Leukocyte-cell adhesion: a molecular process fundamental in leukocyte physiology.

Immunological Reviews 1990, No. 114 Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the author(s)

Leukocyte-Cell Adhesion: A Molecular Process Fundamental in Leukocyte Physiology MANUEL PATARROYO', JACQUELINE PRIETO', JORGE RINCON', TUOMO TIMONEN^ CLAES LUNDBERG\ LENNART LINDBO!/, BIRGITTA ASJO* & CARL G . GAHMBEKG*

INTRODUCTION Leukocytes interact with one another and with vascular endothelial cells to generate immune and infiammatory responses and to traffic throughout the body. These interactions are mediated by soluble factors, physical contact, or both. In blood from healthy donors, practically all leukocytes are free cells, presumably because they are in the resting state. However, on exposure to a stimulus, e.g. antigen for lymphocytes or complement factors and leukotrienes for monocytes and granulocytes, the leukocytes adhere to one another (forming cell aggregates or clusters) or to other cell types such as vascular endothelial cells. This form of physical contact is characterized by fast (firm) stickiness between the cells. It is quickly formed but usually brief, in comparison with the almost permanent intercellular adhesion of solid tissues, and appears to be regulated by the activation state of the cells. The present review will summarize our studies, including recent and unpublished work, in the biology and molecular basis of leukocytecell adhesion and its participation in a variety of leukocyte functions both in vitro and in vivo. We recently published three articles reviewing the literature in this field, an extensive one (Patarroyo & Makgoba 1989a) and two shorter ones (Gahmberg et al. 1988, Patarroyo & Makgoba 1989b).

Manuel Patarroyo, Dept. of Immunology, Karolinska Institutet, Box 60400, S-104 01 Stockholm, Sweden. 'Dept. of Immunology, Karohnska Institute, 'Dept. of Pathology, University of Helsinki, ^Dept. of Inflammation Research, Pharmacia, Uppsala, *Dept. of Physiology, Karolinska Institute, *Dept. of Virology, Karolinska Institute, Stockholm, Sweden, 'Dept. of Biochemistry, University of Helsinki, Helsinki, Finland.

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PHORBOL ESTER-INDUCED LYMPHOCYTE AGGREGATION AN ADHESION-SPECIFIC ASSAY During the 1970s several investigators envisaged that physical contact of leukocytes with cells, such as conjugation of cytotoxic T lymphocytes (CTL) to target cells, constituted a complex interaction between cell surfaces. As a cellular response, adhesion is preceded by recognition of a stimulus followed by cell activation. Consequently, the conjugate formation must require molecules which mediate the stimulus recognition, cell activation and adhesion (reviewed in Patarroyo & Makgoba 1989a). Analysis of each of these interdependent steps required development of specific assays. At the end of 1979, during studies of the sensitivity of Epstein Barr-vinis (EBV)-infected lymphoid cells to cytotoxic lymphocytes, one of us (M.P.) observed that phorbol esters in nanomolar concentrations were able to induce aggregation of cells from lymphoid hnes (Patarroyo et al. 1981). Further studies showed that phorbol esters induced, within minutes of treatment, morphological changes, namely uropod-like structures and membrane ruffles, and cell aggregation in human blood lymphocytes (a T-cell enriched population) (Patarroyo et al. 1982) (Fig. 1). The induction of aggregation, which occurred in either presence or absence of serum, was blocked by metabolic inhibitors, incubation at 4''C, and EDTA, indicating active participation of the cells and a requirement for extracellular divalent cations. Microfilaments, but not microtubules, appeared to participate, since the phenomenon was partially inhibited by cytochalasin B but was resistant to colchicine. Cell surface trypsinization followed by addition of phorbol esters resulted in no cell aggregation, although the characteristic morphological changes were clearly observed. Moreover, the trypsin treatment did not inhibit binding of radioactive phorbol esters to the cells. The protein synthesis inhibitor cyclohexamide was ineffective, while retinoic acid and several protease/esterase inhibitors were inhibitory. By electron microscopy, broad contact areas between lymphocytes and lymphocytes/monocytes were visualized, but not membrane fusion or cytopathic signs. Similar to blood lymphocytes, thymocytes and EBVimmortalizcd nonnal B cells aggregated, in contrast to lymphocytes from several chronic lymphocytic leukemias which did not, although the latter cells were also able to bind phorbol ester molecules (Patarroyo et al. 1983a, 1983b, Eliasson et al. 1983, reviewed in Patarroyo 1982). The structure of these small compounds (500-700 mol.wt), their binding to non-aggregating cells, and the metabolic requirements of the cell aggregation excluded the possibility that this antigen-independent (natural) cell cluster formation was due to a "passive bridging" of phorbol esters between the cells and hence indicated the participation of cell-adhesive moieties. It was concluded that phorbol esters induced a cell-adhesive (binding) phenotype in certain lymphoid cells and that preformed cell surface proteins mediated the cell-cell adhesion

LEUKOCYTE ADHESION: CAMs AND RELEVANCE

69

Figure 1. Scanning electron micrograph of aggregated blood lymphocytes after brief treatment with phorbol ester. Note the uropod-like structures and membrane rufTles of the aggregated cells. (binding). These structures were designated cell-adhesion (binding) molecules (Patarroyo et al. 1983a, 1983b, Eliasson 1983, reviewed in Patarroyo 1982), now referred to as CAMs. In parallel studies other groups demonstrated that phorbol esters rapidly enter cells (Liskamp et al. 1985) and that once in the cytoplasm these compounds behave as diacylglycerol, an endogenous intracellular messenger, and pemianently activate protein kinase C (PKC) (Nishizuka 1984). Thus, phorbol esters induce cellular responses by bypassing cell surface structures which mediate early recognition and activation events (Truneh et al. 1984). These characteristics made the phorbol ester-induced lymphocyte aggregation an adhesion-specific assay, most suiUble for identifying the CAMs with the aid of blocking antibodies. THE Leu-CAM FAMILY (CDIla-c/CDI8) AND ICAM-1 (CD54) As an initial approach to identify membrane events responsible for, or associated with, the phorbol ester-induced intercellular adhesion, the lateral mobility of cell

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surface glycoproteins was analyzed (Patarroyo & Gahmberg 1984). Phorbol esters were found to drastically enhance the lateral redistribution of concanavalin A receptors and the leukocyte common antigen T200, now called CD45, as measured by cap formation, with kinetics and dose-response relationships similar to the one of cell aggregation. Both processes, namely the induced intercellular adhesion and enhanced mobility and redistribution of cell surface glycoproteins, were similarly sensitive to the various inhibitors except EDTA. Identification of the Leu-CAM family Two immunological approaches were then used to identify the CAMs of lymphocytes. The first, used by Edelman (Edehnan 1984) to identify adhesion molecules in neural cells and hepatocytes (N-CAM and L-CAM, respectively), consisted of an inhibition-neutralization assay where Fab fragments of IgG from a polyclonal antiserum to cells inhibited the cell aggregation and were then neutralized by fractions of solubilized membrane proteins. The second approach was to obtain monoclonal antibodies (mAbs) to cell surface antigens which could block the phorbol ester-induced intercellular adhesion. In 1983, a polyclonal antiserum to phorbol ester-treated human lymphocytes was produced. Eab fragments from its IgG were able to block, in a dose-dependent manner, the phorbol ester-induced cell aggregation but not the characteristic morphological changes (M. Patarroyo, unpublished results). These findings, although preliminary, indicated that an immunological approach was suitable. In the same year, mouse mAb 60.3 was reported to define a novel cell surface antigen common to human leukocytes (Beatty et al. 1983). This was the first antibody, after testing more than 100,

TABLE I Cell adhesion antigens and molecules of leukocytes Antigen names OfTicial*

Local

CDl la

TA-1, LFA-1 Mac-1, OKMl, Mol, Leu-15 Uu-M5 LCA-gp90 LB-2, ICAM-1 Tp50, Leu-5, OKTll, LFA-2 LFA-3 Leu-19, NHK-1

CDllb CDllc CDl 8 CD54 CD2 CD58 CD56+^

Apparent molecular weight*" (X lO'*)

Molecule

160 (177) 155(165) 130 (150) 90 (95) 84 (90) 50 (55) 60 220/135

Leu-CAM a a-chain Leu-CAM b a-chain Leu-CAMc a-chain Leu-CAM family ^-chain ICAM-1 T-CAM+ Pan-CAM* N-CAM (isoform)

'Cluster of difTerentiation (CD) nomenclature. **Apparent molecular weight values of each glycopolypqjtide used by Patarroyo and coworkers, and in parenthesis by others groups. ^ Alternative functional names. ^^As suggested by participation of N-CAM in conjugation of NK cells with neiu-al target cells. ,i i


