UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New series No. 456 - ISSN 0346-6612 From the Department of Cell and Molecular Biology University of Umeå

REGULATION OF LEUKOCYTE ADHESIVENESS

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Ilåkan Hedman

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Department of Cell and Molecular Biology University of Umeå

Umeå 1996 REGULATION OF LEUKOCYTE INTEGRIN ADHESIVENESS

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AKADEMISK AVHANDLING som för avläggnade av doktorsexamen i medicinsk vetenskap vid Umeå universitet, offentligen kommer att försvaras i föreläsningssalen, institutionen för mikrobiologi, Umeå universitet, fredagen den 26 januari 1996, kl. 10.00

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Håkan Hedman Institutionen för tillämpad cell- och molekylärbiologi Umeå universitet Umeå 1996 ABSTRACT

REGULATION OF LEUKOCYTE INTEGRIN ADHESIVENESS

Håkan Hedman, Department of Cell and Molecular Biology, University of Umeå, S-90187 Umeå, Sweden.

The adhesive integrin receptors are important for the regulated interactions of leukocytes with other cells and with the extracellular matrix. Adhesion of to their ligands is not constitutive but requires activation. This thesis describes an experimental system for studies of the adhesiveness regu­ lation of p2-integrin LFA-1 and pl-integrin VLA-4. It was used to study the role of kinases and phosphatases for the regulation of lymphocyte integrin adhesiveness, to demonstrate an adhesive potential of membrane microvesi­ cles shed from monocytes, and for isolation and analysis of mutant cells with aberrant regulation.

LFA-1 mediates adhesion between cells by binding to its ligands ICAM-1, ICAM-2 and ICAM-3, and VLA-4 by binding to VCAM-1. Changes in LFA-1 ad­ hesiveness was monitored by evaluating adhesion of cells to magnetic beads coated with a purified ICAM-1 fusion protein. A gene encoding the 5 extra­ cellular domains of human ICAM-1 fused to part of a mouse IgG was con­ structed and expressed in Chinese hamster ovary cells. The gene product was purified from the cell culture supernatant and coupled to magnetic beads. Cells bound the beads in an LFA-1/ICAM-1 dependent m anner and they were used to isolate and quantify cells with activated LFA-1. Monocyte-derived mi­ crovesicles were also found to bind in an LFA-1 dependent manner. A similar system was set up, with VCAM-1 coated beads, to isolate and quantify cells with activated VLA-4.

In human B cells, binding to ICAM-1 and VCAM-1 was activated by protein ki­ nase C activating phorbol ester and by protein kinase inhibitor stau- rosporine, and repressed by protein phosphatase inhibitors okadaic acid and calyculin A. From this we proposed a model for regulation of integrin adhe­ siveness where LFA-1 and VLA-4 is regulated by an integrin regulating protein. When phosphorylated by a staurosporine sensitive ser­ ine/threonine kinase the protein repress integrin function and when de- phosphorylated by protein phosphatase 1 the repression is released.

By an approach of random mutagenesis and selection lymphocyte clones with constitutively active LFA-1 were established. Many of the clones had lost apparent cell surface expression of pi-integrins but expressed wild-type levels of pi mRNA. In immunoprécipitations wild-type 130 kD p 1-chains could not be detected but a novel 30 kD band was found. In western blots however, a 130 kD band was found. Since we showed that neither did pi downregulation per se cause LFA-1 activation, nor did LFA-1 activationper se cause pi down­ regulation, we propose that pi processing and LFA-1 repression are coupled by a common gene product.

Keywords: Lymphocytes; Cell Adhesion; Integrins; LFA-1; VLA-4; ICAM-1; VCAM-1; Microvesicles; Protein Kinases; Protein Phosphatases.

New series No. 456 - ISSN 0346-6612 ISBN 91-7191-144-8 UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New series No. 456 - ISSN 0346-6612 From the Department o f Cell and Molecular Biology University of Umeå

REGULATION OF LEUKOCYTE INTEGRIN ADHESIVENESS

b y

Håkan Hedman

Department of Cell and Molecular Biology University of Umeå

Umeå 1996 Printed in Sweden by Solfjädern Offset AB Umeå 1995 3

CONTENTS

1. ABSTRACT 4

2. PAPERS IN THIS THESIS 5

3. INTRODUCTION TO THE INTEGRIN FAMILY OF ADHESIVE CELL SURFACE RECEPTORS 6 3.1 Identification and role of LFA-1 7 3.2 Integrin structures 7 3.3 Extracellular ligand interactions 9 3.4 Cytoskeletal interactions 11 3.5 Biosynthesis 12

4. INTRODUCTION TO REGULATION OF INTEGRIN ADHESIVENESS 13 4.1 Integrin activation 14 4.2 Conformational changes 15 4.3 Role of 16 4.4 Inside-out signaling 17

5. OBJECTIVES 18

6. RESULTS AND DISCUSSION 6.1 The experimental system 18 6.2 Role of phosphorylation for integrin adhesiveness 20 6.3 Adhesive potential of monocyte microvesicles 20 6.4 Identification of LFA-1 regulating gene products 22 6.5 A model for LFA-1 regulation 23

7. CONCLUSIONS 26

8. ACNOWLEDGEMENTS 27

9. REFERENCES 29 4

1. ABSTRACT

The adhesive integrin receptors are important for the regulated interactions of leukocytes with other cells and with the extracellular matrix. Adhesion of inte- grins to their ligands is not constitutive but requires activation. This thesis de­ scribes an experimental system for studies of the adhesiveness regulation of ß2- integrin LFA-1 and ßl-integrin VLA-4. It was used to study the role of kinases and phosphatases for the regulation of lymphocyte integrin adhesiveness, to demon­ strate an adhesive potential of membrane micro vesicles shed from monocytes, and for isolation and analysis of mutant cells with aberrant regulation. LFA-1 mediates adhesion between cells by binding to its ligands ICAM-1, ICAM-2 and ICAM-3, and VLA-4 by binding to VCAM-1. Changes in LFA-1 adhe­ siveness was monitored by evaluating adhesion of cells to magnetic beads coated with a purified ICAM-1 fusion protein. A gene encoding the 5 extracellular do­ mains of human ICAM-1 fused to part of a mouse IgG was constructed and ex­ pressed in Chinese hamster ovary cells. The gene product was purified from the cell culture supernatant and coupled to magnetic beads. Cells bound the beads in an LFA-l/ICAM-1 dependent manner and they were used to isolate and quantify cells with activated LFA-1. Monocyte-derived microvesicles were also found to bind in an LFA-1 dependent manner. A similar system was set up, with VCAM-1 coated beads, to isolate and quantify cells with activated VLA-4. In human B cells, binding to ICAM-1 and VCAM-1 was activated by protein kinase C activating phorbol ester and by protein kinase inhibitor staurosporine, and repressed by protein phosphatase inhibitors okadaic acid and calyculin A. From this we proposed a model for regulation of integrin adhesiveness where LFA-1 and VLA-4 is regulated by an integrin regulating protein. When phospho- rylated by a staurosporine sensitive serine/threonine kinase the protein repress integrin function and when de-phosphorylated by protein phosphatase 1 the re­ pression is released. By an approach of random mutagenesis and selection lymphocyte clones with consti tu tively active LFA-1 were established. Many of the clones had lost ap­ parent cell surface expression of ßl-integrins but expressed wild-type levels of ßl mRNA. In immunoprécipitations wild-type 130 kD ßl-chains could not be detected but a novel 30 kD band was found. In western blots however, a 130 kD band was found. Since we showed that neither did ßl downregulationper se cause LFA-1 ac­ tivation, nor did LFA-1 activationper se cause ßl downregulation, we propose that ßl processing and LFA-1 repression are coupled by a common gene product. 5

