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ccover.inddover.indd 1 222.10.20142.10.2014 7:29:557:29:55 RegulaƟ on of leukocyte integrin binding to Ig-family ligands

Liisa UoƟ la

Division of Biochemistry and Biotechnology Department of Biosciences Faculty of Biological and Environmental Sciences

and

Integrative Life Sciences Doctoral Program

University of Helsinki

ACADEMIC DISSERTATION

To be presented for public examination with the permission of the Faculty of Biological and Environmental Sciences of the University of Helsinki in the lecture hall B2 of Viikki B-building (VI B LS 2), Latokartanonkaari 7, on 21st November at 12 noon.

Helsinki 2014 Supervisor Carl Gahmberg, professor Division of Biochemistry and Biotechnology Department of Biosciences Faculty of Biological and Environmental Sciences University of Helsinki

Reviewers Jorma Keski-Oja, professor Translational Cancer Biology Research Program Haartman Institute University of Helsinki

Manuel Patarroyo, professor Department of Dental Medicine Karolinska Institutet Stockholm, Sweden

Opponent Francisco Sánchez-Madrid, professor Servicio de Inmunología, Hospital de la Princesa Instituto de Investigación Sanitaria de la Princesa Universidad Autónoma de Madrid Madrid, Spain

Custos Jukka Finne, professor Division of Biochemistry and Biotechnology Department of Biosciences Faculty of Biological and Environmental Sciences University of Helsinki

Dissertationes Scholae Doctoralis Ad Sanitatem Investigandam Universitatis Helsinkiensis

ISBN 978-951-51-0338-3 (paperback) ISSN 2342-3161 (print)

ISBN 978-951-51-0339-0 (PDF) ISSN2342-317X (Online) http://ethesis.helsinki.fi

Layout: Tinde Päivärinta/PSWFolders Oy

Hansaprint Oy, Vantaa, Finland 2014 Le plus grand bonheur de l’étude consiste à trouver les raisons soi-même. René Descartes Table of contents

Original publications Abbreviations Summary REVIEW OF THE LITERATURE ...... 1 1 Introduction to ...... 1 1.1 Interactions of erythrocytes with other cells and ECM ...... 1 1.1.1 Erythropoiesis ...... 1 1.1.2 Erythrophagocytosis, removal of senescent red cells ...... 3 1.1.3 Pathological conditions of adhesion ...... 3 1.2 Leukocyte adhesion ...... 4 1.2.1 Th e immunological synapse ...... 4 1.2.2 Extravasation ...... 6 1.2.3 ...... 7 1.2.4 Pathological conditions involving leukocyte adhesion ...... 8 1.3 Haemostasis and thrombosis ...... 9 2 Th e Ig-superfamily ...... 9 2.1 ICAM family members are important in leukocyte adhesion ...... 9 2.2 ICAM-4 ...... 11 2.2.1 Th e LW blood group antigen is ICAM-4 (CD242) ...... 11 2.2.2 ICAM-4 contains two Ig domains ...... 12 2.2.3 Ligands and functions of ICAM-4 ...... 13 2.3 VCAM-1 ...... 15 3 Leukocyte ...... 15 3.1 Introduction ...... 15 3.2 Integrin structure and conformational changes ...... 16 3.3 LFA-1 (αLβ2, CD11a/CD18) ...... 19 3.4 Mac-1 (αMβ2, CD11b/CD18, CR3) ...... 19 3.5 αDβ2 ...... 20 3.6 VLA-4 (α4β1, CD49d/CD29) ...... 20 3.7 Th e β7 integrins αEβ7 and α4β7 ...... 21 3.8 CR4 (αXβ2, CD11c/CD18, p150.95) ...... 21 3.8.1 Structure ...... 21 3.8.2 Ligands and functions of CR4 ...... 22 4 Regulation of integrin activity ...... 25 4.1 Affi nity/avidity ...... 25 4.2 Inside-out activation of integrins ...... 26 4.3 Outside-in signalling initiated by integrin ligand binding ...... 29 4.4 Phosphorylation ...... 33 4.4.1 β2 integrins ...... 33 4.4.2 VLA-4 ...... 34 4.5 Transdominant regulation of integrins ...... 35 4.5.1 Leukocyte integrins ...... 35 4.5.2 Other integrins in cross talk ...... 36 SUMMARY OF THE STUDY ...... 39 5 Aims of the study ...... 38 6 Experimental procedures ...... 39 7 Results and discussion ...... 40 7.1 Interactions of ICAM-4 with leukocyte integrins (I & II) ...... 40 7.1.1 CR4 is a new ICAM-4 binding partner (II) ...... 40 7.1.2 Characterisation of the interactions between ICAM-4 and CR4 (II) ...... 40 7.1.3 ICAM-4 binds to β2 integrin I domains (I & II) ...... 43 7.1.4 Interactions between ICAM-4 and leukocyte integrins require divalent cations (I & II) ...... 43 7.1.5 Th e role of ICAM-4 in erythrophagocytosis (II) ...... 44 7.1.6 Possible roles of ICAM-4/integrin interactions in red cell life cycle ...... 44 7.2 Phosporylation of αX chain is important for CR4 functions ...... 45 7.2.1 CR4 α-chain is phosphorylated on serine 1158 ...... 45 7.2.2 αX chain phosphorylation is vital for ligand binding ...... 45 7.2.3 Eff ects of αX chain phosphorylation on inside-out and outside-in signalling ...46 7.2.4 Eff ect on αX phosphorylation on phagocytosis ...... 47 7.2.5 Why is this important? ...... 48 7.3 Mechanism of trans-dominant inhibition between β2 integrins and VLA-4 (IV) ...... 48 7.3.1 VLA-4 binding to VCAM-1 is blocked by activated β2 integrins ...... 48 7.3.2 LFA-1/CR4 phosphorylation is essential for transdominant inhibition ...... 49 7.3.3 Signalling through 14-3-3, Tiam1 and Rac-1 leads to VLA-4 inhibition ...... 49 7.3.4 LFA signalling leads to changes in VLA4 phosphorylation and complex formation ...... 50 7.3.5 Transdominant inhibition could be utilised for effi cient therapies ...... 51 7.4 Regulation of leukocyte integrin binding to Ig-family ligands ...... 52 CONCLUDING REMARKS AND FUTURE PERSPECTIVES ...... 53 ACKNOWLEDGEMENTS ...... 54 REFERENCES ...... 56 Original publicaƟ ons

I Ihanus E, Uotila L, Toivanen A, Stefanidakis M, Bailly P, Cartron J-P & Gahmberg CG. Characterization of ICAM-4 binding to the I domains of the CD11a/CD18 and CD11b/ CD18 leukocyte integrins. European Journal of Biochemistry. 270(8): 1710-1723 (2003).

II Ihanus E, Uotila LM, Toivanen A, Varis M & Gahmberg CG. Red cell ICAM-4 is a ligand for / integrin CD11c/CD18: Characterization of the binding sites on ICAM-4. Blood. 109(2): 802-810 (2007).

III Uotila LM, Aatonen M & Gahmberg CG. CD11c/CD18 α-chain phosphorylation regulates leukocyte adhesion. Journal of Biological Chemistry. 288(46): 33494-33499 (2013).

IV Uotila LM, Jahan F, Soto Hinojosa L, Melandri E, Grönholm M* & Gahmberg CG*. Specifi c phosphorylations transmit signals from leukocyte β2- to β1-integrins and regulate adhesion. Journal of Biological Chemistry, in Press (2014). doi:10.1074/jbc.M114.588111

*Th ese authors have contributed equally to the work

Th e articles have been reproduced with the permission of the copyright holders and are referred to in the text by their roman numerals.

ContribuƟ ons

I LU performed protein purifi cation, cell adhesion assays, fl ow cytometry and solid phase ELISA assays together with AT, MS and EI and participated in planning the experiments, analysing the results and writing the manuscript with other authors.

II LU planned and performed or supervised the experiments together with EI, AT and MV and participated in analysing the data and writing the article with EI.

III LU planned the experiments and performed most experiments, analysed the data and wrote the manuscript together with CGG.

IV LU planned the experiments together with MG, performed the experiments together with FJ, LSH, EM and MG, supervised LSH’s work and wrote the article together with MG and CGG. AbbreviaƟ ons

AP-1 Activator protein-1 transcription factor Arf6 ADP-ribosylation factor 6 BCR receptor BSA Bovine serum albumin CalDAG-GEF1 Ca2+ and diacylglycerol-regulated guanine nucleotide exchange factor 1 c-Cbl Casitas B-lineage lymphoma CD Cluster of diff erentiation, a nomenclature system for surface antigens Cdc42 Cell division control protein 42 homolog CNS Central nervous system CR4 4 (leukocyte integrin αXβ2, CD11c/CD18, p150.95) DC-SIGN -specifi c intercellular adhesion molecule-3-grabbing non-integrin Del-1 Developmental endothelial locus-1 DLC-1 Deleted in liver cancer-1 ECM Emp Erythroblast-macrophage protein ERM Ezrin, radixin and moesin FA Focal adhesion FAK Focal adhesion kinase FERM domain 4.1 protein, ezrin, radixin, moesin domain Fn Fibronectin Foxp1 Forkhead fox protein P1 GEF Guanine-nucleotide exchange factor Hb Hemoglobin ICAM Intercellular adhesion molecule IL-2 Interleukin-2 ILK Integrin-linked kinase IP3 Inositol trisphosphate LFA-1 function-associated antigen-1 (leukocyte integrin αLβ2, CD11a/CD18) Lu Lutheran blood group glycoprotein LW Landsteiner-Wiener blood group antigen Mac-1 Macrophage-1 antigen (leukocyte integrin αMβ2, CD11b/CD18, CR3/complement receptor 3) MBP Myelin basic protein MHC Major histocompatibility complex MS Multiple sclerosis NK PH domain Plextrin homology domain PI3K Phosphoinositide 3-kinase PLC Phospholipase C PMN Polymorphonuclear leukocyte/ PSI Plexin-semaphorin-integrin