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which was able to block the phorbol ester-induced aggregation of lymphocytes. IgG and Fab fragments were similarly effective but did not inhibit, as expected, the other simultaneous membrane processes, namely the uropod and ruffle formation and the lateral redistribution of cell surface proteins (Patarroyo et al. 1985a) (Fig. 2D). Since the antibody also reacted with granulocytes, its effect was tested on the phorbol ester-induced aggregation of these cells. Antibody 60.3 was equally inhibitory and did not affect the simultaneous superoxide generation and lysozyme release (Patarroyo et al. 1985b) (Fig. 2P). Interestingly, the phorbol esterinduced adhesion to plastic was also blocked, as was the cell aggregation induced by the ionophore A23187 and the chemotactic tripeptide formyl-methionylleucyl-phenylalanine (FMLP) (Patarroyo et al. 1985b). In the original pubUcation (Beatty et al. 1983), antibody 60.3 was reported to inhibit a large variety of leukocyte functions in vitro and hence was thought to recognize an antigen involved in cell activation. It was also described to inmiunoprecipitate from unfractionated blood mononuclear cells a surface complex of at least three proteins (Beatty et al. 1983). In our studies, the antibody immunoprecipitated two major cell surface glycopolypeptides with apparent molecular weights of 90 000 and 160 000 from lymphocytes, and 92 000 and 155 000 from granulocytes (Patarroyo et al. 1985a, 1985b). A minor component with a molecular weight of 130 000 was also obtained from both cell types. Dissociation of the protein complex by 1% sodium dodecyl sulphate, followed by immunoprecipitation, resulted in precipitation of the 90 000-92 000 (GP90) component only (PatarToyo et al. 1985b). Since the protein complex had similar, although not identical, electrophoretic mobility under reducing and non-reducing conditions, it was concluded that GP90 was recognized by antibody 60.3 and that the larger components, namely GP160, GP155 and GP130, were non-covalently associated to it. It was also concluded that GP90, either alone or associated to the larger glycopolypeptides, mediated leukocyte adhesion. Due to the reactivity of the antibody with lymphocytes (T and B), monocytes and granulocytes, but not with many other cell types (Beatty et al. 1983), these structures were designated leukocytic cell-adhesion molecules (Leu-CAMs) (Patarroyo et al. 1985a, 1985b), following the CAM nomenclature introduced by Edelman (Edelman 1984). The Third International Workshop on Human Leukocyte Differentiation Antigens held in 1986 assigned the cluster of differentiation designation CD18, CDlla, CDllb, and CDllc to GP90, GP160, GP155 and GP130, respectively (Cobbold et al. 1987). Since the apparent molecular weight of the proteins varies with technical conditions, and because several groups have given different names to the same antigens (Table I), resulting in much confusion in the literature, here we shall refer to the antigens by the official CD nomenclature when comparing studies from different laboratories. Association of the glycopolypeptides as heterodimers CDlla/CD18, CDUb/CD18 and CDllc/CD18 with different a chains (CDl la, CD lib, and CDl lc) and a common fi chain (CD 18) implies that

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2. Aggregation of different types of leukocytes induced by phorbol ester treatment and effect of Fab fragments of mAb 60.3 to CD18 on the intercellular adhesion. A-D: blood T cells; E-H: EBV-immortalized normal B cells; I-L: monocytes; M-P: granulocytes; Q-T; Burkitt's lymphoma cell line BL 41. B, D, F, H, J, L, N, P, R, T cells were treated with 60 nM P(Bu)2 for 20 min. C, D, G, H, K, L, O, P. S, T: cells were incubated with 20 /ig/ml of Fab fragments of mAb 60.3. Cells were rotated at 100 rpm at 37°C.


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antibodies to CD18, such as 60.3, defme the whole family of molecules, while antibodies to the distinct a chains define each member of the family. To unify the function of these structures with the official nomenclature we use the following names: CDlla-c/CDI8: Leu-CAM family, CDlla/CD18: Leu-CAMa, CDllb/ CD18: Leu-CAMb and CDlIc/CD18: Leu-CAMc (Fig. 3. and Table I). Other anti-CD 18 antibodies had been previously described (Trowbridge & Omary 1981, Wright et al. 1983, and Sanchez-Madrid et al. 1983) as well as antiCDUa (LeBien & Kersey 1980, Davignon et al. 1981a, Pierres et al. 1982, Sanchez-Madrid et al. 1982, Hildreth et al. 1983), anti-CD lib, (Springer et al. 1979, Breard et al. 1980, Todd et al. 1981) and anti-CDllc (Schwarting et al. 1985) antibodies to either human or mouse antigens. However, the adhesive function of CDlla-c/CD18 had not been demonstrated with these antibodies since adhesion-specific assays had not been developed and, in addition, several of tbe antibodies, particularly those produced early on, were directed to "adhesion irrelevant" epitopes and do not block leukocyte adhesion. Other groups reported results similar to ours in 1985. Amaout et al. (1985) described blocking of chemotactic peptide-induced granulocyte aggregation by a mAb to CDl lb, while Harlan and colleagues reported inhibition of phorbol esterinduced granulocyte aggregation and granulocyte adhesion to cultured vascular endothelial cells by antibody 60.3 (Harlan et al. 1985, Schwartz et al. 1985, Wallis et al. 1985). Moreover, Springer et al. (1985) described blocking of phorbol esterenhanced aggregation of EBV-immortalized normal B cells by antibodies to CDlla and CD18.

The Leu-CAM family Leu-CAMa CD18

Leu-CAMb CDllb

CD1P

Leu-CAMc CD11C

CD1B

LauM5

m

60.3

k

A

GP1W

GPM

GP1SS

{GP1T7)

(OPtS)

(OP test

GPlO (WH)

GPI30 (GP1SD)

Figure 3. Diagram of the Uu-CAM family (CDlla-c/CD18).

GPtO (QP9S}

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In 1980 and the following years, a rare inherited immunodeficiency characterized by recurrent bacterial infections and abnormal leukocyte functions, including neutrophil adhesion, was associated with the absence of a membrane protein of an apparent molecular weight of UO 000-180 000 in these cells (reviewed in Anderson & Springer 1987). Several groups reported in 1984 that leukocytes from these patients lacked CDl la, CDl lb and CD18. Although an association between these molecules and adhesion was suspected, the possibility that other unidentified

-^?J

- ^y 0 £

i'^t

; l.^

•V,*W

g

Figure 4. Adhesion of blood T cells to monocytes and EBV-immortalized normal B cells induced by phorbol ester treatment, and inhibition of the intercellular adhesion by Fab fragments of mAb 60.3 to CD18. a-d: T cells and monocytes (indicated by arrow); e-h: T cells and EBV-immortalized normal B cells (large cells), b, d, f, h: cells were treated with 60nM P(Bu)2 for 20 min. c, d, g, h: cells were incubated with 20 //g/ml of Fab fragments of mAb 60.3.


75

but relevant molecules were concomitantly deficient could not be ruled out. Analysis of this disease, now called Leu-CAM deficiency or leukocyte adhesion deficiency, supports the notion, directly established by adhesion-specific assays and adhesion-blocking mAbs, that CDlla-c/CD18 mediates leukocyte adhesion. Antibodies to mouse CDUa and human CD 18 bad been found to block conjugation of CTL to target cells (Davignon et al. 1981b, Krensky et al. 1984) and to desagglutinate mouse Con A blasts (Pierres et al 1982). These findings did not demonstrate participation of CDlla or CD18 in adhesion itself since altemative explanations could not be excluded, i.e. blocking of receptors for stimulatory ligands or of cell activation, or induction of "negative or off signals" which could alter the cell metabolism or the cytoskeleton (Goldstein et al. 1982, Martz 1987, Mentzer et al. 1985). It had been reported that antibodies to CD4, CD8 and class II molecules were also able to block conjugate formation (Agrwal & Thomas 1984, Biddinson et al. 1984, Landegren et al. 1982) and that growth factors and antibodies to growth factor receptors could weaken adhesion of nonlymphoid cells (Schreiber et al. 1981). Demonstration of simultaneous plasma membrane/cytoskeleton-dependent cellular responses other than adhesion, such as the formation of membrane rufHes and capping of membrane proteins, in presence of antibody 60.3 clearly indicated that the antibody blocked neither binding of the stimulus (phorbol ester) to its (intracellular) receptor nor cell activation. It also excluded the negative signal hypothesis and unambiguously demonstrated the participation of CDlla-c/CD18 in adhesion itself (Patarroyo etal. 1985a, 1985b). In additional studies we analyzed the aggregation of EBV-immortalized normal B cells and of monocytes (Fig. 2), and the adhesion of T lymphocytes to the former cells (Patarroyo et al. 1986, 1988a) (Fig. 4), as well as of each type of leukocyte to vascular endothelial cells (Prieto et al. 1988) (Fig. 5) in presence of phorbol ester. Fab fragments of antibody 60.3 were always inhibitory.

Cellular distribution and structure of CDUa-cjCDlS CDl la, CDl Ib, CDl lc and CD18 appear to be exclusively expressed by leukocytes. CDlla/CD18 (Leu-CAMa) predominates on lymphocytes, CDllb/CD18 (Leu-CAMb) on granulocytes, and CDl lc/CD18 (Leu-CAMc) on macrophages, while monocytes have large proportions of all three molecules (Springer et al. 1987, Patarroyo & Ansotegui 1987, Patarroyo & Makgoba 1989a) (Fig. 6). Granulocytes express some CDl la/CD 18 and CDl lc/CD18 as well, and certain lymphoid subpopulations, such as large granular lymphocytes, express all three heterodimers (Timonen et al. 1988). As measured by the amount of CDlla/ CD 18, two subpopulations of resting T lymphocytes are detected in peripheral blood (Fig. 6C). When they are inhibitory, and in accordance with the cellular distribution of

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Figure 5. Adhesion of ditTerent types of leukocytes lo vascular endothelial cells induced by phorbol ester treatment and effect of Fab fragments of mAb 60.3 to CD 18 on the intercellular binding, a, b: T cells; c, d: EBV-immortalized normal B cells; e, f: monocytes; g, h: granulocytes. b, d, f, h: cells were incubated with 20 /iglml of the Fab fragments. All cells were treated with 60 nM P(Bu)2 for 20 min at 37°C.