2. PAPERS IN THIS THESIS

This thesis is based on the following papers, which will be referred to by their Roman numerals (I-VI).

I. Hedman, Håkan, Henrik Brändén, and Erik Lundgren. 1992. Physical separa­ tion of ICAM-1 binding cells. J. Immunol. Methods 146: 203-211.

II. Hedman, Håkan, and Erik Lundgren. 1992. Regulation of LFA-1 avidity in hu­ man B cells: requirements for dephosphorylation events for high avidity ICAM-1 binding. J. Immunol. 149: 2295-2299.

III. Satta, Natalie, Florence Toti, Olivier Feugeas, Alain Bohbot, Jeanne Dachary- Prigent, Valérie Eschwège, Håkan Hedman, and Jean-Marie Freyssinet. 1994. Monocyte vésiculation is a possible mechanism for dissemination of mem­ brane-associated procoagulant activities and adhesion molecules after stimu­ lation by lipopolysaccharide. J. Immunol. 153: 3245-3255.

IV. Hedman, Håkan, and Erik Lundgren. 1995. Regulation of VLA-4 avidity in hu­ man B cells: requirements for dephosphorylation events for high avidity VCAM-1 binding.Submitted.

V. Hedman, Håkan, and Erik Lundgren. 1995. Generation of mutant cells with consti tu tively active ß2-integrin LFA-1.Submitted.

VI. Hedman, Håkan, Mattias Alenius, and Erik Lundgren. 1995. Coupling between expression of ßl-integrins and function of aLß2.Manuscript. 6

3. INTRODUCTION TO THE INTEGRIN FAMILY OF ADHESIVE CELL SURFACE RECEPTORS.

Virtually all mammalian cells express adhesive integrin receptors. They are het­ erodimers composed of an «-chain non-covalently associated with a ß-chain. There are at present at least 15 mammalian «-chains and 8 ß-chains cloned. They combine to form approximately 21 complete integrin heterodimers (fig. 1). To date, the most studied integrins are the mammalian leukocyte ß2-integrins, the platelet glycoprotein Ilbllla (GPIIbllla), and the prototype integrin - the fibronectin re­ ceptor «5ßl.

A primordial and important role is suggested as mouse egg integrin «6ßl functions as a sperm receptor (Almeida et al., 1995) by binding to the sperm integrin ligand PH-30ß (Biobei et al., 1992). Integrins are found not only in vertebrates but also in arthropods (Bogaert et al., 1987) and nematodes (Gettner et al., 1995), and there is some functional and immunological evidence for expression in fungi as well (Marcantonio and Hynes, 1988; Calderone, 1993). Furthermore, a fibronectin re­ ceptor in the protist Entamoeba histolytica might also be an integrin (Talamas- Rohana et al., 1992). Thus integrins are widely distributed within, and possibly outside, the animal kingdom.

This thesis focuses mainly on the leukocyte integrin LFA-1 but I will try to put the results in context of integrin function in general.

a4-NLA-4^p1 6 3 lGPllblllal«TTh

Figure 1. The integrin receptor family. The figure shows the pairing of mam­ malian integrin heterodimers. Boxes indicate commonly used names for respective heterodimer. References: Hynes, 1992; Danilenko et al., 1995. 7

3.1 Identification and role of LFA-1

LFA-1 was identified by screens for inhibiting cytotoxic T lymphocyte (CTL) mediated killing of target cells (Davignon et al., 1981a; Sanchez-Madrid et al., 1982). The antigen is expressed on most leukocytes (Kiirzinger et al., 1981) and contains a noncovalently associated a- and ß-subunit, where the a-subunit (CDlla, aL) is unique to LFA-1 and the ß-subunit (CD18, ß2) is common to three other leukocyte antigens (fig. 1), (Sanchez-Madrid et al., 1983; Danilenko et al., 1995). LFA-1 mediates CTL-target cell conjugate formation and other intercellular adhe­ sive interactions by binding to its ligands ICAM-1 (Marlin and Springer, 1987), ICAM-2 (Staunton et al., 1989), and ICAM-3 (de Fougerolles and Springer, 1992).

Experiments with inhibitory antibodies have suggested that LFA-1 plays an impor­ tant role in the function of the immune system. Thus,in vitro , anti-LFA-1 anti­ bodies inhibit homotypic aggregation of lymphocytes (Mentzer et al., 1985; Rothlein and Springer, 1986), antigen specific T cell proliferation, the mixed lym­ phocyte response, T-dependent B cell response (Davignon et al., 1981b), and T cell proliferation in response to PHA (Krensky et al., 1983).In vivo, lymphocyte recir­ culation is inhibited to both peripheral lymph nodes and Peyer's patches (Camp et al., 1993). Anti-LFA-1 antibodies are also able to induce tolerance to allografts (Isobe et al., 1992).

The human LFA-1 ß-subunit (CD18, ß2) and a-subunit (CDlla, aL) have been cloned and sequenced (Kishimoto et al., 1987; Law et al., 1987; Larson et al., 1989). Subse­ quently, ß2-homologues have been identified in birds (Bilsland and Springer 1994) and amphibians (Ransom et al., 1993). When the amino acid sequence of the LFA-1 ß-subunit (ß2) was determined it was found to show strong homology to platelet band III (ß3) and chicken fibronectin receptor (ßl), thereby establishing LFA-1 as a member of the integrin family of cell surface receptors.