PtdIns(4,5)P2 Phosphatidyl inositol 4,5-bisphosphate Rac1 Ras-related C3 botulinum toxin substrate 1 Rap1 Ras-related protein 1 RAPL Regulator of adhesion and cell polarization enriched in lymphoid tissues or RASSF5 RBC Red blood cell RhoA Ras homolog gene family, member A SDF-1α Stromal-cell derived factor-1α SLP76 SH2 domain-containing leukocyte phosphoprotein of 76kDa or lymphocyte cytosolic protein2 SYK Spleen tyrosine kinase TCR receptor Tiam1 T-cell lymphoma invasion and metastasis-inducing protein 1 TLR Toll-like receptor VCAM-1 Vascular -1 VLA-4 Very late antigen-1 wt wild type

Three-leƩ er coding for amino acids is used throughout the text

Amino acid 3-letter code 1-letter code Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamic acid Glu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Th reonine Th rT Tryptophan Trp W Tyrosine Tyr Y Valine Val V Summary

Under normal physiological conditions blood cells are usually non-adhesive, but for many of their functions, they need to stick to other cells or to extracellular matrix (ECM). For this purpose, all the haematopoietic cells (red blood cells or RBC or erythrocytes, white blood cells or leukocytes and ) carry on their surface, or in intracellular stores, a variety of adhesion molecules. Th ese molecules intermediate multiple divergent functions, such as old red cell removal from the circulation, migration to the site of infection, blood coagulation or phagocytosis of an invading pathogen by a macrophage. Th e adhesion molecules on blood cells have many requirements that they need to fulfi l in order to maintain a physiological system: they need to stay in an inactive, non-binding state for most of the time, and to be activated and become adhesive only when needed. In addition, they should specifi cally recognise their binding partners or ligands, as unnecessary binding could lead for example to clogging of the blood vessels, autoimmune diseases or allergic reactions. Still one important feature of blood cell adhesion is the ability to let go and release the adhesion, when the cell needs to move forward or continue patrolling the circulation etc. Th e aim of this work was to elucidate the molecular mechanisms behind these adhesion events and, especially to characterize the regulation of certain adhesion molecules. Th e work was initiated by analysis of the interactions between intercellular adhesion molecule-4 (ICAM- 4) and leukocyte integrins. ICAM-4 is a protein belonging to the immunoglobulin superfamily (Ig-SF) of adhesion molecules and it is expressed only on red blood cells and their precursors. It binds to the members of the leukocyte integrin family that are expressed on white blood cells. We discovered a new binding partner for ICAM-4, the leukocyte integrin CR4 (complement receptor 4) and identifi ed the amino acids of of ICAM-4 that are are involved in the binding. We then investigated which part of the leukocyte integrin is responsible for the binding, and which divalent cations are needed for it. Comparison of the binding sites of diff erent members of the leukocyte integrin family on ICAM-4 reveals similarities and diff erences between the family members. Th e interactions between ICAM-4 and leukocyte integrins are probably needed when red cells develop in the bone marrow or when they are removed from the circulation by spleen . An essential question emerged how the phosphorylation of the intracellular part of CR4 regulates its adhesion. Th e exact phosphorylation site was then identifi ed, and the eff ects of this phosphorylation on the known diff erent functions of CR4 were pinpointed. We found out that the phosphorylation is required for cell adhesion and phagocytosis of cells expressing CR4. Phosphorylation was also needed for correct activation of CR4 through an inside-out activation pathway, whereas it was not required for signalling events initiated by leukocyte integrin ligand binding, in the so called outside-in activation. Th ese observations inspired analysis of the interplay between lymphocyte function- associated antigen-1 (LFA-1) or complement receptor 4 (CR4) and very late antigen-4 (VLA-4) that was characterized in the context of T cells. We noticed that LFA-1 activation induced an activation cascade that downregulated the adhesion of VLA-4 to its ligand vascular cell adhesion molecule-1 (VCAM-1) that is expressed on activated endothelium. To understand these events we characterised the molecular mechanisms that mediate this transdominant inhibition between two integrins in a given cell. Th ese analyses of blood cell adhesion from diff erent directions cover the regulation of cell adhesion at several levels: the structural features of the binding partners, the phosphorylation and the intracellular signalling cascades preceding and following the phosphorylation and the transdominant inhibition between diff erent integrins in the same cell. Review of the literature REVIEW OF THE LITERATURE 1 Introduc on to blood cell adhesion Cell adhesion is one of the most fundamental phenomena in the lifespan of multicellular organisms, whether it is a human, mouse or malaria parasite. Adhesion is needed in the fi rst steps of development of a multicellular organism as well as in growth, diff erentiation, immune reactions and neuron diff erentiation and in many other crucial events along the life course of an individual. Adhesive events are also involved in various pathological states, e.g. cancer metastasis and pathogen infection. Stable as well as reversible adhesive events are needed for tissue formation and single cell interactions depending on cell type and function. In blood cells, inducible adhesion is especially signifi cant as the cells need to change their behaviour from non-adherent by-passers to active, adhesive players binding to ECM, other blood cells or to endothelium, oft en in less than seconds. Th e molecules responsible for the adhesive events can be divided into diff erent groups or families according to their size, structure and functions. Th ere are some common traits that all the adhesion molecules share, such as the overall structure that consists of three major domains: the intracellular part associated to the cytoskeleton, the transmembrane part, and the extracellular region that is usually considerably larger than the other parts, and is responsible for the recognition of and binding to external ligands. Traditionally the adhesion molecules responsible for haematopoietic cell adhesions have been divided in three large families: the immunoglobulin (Ig) superfamily, integrins and . Also other molecule families, e.g. , are participating in the adhesion in many tissues. In the current work the focus will be largely on the adhesion events of erythrocytes and leukocytes, and on molecules belonging to the Ig and integrin families. I will especially discuss the roles of erythrocyte ICAM-4 and leukocyte CR4 in the context of red blood cells and leukocytes, respectively. I will also introduce the most important mechanisms contributing to the regulation of blood cell adhesion, such as phosphorylation, intracellular ligand binding and transdominant regulation by other integrins in the very same cell.

1.1 Interac ons of erythrocytes with other cells and ECM Th e life cycle of red blood cells is strictly regulated: red blood cells develop in the bone marrow and aft er maturation and nuclear extrusion the cells move into the blood stream and circulate for about 120 days. Th e senescent cells are removed from the circulation in a process called erythrophagocytosis that is carried out by spleen macrophages. Th e role of red blood cells, the transport and exchange of gases, is generally considered to be disturbed by excessive cell adhesion. However, red blood cells express many molecules that possess adhesive properties or that belong to established adhesion molecule families. Th e novel functions or molecular mechanisms mediated by these adhesion molecules are starting to get elucidated.

1.1.1 Erythropoiesis Red blood cells (RBC) develop and mature in specifi c anatomical structures of the bone marrow called erythroblastic islands (fi gure 1). ereTh the maturing erythroblasts surround the central macrophage and form close contacts with each other, with the central macrophages and with the extracellular matrix. Erythroblastic islands were found in the 1950’s by Marcel Bessis. Th e RBCs mature in several steps that may be recognised by morphology or behaviour of the cells, fi nally 1 Review of the literature leading to massive hemoglobin (Hb) production and nucleus extrusion. Th e maturation of the erythroblasts is guided by the erythropoietin (Epo) hormone that is synthesised in the kidneys (Bonsdorff & Jalavisto 1948), and also by the interactions between erythroblast, the central macrophage and the extracellular matrix (Mohandas & Prenant 1978, Sadahira & Mori 1999, Chasis & Mohandas 2008, Bessis 1958).

AB Erythroblast Central macrophage

7 ICAM-4 αV integrin

2 VLA-4/α4β1 VCAM-1 EMP EMP 1 5 6 Erythroblast 4

3

1 Central macrophage 2 Early erythroblast 3 Late erythroblast Central macrophage 4 Enucleating erythroblast 5 Extruded nucleus 6 Phagocytosed nucleus 7 Young reticulocyte

Figure 1. Erythroblastic island. A. Overview of the central macrophage and diff erentiating erythrocytes. B. Molecular interactions between macrophages and erythroblasts.

Th e interactions between central macrophages and the developing erythroblasts are essential for the proper and suffi ciently rapid development of erythrocytes. Th ese adhesions, and the downstream signalling cascades, lead to changes, protection from apoptosis, enhanced proliferation, cytoskeleton reorganisation and nucleus extrusion of the developing erythroblast. Macrophages participate in the erythropoiesis also by producing cytokines as well as by providing iron for the developing erythroblast and phagocytosing the extruded nucleus in the fi nal stages of erythrocyte maturation. Th e adhesion molecules participating in the interactions have been extensively studied and their signifi cant roles in the multiple steps of red cell maturation are being dissolved (Chasis & Mohandas 2008). Th e surface expression of diff erent adhesion molecules and other cell surface molecules in human and murine cells has been measured at diff erent stages of erythrocyte development. Interesting diff erences have been detected. Th e red cell precursors express many adhesion molecules (e.g. β1 and β2 integrins and intercellular adhesion molecules, ICAMs), but the expression of these adhesion receptors is largely lost when the cells develop and move into circulation (Bony et al 1999, Southcott et al 1999, Liu et al 2010). One of the most important adhesion molecules is Eryhtroblast-macrophage protein (Emp) that is a 30 kDa transmembrane protein expressed on both erythroblasts and central macrophages. It promotes homophilic Emp- Emp adhesion between the two cell types, which enhances nucleus extrusion and inhibition of apoptosis (Hanspal & Hanspal 1994, Hanspal et al 1998). Th e developing erythroblasts also

2 Review of the literature express VLA-4 integrin and the macrophages express its ligand VCAM-1. Th e interaction between these two molecules is needed for the integrity of the eryhtroblastic island (Hamamura et al 1996, Sadahira et al 1995). Another prominent adhesion molecule involved in the erythroblastic island formation is ICAM-4 that has been reported to bind to αV integrins on central macrophage and providing stability to the island structure (Lee et al 2006). ECM , especially fi bronectin (Fn) and laminin are also possibly involved in the regulation processes leading to erythroblast terminal diff erentiation and guiding the mature RBC to the circulation. Th e integrins VLA-4 and VLA-5, which are fi bronectin receptors, are expressed on erythroblasts (Hanspal 1997). Lutheran blood group glycoprotein (Lu) on red cells, in turn can bind to laminins (El Nemer et al 1998).