77

the antigens, antibodies to either CDlla or C D l l b block phorbol ester-induced aggregation of either lymphocytes or granulocytes, respectively, while anti-CD 18 antibodies inhibit both (Table II). Lack of inhibition by the anti-CD lie antibody may be due to the relatively low expression of the antigen and the simultaneous presence of CDl la and CDl lb on the tested cells. The same antibody has been reported to block conjugation of a CTL, which expressed high amounts of CDllc, to the target cells (Keizer et al. 1987). The selective inhibition by the various mAbs is also observed when adhesion of difTerent types of leukocytes to vascular endothelial cells is analyzed (Prieto et al. 1988). Interestingly, phorbol esters also induce adhesion of myelomonocytic cells to artifical substrates such as plastic. This process, which appears to be required for

E z

10

10

10

Relative

10

10

10^

10

10"^

10**

Fluorescence Intensity (Log Scale)

Figure 6. Histogram of the expression of CDlla, CDllb, CDllc, CDl8 and CD54 on different types of leukocytes. A: unfractioned blood mononuclear cells (BMNC); B: BMNC treated with Con A for 3 days; C: a T-cell enriched population; D: EBV-immortalized normal B cells; E: monocytes; F: granulocytes. ( — ) : no mAb; ( ) mAb 60.3 to CD18; ( ) mAb IOT-16 to CDlla; (-.-.-.) mAb 60.1 to CDllb; ( ) mAb Uu-M5 to CDl lc; (-...-...) mAb LB-2 to CD54.

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spreading and migration (chemotaxis) of the cells, is mediated by Leu-CAMb (Patarroyo et al. 1985b, Rosen & Gordon 1987, Patarroyo et al. 1988a, Skoglund et al. 1988a). Recombinant DNA cloning of CD18, CDl la, CDl lb and CDl lc by several groups during recent years (reviewed in Kishimoto et al. 1989) has demonstrated polypeptides with the characteristic features of integral membrane proteins with large extracellular domains and short cytoplasmic tails. The j6-chain (CD 18) contains a cysteine-rich region, and the a-chains (CDlla, C D l l b and CDllc) three sequences homologous to Ca^"^ binding sites in paralbumin, troponin C and calmodulin. By using mouse-human cell hybrids, we located the gene encoding CD18 on human chromosome 21 (Suomalainen et al. 1986). Later on, other groups confirmed this finding and mapped the genes for the three a-chains to chromosome 16 (Kishimoto ct al. 1989). Comparison with other proteins indicates that the Leu-CAM family belongs to a larger family of cell surface receptors referred to as integrins (Hynes 1987). They include the *'VLA" (very late antigen) family, which contains receptors for the extracellular matrix components collagen, fibronectin and laminin (Hemler 1988) and, on the other hand, a vitronectin receptor and the platelet molecule GPIIb/GPIIIa, which constitutes a receptor for fibrinogen, fibronectin and von Willebrand's factor (Hynes 1987). All of these molecules are high molecular weight heterodimers homologous to each other. Many of these cell surface receptors recognize the sequence arginine-glycine-aspartic acid (RGD) in their ligands (Ruoslahti & Piershbacher 1987). However, peptides containing this sequence do not block the phorbol ester-induced leukocyte adhesion (Marlin & Springer 1987,

TABLE II Effect of mAbs to CDI8, CDlla, CDllb. CDllc and CD54 on adhesion among blood mononuclear cells (BMNC). resting T cells (T cells), T-blasts (ConA-T). B-blasts (EBVB). monocytes and granulocytes in presence of phorbol ester* mAb (antigen) t Leukocyte type BMNC T cells ConA-T EBV-B Monocytes Granulocytes

60.3 (CD18)

79 92 .^ . 91 92

IOT-16 Uu-M5 (CDlla) 60.1 (CDllb) (CDllc) LB-2 (CD54) % Inhibition of leukocyte aggregation 40 89 74 73 35 5

4 3

15 1 11 84

7 ND ND 4 5 5

13 3 73 72 32 0

*CeIls were treated with 60nM P{Bu)2 for 20 min al 100 rpm at 37°C. % of cell aggregation in absence ( —) or presence {-\-) of phorbol ester was: BMNC: ( —)3, (-i-)58; T cells: ( - ) l , ( + )31; ConA-T: ( - ) 8 , (-l-)60; EBV-B ( - ) 2 3 , (+)67; monocytes: (-)45, ( + )72; granulocytes: (—)1, ( + )90. *Cells were incubated with 20 /ig/ml of purified mAb (IgG).


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Patarroyo et al. unpublished results). The name "leukocyte integrins" has been recently proposed for CDlla-c/CD18 (Kishimoto et al. 1989). However, most of the other integrins are also expressed by leukocytes. Expression of receptors for extracellular components on hemopoietic cells indicates the importance of leukocyte-matrix interaction {Hemler 1988). We have purified the Leu-CAMs in milligram amounts by immunoaffmity chromatography and gel electrophoresis and partially characterized them. Direct analysis of CD 18 by endo-^-N-acetylglucosaminidase H and endo-j5-N-acetylglycosaminidase F indicated that the polypeptide contains 5-6 complex type Nglucosidic ohgosaccharides (Kantor et al. 1988). Moreover, an immunological mapping of CD 18 was performed by using mAbs (Nortamo et al. 1988). Most of the anti-CD 18 niAbs did not inhibit leukocyte adhesion, and one of them increased the binding of an adhesion-blocking antibody, suggesting induction of a conformational change in the protein. Extracellular divalent cations are required for phorbol ester-induced leukocyte adhesion (Patarroyo et al. 1983a). Although, most adhesion occurs in the presence of only Mg"^"^, a niaximal response is obtained together with Ca"^"^ (Patarroyo & Jondal 1985). We have analyzed binding of the divalent cations to the Leu-CAMs (Gahmberg et al. 1988). Isolated CDlla-c/CD18 from buffy coat cells, as shown in Fig. 7A after Coomassie blue staining, was blotted onto nitrocellulose filters and incubated with^^Ca"^"^. Radioactive Ca^"*^ bound to the a-chain(s) but not to the ^-chain (Fig. 7A). The **^Ca^^ incubation was done in the presence of 20 mM MgCU to reduce nonspecific "'^Ca'^'*^ binding. Interestingly, Mg"^"^ was not able to completely inhibit the binding of Ca^^, which shows that CDll contains Ca^"*^-specific binding sites. Since a radioactive isotope of Mg"^ * is not available, indirect binding studies of Mg^"" to CDl 1/CD18 were performed. A Scatchard analysis with CDl lb/CD18

0.6

B X

0.4 "

0.2

X X X

X

0

too 200 300 400 [CaCL] added (iiM)

500

4

X X

6 8 10 0 1 2 3 Bound (molBa Ca**/moU profain)

Figure 7. Binding of^Ca'^^ to CDlla-c/CD18. A: Binding of^Ca""^ to immobilized CDl la-c/CD18 complex. Insert shows to the left a Coomassie blue-stained gel, and to the right the result obtained after blotting with *^Ca**. B: Binding of ^^Ca"^* to CDllb/CDI8 bound to Sepharose in the absence of Mg*"^. C: Binding of'"Ca'^* toCDlib/CD18 in the presence of 20mM of MgC12.

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PATARROYO ET AL.

was done using varying concentrations of'^Ca'^^ in the absence or presence of 20 mM MgCl;. CDl lb/CD18 was left bound to an anti-CDl lb antibody coupled to Sepharose and controls were done using the same antibody-Sepharose columns without CDl lb/CD18. More high affinity Ca""^ "*• sites were detected in the absence of Mg^^ (Fig. 7B) than in its presence (Fig. 7C), indicating the presence of specific high affinity Mg"^ * sites. We do not know where in the polypeptide chains they are located, and their exact number is still not settled. Little is known about the binding of a-chains to the ^-chain. SDS-treatment resulting in denaturation dissociates the chains from each other. We have recently found that a short treatment at low pH also efficiently splits the al0 association. Fig. 8 shows the results of such an experiment. Periodate/NaB^H4 surface-labelled (Gahmberg & Andersson 1977) leukocyte extracts in Triton X-100-containing buffer were treated at the indicated pH values for 15 min, brought to pH 7.4, and immunoprecipitated with a ^-chain specific mAb. Fig. 8 shows that around pH 4.2 the association of the a-chains to the ^-chain was broken. Interestingly, all CDl 1 polypeptides seem to dissociate from CD18 at similar pH values. This fmding suggests that the binding sites in all three a-chains are similar. Divalent cations do not seem essential because EDTA treatment does not break the af0 complexes. The protein complexes CDl la-c/CD18 also have functions other than adhesion (reviewed in Patarroyo & Makgoba 1989a). These molecules are able to recognize microorganisms such as Staphylococcus epidermidis, Escherichia coli and Histo-

p H 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0

B COa, -CD11C

-CD18 BSA-

OA-

Figure H. Disa»K«uK>p of CD 111'-c/CU'S complexes by low pH treatment. A. Polyacrylamide slab gel of'C-labelleil standard pn^tms: M = myosin, 200 000; PHb = phosphorylase b, 94 000; BSA = bovine serum albumin, 68 000; OA=ovalbumin, 43 000; CA = carbomc anhidrase, 30 000; BF = buffer front. B. polyacrylamide slab gel electrophoresis of immune precipitated CDl 1/CD18 after treatment at indicate pH values.