3.2 Integrin structures

To date, only isolated I domains of integrins have been subject to x-ray analysis (Lee et al., 1995; Qu and Leahy, 1995). Integrin structures have therefore been de­ duced from sequence data and electron microscopic studies. The latter has indi­ cated that integrin heterodimers adopt a mushroom-like structure with two stalks (fig. 2A). 8

.'VA A rfcv

445 631 747

ß2 z h m c ' B 130 327 422 1145 r—I-—i Me2+ I aL I

[DxSxSj(ß2:134-138; ß3:119-123)

1 |^SxSj(aL:137-141) I

IxxDVxxDxxxD/Exl (aIIb:296-306) I— j[J__(aL:422-542)

H S x x x D x x x D 1 (aL:505-513)

Figure 2. Schematic drawings of integrin structures. A, an integrin heterodimer with dimensions as determined by electron microscopic studies by Nermut et al. (1988). B,motifs found in integrinaL and ß2 subunits by Kishimoto et al.(1987), Law et al. (1987),and Larson et al.(1989). In ß2 the four cystein rich repeats are indicated (Cys) and inaL the I-domain(I) and the three repeated putative metal cation-binding sites (Me2+) are indicated. Numbers indicate amino acid numbers in the respective chains according to the above references. C, residues implicated in ligand interactions as discussed in the text. Shown is a schematic of ß2aL, and to which respective ß3 andallb sequences are aligned. 9

The ß-chains share extensive homologies with each other but not with the ex- chains. The processed ß2-chain is predicted to be a 747 amino acid transmembrane protein consisting of a 677 amino acid extracellular domain, a 23 amino acid transmembrane domain, and a short 46 amino acid cytoplasmic domain (fig. 2B). ßl, ß2, ß3, ß5, ß6 all share 56 conserved cysteins in their putative extracellular do­ mains and ß4, ß7 and ß8 share 48, 54 and 50 of those cysteins, respectively (Moyle et al., 1991). The majority of cysteins are located within four cystein-rich tandem re­ peats. The ß-chain cytoplasmic domains are homologous to each other, with the ex­ ception of ß4 and ß8, which appear unrelated to the others (Moyle et al., 1991).

The processed «L-chain is predicted to be 1145 a amino acid transmembrane pro­ tein consisting of a1063 amino acid extracellular domain, 29 a amino acid trans­ membrane domain, and 53a amino acid cytoplasmic domain (fig.2B). Integrina- subunits contain a seven-fold repeat of approximately50 amino acids. The last three or four of these repeats contain putative cation-binding sites with resem­ blance to EF-hands (Hynes,1992). al, a2,aE, aL, «M andaX have an 'inserted' or T domain not present in other a-chains (Shaw et al.,1994). The I domains contain approximately 200 amino acids and are homologous to the ligand-binding repeats in von Willebrand factor. I domains bind cations in a so called, metal ion-depen­ dent adhesion site (MIDAS), (Lee et al.,1995). All human a-chains sequenced so far posses a KxGFFKR motif in the membrane proximal part of the cytoplasmic do­ mains.

3.3 Extracellular ligand interactions

Integrins mediate adhesion of cells by binding to ligands in the extracellular ma­ trix and on other cells in a divalent cation dependent manner. Generally, the affinities between integrins and their ligands are relatively low and adhesion usually requires multivalent integrin-ligand interactions. Recognition is some­ what promiscuous as each integrin usually binds several different ligands, and conversely, a particular ligand often binds several different integrins (tabl.l). Both the a- and ß-chains contribute to binding specificity (tabi.1) and participate in ligand binding. The ligand binding sites of the plateletallbß3 have been mapped by crosslinking studies with peptides from the fibrinogen ligand. Thus, binding of an RGDS peptide has been mapped to residues109-171 in ß3 (D'Souza et al., 1988),while binding of a different fibrinogen peptide maps to residues296-306 in allb (fig. 2C), (D'Souza et al., 1990;D'Souza et al, 1991). 10

Table 1. Integrin ligands a l a2 a3 a5 a4 aE FN40, ßl CN, LN CN, LN FN, LN FN VCAM-1 CN aa4 acx4 ß7 bMadC-l cE-cad

aL aM aX a d a l l b aV iC3b, FG, VN, FG, ß2 ICAM-1 X, eHN, FG, iC3b, hICAM-3 ß3 FG, FN, vWF, ICAM-2 fDP, fDP vWF, TSN, FN, dICAM-3 ICAM-1 VN, TSN OP, CN siCAM-2

Ligands for 13 of the 21 described mammalian integrins according to Hynes (1992) except where references are indicated. CN, collagens; LN, laminin; FN, fibronectin; FN40, 40 kD chymotryptic fibronectin fragment; MadC-1, MadCAM-1; E-cad, E-cad- herin; X, factor X; HN, heparin; DP, denatured proteins; vWF, von Willebrand fac­ tor; VN, vitronectin; TSN, thrombospondin; OP, osteopontin. References: aAltevogt et al., 1995, bBerlin et al., 1993, cCepek et al., 1994, dde Fougerolles and Springer, 1992, eDiamond et al., 1995, fDavis, 1992, §Xie et al., 1995, hDanilenko et al., 1995.

The ß3 residues 109-171 lies within a segment that is highly conserved among the ß subunits. Furthermore, the sequence DxSxS (residues 119-123) is completely con­ served among all ß subunits. This is part of the so called MIDAS motif (see below) that is suggested to coordinate metal ions and participate in ligand binding (Lee et al., 1995). Consequently, replacing point mutations of D119, S121, and S123 abol­ ishes fibrinogen and RGD binding (Bajt and Loftus, 1994). Similarly, mutations of the corresponding ß2 residues D134 and S136 abolish LFA-1 and Mac-1 mediated cell adhesion, while mutation of S138 results in loss of ß2 surface expression (Bajt et al., 1995).

Residues 296-306 ofallb contain the second of the four repeated putative cation binding sites withinallb. There are also indications that the two first metal bind­ ing repeats of aL may participate in the binding of LFA-1 to ICAM-1 (Stanley et al., 1994). However, for I-domain containing a-chains, the I-domain itself is the best characterized region directly implicated in ligand recognition (Diamond et al., 1993; Huang and Springer 1995). Isolated I-domains bind metal ions (Michishita et al., 1993) and ligands directly (see for example: Randi and Hogg, 1994; Ueda et al., 1994; Zhou et al., 1994; Xie et al., 1995). Mg^+ binding to the I-domain forms five co­ ordinations with the MIDAS site, thus leaving an extra ”available" coordination 11

site free (Lee et al., 1995). This site is predicted to be coordinated by oxygenated residues in integrin ligands (Lee et al., 1995). In the case of ICAM-1, this is likely to be residue E34 which is essential for ligand binding (Huang and Springer, 1995). Thus, the Mg^+ seems to directly bridge the integrin I-domain with the in­ tegrin ligand. Surrounding the Mg 2+ several residues have been mapped that do not coordinate the metal ion but instead define a binding interface on the LFA-1 I-domain (Huang and Springer, 1995).

