- positive cells are exposed to HA. It is not clear to what extent such CD44-mediated adhesion occurs in uiuo or whether it occurs at all. Nevertheless, there is at least the possibility that CD44-CD44 interactions could contribute to tumor cell survival or movement. In this connection, CD44 has been reported on vascular endothelium (Pals et al., 1989a) and umbilical vein endothelial cells (Oppenheimer-


Marks et al., 1990) and a “CD44-like” molecule has been reported on a chemically transformed cell line derived from bovine aorta (Bour- guignon et al., 1992). Thus the possibility exists that tumor cells bearing HA or CD44 itself could bind to-CD44 on the walls of blood vessels and that, therefore, CD44 could be of importance in ad- herence of tumor cells to blood vessel walls and in intravasation or extravasation of tumor cells from the circulation. This is all, however, entirely speculative and there is no experimental evidence.

B. CD44 EXPRESSION AND METASTASIS There are many reports of CD44 expression in human tumor cell

lines (Nemec et al., 1987; Picker et al., 1989a; Stamenkovic et al., 1989; Quackenbush et al., 1990; Hoffinann et al., 1991; Dougherty et al., 1991; Kuppner et al., 1992; Jackson et al., 1992; Heider et al., 1993; Koopman et al., 1993). The pattern of expression in cell lines is variable, with some cell lines expressing no CD44, some expressing only the hematopoietic form, and others expressing various higher molecular weight isoforms. It is difficult to discern a clear pattern in these studies. Some of these differences may reflect differences in CD44 expression on the tissues from which the cell lines were derived (Picker et al., 1989a; Heider et al., 1993); however, this does not seem to be the case in every instance (Heider et al., 1993). In long-term cultured cell lines, it is not always certain that the CD44 expression pattern is the same as that of the tumor from which the cell line was derived.

CD44 expression on some, but not all, primary tumors may be enhanced over the level generally seen on the corresponding normal cell type. Also, differences in the expression of isoforms may be seen when normal and tumor tissue is compared. Although no direct cause-and-effect relationship can be demonstrated, there are reports that increased levels of CD44 expression in non-Hodgkin’s lymphoma are correlated with increased tumor “aggressiveness.” Pals and colleagues (Pals et al., 198913; Horst et al., 199Oc) and Jalkanen and colleagues (1991) examined CD44 expression in non-Hodgkin’s lymphoma and found a statistically significant relationship between high levels of CD44 expression and clinical stage, tumor spread, and a poor response to treatment. A large proportion, although by no means all, aggressive non-Hodgkin’s lymphomas express an antigenic determinant encoded by exon 10, indicating that these cells express a variant isoform (Koopman et al., 1993). This variant isoform is not expressed at significant levels on resting lymphoctyes, although transient expression has been seen when lymphocytes are


activated (Arch et al., 1992; Koopman et al., 1993). CD44 has also been reported to be present on 95% of tumor cells from childhood acute lymphoblastic and acute myeloblastic leukemia (Kreindler et al., 1990). A series of primary gastrointestinal malignant B cell lymphomas showed a heterogeneous pattern of CD44 expression that did not indicate a role for CD44 in the localization of these tumors (Moller et al., 1991).

In tumors of the breast and colon, enhanced expression of CD44 variant isoforms is seen as compared to the corresponding normal tissue (Matsumura and Tarin, 1992; Heider et al., 1993). The significance of these Observations is uncertain and clearly isoform expression does not correlate with metastasis. Variant isoform expression is seen in breast and colon carcinoma with and without metastases (Matsumura and Tarin, 1992; Heider et al., 1993) and most cases of adenomatous polyps examined also show expression of variant CD44 isoforms (Heider et al., 1993). CD44 expression on a series of brain tumors was variable, with high-grade gliomas showing strong expression (Kuppner et al . , 1992). As pointed out above (Section 11), it is possible that enhanced CD44 expression and/or isoform expression in tumor cells may reflect the activation state of the cell in which the neoplastic event occurred. Clearly, relating observations on quantitative levels of CD44 expression and the expression of CD44 isoforms to the metastatic behavior of tumors in vivo requires the development of meaningful experimental models (Fidler, 1990; Rettig, 1992).

Birch and colleagues (1991) used fluorescence-activated cell sorting to isolate mutants expressing high levels of CD44 from the human melanoma cell line LT1. Stable clones could be isolated that expressed about five- to sevenfold higher levels of the hematopoietic form of CD44 relative to CD44’” clones. Compared to the clones expressing lower levels of CD44, these CD44hi clones showed an enhanced ability to adhere to immobilized HA, enhanced homotypic aggregation, and an increased ability to migrate in a “wounded” monolayer. Although both CD44hi and CD44l” clones showed a tumor incidence approaching 100% when injected subcutaneously, the CD44hi clones showed an increased capacity for lung colonization when injected intravenously (only a single cell dose was examined in these experiments). The mechanism responsible for this enhanced lung colonization is uncertain; however, HA has been shown to b e present in perivascular areas of the lung (Underhill, 1989), whereas CD44 is present in alveolar macrophages (Green et al., 1988; Under- hill, 1989) and on alveolar lining epithelium (Picker et al., 1989a).