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plasma capsulatum without the participation of serum components, and CDllb/ CD 18 also serves as a receptor (CR3) for iC3b, a breakdown product of the third component of complement, for factor X and fibrinogen, two coagulation molecules, and for zymosan, a yeast cell extract (Table III). When compared, the CAM and CR functions of CD 11 b/CD 18 are cleady distinct, since antibodies to different epitopes of this molecule can inhibit only one of the functions. CDl laC/CD18 also appear to mediate cell activation and signaling. Accordingly, CDl lb/ CD18-dependent adhesion enhances degranulation in granulocytes (Schleiffenbaum et al. 1989).

lCAM-1 (CD54) The phorbol ester-induced adhesion of leukocytes to CD18-negative cells, such as vascular endothelial cells (Harian 1985, Wallis et al. 1985), indicated that the intercellular adhesion did not require a like-like (homophilic) interaction between CAMs on adjacent cells, and suggested the participation of other adhesion molecules distinct from the Leu-CAM family CD54 was originally described by Clark and colleagues (Clark & Yokochi 1984, Clark et al. 1986) as a surface antigen on human activated lymphocytes (BB-l/LB-2 antigen). Thereafter, the adhesive function of this structure was demonstrated by Rothlein et al. (1986) and Patarroyo et al (1987) by inhibition of the phorbol ester-enhanced aggregation of EBV-immortalized normal B cells with different mAbs. ICAM-1 (intercellular adhesion molecule-1) is weakly expressed by some blood mononuclear cells, namely B lymphocytes, NK cells and monocytes. However, stimulation with lectins increases and induces the expression on B and T cells, respectively (Clark et al. 1986, Dustin et al. 1986, Patarroyo et al. 1987) (Fig. 6). Although practically absent from granulocytes, myeloblastoid, monoblastoid and erythroleukemia cell lines also display the antigen, and certain non-hematopoietic cells, such as vascular endothelial cells, are strongly positive. By staining of frozen sections CD54 is detected mainly on tissue macrophages, germinal center dendritic cells, and vascular endothelium. Its expression is selectively induced or increased, within hours, by inflammatory mediators such as interferon, interleukin 1, and tumor necrosis

TABLE III Cell adhesion molecules and their ligands Molecule

Adhesive ligand

Other ligands

Leu-CAMa (CDlla/CD18) Leu-CAMb (CDl lb/CD18) Leu-CAMc (CDl lc/CD18) T-CAM (CD2) L-CAM (CD56)

lCAM-1 (CD54), ICAM-2 ? ? Pan-CAM (CD58) L-CAM (CD56)

iC3b,Fg,Fx >C3b

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PATARROYO ET AL. t

factor (reviewed in Patarroyo & Makgoba 1989a). ICAM-1 is a single cell surface glycoprotein with a heterogeneous apparent molecular weight ranging from 76 000 to 114 000 in different cell types. Cloning and sequencing of this heavily glycosylated protein demonstrated an integral membrane protein with no RGD motifs, homologous to N-CAM, and thus a member of the immunoglobulin supergene family. Its gene was mapped to human chromosome 19. ICAM-1 mediates not only adhesion among B blasts but also aggregation of T blasts and monocytes (Table II). Purified ICAM-1, but not glycophorin, reconstituted into lipid vesicles and bound to artifical surfaces supported the adhesion of Leu-CAMa-positive lymphoblasts but not of Leu-CAM-negative cells (Marlin & Springer 1987). As with phorbol ester-induced lymphocyte aggregation, adhesion of the lymphoblasts to ICAM-1-coated surfaces is temperature-dependent and requires cellular metabolism, an intact cytoskeleton, and the presence of divalent cations, mainly Mg^"^. Moreover, this adhesion is blocked by antibodies to either CDl la, CD18 or CD54. Altogether these results indicate that ICAM-1 is a ligand for Leu-CAMa (Table III). On the other hand, inhibition of FMLPinduced adhesion of granulocytes to endothelial cells by pretreating the latter cells with an antibody to ICAM-1 (Smith et al. 1988) suggests interaction of this molecule also with Leu-CAMb. Recently, we (Prieto et al. 1989) and Horley et al. (1989) identified mouse ICAM-1 by using a rat mAb to MALA-2, a mouse lymphocyte activation antigen (Takei 1985). In other studies, CD54 was found to constitute the major human rhinovirus receptor (Greve et al. 1989, Staunton et al. 1989b) and a receptor for Plasmodium falciparum (Berendt et al. 1989). Since antibodies to ICAM-1 do not block all CDlla/CD18-dependent adhesion of lymphoid cells (Rothlein et al. 1986) (Table II), the existence of an additional ligand for Leu-CAMa has been suspected. The recently described ICAM-2 molecule could be such a ligand (Staunton et al. 1989a). The putative adhesive ligands for Leu-CAMb and Leu-CAMc have not been definitely identified. Regulation and mechanisms of Leu-CAM-ICAM-1-mediated adhesion A large variety of stimuli induce leukocyte adhesion to cells (reviewed in Patarroyo & Makgoba 1989a). We have recently found that HjOj, an endogenous compound produced by myelomonocytic cells, rapidly activates a Leu-CAMbdependent adhesion of monoblastoid cells to plastic (Skoglund et al. 1988a), and that the adhesive responses of both granulocytes and vascular endothelial cells induced by leukotriene B4 are CD 18-dependent (Lindstrom et al. 1990). It is likely that several cell surface molecules transduce signals which activate the LeuCAMs. In agreement with this concept, an antibody to the major histocompatibility


83

complex (MHC) class II molecules was foutid to enhance the aggregation of EBV-immortalized normal B cells (Patarroyo et al. 1986), and cross-linking of the T-cell receptor results in stimulation of a transient Leu-CAM-mediated adhesion in T lymphocytes (Dustin & Springer 1989). The leucocyte adhesion to cells depends on the availability of either LeuCAMa, Leu-CAMb or Leu-CAMc and their respective ligands on the cell surface (reviewed in Patarroyo & Makgoba 1989a). Unstimulated granulocytes and monocytes have, in addition to the cell surface Leu-CAMs, intracellular pools of CDllb/CD18 and CDnc/CD18. These stored molecules are translocated to the plasma membrane within a few minutes during stimulation with phorbol esters and other agents. Several groups have demonstrated that this upregulation is not necessary for adhesion to occur (reviewed in Patarroyo & Makgoba 1988a, SchelifTenbaum et al. 1989). Moreover, induction oflymphoblast aggregation by phorbol ester occurs without a quantitative increase in the cell surface expression of Leu-CAMa and ICAM-1. Thus, the increased cell aggregation appears to be the result of a qualitative, rather than quantitative, change in the adhesion molecules. Indeed, the metabolic requirements for the adhesion of Leu-CAMapositive lymphoblasts to purified lCAM-1 indicates that the Leu-CAM has to be activated. A reorientation and/or a redistribution of preformed cell surface proteins was originally proposed as an event(s) responsible for the phorbol esterinduced lymphocyte aggregation, since trypsination of unstimulated cells followed by addition of phorbol esters resulted in no cell aggregation (Patarroyo 1982, Patarroyo et al. 1982, 1983b). The sensitivity to cytochalasin B also indicated that functional microfilaments, which control the mobility of cell surface proteins, were necessary. In a subsequent study, the cell aggregation was temporally associated with increased lateral mobility of cell surface glycoproteins (Patarroyo & Gahmberg 1984). It was then postulated that phorbol esters first induced a reorientation/conformational change of CAMs to initiate the intercellular binding and that recognition of complementory CAMs on adjacent cells was followed by lateral redistribution of proteins to the area cell-cell contact to strengthen the intercellular binding and facilitate intercellular communication through cell surface receptors. Accordingly, antibody 60.3 reacts with practically all unstimulated blood lymphocytes, and phorbol ester enhances capping of CD 18 (Patarroyo et al., unpublished results). A conformational change of Leu-CAMs may be necessary for their transition from an inactive to an active state (reviewed in Patarroyo & Makgoba 1989a). This is supported by the reactivity of particular mAbs with either CDlla or C D l l b only on activated leukocytes. A unique mAb to an epitope on CDl la, C D l l b and CDllc induced by Mg+^ binding was recently reported by Dransfield & Hogg (1989). Interestingly, inhibition of cellular metabolism also reduced expression of this epitope. The conformational change(s) and/or redistribution of Leu-CAMs may be due to phosphorylation of these and/or associated stnic-

84

PATARROYO ET AL.