Taken together, several sites in integrina- and ß-chains have been implicated in ligand recognition. They all contain cation binding motifs and are: (i), the «- chain, I-domain, MIDAS motif, (ii), some of the putative cation-binding «-chain repeats, and (iii), sites in the ß-chains with similarities to the MIDAS motif (summarized in fig. 2C).

3.4 Cytoskeletal interactions

Integrins are so termed due to their integral membrane nature and probable role as a link between the extracellular matrix and the cytoskeleton (Tamkun et al., 1986). When fibroblasts grown in vitro form stable adhesions to the substrate, in­ tegrin receptors concentrate in adhesion sites termed focal adhesions. In these regions, where cytoskeletal proteins such as and «-actinin also co-localize, large bundles of actin microfilaments, termed stress fibers, are anchored (reviewed in Burridge et al., 1988). For the «5ßl fibronectin receptor, the ßl cyto­ plasmic domain is necessary and sufficient for localization to focal adhesions (LaFlamme et al., 1992).

Three ßl amino acid clusters have been implicated in cytoskeletal association and focal adhesion localization (fig. 3) (Reszka et al., 1992). Direct interactions of pu­ rified proteins with ß-chain cytoplasmic peptides have been demonstrated for ßl and «-actinin (Otey et al., 1990; Otey et al., 1993), ß2 and «-actinin (Pavalko and LaRoche, 1993), ß2 and filamin (Sharma et al., 1995), and, ßl and focal adhesion ki­ nase (FAK), (Schaller et al., 1995), (fig. 3). However, it has been observed that talin (Horwitz et al., 1986), vinculin (Sharma et al., 1995) and paxillin (Schaller et al., 1995), also directly or indirectly interact with integrins. The complexity is illus­ trated by talin which binds to integrin (Horwitz et al., 1986), vinculin (Gilmore et al., 1993) and FAK (Chen et al., 1995). FAK, in turn, binds integrin directly (Schaller et al., 1995), and vinculin binds F-actin (Johnson and Craig, 1995) and «- actinin (Belkin and Koteliansky, 1987), the latter of which binds integrins di- - pl25FAK pi- ß2------a-actinin ß2------filamin

Figure 3. Amino acid sequences of ß-chain cytoplasmic domains. Shown are ßl residues K752-K798 (Argraves et al., 1987), ß2 residues K724-S769 (Kishimoto et al., 1987), and ß3 residues K716-T762 (Fitzgerald et al., 1987). Amino acids identical between chains are boxed. Lines above sequences, indicate regions involved in fo­ cal adhesion localization. Lines below sequences indicate peptides that have been shown to directly interact with respective protein (see text for references). (-) and (+) indicates regulatory sequences discussed in section 4.4. rectly. Thus, only future work will tell what the significant molecular interac­ tions between integrins and the cytoskeleton are.

3.5 Biosynthesis

Integrin a- and ß-chains are synthesized as transmembrane precursor polypep­ tides which associate to form aß heterodimers in the endoplasmic reticulum. The chains must dimerize before maturation and export to the cell surface. This is manifested in two human genetic diseases, leukocyte adhesion deficiency I and Glanzmann's thrombasthenia.

In leukocyte adhesion deficiency I the leukocytes are deficient in the ß2 integrins LFA-1, Mac-1 and pl50,95. This is due to different mutations in the ß2 gene which results in the inability of the correspondinga chains, aL, aM, andaX, to be trans­ ported to the cell surface (Anderson and Springer, 1987).

Similarly, the molecular basis for variants of Glanzmann’s thrombasthenia are de­ fects in either the allb or the ß3 gene (Coller et al., 1991). In either case, the «IIbß3 complex is absent from the cell surface. However, since normal platelets express allbß3 and«Vß3 (but not ß5, ß6,or ß8), defects inß3 also results in the ab­ sence of aV on the surface (Coller et al., 1991). Thus, single chain integrin molecules are not normally found at the cell surface, and excess subunits are re­ tained in the endoplasmic reticulum and eventually degraded (Heino et1989). al.,

In the endoplasmic reticulum, thea6- andß 1-chains have been demonstrated to associate with the chaperone calnexin prior to heterodimer assembly (Lenter and 13

Vestweber, 1994). Calnexin binds to proteins with monoglucosylated N-linked car­ bohydrate chains which are thereby retained in the endoplasmic reticulum. Fol­ lowing proper folding the proteins are deglucosylated and released from calnexin (Hebert et al., 1995). The cytoskeletal protein talin (see sect. 3.4) has also been found to be important for correct folding of the «5ßl complex (Albigès-Rizo et al., 1995). Notably, calreticulin which interacts with the «-chain KxGFFKR motif is homologous to calnexin and might also act as a chaperone (Nauseef et al., 1995).

4. INTRODUCTION TO REGULATION OF INTEGRIN ADHESIVENESS

Cellular adhesion is a dynamic process which is often regulated by differential integrin adhesiveness. However, cell adhesion is complex and may involve several steps. A multistep model for leukocyte-endothelial cell recognition has been pro­ posed (Butcher, 1991), and in fig. 4, a modified, and more general model for cell adhesion is shown. With slight modifications it is likely to apply to most cell adhe­ sion events and it will help in understanding adhesive events such as cell aggre­ gation. The model has two important consequences. First, since the steps are se­ quential, and not parallel, later events will depend on earlier steps. I.e., failure of, for example, step one will in turn abolish steps two and three. Second, since it is a multistep model, different molecules and mechanisms can be effective at different steps.

In leukocyte endothelial recognition, the first step is reversible adhesion medi­ ated by selectins binding to their carbohydrate ligands (Butcher, 1991). In more general terms, initial contacts between cells are likely to involve recognition of carbohydrate structures. This is for topological reasons, since cell surface carbo­ hydrate chains are extended structures that reach out far from the cell surface. These interactions are required to overcome electrostatic repulsive forces (Bell et al., 1984), and allow for tight adhesion. Thus, carbohydrate binding lectins, like the selectins and molecules of the newly described sialoadhesin family (Kelm et al., 1994), are candidates to mediate the initial contacts between cells. Interest­ ingly, as discussed by Cyster and Williams (1992), certain antibodies reported to induce integrin mediated cell aggregation, might do so by providing the initial contact-step by direct cross-linking of cells. As the model stands, integrins are in­ volved in steps two and three, however, different mechanisms are of importance at the two steps. Step one: Step two: Step three: labile contacts firm adhesion adhesion strengthening

Figure 4. A three step model for cell adhesion. Upon encounter, labile contacts are made (step one), which are required to overcome electrostatic repulsive forces. These contacts are likely to involve recognition of carbohydrate structures. Thereafter, firm adhesion is established (step two) and closer contact is made. Firm adhesion involves interactions between integrins and their ligands. Adhesion strengthening (step three) involves anchoring of adhesion molecules to the cy- toskeleton, recruitment of more adhesion receptors to the area of contact, and cell shape changes resulting in flattening and spreading of the cell.