1.1.2 Erythrophagocytosis, removal of senescent red cells Aft er 120 days in the circulation, the senescent erythrocytes are removed from the circulation primarily by spleen macrophages and the liver in an action called erythrophagocytosis (reviewed in Antonelou 2010). Th e mechanisms how senescent red cells are recognised by splenic macrophages are poorly understood. Several changes contributing to the removal appear in red blood cells during ageing. Th e major changes observed in senescent erythrocytes include changes in size, density, volume and morphology of the cells, loss of membrane asymmetry, desialylation of membrane sialoglycoconjugates, formation of senescent cell antigens and increase in the membrane-bound immunoglobulins and complement component . Th e microvesiculation of the red cell plasma membrane is increased in the older red cells, leading to loss of membrane and its constituents and decreasing the membrane fl exibility but, on the other hand, enabling the disposal of denatured proteins. Probably the most important player in the senescence signalling is oxidative stress. It causes changes in Band 3 cell surface protein, creating neo-antigens (or senescence antigens) that are recognised by autologous IgG and complement component C3, and leads to phagocytosis of the red cells by macrophages that are able to bind these molecules. Another consequence of oxidative stress is the activation of pro-apoptotic components (especially caspase 3). Hemoglobin as well is modifi ed and the interactions with plasma membrane components like Band 3 and cytoskeletal spectrin are enhanced, increasing the deformability of the cells. Many hypotheses for the ultimate erythrophagocytosis signal have been put forward, but none have proven to be exclusively responsible for the senescence signals (Aminoff et al 1992, Bratosin et al 1998, Antonelou et al 2010).

1.1.3 Pathological condi ons of red blood cell adhesion In addition to the physiological situations described above, red cells may become too adherent in some pathological conditions. In sickle cell anaemia, a mutation in the Hb gene leads to expression of HbS, a poorly soluble and easily precipitating and polymerising form of Hb. Due to the long strands of Hb, the RBC become sickle-shaped and may block the microcirculation, causing hypoxia and acute pain episodes in diff erent tissues. Molecules responsible for the increased adhesion of sickled cells to endothelium appear to be red cell VLA-4 (binding to endothelial VCAM-1), red cell Lu (binding to ECM laminin α5) and ICAM-4 (binding to endothelial αV integrins) (Zennadi et al 2012, Wautier & Wautier 2013). Other pathophysiological conditions where erythrocytes display increased adhesion to the endothelium include e.g. diabetes mellitus and polycythemia vera. In malaria, the Plasmodium

3 Review of the literature falciparum –infected red cells adhere also abnormally to the endothelium, which facilitates the dissemination of the parasite (Wautier & Wautier 2013).

1.2 Leukocyte adhesion Leukocytes or white blood cells are the cornerstone of a functional immune system, participating in both adaptive and innate immune functions. Adhesion events are essential in virtually all events associated with immunological reactions. Together the leukocytes form a functional immune system that can protect the system against foreign pathogens. Leukocytes are divided in several subgroups. may be further divided in antibody-producing B cells, T cells that assist in the activation and regulation of B cells (T helper cells) or participate in infected cell killing (cytotoxic T cells), and natural killer (NK) cells. are phagocytes, able to bind and engulf infected or apoptotic cells. Th ey also give rise to macrophages and dendritic cells, when escaping the blood stream. form a group of three cell types; eosinophils that take care of parasites and participate in allergic reactions, that release histamine for the good and the bad, and that are the fi rst cells to arrive to sites of infl ammation in large numbers, starting to engulf bacteria and fungi.

1.2.1 The immunological synapse Th e immunological or immune synapse (fi gure 2) is a structural entity formed between a T cell and a professional antigen presenting cell (APC). It is needed for the development of T cell responses in the lymph nodes, where T cells are activated for proliferation and diff erentiation in order to generate eff ector and memory T cells. Th e intracellular events leading to this outcome are initiated when a T cell receptor (TCR) recognises and binds to a bacterial or other foreign peptide presented on the surface of an APC. Th e signalling pathways involved show a branched, rather than a top-down network of molecules interacting with and activating each other, and lead to the numerous functions of T cells. Th e immunological synapse was described at the end of the last millennium (Dustin et al 1998). Another name used for the immunological synapse is supramolecular adhesion complex (SMAC, Monks 1998). It consists of two distinctive, ring-like molecular assemblies, called cSMAC (central SMAC) and pSMAC (peripheral SMAC). TCR, co-receptors CD4 and CD8 as well as additional molecules involved in T cell activation are clustered in the cSMAC, whereas especially LFA-1 integrin can be found in the pSMAC (Monks et al 1998). LFA-1 activation and subsequent binding to its ligand (ICAM-1) is required for the formation of a stable immune synapse. Th e sustained interaction between the two cells, in turn, allows the prolonged signalling needed for the appropriate T cell activation and proliferation. A third area, the distal SMAC (dSMAC) with concentrated negative regulators such as CD45, has been described later (Huppa & Davis 2003, Springer & Dustin 2012). Th e binding of TCR to the peptide associated with the major histocompatibility complex (MHC) leads to phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) on the cytoplasmic moieties of CD3γ, CD3δ, CD3ε and CD3ζ polypeptide chains. Th e kinase responsible for CD3 ITAM phosphorylation is Lck that is brought to the vicinity of TCR by CD4, a co-receptor binding to the MHC molecule. ITAM phosphorylation leads to binding of the zeta-chain associated protein kinase of 70 kDa (ZAP70) to the phospho-ITAMs and its subsequent phosphorylation by Lck. Activated ZAP-70 in turn phosphorylates the linker for activation of T cells (LAT). LAT is a scaff old protein bound to the plasma membrane which,

4 Review of the literature when activated, recruits many other signalling proteins and off ers a base for the construction of a LAT signalosome. From this multimolecular complex the signalling continues further, branching in directions that result in the variety of outcomes of the T cell activation, such as integrin activation and cell adhesion, gene expression and actin reorganisation (Huppa & Davis 2003, Dustin 2009, Brownlie & Zamoyska 2013). Recent studies have shown further interesting aspects of the immune synapse and its structure. Th e observation that the immune synapse, and especially the centrosome structure and intracellular vesicular transport, of a cytotoxic T cell (CTL) encountering tumor or virally infected cells are surprisingly similar to the structure of primary cilia, has led to new insights into the regulation of vesicular transport (Finetti & Baldari 2013). Like in primary cilia, the hedgehog signalling has been shown to have a vital role in the regulation of the vesicular transport and the killing of target cells by CTL. Interestingly, in contrast to the earlier identifi ed hedgehod signalling pathways, the Indian hedgehog homolog (Ihh) molecule needed for signalling is produced by the T cell itself, and the signalling events are intracellular, without a need for extrinsic signalling molecules (de la Roche et al 2013). Another interesting feature of the immune synapse is the release of extracellular vesicles or exosomes by the T cell. Th ey are instantly engulfed by the APC but their function remains to be elucidated, although general cell activation seems to occur upon vesicle uptake. In any case, the contents of the vesicles is strictly regulated and controlled and thus, most likely play a role in the cell-cell signalling events required for the immunological processes (Choudhuri et al 2014, Gutierrez-Vazquez et al 2013).

A T cell B

CD45 CTLA-4 TCR LFA-1 complex CD44 CD43 CD2 CD28

CD4/CD8 ICAM-1

CD48/ Peptide- CD59 CD80/ MHC CD86 APC

Figure 2. Schematic description of the immunological synapse and the molecular distribution within T cell and APC (A) and in the membrane plane of T cell (B). DSMAC, distal SMAC, pSMAC, peripheral SMAC, cSMAC, central SMAC. Adapted from Huppa & Davis 2003.

5 Review of the literature 1.2.2 Extravasa on To perform their immunological functions, the leukocytes need to leave the bloodstream, some of them several times during their life cycle. Th e exit, called extravasation or transendothelial migration, may take place in the lymph nodes, where lymphocytes meet peptide-MHC complexes on the surface of APCs. Another extravasation site is the site of infection or injury, where the leukocytes move to the infl amed tissue in a well-defi ned sequence (Hyduk & Cybulsky 2009). Th e traditional view of the extravasation includes the following steps: rolling, activation, arrest and migration (Dutrochet 1824). Later more refi nement to the model has been acquired, but the basic steps of the extravasation cascade remain the same (fi gure 3).

Activation of Tethering and Integrin- Paracellular Transcellular endothelial cells Firm adhesion rolling mediated rolling migration migration by chemokines

Chemokines stored in intracellular vesicles

L- LFA-1 PSGL-1 VLA-4 Mac-1 P-selectin VCAM-1 CR4 E-selectin LFA-1 ICAM-1 Mac-1 ICAM-2 JAM-1 PECAM-1

Figure 3. . Essential steps and molecules involved.