tures, such as cytoskeleton proteins, mediated by PKC, as phorbol esters are potent activators of this kinase, and inhibitors of PKC block the phorbol esterinduced lymphocyte aggregation {Patarroyo & Jondal 1985, Dustin & Springer 1989). Indeed, phorbol esters have been found to induce phosphorylation of the ^ chain (CD 18) of Leu-CAMa and Leu-CAMb, and the phosphorylated amino acid was identified as serine. Low concentrations of phorbol ester induce LeuCAMb-dependent adhesion of monoblastoid U937 cells to plastic without translocating the kinase from the soluble to the particulate fraction of the cells, a phenomenon observed at higher doses (Skoglund et al. 1988b). The meaning of this finding and its relationship to the phosphorylation of CD 18 are poorly understood. The Leu-CAM b-mediated adhesion of myelomonocytic cells to artifical substrates, such as plastic or glass, is intriguing. This process may be due to either a "non-specific" interaction of Leu-CAMb with a large variety of structures or, more likely, a specific interaction with a ligand which is secreted by the stimulated leukocyte and attached to the substrate. The precise function of each subunit of the Leu-CAM family in the adhesion process is inknown. Since the a chains are distinct, they may be responsible for the apparent specificity of adhesion. Since many antibodies to CDlla-c/CD18 do not block adhesion, only a particular region of each Leu-CAM must mediate the process. The exact localization of the adhesive sites is under investigation. It is unlikely that antibodies such as 60.3, which strongly react with resting, nonadherent leukocytes, recognize adhesion sites. Most likely these antibodies bind very close to the adhesive site(s) and inhibit adhesion by steric hindrance. Under physiological conditions, leukocyte adhesion is temporary, implying that cell de-adhesion also occurs. In analysis of kinetics, the Leu-CAMa-dependent adhesion oflymphocytes stimulated by cross-linking of the T-cell receptor peaked at 10 min and disappeared by 30 min {Dustin & Springer 1989), in contrast to the phorbol ester-stimulated adhesion which was maximal at 10 min and remained elevated for several hours.

THE CD2-CD58 ADHESION PATHWAY In 1981, three groups of investigators reported the identification of a novel human T-cell surface antigen, now called CD2 {Table I), and its participation in the rosette formation of human blood lymphocytes with sheep erythrocytes by using blocking mAbs (reviewed in Springer et al. 1987, Patarroyo & Makgoba 1989a). This in vitro assay appeared to simulate a physiological phenomenon, since human thymocytes and lymphoblasts, but not resting blood lymphocytes, could also bind human erythrocytes. Subsequently, anti-CD2 antibodies were found to block almost completely the autologous rosette formation, and to some extent, binding of thymocytes and T lymphocytes to human tumor cells, and conjugate formation


85

between cytotoxic T cells and their targets. CD2, a single cell surface glycoprotein with an apparent molecular weight of 40 000-55 000 mainly found on T cells, appears to be functionally linked to a calcium channel as demonstrated by certain anti-CD2 mAbs which induce T-cell activation. By screening mouse mAbs for inhibition of human T lymphocyte-mediated cytotoxicity. Sanchez-Madrid et al. (1982) found CD58, a cell surface glycoprotein of 55-70 kDa, widely distributed on both hemopoietic and non-hemopoietic tissues (Table I). In contrast to antibodies to CD2, the anti-CD58 mAb appeared to block by binding to the target cell, but similarly it inhibited the conjugate

figure 9. Roselling of Jurkat T cells to autologous erythrocytes and effect of lnAb y.o to CD2. A: no mAb; B: 20 /Jg/ml of mAb 9.6 (similar inhibition was obtained with mAb TS2/ 9 to CD58). Jurkal cells and leuraminidase-treated erythrocytes (5 units for 30 min at 37''C) were mixed at 1:50 ratio and incubated on ice for 2 h after centrifugation. Antibody 9.6 was adri-^d to the cells bef' - mixture.

8©

PATARROYO ET AL.

between effector and target cells. CD58 is expressed on both leukocytes and erythrocytes, and rosetting of human T cells with autologous erythrocytes is mediated by CD58 on the latter cells. Both C D 2 and CD58 have been cloned and the molecules have been found to share significant homology to one another. Recent studies using purified CD2 and CD58 have demonstrated high-affinity binding between these two molecules, as receptor ligand pair (Table III). In addition, the adhesive function of human C D 2 was unambiguously demonstrated by using a C D 2 which lacks a functional cytoplasmic signal transducing element (Moingeon et al. 1989). We and others use the rosette formation of Jurkat T cells with autologous erythrocytes to analyze the CD2-CD58 adhesion pathway (Fig. 9). This physical interaction is insensitive to anti-CD 18 or -CD54 mAbs (Rincon & Patarroyo 1990). On the other hand, the phorbol ester-induced aggregation of either T or B cells is resistant to anti-CD2 or CD58 mAbs (Patarroyo et al. 1985a, Patarroyo &

A

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10'

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Fluorescence Intensity (Log. Scale) Figure 10. Histogram of the expression of CD2, CD58, CD18 and CD54 on different types of leukocytes, and erythrocytes. A: unfractionated blood mononuclear cells; B; blood lymphocytes; C: EBV-immortalized normal B cells; D: monocytes; E: granulocytes; F: erythrocytes. ( — ) no mAb; ( ) mAb 9.6 to CD2; ( ) mAb TS2/9 to CD58; ( — ) mAb 60.3 to CD18; (-..-..-) mAb LB-2 to CD54.


87

Ansotegui 1987). Shaw et al. (1986) have reported that CTLs use both antigenindependent adhesion pathways: the Leu-CAM pathway, which requires divalent cations and is temj>erature-sensitive, and the CD2 pathway, which does not require divalent cations and is temperature-insensitive. Activation-related epitopes of CD2 and a higher density of this molecule are expressed on thymocytes and activated mature lymphocytes when compared with resting hlood lymphocytes. The role played by this apparent conformational change and the quantitative increase of CD2 in the cell adhesion is poorly understood. The reactivity of mAbs to CD2 and CD58 with different types of leukocytes and erythrocytes is shown in Fig. 10. Although CD2 has been reported to be selectively expressed by T and NK cells, we have observed reactivity of several anti-CD2 mAbs with monocytes (Rincon & Patarroyo 1990) (Fig. lOD). Although alternative explanations can not be excluded, this finding suggests that CD2 may also be expressed by cells other than T lymphocytes, a concept supported by the detection of CD2 on murine B cells (Yagita et al. 1989). Alternative functional names for CD2 and CD58 are T-CAM and Pan-CAM, respectively (Tables I and III). ADHESION PATHWAYS IN ENDOGENOUS AND IL-2-ACTIVATED NATURAL KILLING Antibodies to the various CAMs inhibit several leukocyte functions, including lymphocyte-mediated cytotoxicity (Beatty et al. 1983, Axberg et al. 1987, reviewed in Springer et al. 1987, Patarroyo & Makgoba 1989a). Natural cellular cytotoxicity is mediated by natural killer (NK) cells as well as so-called lymphokineactivated killer cells that comprise both IL-2-activated NK cells and non-MHC restricted T-killer cells. NK activity can roughly be divided into four phases; namely, the binding of the effector to the target, triggering of the cytolytic machinery, secretion of cytotoxic factors, and finally the killer cell independent phase of lysis. Surface molecules by which NK cells recognize target cells have been characterized only partially. The actual antigen-recognizing "NK-cell receptor(s)" has remained hypothetical so far, and a clear-cut distinction between molecules mediating the binding and triggering phases may be artificial. We have studied the involvement of the Leu-CAMs, ICAM-1, CD2 and RGD-recognizing matrix protein receptors in the adhesion and cytotoxicity of both endogenous and IL-2-stimulated highly purified NK cells (Timonen et al. 1988, 1990, Heiskala etal. 1990). Effect of mAbs to CDlla-clCD18. CD54 and CD2 and of RGD-containing peptides on NK function As shown in Fig. 11, mAbs against the adhesion-relevant epitopes of Leu-CAMs, ICAM-1 and CD2 inhibit the binding of endogenous NK cells to MOLT-4

PATARROYO ET AL.

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Figure 11. Effect of mAbs to adhesion relevant epitopes of CDlla, CDllb, CDllc, CD18, CD54 (ICAM-1) and CD2 on the binding of highly purified CD3-negalive large granular lymphocytes to MOLT-4 (-IL-2) and COLO ( + IL-2) target cells. C: control mouse IgG. The numbers represent clusters of differentiation (CD). All antibodies were tested at 5 /ig/ ml concentration, and recombinant IL-2 (Biogen, Geneva, Switzerland) was used at 500 U/ml for 3 days to stimulate 1 x 10^ CD3-LGL cells/ml. Results from experiments with eight different donors (I-VIII).


89

lymphoma cells, and that of IL-2-activated killer cells to COLO adenocarcinoma cells. The effects of individual anti-a-chain (CDlla, C D l l b and CDllc) antibodies are additive, and as a mixture they inhibit NK binding as much as the mAb to their common ^ chain (CD 18). The results indicate that all three members of the Leu-CAM family participate in NK activity. The combination of antibodies to CD 18, CD54 and CD2 eradicated the adhesion of endogenous NK cells in all experiments and that of IL-2-activated killer cells in 4 out of 7 cases. The same antibody combination also effectively blocked the cytotoxic activity of both endogenous and IL-2-activated NK cells (Fig. 12). These results indicate that the major adhesion molecules of NK cells against MOLT-4 and COLO target cells are Leu-CAMa, Leu-CAMb, Leu-CAMc, lCAM-1 and CD2. However,-when anchorage-dependent fibroblasts and renal parenchymal cells are used as targets for NK cells, the pattern of inhibition by the mAbs is different. Although the binding of NK cells is somewhat inhibited by the mixture of mAbs against CD 18, CD56 and CD2 (Fig. 13A), cytotoxicity is not affected by these antibodies (Fig. 13B). In contrast, the cytotoxicity was quite efficiently blocked by the RGDcontaining peptide GRGDS (Fig. 13B), and mAbs to CD 18, CD54 and CD2 showed an additive efiect in the inhibition. This pattern of inhibition by the reagents suggests that RGD-recognizing receptors are the major adhesive structures of NK cells towards fibroblasts and renal parenchyma! cells and that LeuCAMs and CD2 adhesion pathways play a supportive role, apparently less dramatically than in their contribution to NK activity against MOLT-4 and

+IL-2

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Figure 12. Inhibition of natural killer activity by individual mAbs and their combination. A 4-h chromium-release assay in which CD3-negative LGL were tested at three different efTector: target cell (E:T) ratios against MOLT-4 (-rL-2), and IL-2 activated CD3-negative LGL against COLO ( + IL-2).