4.1 Integrin activation

Integrins on platelets and cells that circulate in the vascular system, are not ad­ hesive unless activated by appropriate stimuli. Thus, platelet integrin adhesive­ ness is activated by stimuli signaling vascular injury such as collagen and thrombin, while leukocyte adhesiveness is activated by a variety of stimuli, re­ flecting the variety of roles played by leukocyte integrins.

For example, as previously discussed (sect. 3.1), LFA-1 mediates conjugate for­ mation between CTL’s and target cells. CTL's recognize the targets by means of the T cell receptor, and hence, stimulation of the T cell receptor complex activates LFA-1 (Dustin and Springer, 1989). Adhesiveness of LFA-1 and Mac-1 is also acti­ vated b>", for example, chemoattractants when binding to respective seven trans­ membrane G-protein coupled receptor (reviewed in Springer, 1994). 15

The integrin activation step is a rapid process. Dynamic measurements demon­ strate that the platelet integrinallbß3 becomes fully activated within less than 1-3 seconds after platelet stimulation (Frojmovic et al., 1991). At the single cell level, platelet activation is not a graded response but appears quantal. I.e., single resting platelets are triggered at critical agonist concentrations to fully activate all of the respective allbß3 fibrinogen receptors (Frojmovic et al., 1994). Activation of platelets involves a transition from no measurable fibrinogen binding, to active ligand bindingallbß3 fibrinogen receptors (Marguerie et al., 1979). Similarly, ac­ tivation of LFA-1 adhesiveness involves a 200-fold increase in affinity of LFA-1 for ICAM-1 (Lollo et al., 1993). As will be discussed in the next section, activation of integrin adhesiveness involves not only an increased affinity for ligand, but also conformational changes and anchoring to the cytoskeleton.

4.2 Conformational changes

Specific monoclonal antibodies have provided evidence for different integrin conformations. For example, the PACI monoclonal recognizes a neoepi­ tope on «IIbß3 after platelet stimulation (Shattil et al., 1985). PACI binds only the activated form of allbß3, and in doing so, competes for binding with RGDS pep­ tides. Similarly, the AC7 which was raised against theß3 residues 109-128, reacts with activated platelets only, and competes with RGDF peptides for binding (Andrieux et al., 1991). This indicates that activationallbß3 of involves exposure of the MIDAS-like RGD binding site in theß3 chain (about MIDAS, see sect. 3.3). Recently has been reported the monoclonal antibody CBRM1/5 which recognizes an activation neoepitope on the ß2 integrin Mac-1 (Diamond and Springer, 1993). Interestingly, the neoepitope recognized by CBRM1/5 maps to the «M-chain ligand binding I-domain (Diamond and Springer, 1993).

Concerning propagation of conformational changes, it is interesting that the monoclonal antibody anti-LIBS2 recognizes a ligand induced binding site at a membrane proximal region ofß3 (Du et al., 1993). The allbß3 ligand binding site and anti-LIBS2 epitope are about16 nm apart, but nevertheless, binding of anti- LIBS2 and RGDS peptide is cooperative. I.e., ligand binding promotes anti-LIBS2 binding and anti-LIBS2 enhances ligand binding. This provides evidence for long range propagation of conformational changes in integrins.

Taken together, activation of integrin adhesiveness might involve long range propagation of conformational changes, resulting in exposure of hidden ligand 16 binding sites. In addition to induce conformational changes, activation of integrin adhesiveness also involves cytoskeletal anchoring of the adhesive receptors.

4.3 Role of cytoskeleton

Activation of lymphocytes with protein kinase C activating phorbol esters or CD3 cross-linking triggers transmembrane proximity between LFA-1 and microfila­ ments (Poo et al., 1994), colocalization of LFA-1 and talin (Burn et al., 1988), cap­ ping of LFA-1 and colocalization with F-actin (Haverstick et al., 1991), and associa­ tion of LFA-1 with the detergent insoluble cytoskeletal fraction (Pardi et al., 1992). Furthermore, treatment of cells with actin depolymerizing cytochalasins often inhibit the adhesive event itself (see for example: Marlin and Springer, 1987). However, the inhibitory effects of cytochalasins on adhesion are uncoupled from effects on the affinity state of the adhesive receptors (Faull et al., 1994; Peter and O'Toole, 1995). In fact, cytochalasins might even enhance the integrin affinity for soluble ligands (Elemer and Edgington, 1994). What then, is the role of the cy­ toskeleton?

The cytoskeleton is involved in the adhesion-strengthening step discussed previ­ ously (step 3, fig. 4). Thus, following initial adhesion of fibroblasts to fibronectin, the adhesion strength is increased more than ten-fold in a process that is inhib­ ited by cytochalasin b (Lotz et al., 1989). The mechanisms for cytoskeleton medi­ ated adhesion strengthening may be several-fold. Firstly, the cytoskeleton can mediate recruitment of adhesive receptors to the area of cell contact (LaFlamme et al., 1992) and thereby increase the number of adhesive bonds. Secondly, associa­ tion of integrins with the cytoskeleton may increase the strength of the adhesive bond itself, by preventing "uprooting” of the receptors. Uprooting of integrins may be physiologically relevant, since, for example, motile fibroblasts during mi­ gration leave behind a significant portion of their «5ßl fibronectin receptors on the adhesive substrate (Regen and Horwitz, 1992). Thirdly, mechanical coupling of integrins to each other and stiffening of the cell could help in preventing "peel­ ing" of the cell from the substrate (Lotz et al., 1989; Tözeren et al., 1992). Finally, under conditions of hydrodynamic flow, cytoskeleton mediated cell flattening may reduce the shear forces subjected to the cell.

Thus, the cytoskeleton may be able to mediate enhancement of adhesion strength without increasing the actual affinities between the adhesive receptors and their ligands. 17

4.4 Inside-out signaling

How are intracellular signals translated into conformational changes in the ex­ tracellular domains of integrins? This so called, inside-out signaling, is cell type specific and dependent on the cytoplasmic tails of the integrin chains. In CHO cells, endogenously expressed «5ßl fibronectin receptors bind fibronectin consti- tutively with high affinity, but in the same cells, ectopically expressed allbß3 do not appear in a high affinity conformation (O'Toole et al., 1994). However, in chimeric integrins expressed in CHO cells, «5ßl cytoplasmic tails confer a high affinity conformation to the allbß3 extracellular domains (O'Toole et al., 1994).