Sy et u1. (1991) examined the rate of tumor growth and metastatic


ability of the human Burkitt lymphoma Namalwa transfected with either the hematopoietic or epithelial forms of human CD44 and injected into nude mice. Transfectants for the hematopoietic form bound HA much more efficiently than transfectants for the epithelial form (see Section 111,C). The transfectants for the hematopoietic form showed an increased rate of tumor development when injected subcutaneously or intravenously and a somewhat greater tendency to metastasize, especially to the bone marrow, than untransfected Na- malwa cells or transfectants for the epithelial form of CD44. Although these results do correlate with ability to bind HA, the mechanism responsible for the increased frequency of tumor takes and metastasis is unclear. Treatment of the mice with a soluble CD44-immunoglobulin fusion protein at the time of tumor injection and at intervals thereafter suppressed the incidence of tumors (Sy et al., 1992), suggesting that binding to HA may be involved. The mechanism of this latter inhibition is unclear, however, because the fusion protein dis- appears from the circulation of these mice within minutes after injection and it seems unlikely that at the doses used all possible binding sites in the animal are saturated.

Gunthert and colleagues have used as a model the rat pancreatic adenocarcinoma cell line BSp73 (Gunthert et al., 1991). A metastatic cell line and a nonmetastatic cell line have been established that either do or do not form metastases in the lymph node and the lungs when cells are injected into the footpad and the local tumors are removed 10 days later. The metastatic cell line expresses a unique antigenic determinant, which expression cloning showed was related to the presence of a CD44 isoform containing an alternatively spliced exon 10 (see Fig. 2; Gunthert et al., 1991; Arch et al., 1992; Seiter et al., 1993; Herrlich et al., 1993). Transfection of a CD44 construct expressing this isoform into the nonmetastatic cell line conferred metastatic behavior on it (Gunthert et al . , 1991; Seiter et al., 1993). Antibodies specific for the variant isoform inhibited metastatic spread when injected with the tumor, but not when injected later, suggesting that the antibodies interfered with migration from the site of injection in the footpad to the local lymph node (Reber et al., 1990; Seiter et al., 1993). Because the antibody is not directly cytotoxic or cytostatic, at least in uitro, it may act by interfering with interactions between the tumor cell and other cells and/or the ECM, rather than acting to enhance effector immune mechanisms (Seiter et al., 1993).

At least in this model system, expression of a particular CD44 isoform does seem to be essential for metastasis. How general this result


will turn out to be and how it relates to the other studies in which higher levels of the hematopoietic form of CD44 correlated with tu- morigenicity and/or metastatic ability are uncertain. At least one other series of metastatic cell lines in the rat also expresses this isoform (Gunthert et al., 1991) and transfection of the isoform into a rat fibrosarcoma cell line confers metastatic capability (Herrlich et al., 1993). As discussed above, however, there is not yet enough informa- tion on the pattern of isoform expression in human tumors and tumor cell lines to allow generalizations to be made regarding the importance of CD44 isoform expression in metastasis. The expression of these isoforms in nonmetastatic, and even noncancerous, cells indicates that cellular factors other than CD44 isoform expression must contribute to metastasis. How the presence of the extra exon affects ligand recognition by CD44 is unclear as well (Gunthert et al., 1991; Herrlich et al., 1993). Although the alternatively spliced exons found in the nonconserved domain have potential sites of O-glycosylation and chondroitin sulfate addition, it is not clear that cells expressing only exon 10 have any additional posttranslational modifications not found in the standard CD44 molecule (Herrlich et al., 1993). Whether the presence of this exon confers new ligand-binding specificities is unknown. Interestingly, the cytoplasmic domain does not appear to contribute to metastatic behavior, because transfectants of deletion constructs lacking this domain but including exon 10 exhibit metastatic behavior (Herrlich et al., 1993).

Although there are clearly many unanswered questions, these studies and the others discussed above have stimulated much interest in the details of CD44-ligand recognition. For a more coherent picture to emerge, it will be necessary to develop methodology that will relate advances in the molecular biology of CD44 to biologically meaningful models of tumor metastasis.

VII. Summary

It is now generally accepted that CD44 is a cell adhesion receptor and that hyaluronan is one of its ligands. Like many cell adhesion receptors, CD44 is broadly distributed, and its ligand, hyaluronan, is a common component of extracellular matrices and extracellular fluids. Yet a great variety of responses has been reported to result from CD44 ligation. These include cell adhesion, cell migration, induction (or at least support) of hematopoietic differentiation, effects on other cell adhesion mechanisms, and interaction with cell activation signals.


This diversity of responses indicates that downstream events following ligand binding by CD44 may vary depending on the cell type expressing CD44 and on the environment of that cell.

CD44 is expressed on cells in the early stages of hematopoiesis and has been shown to participate in at least some aspects of the hematopoietic process. In mature lymphocytes, CD44 is upregulated in response to antigenic stimuli and may participate in the effector stage of immunological responses. Along with other adhesion receptors that show alterations in expression after activation, CD44 probably contributes to differences in the recirculation patterns of different lymphocyte subpopulations.

CD44 ligand-binding function on lymphocytes is strictly regulated, such that most CD44-expressing cells do not constitutively bind ligand. Ligand-binding function may be activated as a result of differentiation, inside-out signaling, and/or extracellular stimuli. This regulation, which in some situations can be rapid and transient, potentially provides exquisite specificity to what would otherwise be a common interaction.

CD44 is not a single molecule, but a diverse family of molecules generated by alternate splicing of multiple exons of a single gene and by different posttranslational modifications in different cell types. It is not yet clear how these modifications influence ligand-binding function. The significance of the multiple isoforms of CD44 is not under- stood, but association of some isoforms with malignancies has been observed. And in at least some experimental systems, a contribution of CD44 isoforms to metastatic behavior has been demonstrated.

ACKNOWLEDGEMENTS We would like to thank all ofour collegues who shared their unpublished manuscripts

with us. This work was supported by NIH Grants CA-13287 and AI-31613 and NSF Grant DCB-8900579 to R. Hyman, NIH Grants AI-19884 and AI-20069 to P. W. Kincade, National Cancer Institute Core Grant CA-14195 to the Salk Institute, and by the Hansen Foundation.

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