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COLO target cells. Clearly, the participation of RGD-recognizing receptors of NK cells is a prerequisite for lysis of fetal fibroblasts and renal parenchymal cells. It is possible that these structures serve as triggering receptors or, alternatively, that they establish a sufficiently firm contact between effector and target cells for the actual triggering through as yet unidentified NK-cell receptor(s). Inactivation ofNK via CDl}ajCD18 NK cells are inactivated by NK-resistant target cells via a cell-to-cell contactmediated mechanism (Heiskala et al. 1987). The inactivation phenomenon is particularly prominent when anchorage-dependent monolayers of benign cells are exposed to NK cells (Heiskala & Timonen 1987), and thus it may direct the

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Figure 13. Effect of combinauon of mAbs to CD2, CD18 and CD54 (ICAM-1) and GRGDS on tbe binding (A) and cytotoxicity (B) of purified LGL to fetal fibroblast monolayers. The mAbs and GRGDS were present in the assays at 10 /ig/ml and 0.5 mg/ml concentrations, respectively.


91

cytotoxic force of naturally cytotoxic lymphocytes towards malignant cells. As the inactivation is strictly dependent on a contact between NK cells and the inactivating monolayers, we have explored the hypothesis that the inactivation signal would be transduced through the adhesion molecules. Purified NK cells were incubated with mAbs to the adhesion-relevant epitopes of CD]la-c/CD18, CD54 and CD2. With the antibodies against CDlla, inhibition of lysis resulted (Fig. 14), suggesting that the inactivation signal is transmitted through CDlla/ CD18. It should be emphasized that no downregulation of binding occurred during the inactivation, probably due to the fact that the other adhesion molecules were operative even if Leu-CAMa was blocked. Furthermore, the emergence of the inactivation required several hours of incubation in cell culture conditions, and therefore it differed from mere steric hindrance of the adhesion-relevant epitope of CDl la. Moreover, it has been recently shown that solid phase-coupled immunoafTinity-purified CD54 also downregulates NK activity, indicating that

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150-

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C. 100 200 300 450 Figure 14. Effect of anti-CDl la mAb (IOT-16) on the cytoxicity of purified LGL. Lymphocytes were preincubated overnight in cell culture conditions with the antibody at the indicated concentrations (/ig/ml), washed and subsequently tested for cytotoxic activity against K562. This treatment did not affect the binding of LGL to K562.

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the block observed with anti-CD 11a antibodies is not due to a nonspecific phenomenon, such as internaUzation of immunocomplexes (O. Carpen, personal communication). The downregulation of NK activity through CDl la has led us to hypothesize that the continuously cytotoxic NK cells are inactivated by contact with such target cells that are bound by cytotoxic lymphocytes but do not express the relevant antigens recognized by the NK-cell receptor(s). NK cells may thus screen the environment through contact formation, and leave healthy cells intact through the downregulation via CDlla/CD18. Several candidates have been presented for the actual NK cell receptor that would activate the cells after or during the contact formation. The available information suggests that non-MHC restricted natural cytotoxicity can be triggered through many parallel pathways, including CD16, CD2 as well as other less characterized lymphocyte surface molecules (Lopez &Ades 1989). In separate studies, CDlla-c/CD18 were found to participate as CAMs in the increased natural killing of iC3b-coated target cells (Ramos et al. 1989), and both ICAM-1 and MHC class I molecule on tumor cells participated in the cytotoxicity by autologous lymphocytes (Vanky et al. 1990). CD56-CD56 adhesion pathway N-CAM mediates adhesion between neural cells by a homophilic (like-like) molecular interaction (Edehnan 1984). CD56, a 200-220/135 kDa glycoprotein that is mainly expressed by NK cells and a minor subset of T lymphocytes (Table I), has been found to be an isofonn of N-CAM (Lanier et al. 1989). During the preparation of this manuscript, Nitta et al. (1989) reported a synergistic inhibitory effect of an anti-CD56 mAb together with antibodies to CDl la and CD58 on the conjugation of NK cells and CD56-positive (glioblastoma) target cells. Although preliminary, this result suggests that N-CAM mediates another cell-adhesion pathway in leukocytes, when both effector and target cells express the molecule (Table III).

LEUKOCYTE ADHESION IN VIVO Physical interaction between leukocytes and vascular endothelium appears to participate in granulocyte emigration, lymphocyte homing and recirculation, and leukocyte-dependent vascular injury (Harlan 1985). Taking advantage of the cross-reactivity of some mouse anti-human mAbs with leukocytes from other species, several groups have investigated the participation of CAMs in leukocyte adhesion in rabbits, cats and other animals (reviewed in Patarroyo & Makgoba 1989a). The fundamental role of CDlla-c/CD18 in leukocyte functions in vivo was


93

demonstrated by Arfors et al. (1987) by using mAb 60.3 to CD18. Granulocyte accumulation, as measured by myeloperoxidase, following i.d. injections of purified inflammatory substances, i.e. FMLP, LTB4 and C5a, was totally abolished by pretreatment of the rabbits with the antibody (Fig. 15). Moreover, granulocytedependent plasma leakage induced by these mediators was also inhibited, whereas the granulocyte-independent plasma leakage induced by histamine was unaffected. This in vivo function of CDl la-c/CD18 has later been confirmed in similar models (Price et al. 1987, Nourshargh et al. 1989, Barton et al. 1989, Lindbom et al. 1990, Lundberg & Wright 1990). The inhibition of inflammatory responses by anti-CD 18 mAbs in vivo shows a strong dose dependency. With 0.03 mg antiCD 18 mAb/kg, a 40% reduction in granulocyte accumulation was observed in skin sites injected with zymosan-activated serum (ZAS), and a > 9 5 % inhibition with 1 mg/kg (Lundberg & Wright 1990). Granulocyte-dependent plasma leakage was similarly inhibited in a dose-dependent way by the antibody. It is unlikely that the inhibitory effect of anti-CD18 mAbs in vivo is due to a complement-dependent cytotoxic effect since F(ab')2 fragments of the antibodies are as effective as the whole IgG molecules (Lundberg & Wright 1990). Intravital microscopy of the rabbit tenuissimus muscle superfused with inflammatory mediators has clearly demonstrated that the effect of the anti-CD 18 mAbs relies on inhibition of leukocyte adhesion to post-capillary venular endothelium (Arfors et al. 1987, Lindbom et al. 1990) (Fig. 16). Lack of the adhesive function precludes

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Figure 15. Granulocyte accumulation, as measured by myeloperoxidase, in inflammatory skin lesions from the dorsal region of the rabbit. The animals were pretreated with either saline (control) or mAb 60.3 to CD18 (2mg/Kg) i.v. 10 min prior to the experiment. Mean + SD.

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further steps in the chemotactic response of the stimulated leukocytes, and thus prevents these cells from reaching the interstitial space. Rolling of leukocytes along the venular endothelium was unaffected by anti-CD 18 mAb treatment, indicating that this physical interaction between leukocytes and endothelial cells has a different molecular basis. The "marginating pool" of granulocytes also appears to be independent of CDl la-c/CD18, since injection of epinephrine into a Leu-CAM-deficient patient caused a normal rise in the number of circulating granulocytes (Buchanan et al. 1982). Moreover, CDl la-c/CD18 does not participate in the transient neutropenia caused by systemically injected chemoattractants (Lundberg & Wright 1990). Activation and accumulation of granulocytes are known to cause tissue damage in various inflammatory disorders. Consequently, anti-CD 18 mAb treatment has been shown to reduce granulocyte accumulation and to be beneficial in the outcome of ischemia/reperfusion injury (Hernandez et al. 1987, Vedder et al.

Figure 16. In vivo micrographs of venules in the rabbit tenuissimus muscle before (u|i]Hr panels) and 15 min after (lower panels) topical application of zymosan-activated serum (ZAS). A: animal pretreated intravenously with saline. B: animal pretreated with mAb 7E4 to CD18 (5 mg/Kg). Note profund adherence of leukocytes in the saline-treated animal (lower left) and lack of adherent cells in the antibody-treated animal (lower right) after ZAS stimulation. (From Lindbom et al. 1990).