Th e fi rst step in the transmigration cascade is the activation of endothelial cells by infl ammatory cytokines, which leads to rapid expression of chemokines, adhesion molecules and lipid chemoattractants on the luminal surface of the endothelial cells (Bevilacqua 1993). Th e next step is rolling of the leukocytes along the surface of the endothelium, mediated by selectins (adhesion molecules that adhere to carbohydrate moieties) and their ligands. L-selectin and P-selectin glycoprotein ligand (PSGL-1) are mostly expressed on the leukocytes, whereas P- and E-selectins are expressed on the infl amed endothelium. Binding of selectins to their ligands induces signalling both in the endothelial cells and in leukocytes, leading to e.g. activation of integrins (Simon et al 2000, Ley et al 2007). Following these steps, the leukocytes start the integrin-mediated rolling. Th e integrin thought to be mainly responsible for the monocyte and T cell rolling is VLA-4 and it binds to its ligand VCAM-1, that is strongly expressed on the surface of the infl ammation-activated endothelial cells (Imai et al 2010). In neutrophils the rolling is mediated by the selectins and the interactions of lymphocyte function associated antigen-1 (LFA-1) with ICAM-1 and -2 on the endothelium (Hakkert et al 1991, Springer 1990, Gahmberg 1997).

6 Review of the literature

Th e next step is the high-affi nity binding and the rapid arrest of the leukocytes at the site of infl ammation. In addition to previously mentioned selectins, also chemokines excreted by the endothelium are able to activate adhesion. Chemokines bind to the G-protein coupled receptors on leukocytes, which results in rapid intracellular signalling events, fi nally leading to activation of leukocyte β2 family integrins and subsequent binding to their ICAM counterreceptors expressed on endothelial cells. Aft er binding to their ligands, the integrins start a signalling cascade in the leukocyte, which causes further reinforcement of the leukocyte-endothelium adhesion through changes in the actin cytoskeleton (Gahmberg 1997, Ley et al 2007, Hogg et al 2011). Th e eff ector lymphocytes, contrary to naïve or memory lymphocytes, do not need chemokine induction in order to arrest on the endothelium. Instead, they express high numbers of leukocyte integrins that are easily outside-in activatable (Shulman et al 2011, Lek et al 2013). Finally the cells start to migrate through the endothelial cell layer, the basement membrane and the pericytes surrounding the vessels in most areas of the body. Crawling cells form protrusions penetrating the endothelial cell layer and show a polarised morphology. In the leading edge, the cell has a structure called lamellipodium that is a site of intensive dynamic actin cytoskeleton reorganisation. Filopodia are structures that extend even further onwards to the direction of the migration from the lamellipodia. In the lamellipodia, constant formation of focal complexes takes place. Th ey are adhesive units consisting of integrins, talin and other associated molecules. In case the focal complex adheres to the endothelium or ECM, it collects around it a structure called focal adhesion (FA), a reasonably stable adhesion site between the migrating cell and the endothelium or ECM. Th e cell utilises FAs as anchoring sites while dragging the cell body along the surface. In the rear, the cell has a structure called uropod, where FAs are dissolved and the adhesion molecules are recycled to the leading edge or to FAs (Ley et al 2007). Migrating cells use a somewhat diff erent set of adhesion molecules than the ones used in rolling and adhesion, including integrins and proteases. Two routes for the transendothelial migration have been suggested: paracellular (between the cells), where the endothelial cell activation and their contacts with leukocytes induce opening of the interendothelial cell junctions and promote leukocyte migration towards the cell-cell junctions. Th e other route is transcellular (through the cells) migration that takes place at sites where the thickness of the endothelium has diminished. Chemokines are secreted from intracellular endothelial stores to guide the leukocytes on their way (Shulman et al 2011). Migration is facilitated by a system called vesiculo-vacuolar organelles, which form a channel structure through the endothelial cell, leading the leukocyte to the extracellular space. In order to get through the basement membrane below the endothelial cells, yet another set of integrins and proteases degrading the extracellular matrix is needed. Once in the tissue, the cells follow a gradient of chemoattractants to orient themselves in the tissue (Ley et al 2007).

1.2.3 Phagocytosis Phagocytosis is a means of the innate immunity system to get rid of invading pathogens and to process them further, if needed, for the antigen presentation to lymphocytes. It is also important in the removal of apoptotic cells from the system. Cells responsible for phagocytosis (phagocytes) are macrophages, granulocytes and dendritic cells. Th e phagocytic process is initiated when phagocyte cell surface receptors encounter and bind to a microbial or other foreign surface. Th is leads to the formation of a phagosome, an endocytic vesicle. Most common receptors involved in the phagocytosis are Fc receptors (FcR), toll-like receptors (TLR), complement receptors 1-4 (CRs, of which CR3 and CR4 are leukocyte integrins) and scavenger receptors. Interestingly, the 7 Review of the literature type of phagocytosis depends on the receptor responsible for the recognition of the pathogen/ foreign structure, due to diff erent signalling pathways associated with diff erent receptor families (fi gure 4). Th e Fc-receptors induce a signalling pathway leading to “reaching phagocytosis”, whereas complement receptors induce ”sinking phagocytosis” (Caron & Hall 1998, van Lookeren Campagne et al 2007, Dupuy & Caron 2008, Underhill & Goodridge 2012).

Figure 4. Presentation of diff erent phagocytosis types and macropinocytosis. From Underhill & Goodridge 2012. Figure is reprinted with kind permission of the copyright holders.

1.2.4 Pathological condi ons involving leukocyte adhesion In many pathological conditions the adhesive ability of the leukocytes, or some subset of them, is altered. Oft en the leukocyte adhesion is too active, enabling the leukocytes to attack self tissues as they were foreign pathogens. Th e activated immune cells accumulate in the target tissues in an uncontrolled way, leading to pathological autoimmunity. For example in multiple sclerosis (MS), the activated immune cells are able to penetrate through the blood-brain barrier (BBB) into the brain and the central nervous system. Once there, they induce an infl ammatory state and attack the myelin sheath protecting the neurons. Th is causes lesions in the central nervous system (CNS), leading to symptoms like fatigue, imbalance, loss of mobility, sensory symptoms, visual problems and pain. Other examples of diseases where leukocyte adhesion is impaired are asthma, reperfusion syndromes, arthritis, neuroinfl ammatory diseases, autoimmune diabetes, organ transplant rejection, , Crohn’s disease and ulcerative colitis (Hilden et al 2006, Millard et al 2011).

8 Review of the literature 1.3 Haemostasis and thrombosis Haemostasis starts when the endothelium is injured and the platelets are activated and start to aggregate and bind to the subendothelium, forming a plug to stop the bleeding. Fibrinogen is spliced into fi brin which forms fi brin fi bres and a fi brin clot, where platelets and other cells are trapped, further strengthening the blood clot. While haemostasis is the physiological response to an injury, thrombus is the result of pathological clot formation due to excessive activation of haemostasis (Rasche 2001, Versteeg et al 2013).

2 The Ig-superfamily

Immunoglobulin superfamily (IgSF) of adhesion molecules consists of numerous adhesion molecules containing one or more immunoglobulin (Ig) domain on their extracellular part. Some family members contain also other domains needed for adhesion or other functions.

2.1 ICAM family members are important in leukocyte adhesion For leukocyte functions, especially the ICAM family as well as VCAM-1 are essential. ICAM molecules all share the common ligands, the β2 family of leukocyte integrins and VCAM-1 binds to α4 integrins VLA-4 (α4β1) and α4β7. Th e structure of the ICAMs as well as VCAM-1 consists of a various number (2-9) of immunoglobulin (Ig) domains, a transmembrane domain and a short cytoplasmic part. Th e Ig-domains are held in tight conformation with intra-domain cysteine bridges. In fi gure 5, the schematic structures of the ICAM-1 to -5 and VCAM-1 along with the possible glycosylation sites are depicted. Th e ligand binding site is oft en in the fi rst, outermost domain, but also other domains participate in the binding of some ligands (e.g. D3 of ICAM-3 binds to Mac-1). All the ICAMs as well as VCAM-1 are heavily N-glycosylated on their extracellular domains. ICAMs are also genetically linked so that the genes for all the other ICAMs than ICAM-2 reside on the human 19p13.2-13.3, the ICAM-2 gene is on chromosome 17g23-25. Th e VCAM-1 gene is on chromosome 1p21.2 (Cybulsky et al 1991, Gahmberg 1997, Gahmberg et al 2008).

9 Review of the literature

NH2

NH2

NH2 NH2

NH2 NH2

COOH COOH COOH COOH COOH COOH ICAM-1 ICAM-2 ICAM-3 ICAM-4 ICAM-5 VCAM-1

Figure 5. Schematic structures of ICAM family members and VCAM-1. Ig domains are depicted with open loops and N-glycosylation sites with black triangles. S-S-bonds are shown with a dotted line.