Maintenance of the high affinity state is an active process that requires metabolic energy. Both negative and positive elements are present in the a and ß cytoplas­ mic tails. Repression of the high affinity state in intact allbß3 is dependent on the highly conserved membrane proximal sequences GFFKR in allb (O'Toole et al., 1991) and LLITIHD in ß3 (Hughes et al., 1995), (fig. 3). If either of these sequences are deleted from respective chain it results in receptors with a constitutively high affinity conformation. However, these deletion mutant receptors are not depen­ dent on metabolic energy for their high affinity states, indicating a "locked” con­ formation that is not subject to modulation by intracellular signals (O'Toole et al., 1994).

De-repression of intact integrins requires the ß-chain cytoplasmic domain, C- terminal of the residues corresponding to LLITIHD in ß3 (fig. 3). Of particular significance is a conserved NPXY motif, three consecutive residues corresponding to the TST motif in the ß3 subunit, and the C-terminal Y-residue (O'Toole et al., 1995). Interestingly, overexpression of isolated cytoplasmic domains of ßl and ß3 subunits inhibit induction of the high affinity state of intact integrins (Chen et al., 1994). This is consistent with a competition for limiting amounts of cytoplasmic factors that bind to the cytoplasmic domains of ß-chains and thereby de-repress the integrin.

The high degree of conservation in the regions involved in regulation of «IIbß3 (fig. 3) implies similar modes of regulation in other integrins as well. Thus, in LFA-1, deletion of the GFFKR motif in the aL-chain confers a high affinity con­ formation on chimeric receptors with

5. OBJECTIVES

We wanted to elucidate the signal transduction pathways involved in regulation of leukocyte integrin adhesiveness and eventually to determine the molecular mechanisms mediating so called, "inside-out signaling”. I.e., the molecular mech­ anisms translating intracellular signals into conformational changes in the ex­ tracellular domains of integrins. But first, we had to establish an appropriate ex­ perimental system to assay adhesiveness of leukocyte integrins.

6. RESULTS AND DISCUSSION

6.1 The experimental system

In paper I we established an efficient experimental system. We needed a simple and reproducible assay to specifically monitor changes in LFA-1 adhesiveness and we took a novel approach to produce large quantities of the LFA-1 ligand ICAM-1. ICAM-1 is an integral membrane protein composed of five extracellular im­ munoglobulin-like domains, a transmembrane domain and a short cytoplasmic tail (Simmons et al., 1988; Staunton et al., 1988). Based on the assumption that im- munoglobulin-like domains possessed structural integrity we generated a hybrid gene, coding for the five ICAM-1 extracellular domains fused to a hinge region and two immunoglobulin domains from a mouse IgG (fig. 5A).

During the course of the study, the realization of a similar approach was reported by Capon et al. (1989). They reported the successful expression of chimaeric pro­ teins composed of the four immunoglobulin like domains of CD4 fused to im­ munoglobulin domains and a hinge region from a human IgG. The ICAM-1 fusion protein was expressed in Chinese hamster ovary cells, and due to the deletion of the transmembrane and cytoplasmic domains, the gene product was secreted to the medium. The mouse IgG part of the fusion protein made it easy to purify by affin­ ity chromatography. 19

m 0 S M m r n m m m m m m . C H 3

10 nm

LFA-1 ICAM-1 5^m

Figure 5. Schematic representation of the experimental system. A, the ICAM-l-IgG fusion protein is composed of the five extracellular domains of ICAM-1 (D1-D5) fused to the hinge region (H) and constant heavy domains two and three (CH2, CH3) of mouse IgGZb. Approximate dimension of ICAM-1 is taken from Staunton et al. (1990) and is in agreement with dimensions of single immunoglobulin-like do­ mains of 4x2.5x2.5 nm. B, LFA-1 expressing lymphocyte and ICAM-1 coated mag­ netic beads are shown to scale. The molecules ICAM-1 and LFA-1 are indicated but not drawn to scale. Cells with activated LFA-1 bind the beads and can be separated from un-activated cells magnetically.

To our surprise, we could not demonstrate specific binding of soluble, 125i_[abeled, ICAM-1 fusion protein to LFA-1 expressing cells. We speculated that this might be due to an interaction of relatively low affinity, and indeed, this has later been demonstrated (Lollo et al., 1993). Instead, we coated the ICAM-1 protein on mag­ netic beads (fig. 5B), and then, ICAM-1 and LFA-1 specific adhesion of cells could be monitored. The assay was simple and reproducible. With minor variations the same protocol has been used throughout the study: 5xl04 cells in 50 m-1 medium was incubated for ten minutes at 37°C before addition of 1x10^ ICAM-1 coated beads. Binding was performed for seven minutes followed by magnetic isolation of beads and adherent cells. The method proved useful for monitoring changes in LFA-1 adhesiveness (I; II; IV-VI), to demonstrate active LFA-1 on microvesicles (III) and for isolation of rare mutants with consti tu tively active LFA-1 (V). Furthermore, a modification with VCAM-1 coated beads was used to monitor changes in VLA-4 ad­ hesiveness (IV). 20

6.2 Role of phosphorylation for integrin adhesiveness

In paper II and IV we investigated the role of kinases and phosphatases for regu­ lation of LFA-1 and VLA-4 adhesiveness. The major findings of paper II were that LFA-1 could be activated by the protein kinase inhibitor staurosporine and in­ activated by the protein phosphatase inhibitor okadaic acid. Consequently, we could show that protein kinase C activation was not required for induction of the LFA-1 adhesive state. A model was proposed (paper II: fig. 6) where LFA-1 was regulated by the phosphorylation state of an LFA-1 regulating protein. Phospho­ rylation by a staurosporine sensitive serine/threonine kinase induced the non­ adhesive state, while de-phosphorylation by protein phosphatase 1 (PP1) or PP2A induced an adhesive state. To distinguish between PP1 and PP2A, in paper IV we compared the potency of okadaic acid with a different protein phosphatase in­ hibitor, calyculin A. We could conclude that PP1 was most likely the serine- threonine phosphatase regulating LFA-1 adhesiveness.

In spite of the fact that protein kinase C was not required, protein kinase C acti­ vating phorbol esters nevertheless activate LFA-1. Apparently, this was not by inhibiting the kinase proposed to in-activate LFA-1 (II). Thus, protein kinase C might activate LFA-1 either by activating PP1 or by a mechanism distinct and independent of the pathway proposed in the model. Support for the former model came when it was shown that protein kinase C could activate PP1 (Srinivasan and Begum, 1994.).