95


1988, Simpson et al. 1988, Schierwagen et al. 1990) as well as in bacterial meningitis (Tuomanen et al. 1989) in experimental animals. Little is known about the in vivo relevance of the CDl la-c/CD18 complex in the interaction of lymphocytes with vascular endothelium. Hamann et al. (1988) showed that anti-CDlla mAb treatment of radioactively labelled lymphocytes reduced their localization in lymph nodes and Peyer's patches by 40-60%. We have observed a 200% increase in the number of peripheral blood lymphocytes 24 hours after i.v. injection of anti-CD18 mAbs in rabbits (Lindbom et al. 1990) (Fig. 17). This redistribution of lymphocytes appears to be due to interference with lymphocyte homing since the lymphocyte count in lymph fluid drops in parallel (C. Lundberg, unpublished observation). Thus, CDlla-c/CD18 has an acessory role in lymphocyte homing, the specificity of which appears to be determined by the "homing receptors". We have further found that tissue swelling in response to a delayed-type hypersensitivity reaction (DTH), which is mainly T-lymphocyte mediated, is reduced by 65% in rabbits pretreated with anti-CD 18 mAbs (Lindbom et al. 1990). Participation of ICAM-1 in vivo has been also demonstrated. Barton et al. (1989) reported that pretreatment of rabbits with an anti-CD54 mAb inhibited neutrophil migration into phorbol ester-induced inflamed lungs by more than 60%.

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Figure 17. Number of circulating mononuclear and polymorphonuclear leukocytes in peripheral blood during a 72-h period after intravenous treatment with mAb IB4 to CD18 (3 mg/kg). Values represent mean-l-SD of 6 animals. (From Lindbom et al. 1990).

PATARROYO ET AL.

Leu-CAMs AND HIV-INFECTION

Cell fusion and formation of multinucleated giant cells (syncytia) with progression to cell death is a characteristic manifestation of the cytopathic efiect induced by the human immunodeficiency virus (HIV) in infected CD4-positive cells (Valentin et al. 1990). The syncytia formation depends on the interaction of envelope protein gpl20-expressing cells with neighboring cells bearing the surface molecule CD4. HIV infection of monoblastoid U937 clone 16 cells results in a rapid cell aggregation followed by virus replication, pronounced syncytium formation and cell death (Fig. 18A and B). To determine the participation of CDl la-c/CD18 in these phenomena, we have investigated the effect of mAbs 60.3 and 1B4 to CDI8. Treatment of the cells with either of these mAbs prior to HIV-infection and continuous presence of the antibodies in the culture medium completely inhibited the cell aggregation and cytopathic effects (Table IV and Fig. 18C). Moreover, the number of infected cells remained at a very low level for the entire culture period (Valentin et al. 1990). The selective participation of CDlla-c/CD18 in the HIV-induced syncytium formation was demonstrated by replacing mAb 60.3 by W6J32, a mAb of the same isotype but directed to MHC class I molecules. Table IV shows no difference between cells cultured in presence of mAb W6/32 and control cells. In both cultures, the HIV infection resulted in strong syncytia formation and cell death. Similar inhibition of HIV-induced syncytia formation in T cells by mAbs to either CDl la or CD 18 was recently reported by Hildreth & Orentas (1989). Thus, antibodies to CD 18 specifically interfere with HIV infection and HIVinduced cell fusion. The inhibition is not due to interference with the function of CD4 as a receptor for HIV since none of the anti-CD 18 mAbs blocks the binding

Figure 18. HIV-infected U937 clone 16 cells cultured in Libsoiice or presence of mAb 60.3 to CD18. A: infected cells cultured in medium alone showing cell aggregation; B; the same cells showing syncyliutn formation (multinucleated giant cells); C; infected cells pretreated and ciUtured in presence of mAb 60.3

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PATARROYO ET AL.

of purified gp 120 to the cell surface (Valentin et al. 1990). Altogether, the results indicate participation of CD 11 a-c/CD 18, in addition to CD4, in an early step of HIV infection and syncytium formation. They also suggest that intercellular adhesion contributes to virus transmission from cell to cell and may be an important mechanism for virus spreading.

ABSENCE OF CAMs IN BURKITT'S LYMPHOMA (BL) Anti-CD 18 antibodies inhibit induction of mononuclear leukocyte aggregation and the following IL-2 production and lymphocyte proliferation (Beatty et al. 1983, Lindqvist et al. 1987). Thus, intercellular adhesion appears to modulate lymphocyte growth. In 1982, one of us (M. P.) proposed that adhesion (binding) between different mononuclear leukocytes could mediate the intercellular communication required for the control of lymphocyte proliferation and maturation (Patarroyo 1982, Patarroyo et al. 1983 a,b). Consequently, we postulated that lack of a cell-adhesive phenotype or expression of abnormal adhesion patterns might free transformed lymphocytes from this control and thus contribute to leukemogenesis and lymphomagenesis (Patarroyo et al. 1986). Folllowing the preliminary finding that cells from an EBV-positive BL line expressed lower amounts of CD 18 than EBV-immortalized normal B cells from a lymphoblastoid cell line (LCL) (Patarroyo et al. 1987), we analyzed the cell surface expression of the Leu-CAM family and lCAM-1 in 10 LCLs and 10 BL lines (Patarroyo et al. 1988b). Leu-CAMa, the most abundant member of the family on LCLs, as well as Leu-CAMb and Leu-CAMc, were absent, or expressed at low levels, on all BL lines, whereas three BL lines expressed low amounts of ICAM-l, although the difference in density of this molecule was less extreme than that of Leu-CAMa. BL hnes and LCLs derived from the same patient also display the contrasting expression of Leu-CAMa. As expected, phorbol ester could not induce aggregation of Leu-CAM-negative BL hnes (Fig. 2). In additional studies we have found also low expression of CD58 on the tumor cells (Rincon et al., unpublished observations). Although LCLs express Leu-CAMa, lCAM-1 and CD58 in rather equal amounts, in BL lines Leu-CAMa is expressed at the lowest level, followed by CD58 and ICAM-I. Related studies, including analysis of frozen sections of tumor biopsies, by other groups have provided similar results (reviewed in Patarroyo & Makgoba 1989a). Thus, this EBVassociated B-cell malignancy lacks, or has very low levels of, the CAMs that are otherwise largely expressed on EBV-transformed normal B cells. The latter cells, which are activated by the virus and become adhesive, can be obtained from the blood of patients with infectious mononucleosis, a benign and self-limiting lymphoproliferative disease. Absence of CAMs renders the defective cells unable to conjugate with T lymphocytes, a phenomenon involved in T-cell stimulation and lymphocyte-mediated cytotoxicity.


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The absence of Leu-CAMa, and probably of ICAM-1 and CD58, in BL cells appears to be due to a downregulation of the molecules and may help the transformed cell to escape from the immunological control imposed by normal lymphocytes. We have postulated that deregulated expression of the c-myc oncogene, a characteristic finding in BL cells, contributes to the downregulation (Patarroyo et al. 1988b). In further studies we induced expression of Leu-CAMa, but not of Leu-CAMb or Leu-CAMc, in BL lines by prolonged (3 days) treatment with phorbol ester (Fig. 19). Upregulation of ICAM-1 and CD58 was observed in some, but not all, BL lines, and only sometimes. These results indicate that BL cells, when properly stimulated, are able to express Leu-CAMa. They also suggest that the synthesis of this molecule involves prolonged PKC activation, and is regulated differently from ICAM-1 and CD58. Analysis of CAM-mRNA expression is in progress. We have not succeeded in inducing Leu-CAMa expression by treatment with various cytokines, including IL-4. Recently, Rousset et al. (1989) reported induction of Leu-CAMa and CD58 in a BL line by treatment with IL-4. CONCLUDING REMARKS CAMs have been identified in leukocytes by using adhesion-specific assays and blocking mAbs. To conclude that all surface molecules involved in physical

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O — O CD18

# — • CDlla A — A CDllb A CD11C 75-- •

Time (hours) Figure 19. Itiduction of expression of CDlla/CD18 in Burkitt's lymphoma cells by prolonged treatment with phorbol esters. BL 41 cells were incubated with P(Bu): (60nM) for different periods of time. AnUbodies used: CDlla (H12), CDl lb (60.1), CD! lc (Leu-M5) and CD18 (60.3).

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contacts between cells, such as conjugation of CTL and target cells, are CAMs is incorrect, since several of the molecules may instead mediate stimulus recognition and cell activation. Excluding the CAMs, antibodies to at least 10 different cell surface molecules have been reported to block conjugation of leukocytes with cells (reviewed in Patarroyo & Makgoba 1989a) including CD4, CD8, class II molecules, Thy-1, the T-cell receptor, "homing receptors", "vascular addressins" and ELAM-1. Although some of these molecules appear to recognize complementary structures on the surface of adjacent cells, as the CAMs do, and somehow participate in conjugate formation, it is unclear whether their intermolecular bonds directly provide the fast (firm) stickiness between the two cells characteristic of adhesion under physiological conditions. Thus, several of these structures may be receptors for membrane-bound ligands, the recognition of which is necessary for triggering the Leu-CAM-ICAM-1 and/or CD2-CD58 adhesion pathways, as demonstrated for the T-cel! receptor (Dustin & Springer 1989). Similarly, LeuCAMa has been reported to have an "accessory role" in the physical interaction between lymphocytes and high endothelium mediated by the homing receptors. Alternatively, these molecules may be cell surface enzymes required for cell activation. Recently, enzymatic activity has been demonstrated for several CDs (Kenny et al. 1989). Another possibility is that these structures transduce "negative signals" into the leukocyte. On the other hand, ELAM-1 could be a novel CAM since this molecule participates in CDl 1 a-c/CD 18-independent granulocyte adhesion to endothelial cells (Dobrina et al. 1989). High binding strength is a major parameter of leukocyte adhesion and can be measured by using biophysical approaches. Binding of lymphoid cells to transfected cells expressing high levels of either CD4 or CD8, through MHC class II and I molecules, respectively, on the former cells has been also reported. However, the interactions were of low avidity. In contrast, the Leu-CAM-ICAM-1 adhesion stimulated by phorbol esters is so strong that mechanical dissociation of the cell aggregates produces fragmentation of the cells before they de-adhere (M. Patarroyo, unpublished observation). To determine the exact function of all these other molecules, dissecting and specific functional assays are needed. Anti-CAM antibodies have been reported to block a large variety of complex leukocyte functions (induction of lymphocyte proliferation and differentiation, lymphocyte-mediated cytotoxicity, phagocytosis of large particles, chemotaxis etc). Although inhibition of the physical contact/adhesion has been demonstrated in most cases, blocking of additional unknown functions of these molecules is not excluded. Thus, cell adhesion is a fundamental process in the physiology of T and B lymphocytes, NK cells, monocyte-macrophages, and granulocytes. Since the process is required for most leukocyte functions, blocking of the adhesion, by mAbs or synthetic peptides that mimic the CAMs, has a large therapeutic potential. Such agents may suppress rejection of transplanted organs as well as inflammation