ICAM-1 (CD54) was the fi rst described member of the ICAM-family (Rothlein et al 1986, Patarroyo et al 1987, Marlin & Springer 1987). It is expressed at low levels on resting leukocytes, endothelia and other tissues, but infl ammatory stimuli, such as TNF, IFN-γ or bacterial lipopolysaccharide (LPS) induce increased expression (Dustin et al 1986, Nortamo et al 1991). Its expression is relatively low on mature T and B cells, but clearly higher on lymphoblasts (Rothlein et al 1988, Prieto et al 1989). ICAM-1 is involved in various adhesion events during the lifecycle of the leukocytes, such as T cell and leukocyte extravasation (Shaw et al 1986, Dustin & Springer 1988). It can serve as a receptor for several pathogens that use it to infect host cells (rhinovirus, Plasmodium falciparum) (Greve et al 1989, Berendt et al 1989). ICAM-1 extracellular domains adopt an L-like structure that is bent between domains three and four (Kirchhausen et al 1993). It has also been shown to form dimers (Miller et al 1995, Reilly et al 1995). In addition to the integrins, ICAM-1 binds fi brinogen (Fg) which may be used as a cross-linker to bind leukocytes through their Fg-binding integrins (Languino et al 1993). ICAM-1 is also essential in the formation and maintenance of immune synapses, where it binds the LFA-1 integrin expressed on T cells (Monks et al 1998). ICAM-2 (CD102) was cloned in 1989 (Staunton et al 1989), the protein was characterised in the beginning of the 1990s (de Fougerolles et al 1991, Gahmberg et al 1991) and it is the only ICAM expressed on platelets (Diacovo et al 1994). Its expression is constitutively low on lymphocytes as well as monocytes, whereas on endothelial cells it shows higher expression levels. Its expression level is, however, not inducible by cytokines or other infl ammatory stimuli (Nortamo et al 1991, de Fougerolles et al 1991). With two Ig-domains, its molecular weight is 55 kDa. An interesting observation was the discovery of an ICAM-2 derived peptide (called P1) that

10 Review of the literature was able not only to inhibit LFA-1 binding to the endothelium, but also to stimulate leukocyte integrins on a number of diff erent cell types (Li et al 1995, Xie et al 1995, Kotovuori et al 1999). Th e ICAM-2 cytoplasmic part binds to α-actinin (Heiska et al 1996). Th e crystal structure of the ICAM-2 extracellular domain has been solved and it shows similarities between the ICAM family but diff erences when compared to VCAM-1 and MAdCAM-1 that bind to I-less integrins (Casasnovas et al 1997, Casasnovas et al 1999). ICAM-3 (CD50) has high homology with ICAM-1 on the extracellular domain level but diff ers in the cytoplasmic domain sequence (de Fougerolles & Springer 1992, de Fougerolles et al 1993). It is also expressed on T cells and has been reported to be an important ligand of dendritic cell-specifi c intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) expressed on dendritic cells (Geijtenbeek et al 2000, Bogoevska et al 2007). ICAM-3 also binds to αDβ2 integrin (Van der Vieren et al 1999). Its expression is high on resting leukocytes, but it is not expressed on endothelial cells (Van der Vieren et al 1995). Its most important roles are suggested to be in the leukocyte-leukocyte interactions like B cell activation by T cells, or T cell activation by APCs (Gahmberg 1997, Hayfl ick et al 1998). ICAM-4 or LW blood antigen is expressed only on erythrocytes and their precursors. Its primary function has remained unclear, but it has been implicated in several adhesive functions of RBC, such as erythropoiesis, erythrophagocytosis and in sickle cell disease and deep vein thrombosis (see chapter 2.2). ICAM-5 is the latest member of the ICAM family to be discovered (Mori et al 1987, Tian et al 1997). It is only expressed in the telencephalon-derived regions of the brain and specifi cally on the neuronal somas and dendrites (Yoshihara et al 1994, Benson et al 1998). It is able to bind to various diff erent receptors, of which LFA-1 was the fi rst to be discovered (Tian et al 1997, Tian et al 2000a). Other ligands are presenilin (Annaert et al 2001), ERM proteins (ezrin, radixin, moesin) (Furutani et al 2007), vitronectin (Furutani et al 2012), α-actinin (Nyman-Huttunen et al 2006) and β1 integrins (Ning et al 2013). In addition, it shows homophilic binding (Tian et al 2000b). Th e three dimensional structures of fi rst two domains (Zhang et al 2008) and domains 1-5 (Gahmberg et al 2014) have been solved. It is important in both neuronal development and immunological functions, and it is the fi rst known negative regulator of spine (dendritic protrusions that may develop into synapses) development (Tian et al 2009). It also takes part in the maintenance of the immune privilege in the CNS (Tian et al 2008). Recently, it has been observed to regulate synapse formation through interactions with β1 integrins (Ning et al 2013). 2.2 ICAM-4 2.2.1 The LW blood group an gen is ICAM-4 (CD242) LW and Rh blood group antigens were discovered simultaneously and they were mixed up with each other for some time. Th e blood group antigen was named fi rst as Rh (Landsteiner & Wiener 1940), but was later renamed LW according to its fi nders (Levine et al 1963). Th e RhD antigen was identifi ed as a separate protein of 300 kDa (Gahmberg 1982). Th e LW blood group consists of LWa and LWb antigens that can be recognised by anti-LWa and anti-LWb antibodies (Sistonen et al 1983). Rh and LW are closely related on a phenotypic level: the amount of LW expressed is dependent on the expression of RhD antigen. RhD+ cells express more LW, whereas RhD- cells have less LW on their surface. Rhnull cells don’t express any LW (Sistonen et al 1983, Giles 1980). LW and Rh –complex has been characterized further and the two proteins were discovered to form a non-covalent complex that is transported to the cell surface together (Mallinson et al

11 Review of the literature

1986, Bloy et al 1989, Hermand et al 1996). Later, a macrocomplex consisting of ICAM-4/LW, Band 3, Rh and many other red cell membrane proteins, taking care of gas exchange, has been reported (Bruce et al 2003). Most Europeans are of LW(a+b-) phenotype, and only less than 1% are LW(a+b+), but Finns are diff erent: about 5% of the population are LW(a+b+) and 0,1% are LW(a-b+), which is very rare elsewhere (Sistonen et al 1981, Sistonen & Tippett 1982, Sistonen et al 1983). Th e expression of the LW antigen may temporarily decrease during immunological anomalies and simultaneously anti-LW antibodies may be detected (Chown et al 1971, Perkins et al 1977, Parsons et al 1994, Komatsu & Kajiwara 1996). One case of haemolytic disease of the foetus and newborn (HDFN) has been reported to be caused by anti-LW autoantibodies from the mother (Davies et al 2009). When the expression of the LW antigen was analyzed at the cDNA level, two diff erent forms were discovered: one of 270 amino acid polypeptide and another of 236 amino acid residues that did not contain the transmembrane and cytoplasmic parts. Th ese two were predicted to be the membrane bound and secreted forms (Mallinson et al 1986, Bailly et al 1994). Th e full-length LW antigen is a 42 kDa glycoprotein that is connected to the cytoskeleton and associated with Rh expression (Mallinson et al 1986, Bloy et al 1989). It needs intramolecular disulfi de bonds to be antigenic (Konigshaus & Holland 1984). Th e soluble form was reported in mice by (Lee et al 2003) and in by (Choi et al 2013). Both forms are expressed only on erythrocytes and their precursors (Bailly et al 1994). ILW has 4 possible N-glycosylation sites and consists of 2 extracellular Ig domains, a transmembrane domain (21 amino acids) and a short cytoplasmic tail of 12 amino acid residues. Th e diff erence between LWa and LWb is a one-nucleotide change on the DNA-level (A308G), leading to a Gln70Arg change in the amino acid sequence (Hermand et al 1995). Th e structure and sequence of LW was demonstrated to be homologous to the intercellular adhesion molecule family, and it could also bind to leukocyte integrins as all the other members of the ICAM family, so LW was renamed again, this time as ICAM-4 (Bailly et al 1994, Bailly et al 1995, Hermand et al 1995, Hermand et al 1996).

2.2.2 ICAM-4 contains two Ig domains A three dimensional model based on the crystal structure of ICAM-2 revealed the structure of ICAM-4. It consists of two Ig domains (D1 & D2), each of which consists of two β-sheets. Th e fi rst sheet of D1 consists of strands ABED (in D2: ABE) and the second sheet of strands CFG (D2: C’CFG) (Hermand et al 2000). Th e comparison of the amino acid sequence to other ICAMs revealed interesting diff erences inside the family. Importantly, Glu34 and Gln73

(numbering from ICAM-1), that are important in other ICAMs adhering to their β2 integrin family ligands, are lacking, and they are replaced by Arg52 and Th r91 in ICAM-4. Replacing these with glutamate and glutamine, respectively, does not improve binding to LFA-1 and even inhibits binding to Mac-1. On the other hand, N-glycosylation at position 48 of ICAM-4 is not present in other ICAMs (Hermand et al 2000). A homologue to human ICAM-4 has been found in mouse red cells (Lee et al 2003).

12 Review of the literature

LFA-1 binding surface Mac-1 binding surface CR4 binding surface

Figure 6. Th e structure of the extracellular domain of ICAM-4. Amino acid residues participating in leukocyte integrin binding are marked. CR4 binding is introduced in chapter 7.1. From Ihanus et al 2007.

2.2.3 Ligands and func ons of ICAM-4 ICAM-4 can bind to the LFA-1 and Mac-1 leukocyte integrins (Bailly et al 1995). Th e binding sites of these integrins on ICAM-4 have been characterized by site-directed mutagenesis and the important amino acid residues which participate in the integrin/ICAM-4 interactions have been pinpointed. Th e studies revealed that the two integrins bind to distinct but overlapping sites on ICAM-4. Interestingly, the outermost domain of ICAM-4 (D1) is enough for binding to LFA-1, whereas both D1 and D2 are needed for binding to Mac-1 (Hermand et al 2000). ICAM-4 diff ers notably from other ICAM family members due to its promiscuous nature.

Other members of the family bind almost exclusively to 2 family integrins, whereas ICAM-4 interacts with many diff erent integrins. ICAM-4 binding partners include αIIb3 on activated platelets (Hermand et al 2003, Hermand et al 2004), V3 (Hermand et al 2004), V1, V5 and VLA-4/41 integrins (Spring et al 2001, Lee et al 2003, Mankelow et al 2004), although in other reports the interaction between VLA-4 and ICAM-4 could not be detected (Hermand et al 2004). Th e integrins capable of serving as ICAM-4 ligands are expressed on a great variety of cell types (leukocytes, platelets, endothelial cells), suggesting a big diversity in the possible roles of ICAM-4.