In paper IV we extended the study to VLA-4 by assessing binding of cells to VCAM-1. We found that the model for LFA-1 regulation was applicable to VLA-4 as well. The generality of the model is further supported as okadaic acid and caly­ culin A have been found to also inhibit activation of platelet allbp3 (Sakon et al., 1993). A modified and updated version of the model proposed in paper II is shown in fig. 6.

6.3 Adhesive potential of monocyte microvesicles

In paper III the procoagulant activity of activated monocytes was investigated, and in relation to this thesis, the adhesive potential of monocyte-derived mem­ brane microvesicles was evaluated. When stimulated by lipopolysaccharide (LPS), IL-1, or TNF, monocytes are induced to exhibit procoagulant activities by express­ ing tissue factor and expose anionic phospholipids. Interestingly, the major part of procoagulant activity from LPS stimulated monocytes was detected in the cell 21

0 INTEGR»*: s œ s æ s i a ^ » ■ i i i i i i i PROTEIN-1 J -//■ ÉÌÌIÌÌÉ£1 : ♦ ^ ^Ser/Thr Kinase^) ^ ♦ NON-ADHESIVE ADHESIVE INTEGRIN INTEGRIN

Figure 6. Hypothetical model for the adhesiveness regulation of integrins LFA-1 and VLA-4. Integrin adhesiveness is regulated by the phosphorylation state of the integrin regulating protein-1. The integrin regulating protein-1 is dephosphory- lated by active PP1 (PPla) which results in an integrin adhesive state, and is phosphorylated by a serine/threonine kinase, which results in a non-adhesive state. Protein kinase C (PKC) converts inactive PP1 (PPli) to PPla- PP1 is inhibited by okadaic acid (OA) and the serine/threonine kinase is inhibited by stau- rosporine (SS).

culture supernatant and associated with shed microvesicles (III). The microvesi­ cles expressed LFA-1 and had a true adhesive potential, i.e., bound ICAM-1 in an LFA-1 dependent manner. Thus, monocyte membrane vesicles could potentially be responsible for dissemination of procoagulant activities and participate in en­ dothelium stimulation.

Furthermore, Tabibzadeh et al. (1994) demonstrated by using the experimental system described in paper I, that microvesicles shed from lymphocytes could con­ fer active LFA-1 to previously LFA-1" cells. Thus, membrane microvesicles shed from leukocytes appear to carry LFA-1 in its active conformation. This interesting finding has implications on LFA-1 regulation as well as on the potential physio­ logical role of shed vesicles. Concerning the LFA-1 regulation, it will be interest­ ing to further biochemically characterize shed membrane vesicles. How is the cytoskeleton organized? Does the unusual lipid composition influence LFA-1 ac­ tivity? Can LFA-1 be regulated in these entities, and if so, how? 22

6 . 4 Identification of LFA -1 regulating gene products

In paper V and VI we initiated a genetic approach for identification of LFA-1 regulating gene products. The approach was to generate lymphocyte clones with consti tu tively active LFA-1 by means of random mutagenesis and selection. These clones could then be used for identification of mutated genes which would hope­ fully represent key regulators of LFA-1 activity (fig. 7). To this end, we succeeded in the first part, i.e., to generate, select, and clone mutant cells with aberrant (or no) LFA-1 regulation (paper V).

One clone, termed HAP4, was studied in more detail and was found to have consti- tutively adhesive LFA-1. Furthermore, binding of LFA-1 to ICAM-1 was insensitive to inhibition by previously described pharmacological inhibitors of LFA-1. A pos­ sible explanation being that HAP4 had lost an LFA-1 repressor.

cDNA sequencing (8)

Ç Normal cell line ^)- mRNA cDNA expression library (4 )

Mutagenesis (1 ) Transfection of clone (5) PCR o f cDNA (7 )

ICAM-1 binding (2 ) Expansion ICAM-1 binding (6 )

Aberrant cells Cloning (3) lormal cells (revertants) Aberranrcells

Figure 7. Strategy for genetic identification of LFA-1 regulating proteins. A nor­ mal cell line is subjected to random mutagenesis (1) followed by selection for aberrant ICAM-1 binding (2). In paper V, HPB-ALL cells were mutagenized by EMS treatment and selected for constitutive ICAM-1 binding by the method described in paper I. After a number of selection cycles the aberrant cells are cloned (3). Epstein-Barr virus-based cDNA expression libraries (Margolskee et al., 1988) are made from normal cells (4) and transfected into an aberrant clone (5). The trans­ formed clone is then subjected to ICAM-1 binding (6) and revertants are selected. cDNA from the revertants is PCR-amplified (7), ligated into expression vector, fol­ lowed by new cycles of transfection / selection / PCR-amplification. When suffi­ cient enrichment of "rescuing" cDNA is achieved, cDNA is cloned, and eventually sequenced (8). 23

HAP4 was further analyzed and appeared similar to the parental cells by a variety of criteria. However, HAP4 had lost apparent expression of cell surface ßl-inte- grins. This phenomenon was studied in paper VI. By studying several mutant clones we found a correlation between LFA-1 activation and absence of ßl-inte- grins. In HAP4 the ß 1-deficiency was found, not to be due to reduced ßl-mRNA levels. Instead, aberrant folding, or processing, of ßl was observed. A polyclonal antiserum recognized ßl from denatured samples but failed to do so in detergent lysates. In addition, a novel 48 kD, ßl-cross-reactive species, was found to be ex­ pressed in HAP4.

Thus, we demonstrated the feasibility of generating mutant cells with aberrant LFA-1 regulation by an approach of random mutagenesis and selection. The next step will be to identify the relevant mutations. Since the observed phenotype appeared to be coupled to apparent ßl-deficiency, an alternative cloning strategy might be investigated. This would involve expression cloning as outlined (fig. 7), but selecting for revenant ßl expression instead of selecting for a revertant adhesion-phenotype.

6.5 A model for LFA-1 regulation

From what I have discussed in this thesis, I propose the following unifying model to account for my observations. A gene product, integrin regulating protein-2 (IRP-2), interacts with residues in the cytoplasmic domains ofa - and ß-chains of VLA-4 and LFA-1 (fig. 8). These interactions result in (i), proper heterodimer for­ mation during VLA-4 biosynthesis allowing for integrin export to the cell surface (fig. 9A), and (ii), repression of the adhesive conformation of LFA-1 (fig. 9B). The interaction between IRP-2 and LFA-1 is subject to post-translational control. Thus, Ser/Thr dephosphorylations by PP1 releases the interaction, and frees LFA-1 to assume the adhesive conformation. If IRP-2 is the direct target for PP1, this would imply that IRP-2 is identical to the IRP-1 discussed previously (6.2). In that case, the role of the ß2-tail TTT-sequence and the C-terminal phenylalanine, could be to recruit PP1 into proximity of the IRP.