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in immune diseases or in hypovolemic and endotoxic shock. Further adhesion studies with mononuclear leukocytes may contribute to a better understanding of the genesis of lymphoid organs and their cellular composition and the pathophysiology of hemopoietic malignancies. The therapeutic implications of these observations on leukocyte adhesion remain a challenge for future basic and clinical research. ' SUMMARY Leukocyte-cell adhesion is a form of physical contact characterized by fast (firm) stickiness between the cells. To analyze the biology and molecular basis of this process, an adhesion-specific assay was developed: the phorbol ester-induced aggregation of human lymphocytes. This rapid and antigen-independent intercellular adhesion requires cellular metabolism, an intact cytoskeleton and extracellular divalent cations, and is mediated by preformed cell-surface proteins referred to as CAMs. Phorbol ester also induces aggregation of monocytes and granulocytes, as well as adhesion of T lymphocytes to either B cells or monocytes and of the leukocytes to vascular endothelial cells. By using the adhesion-specific assay and blocking monoclonal antibodies, several CAMs have been identified, namely the Leu-CAM family (CDlla-c/CD18) and ICAM-I (CD54). The LeuCAM family is composed of Leu-CAMa (CDlla/CD18), Leu-CAMb (CDllb/ CD18) and Leu-CAMc (CDl lc/CD18), three glycoprotein heterodimers made of a common ^-chain and distinct a-chains. ICAM-1 is an adhesive ligand for LeuCAMa. Expression and use of the various CAMs is selective in different types of leukocytes. The Leu-CAMs have been purified and partially characterized. CD18, whose gene is on human chromosome 21, contains 5-6 N-linked complextype oligosaccharides, and CDl 1 binds Ca^^. Another adhesion pathway is mediated by CD2 and CD58. CD2, a glycoprotein selectively expressed by T cells, is a receptor for CD58, a cell-surface adhesive ligand with broad tissue distribution. Antibodies to the latter CAMs do not block the phorbol ester-induced lymphocyte aggregation. Adhesion is involved in a large variety of leukocyte functions. Anti-Leu-CAM antibodies block induction of IL-2 production and lymphocyte proliferation. Lymphocyte-mediated cytotoxicity is also inhibited. Endogenous NK and LAK cells use Leu-CAMs, ICAM-l and CD2, and sometimes RGD receptors, to bind and kill tumor cells. Endogenous compounds such as HjOj and LTB4 also induce Leu-CAM-dependent adhesion in monocytoid cells and granulocytes, respectively, and degranulation of the latter cells is enhanced by the adhesion process. Homologous CAMs have been identified in rabbit and mouse. In in vivo studies in the former species, anti-Leu-CAM antibodies block adhesion of leukocytes to vascular endothelium and thereby their migration into extravascular tissues. The antibodies thus inhibit granulocyte accumulation and plasma leakage in inflammatory

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lesions, and induce lympho- and granulocytosis, indicating that cell-adhesion contributes to the distribution of leukocytes in the body. In a separate study, LeuCAMs were found to participate in the cytopathic effects, including syncytium formation, induced by HIV in human monocytoid cells. Cells from Burkitt's lymphoma, a B-cell malignancy associated witb EBV, lack or have very low levels of the CAMs that are otherwise largely expressed on EBV-transformed normal B cells. The absence of Leu-CAMa, ICAM-1 and CD58 on BL cells may help these cells to escape from the immunological control imposed by normal lymphocytes. Treatment of the tumor cells with phorbol ester for 3 days induces expression of Leu-CAMa. ACKNOWLEDGMENTS The authors wish to thank Ms I. Lindfors and I. Axberg for typing the manuscript, and Drs P. Beatty, E. Clark and S. Wright for providing mAbs 60.3, LB-2 and IB4, respectively. The research work of M. Patarroyo was supported by the Swedish Cancer Society, the Medical Research Council and the Karolinska Institute. L. Lindbom is supported by the Medical Research Council and C. G. Gahmberg by the Academy of Finland and the Sigrid Juselius Foundation. REFERENCES Agrwal, N. & Thomas, D. W. (1984) A role for la antigens in tbymocyte binding by macrophages. Cell Immunol. 84, 352. Anderson, D. C. & Springer, T. A. (1987) Leukocyte adhesion deficiency: an inherited defect in the Mac-1, LFA-1, and pl50, 95 glycoproteins. Ann. Rev. Med. 38, 175. Arfors, K. E., Lundberg, C , Lindbom, L., Lundberg, K., Beatty, P. G. & Harlan, J. M. (1987) A monoclonal antibody to the membrane glycoprotein complex CD18 inhibits polymorphonuclear leukocyte accumulation and plasma leakage in vivo. Blood 69, 338. Araaout, M. A., Raymond, M. H., Todd III, R. F., Dana, N. & Colten, H. R. (1985) Increased expression of an adhesion-promoting surface glycoprotein in the granulocytopenia of hemodialysis. N. Engl. J. Med. 312, 457. Axberg, L, Ramstedt, U., Patarroyo, M., Beatty, P. G. & Wigzell, H. (1987) Inhibition of natural killer cell cytotoxicity by a monoclonal antibody directed against adhesionmediating protein gp 90 (CD 18). Scand. J. Immunoi 36, 547. Barton, R., Rotlein, R., Ksiazek, J. & Kennedy, C. (1989) The effect of anti-intercellular adhesion molecule-1 on phorbol ester-induced rabbit lung inflammation. /. Immunol. 143, 1278. Beatty, P. G., Ledbetter, J. A., Martin, P. J., Price, T. H. & Hansen, J. A. (1983) Definition of a common leukocyte ceU-surface antigen (Lp95-150) associated with diverse cellmediated immune functions. / Immunol. 131, 2913. Berendt, A. R., Simmons, D. L., Tansey, J., Newbold, C. I. & Marsh, K. (1989) Intercellular adhesion molecule-1 is an endothelial cell adhesion receptor for plasmodium falciparum. Nature 341, 57. Biddinson, W. E., Rao, P. E., TaUe, M. A., Goldstein, G. & Shaw, S. (1984) Possible involvement of the T4 molecule in T cell recognition of class II HLA antigens; evidence from studies of CTL-target cell binding. J. Exp. Med. 159, 783.


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of endothelial cells and of neutrophils induced by leukotrine B4 are mediated by leukocyte adhesion protein CD18. (submitted for publication). Liskamp, R. M. J., Brothman, A. R.. Arcoleo, J. P., Miller, O. J. & Weinslein, I. B. (1985) Cellular uptake and localization of fluorescent derivatives of phorbol ester tumor promoters. Biochem. Biophys. Res. Comm. 131, 920. Lopez, C. & Ades, E. eds. (1989) Natural Killer cells and host defences. Karger, Basel. Lundberg, C. & Wright, S. D. (1990) Relation of the CD11/CD18 family of leukocyte antigens to the transient neutropenia caused by chemoattractants. (submitted for publication) Marlin, S. D. & Springer, T. A. (1987) Purified intercellular adhesion moleculc-l (ICAM1) is a ligand for lymphocyte function-associated antigen 1 (LFA-1). Cell 51, 813. Martz, E. (1987) LFA-l and other accesory molecules functioning in adhesion of T and B lymphocytes. Hum. Immunol. 10, 3. Mentzer, S. J., Gromkowski, S. H., Krensky, A. M., Burakoff, S. J. & Martz, E. (1985) LFA-1 membrane molecule in the regulation of homotypic adhesions of human B lymphocytes. J. Immunol. 135, 9. Moingeon, R, Chan, H., Wallner, B. R, Stebbins, C , Frey, A. Z. & Reinherz, E. L. (1989) CD-2-mediated adhesion facilitates T lymphocyte antigen recognition. Nature 339, 312. Nishizuka, Y. (1984) The role of protein kinase C in celt surface signal transduction and tumor promotion. Nature 308, 693. Nitta, T., Yagita, H., Sato, K. & Okumura, K. (1989) Involvement of CD56 {NKH-1/Leu19 antigen) as an adhesion molecule in natural killer-target cell interaction. /. Exp.

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