Erythropoiesis Th e interactions between the central macrophage and the maturing erythroblast are considered essential for the formation of the erythroblastic islands and for erythropoiesis. An ICAM-4 knock-out mouse has been made. ICAM-4/V integrin interaction was found to play a role in erythroblastic island formation and erythroblast development (Lee et al 2006). ICAM-4 probably also interacts with the β2 integrins on the central macrophages (Bailly et al 1995, Hermand et al 2000). Th e soluble form of ICAM-4 has been detected and it plausibly participates in the

13 Review of the literature detachment of mature red cells from the erythroblastic island central macrophage (Lee et al 2003). ICAM-4 – VLA-4 –interactions could be also seen, although not in all occasions, possibly connecting neighbouring erythroblasts (Spring et al 2001, Hermand et al 2004). Th e expression pattern of ICAM-4 proposes an important function during erythrocyte diff erentiation, as the ICAM-4 expression rapidly increases in the beginning of erythropoiesis and, aft er some time, gradually decreases to the level of the mature erythrocyte at the time of reticulocyte stage (Southcott 1999, Bony et al 1999). Similar studies have been conducted in mice and they show an even more pronounced decrease of ICAM-4 during the fi nal maturation of the reticulocytes (Chen et al 2009, Liu et al 2010). An interesting report about in vitro erythropoiesis by Choi et al indicates that ICAM-4 would have a signifi cant role in erythropoiesis in the absence of erythroblast – macrophage contact. ICAM-4 interaction with Deleted in liver cancer-1 (DLC-1, a Rho-GTPase-activating protein) seems to enhance cell survival and nucleus extrusion in these conditions, and the addition of soluble ICAM-4 can induce erythropoiesis in vitro (Choi et al 2013).

Erythrophagocytosis Th e binding of ICAM-4 to leukocyte integrins expressed in macrophages might clarify some of the controversy concerning the recognition and uptake of senescent red blood cells in spleen.

Indeed, the phagocytosis of senescent red cells is ICAM-4/2-dependent (Toivanen et al 2008)

Haemostasis Th e role of red blood cells in haemostasis has, already in the beginning of the last century, been suggested to be more active than just getting trapped in the thrombus through the fi brin network. In fact, there are some indications of RBCs playing an active role in the formation and/ or removal of a blood clot, binding actively to the platelets and leukocytes (Andrews & Low

1999). ICAM-4 is known to bind integrin IIb3 on activated platelets so it may take part in these adhesion events (Hermand 2003). ICAM-4/integrin interactions have also been implicated in deep vein thrombosis, where red cells bind to neutrophils at low shear rates (Goel & Diamond 2002).

Sickle cell anaemia In sickle cell anaemia, ICAM-4 mediates the abnormal adhesion of red cells to endothelium. ICAM-4 on sickled but not on normal cells can be activated by epinephrine (adrenaline) through the adrenergic pathway to mediate adhesion to endothelial cell αVβ3. Aft er epinephrine activation, the atypical activation of ERK1/2 results in activation of PKA and tyrosine kinase p72syk, which in turn leads to phosphorylation of ICAM-4 and its abnormal adhesion to endothelium. In normal red cells the serine phosphorylation of ICAM-4 is negligible (Zennadi et al 2004, Zennadi et al 2007, Zennadi et al 2012). Sickle cells were also found to induce adhesion of leukocytes (lymphocytes and monocytes) to the endothelium, probably by activating them, leading to increased adhesion (Zennadi et al 2008). Th e sickled red cells have been reported to bind to endothelium-adherent leukocytes in infl amed venules (Turhan et al 2002). Peptides based on the regions of ICAM-4 that bind to V-integrins expressed on the endothelium, as well as αVβ3 integrin agonists, inhibit sickle red cell-endothelial interactions and vaso-occlusion in the microcirculation (Kaul et al 2006, Finnegan et al 2007).

14 Review of the literature 2.3 VCAM-1 VCAM-1 (CD106), a member of the IgSF, is a cell surface protein expressed by activated endothelial cells and certain leukocytes such as macrophages. VCAM-1 expression is induced by IL-1β, IL-4, TNF-α and IFN-γ (Bevilacqua 1993, Zhang et al 2011, Min et al 2005). VCAM-1 binds to leukocyte integrins VLA-4 and α4β7 (Kilger et al 1997, Newham et al 1997). VCAM-1 binding to VLA-4 supports leukocyte tethering and rolling on the endothelium (Alon et al 1995). VCAM-1 is also expressed in bone marrow and lymph nodes, where it participates in the control of the leukocyte homing (Cook-Mills et al 2011). Contrary to the ICAM family of adhesion molecules, VCAM-1 may be expressed in two diff erent splice variants, one having 7 and one having 6 Ig domains (in the shorter version, D4 is lacking) (Cybulsky et al 1991). Th e structure of the domains one and two has been solved (Jones et al 1995, Wang et al 1995).

3 Leukocyte integrins 3.1 Introduc on Integrins form a reasonably large and diverse family of adhesion molecules that are heterodimeric type I transmembrane proteins. Th ey have a large extracellular ligand binding domain and a shorter cytoplasmic domain (except β4 that has longer cytoplasmic part) that does not have enzymatic activity, but it off ers binding site for numerous intracellular adaptor and scaff old proteins. An integrin molecule consists of an α and a β subunit and to date there are 18 α chains and 8 β chains expressed in humans. Most α chains can combine with several β chains and vice versa, which raises the number of heterodimers known to 24 (see fi gure 7 for overview of the presently known α/β combinations and the nomenclature). Th e general functions of the integrins are cell adhesion and migration and participation in the signalling cascades leading to cell diff erentiation, proliferation and angiogenesis or to programmed cell death (Tan 2012). Leukocytes express a variety of integrins, depending on their maturation and activation status and the cell type. Th e principal integrins on leukocytes are the four members of the CD18

(β2) family and VLA-4 (α4β1, CD49d/CD29) (Gahmberg 1997, Chigaev & Sklar 2012, Patarroyo et al 1990). Other integrins expressed exclusively on leukocytes are α4β7 (or LPAM) (Ruegg et al

1992) and αEβ7 (Andrew et al 1996). Leukocytes express also a number of β1 integrins (α1β1-α6β1), the expression of which is not specifi c to leukocytes. Th e functions of integrins are not only to adhere in response to diff erent stimuli, but also to function as a signalling molecules themselves. Integrins have the unique ability to transduce signals in two directions (i.e. inside-out and outside-in). Inside-out activation of integrins happens when a non-integrin receptor (e.g. TCR, chemokine receptor, LPS receptor) gets activated upon ligand binding and starts a signalling cascade involving various cytoplasmic signalling and adapter proteins. Some of these proteins also reach the integrin cytoplasmic parts and bind there, enabling a conformational change leading to ligand binding in the extracellular part of the molecule or integrin clustering on the plasma membrane. Outside-in activation may take place when external ligand binds to integrin (see also chapter 4, Regulation of integrin activity).

15 Review of the literature

α1 α2 CD49a CD49b α11 GPIa α1β1, VLA-1 laminin & collagen α10 α11β1 α2β1, VLA-2 collagen α3 α10β1 α9 CD49c αE α3β1, VLA-3 β7 αEβ7 CD103 α9β1 β1 αIEL α4β7 CD29 α4β1, VLA-4 α8β1 α8 GPII α VCAM-1 α4 CD49d α7β1 α5β1, VLA-5 αL fibronectin CD11a α6β1, VLA-6 α7 αVβ1 α5 αLβ2, LFA-1, laminin CD49e CD11a/CD18 αM αMβ2, Mac-1, ICAMs 1 to 5 CD11b α6 αV CD11b/CD18, CR3 CD49f CD51 iC3b, ICAMs GPI α6β4 β2 αxβ2, CR4, αX CD18 CD11c/CD18, p150.95 CD11c β4 αVβ3, CD51/CD61 iC3b, ICAMs CD104 αVβ8 vitronectin αVβ6 αDβ2 αVβ5 β3 αD CD11d β8 CD61 β6 β5 GPIII α αIIbβ3, GPIIb-IIIa, αIIb CD41/CD61 CD41 fibrinogen GPIIb

Figure 7. Overview of reported integrin dimers, their primary ligands and most common nomenclature.

3.2 Integrin structure and conforma onal changes Th e integrin extracellular part consists of two polypeptides (α and β) forming one functional unit (see fi gure 8A for general structure). Th e α chains may be divided in two categories: nine α chains have an inserted or I domain (also called A domain) in their extracellular region, serving as the ligand-binding site, and the other nine alphas do not have this domain (they bind ligands with their β-chain I-like domain). All integrin alpha chain ectodomains consist of a seven-blade β-propeller, a thigh domain (resembling an Ig-domain) and two calf domains that are each composed of two antiparallel β-sheets. Th e I domain is inserted between the blades two and three of the β-propeller. Th e structure of the β chain extracellular moiety is composed of a plexin- semaphorin-integrin (PSI) domain, an I-like domain, a hybrid domain (inside of which the I-like domain is inserted), four cysteine-rich EGF domains and the membrane-proximal β-tail domain (Campbell & Humphries 2011). Especially in haematopoietic cells (platelets, leukocytes, even erythrocytes), the integrins in the resting cells are in an inactive state, but they need to be rapidly and accurately activated to bind their ligands as well as “turned off ” when needed. ereTh are at least two ways to control the ligand binding capacity of a certain cell: regulation of integrin affi nity and integrin avidity. Affi nity means the capability of a single molecule to bind its ligand and is controlled by the conformation of the integrin. Avidity, on the other hand is the measure of a number of clustered integrins on a given cell to bind to their ligand (Dustin et al 2004).