It follows that (mutational) inactivation of IRP-2 results in (i), failure of VLA-4 heterodimer formation, and (ii), constitutively active LFA-1. This is what we have observed for the HAP4 clone (paper V and VI). Conversely, interruption of the interaction between IRP-2 and VLA-4 only, by deletion of appropriate residues in the a4ß 1-tails, would result in failure of VLA-4 heterodimer formation (but not in LFA-1 activation). Interruption of the interaction with LFA-1 only, by deletion of 24

yx/xsx/x/> AWWOÄWvW ysyxy-j/i^ ß1 *c^752K L L H I I f f R R • * ■ >/NXV'.V'.

r • a4*^968 U f i r * K B\Q Y K- >>>CS>OvW ;AXv»^> sx/x/xw iWv'ïv'W IRP-2

ys/x/x/s/> yxsx/x/xsyx/x/x/&

ß 2 ^ 7 2 4 KAUHLS-DiLRE- I . .vCvN ^.v.i i-.-.-.-.-.-.-.-.-.-.-.-.w.-.-.-.-.w.-.-.-.l yN/N/.xWO B AWiWyxsw&iï aL*^vlÒ88K V G r T K #VN L K E* AWiW >Wv\W IRP-2

Figure 8. Hypothetical model for interactions between integrin cytoplasmic do­ mains with an integrin regulating protein. The hypothetical integrin regulating protein-2 (IRP-2) is proposed to interact with the indicated cytoplasmic residues of a- and ß-chains of VLA-4 (A), and LFA-1 (B).

appropriate residues in the aLß2-tails, would result in activation of LFA-1 adhe­ siveness (but not in VLA-4 deficiency). Interestingly, deletion of the a4 residues KAGFFKR results in failure of VLA-4 heterodimer formation (Kassner et al., 1994) and deletions of aL residues KVGFFKR results in LFA-1 activation (Peter and O’Toole, 1995).

If the model holds true, expression cloning of cDNA’s that correct the ßl-defi­ ciency in the HAP4 clone will yield a gene product that also regulates LFA-1 adhe­ siveness by repressing the active conformation of LFA-1. 25

Association

export

Ser/Thr Kinase

Figure 9. Proposed roles of the hypothetical integrin regulating protein-2 (IRP). IRP-2 is proposed to act as a chaperone during VLA-4 biosynthesis (A), and as a repressor for LFA-1 adhesiveness (B). A: IRP-2 interacts with the cytoplasmic do­ mains of newly synthesized «4 and ßl-chains, and so, participates in association of the a4ßl heterodimer, which is a prerequisite for integrin export to the cell sur­ face. B: IRP-2 interacts with the cytoplasmic domains of cell surface LFA-1, keep­ ing LFA-1 repressed in a non-adhesive conformation. Ser/Thr-dephosphoryla- tions by PP1, directly or indirectly, releases the repression, and frees LFA-1 to as­ sume the adhesive conformation. A Ser/Thr kinase reverses the action of PP1, which results in LFA-l-IRP-2 complex formation, and repression of LFA-1 adhe­ siveness. 26

7. CONCLUSIONS

An experimental system was set up for separation of cells with adhesive ß2-inte- grin LFA-1 receptors, by means of magnetic beads coated with the LFA-1 ligand ICAM-1. The system was exploited to study regulation of integrin adhesiveness in leukocytes, and it was found that:

• Adhesiveness of LFA-1 was activated by the protein kinase inhibitor stau- rosporine and inactivated by the protein phosphatase inhibitor okadaic acid. This suggested an involvement of a serine/threonine kinase in repression of LFA-1 function, and of protein phosphatase 1 in de-repression.

• The ßl-integrin VLA-4 appeared to be regulated in a similar manner.

• Monocyte-derived membrane microvesicles with procoagulant activities were found to possess functional adhesive LFA-1 receptors.

• Mutant cells with constitutively active LFA-1 could be isolated by an ap­ proach of random mutagenesis and selection.

• A phenotype was identified, with constitutively active LFA-1 and appar­ ently abnormal processing of ßl-integrins, resulting in failure of ßl cell surface expression. 27

8. ACKNOWLEDGEMENTS

This work was carried out at the Department of Cell and Molecular Biology, University of Umeå.

I wish to express my gratitude to:

My supervisor Erik Lundgren, for letting me in, teaching me sterile technique (it is true, he did!), and from then on, showing faith in me and my ideas, and for continuous support.

The inhabitants of lab 35, Håkan Persson (you look better in women’s clothes), Christina Thylén, and Thomas Bergman, for being good friends. Information from Kristina Ruth has been a continuos source of amusement, as has Marc Pilon's contributions, which adds a dynamic dimension to everyday’s life.

The senior's who helped me in the beginning. Especially, Fredrik Ivars for his optimism, and Martin Gullberg for his realism.

The junior who helped me in the end, i.e., Mattias Alenius, for a great job.

Paschalis Sideras, for valuable advice, Iwor Tittawella, for interesting dis­ cussions, and Richard Craven, for help with the Englisch in this thesis.

Present members of the ELN-group, Erik, Thomas, Anna, Christina, Lelle, Mattias, Mia, Shu, Karin, Gundars, Anders, Ingrid, Irina, Håkan and Rutan, for sharing interesting cakes and/or data.

Previous members, Henrik Brändén, for contributing to the diversity of the de­ partment and for excellent cakes and food, and Åsa E, for Southern Comfort.

Those who kept me updated on different subjects: Kate, Gundars, Richard, Helena, Xiao-Qi, Thomas, Shu, Johan, Stefan, Nicklas, Magnus, Martin, Lars, Erik, Åsa, Ulrika, Irene, Sara, Jacqueline, and Paschalis.

Gull-Britt and Karin Ö, for providing high-quality products only, and Karin E, for early morning entertainment.

Jonny and Bengt-Erik for excellent service and technical assistance. 28

The adventurer, Peter Törnkvist, for engineering hazardous and exciting enterprises, and for being the best of companions throughout.

My mother, Ingrid, and my parents-in-law, Eva and Egon, for satisfying meals and for providing open harbours for the kids.

My family, Maria, Harry and Ture, for love and joy.

This work was supported by grants from the Swedish Cancer Society, the Swedish Board of Technical Development, Lion's Cancer Research Foundation, University of Umeå, the Swedish Medical Research Council, and the J.C. Kempes Memorial Foundation. 29

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