16 Review of the literature

A B α 1 2 β Extended Extended I Headpiece closed Headpiece open domain PSI 3 Hybrid 4 I-like Bent domain 5 Headpiece closed 6 Hybrid 7 1 2 Thigh 3 Calf-1 4 β-tail Calf-2 domain TM TM

Tail Tail P P P P P

Figure 8. Integrin extracellular part structure. A. Domain organisation in an α chain I domain containing integrin. B. Th ree conformations of I-domain containing integrins. Domains are colour-coded as in A. Adapted from Hogg et al 2011.

Th e conformation of the integrins and their affi nity towards the ligands are tightly interconnected. Integrins are considered to adopt three diff erent conformations of the extracellular part: bent conformation, extended with a closed headpiece and extended with an open headpiece (see fi gure 8). It has been proposed that the intracellular signalling (from e.g. TCR) leads to the separation of the α and β cytoplasmic domains from each other, which transduces the signal to the extracellular part of the integrin, extending the conformation and off ering a site for the ligand to bind (Hogg et al 2011, Springer & Dustin 2012). Th ere has been controversy about the existence of the intermediate conformation and its ligand binding abilities and even suggestions of the bent conformation being able to bind ligand (Hogg et al 2011, Springer & Dustin 2012, Feigelson et al 2010). Nevertheless, the extended-open conformation has been unanimously shown to bind ligand with high affi nity. Th e understanding of the relations between integrin structure and function constantly increases as new structures are being resolved.

Th e crystal structures of many I domains have been solved, like αM (Lee et al 1995) αL (Qu

& Leahy 1995), α2 (Emsley et al 1997), α1 (Salminen et al 1999), αX (Vorup-Jensen et al 2003) and they show changes in conformations in the presence of diff erent divalent cations (like Mg2+ or Mn2+), ligands or peptides derived from the ligands. Th is, indeed, has revealed the importance of the MIDAS (metal-ion dependent adhesion site) sequence DxSxS in the ligand binding of the I-domain containing integrins. Th e crystal structures of the whole extracellular parts of αVβ3

(Xiong et al 2001), αIIbβ3 (Zhu 2008) CR4/αxβ2 (Xie 2010) and α5β1 (Nagae et al 2012) have also been solved, and negative-stain electron microscopy images of e.g. CR4 (Chen et al 2010) have been obtained. Th ese reveal the importance of the conformational regulation of the integrins. One important feature of ligand binding is the opening of the headpiece that is needed for the high-affi nity state of the integrin. Opening of the I-less integrin (αIIbβ3) has been resolved in the crystal structure level and it involves eight separate steps (Zhu et al 2013). Headpiece opening

17 Review of the literature of the α chain I domain containing integrins requires interplay between the α I domain and the

β chain I-like domain. A conformational change occurs when the α7 helix of the α I domain is pulled downwards to contact the β I domain. An interaction between a glutamate and the β chain β propeller - I domain interface exposes the ligand-binding site in the I domain of the α chain. Importantly, the β chain hybrid domain swings out to create an overall conformation with open legs (Xie et al 2010). Integrin intracellular domain structure is of utmost importance in the regulation of integrin functions. Th is part relays information both from the inside of the cell to the outside, and from the extracellular domain of the integrins towards the inside of the cell. Some common traits in the α and β chains have been reported (fi gure 9), probably the most important being the GFFKR sequence in the membrane-proximal part of the α chains and the β chain membrane proximal NPxY/F and membrane distal NxxY/F, as well as the serine/threonine-rich sequence between these two (Ylänne 1995). Multiple proteins can bind to these areas (Calderwood et al 2003, Takala et al 2008). Th e NMR structures of a number of cytoplasmic tails have been obtained:

αLβ2 (Bhunia et al), αMβ2 (Chua et al 2011), αXβ2 (Chua et al 2012), αIIb (Vinogradova et al 2000) and α4 (Chua et al 2013). A comparison of the α chain conformations shows large diff erences at the C-terminus of the cytoplasmic domains, although the membrane-proximal structures form similar conserved helix structures. Th e α and β chains make numerous contacts with each other through ionic and hydrogen bonding in the helical area close to the membrane, but the membrane-distal parts vary and the tails move more freely in solution. Th e αL tail, being the longest, makes an exception as it is packed in three helices held together by salt bridges and/or hydrogen bonds. Th e helices form a large negatively charged surface that is able to bind metal ions (Bhunia et al 2009). Th ese fi ndings suggest that the α chain cytoplasmic parts off er a great deal of variety to the integrin regulation mechanisms and probably determine the specifi city of the signalling. Th e reported phosphorylation sites of αL, αM and αX cytoplasmic tails as well as

β2 tail are situated outside the membrane-proximal helices and are thus available for kinases or phosphatases for phosphorylation as well as for other cytoplasmic molecules for interactions (see chapter 4.4 on phosphorylation and 4.2 and 4.3 about cytoplasmic binding partners).

˞/&'D.9*)).51/.(.0($*5*931*,3$('6(4/$6*4($*'3*&/.3/+(.'6(6***.' 1126 ˞0&'E./*)).54<.'006(**33*$(34 1158 ˞;&'F.9*)).54<.(00(($1*4,$3(1*74736336(.

˞'&'G./*)).5+<.(0/('.3('7$7)*6*'')6&9$3193/6 988 ˞&'G.$*)).54<.6,/4((155'6:6<,16.61'' 735 745 756 758 β2 &'.$/,+/6'/5(<55)(./.64:11'13/).6$7779013.)$(6 783 785 788 β1 &'.//0,,+'55()$.)(.(.01$.:'7*(13,<.6$9779913.<(*.

Figure 9. Cytoplasmic sequences of integrins expressed in leukocytes (αL, αM, αX, αD, α4, β1, β2). Reported phosphorylation sites are shown in red and amino acids are numbered. Th e NPXY/F sequence of β chains (see chapter 4) is shown in blue.

18 Review of the literature 3.3 LFA-1 (αLβ2, CD11a/CD18) LFA-1 is the most widely expressed integrin on leukocytes and probably the most studied member of the β2 integrin family. It was discovered already in the 1980’s and later found to belong to the 2/CD18 leukocyte diff erentiation antigen family that consisted of LFA-1, CR3 and p150,95 (Davignon et al 1981, Beatty et al 1983, Sanchez-Madrid et al 1983, Micklem & Sim 1985, Springer & Anderson 1986). It was found to be an adhesion protein in 1985 (Patarroyo et al 1985a). It binds to all the fi ve members of the ICAM-family (Patarroyo et al 1987, Marlin & Springer 1987, Staunton et al 1989, de Fougerolles & Springer 1992, Bailly et al 1995, Tian et al 1997), to junctional adhesion molecule-1 (JAM-1) (Ostermann et al 2002), to E-selectin (Kotovuori et al 1993) and to collagen (Lahti et al 2013). Its most relevant functions are to take part in cell-cell interactions between leukocytes and endothelial cells in the extravasation of leukocytes to the tissues at the site of infl ammation, between leukocytes and antigen presenting cells in the lymph nodes and between leukocytes and infected target cells to be destroyed (Kavanaugh et al 1991, Scheeren et al 1991, Davignon et al 1981). Th e role of LFA-1 in the immunological synapse is to off er stabilisation to the cell-cell interaction, but it is also fundamental in the formation of the synapse/SMAC structure (Monks et al 1998, Grakoui et al 1999). LFA-1 in T cells, and especially T cell lines like Jurkat, has been exploited to study various integrin-related signalling, activation and adhesion related issues.

3.4 Mac-1 (αMβ2, CD11b/CD18, CR3) Mac-1 is expressed on granulocytes, NK-cells and macrophages, and on γδ T cells (Springer et al 1979, Sanchez-Madrid et al 1983, Graff & Jutila 2007). It was fi rst discovered as an antigen on the surface of macrophages (hence the name macrophage-1 antigen) and later researchers realised it was the same molecule as complement receptor 3, found some time ago (Sanchez-Madrid et al 1983).

Together with two other β2 integrins, CR4 (αXβ2, CD11c/CD18) and αDβ2 (CD11d/CD18), it forms a quite homologous group, whose amino acid sequence, ligands and functions diff er from

LFA-1. Nonetheless, Mac-1 shares the common traits of β2 family, such as the I domain as the binding site for iC3b and the fact that the binding is temperature and divalent cation-dependent (Ueda et al 1994). Mac-1 ligand repertoire includes cell adhesion molecules as well as ECM molecules. In addition to complement receptor functions and binding to iC3b (Beller et al 1982), Mac-1 was reported soon aft er its discovery to mediate granulocyte cell-cell and cell-substrate adhesion, that was unrelated to iC3b binding function (Patarroyo et al 1985b). Other Mac-1 ligands include fi brinogen (Wright et al 1988), ICAM-1 (Diamond et al 1990), ICAM-2 (Xie et al 1995), low density lipoprotein (LDL) receptor (Spijkers et al 2005), MMP9 (Stefanidakis et al 2003) and JAM-3 (Santoso et al 2002). Th is vast repertoire of ligands probably refl ects its various functions. Th e role and regulation mechanisms of Mac-1 in phagocytosis have been clarifi ed extensively (reviewed in Dupuy & Caron 2008). Insights on Mac-1-mediated phagocytosis may shed a also on other signalling and regulation mechanisms concerning β2 integrins or integrins in general (Lim & Hotchin 2012). Some Mac-1 ligands, like uPAR and its ligand uPA and MMP9 refer to a role in ECM degradation and remodelling (Pluskota et al 2004, Stefanidakis et al 2009). An interesting feature of Mac-1 that has not been reported for the other family members is its function in negative regulation of the immune system. Many of the fi ndings of these functions have been revealed with the use of Mac-1 knockout mice or cell lines. When studying

19 Review of the literature the function of β2 integrins in APCs, it was found (Varga et al 2007) that, in fact, CD18-defi cient APCs are fully capable of antigen presentation and T cell activation and it seems that the integrins in wt DCs are in an inactive, non-functional state. Interestingly, when DC integrins were activated, T cell activation was inhibited. In macrophages, the suppression of Mac-1 activity led to enhanced antigen presentation, suggesting a repressive role for the Mac-1 in antigen presentation. In another study, Mac-1 on APC was found to suppress Th 17 diff erentiation, -/- which led to peripheral immune tolerance. In the same study, αM cells increased the IL-6 production, leading to diff erentiation of naïve T cells towards Th 17 cells and further inhibition of immune tolerance formation (Ehirchiou et al 2007). Th e interaction of Mac-1 on DC with ICAM-1 on lymphatic endothelial cells results in diminished coreceptor CD86 expression on DCs and further to decreased T cell proliferation (Podgrabinska et al 2009). Mac-1 is also able to regulate signals coming through TLR. TLR ligand binding leads to activation of Mac-1, which in turn leads to feedback regulation of TLR, leading to repression of TLR signalling and reduced proinfl ammatory cytokine production (Han et al 2010, Yee & Hamerman 2013).

In systemic erythematosus (SLE), the mutation Arg77His of the αM chain in its β propeller domain (that resides next to the ligand-binding I domain) has been implicated. Th is mutation leads to a decrease in ICAM-1, ICAM-2 and iC3b binding and phagocytosis. On the other hand, IL-6 release is increased. Th ese fi ndings may indicate that the negative regulation function of Mac-1 is inhibited (MacPherson et al 2011). Mac-1 is involved in MS, binding to myelin basic protein (MBP). It probably leads to phagocytosis and presentation of MBP peptides to T cells, thus eliciting an autoimmune reaction (Bullard et al 2005, Stapulionis et al 2008).

3.5 αDβ2

αDβ2 is the most recently found leukocyte β2 integrin. It is expressed on high level on foamy macrophages in aortic strikes, on certain tissue macrophages and on certain subgroups of circulating CD8+ T cells (Van der Vieren et al 1995, Danilenko et al 1995). Its expression is increased upon monocyte diff erentiation into macrophages (Noti 2002) and cell activation (Van der Vieren et al 1995). αDβ2 binds ICAM-3 (Van der Vieren et al 1995) and, when expressed on eosinophils, VCAM-1 (Grayson et al 1998). VCAM-1 binds to the I domain (Van der Vieren et al 1999). As the high homology with Mac-1 suggests, αDβ2 has been reported to bind to many of Mac-1 ligands, such as vitronectin, fi bronectin, plasminogen (Yakubenko et al 2006). It is also involved in the migration of monocytes and macrophages, and the amount of αDβ2 on the cell surface seems to control the retention of the macrophages at the infl ammatory sites. Th e more

αDβ2 the cells have on their surface, the tighter is their adhesion (Yakubenko et al 2008). 3.6 VLA-4 (α4β1, CD49d/CD29)

VLA-4 or very late antigen-4 is a β1 family integrin, consisting of α4 (CD49d) and β1 (CD29) polypeptide chains (Hemler et al 1990). Its ligands are VCAM-1 (Chuluyan & Issekutz 1993) and fi bronectin. It is expressed on many haematopoietic cell types such as T cells, B cells, monocytes, NK cells, eosinophils and neutrophils as well as on haematopoietic stem and progenitor cells. It plays an important role in the regulation of the hematopoietic events, especially in the release of the maturing cells into the circulation. It is dispensable for the haematopoiesis itself, as cell diff erentiation or development of the cells does not seem to require VLA-4, but essential for homeostasis, taking care of keeping the immature cells in the bone marrow until they are ready to be released. Even though other α subunits dimerize with β1 (e.g. α6, laminin receptor),

20 Review of the literature they do not have an eff ect on homing of adult cells. Th e functions of VLA-4 are regulated by conformational changes. α4 lacks an I domain and is more easily activated to bind ligand than LFA-1 (Imai et al 2010, Chigaev & Sklar 2012).

3.7 The β7 integrins αEβ7 and α4β7

Th e β7 integrins α4β7 and αEβ7 are also leukocyte-specifi c. α4β7 was fi rst described as a fi bronectin receptor and soon it was observed to bind also VCAM-1 and MAdCAM. It mediates homing to the mucosal sites, but also leukocyte homotypic binding. αEβ7 is expressed on the mucosal CD8+ T cells and on some other T cell subsets (such as certain regulatory T cells) and its functions are implicated in the mucosal homing of T cells, due to its ability to bind E-selectin expressed on mucosal endothelial cells. Later αEβ7 has been implicated in allograft rejection reactions (Ruegg et al 1992, Berlin et al 1993, Hamann et al 1994, Kilshaw & Higgins 2002).

3.8 CR4 (αXβ2, CD11c/CD18, p150.95) p150.95 (nowadays also known as αXβ2, CD11c/CD18 or complement receptor 4, CR4) was reported some thirty years ago to belong to the “LFA-1, Mac-1, p150.95 family” (Sanchez-Madrid et al 1983, Micklem & Sim 1985, Springer et al 1986, Lanier et al 1985) and later it was found to be the same molecule as the previously described complement receptor 4 (CR4) (Myones et al 1988). Its amino acid sequence was solved some years later and it displayed high similarity with the Mac-1 integrin (Corbi et al 1987). CR4 is expressed on monocytes, macrophages, dendritic cells, granulocytes and on some subsets of T and B cells (Hogg et al 1986, Keizer et al 1987a, Freudenthal & Steinman 1990, Postigo et al 1991, Miller et al 1986) and it is considered to be a marker of hairy cell (HCL) cells (Schwarting et al 1985, Miller et al 1987b). It is mobilised from intracellular vesicular stores upon monocyte and granulocyte activation (Springer & Anderson 1986, Miller et al 1987a). Th e expression was noticed to be partly similar to that of Mac-1, but some diff erences were discerned. Later the reports on the expression of CR4 on diff erent cell types have shown the distinctive expression pattern of CR4. For example, CR4 is more strongly expressed on tissue macrophages, compared to blood monocytes or neutrophils, whereas Mac-1 is not that heavily expressed on macrophages (Myones et al 1988). Th e expression level of CR4 has been reported to diff erentiate mature DC from the immature (O’Doherty et al 1994). CR4 is also expressed on cytotoxic pulmonary T cells aft er a RSV (respiratory syncytial ) infection that induce virus- specifi c (antiviral) cytotoxicity, e.g. production of IFNγ (Beyer et al 2005). In the brain, CR4 is expressed on that are considered to resemble macrophages and have similar functions.

3.8.1 Structure Th e extracellular part of CR4 was the fi rst reported structure of an integrin with an α chain I domain (Xie et al 2010), and it revealed more detailed information about the relations between the structure and the activity of the integrin. It seems that the α chain I domain is able to adopt three diff erent conformations, and that these conformations can fl exibly be combined to the two β chain I domain conformations (Xie et al 2010) (fi gure 10). eTh α chain I domain structure was published earlier and it shows the importance of isoleucine in position 314. Th is residue is normally in a hydrophobic socket, which locks the I domain in an inactive conformation. When mutated to glycine, an open, ligand-binding conformation is induced. Binding of the iC3b ligand to the I domain is cation (Mg2+) dependent (Vorup-Jensen et al 2003). Recently, the opening of

21 Review of the literature the headgroup has been studied in even more detail, and a metastable intermediate that probably does not exist in vivo, was found. It reveals a hypothetical mechanism of rapid activation of the conformation through “cocked” conformations of β chain I domain, bound or not bound to an internal ligand from α chain (Sen et al 2013). Th e structure of the intracellular part of CR4 has been solved by NMR. Comparison with other integrin cytoplasmic parts (LFA-1 and Mac-1, VLA-4, αIIbβ3) shows some similarities but also diff erences between the integrins. Th e membrane proximal part of αX chain forms an α helix, interacting on its polar face with the membrane-proximal part of the β2 chain. Th e rest of the αX cytoplasmic part forms an irregular loop, part of which folds back to contact the α helix of the same chain. Th e C-terminal part of the β2 cytoplasmic part also has a relatively long irregular conformation (Chua et al 2012). One important feature of CR4 i its resistance to activation. Th is was found in experiments where chicken/human chimeras of CR4 were tested for the binding of iC3b (Bilsland et al 1994).

Th e diffi culty to activate αXβ2 is due to extremely tight intersubunit interaction/restraint between

αX and β2 chains. It has been reported that especially the residues in the N-terminal cysteine-rich PSI domain (plexin/semaphoring/integrin) and in the C-terminal cysteine-rich EGF-repeats 2 and 3 are involved in these interactions (Zang & Springer 2001).

Figure 10. Th e structure of

αXβ2. Th e binding sites of some important inhibiting or activating antibodies are indicated. From Xie et al 2010. Figure is reproduced with kind permission of the copyright holders.

3.8.2 Ligands and func ons of CR4 CR4 binds to various cellular, soluble and extracellular matrix (ECM) ligands. Th e binding sites of many known antibodies used in functional assays (Lu et al 2001, Xie et al 2010, Chen et al 2010) have been solved, which is of great help in the studies concerning the interplay between structure and function. Earliest studies on CR4 and the whole “LFA-1, Mac-1, p150.95 family” 22 Review of the literature described the diff erent roles of the family members. Th e fi rst described role was in granulocyte and monocyte adhesion, phagocytosis, migration and cytotoxicity (Anderson et al 1986, Keizer et al 1987a, Keizer et al 1987b). Probably the most important or at least most studied ligand of CR4 is the complement component iC3b. Recognition of this molecule by CR4 mediates the phagocytosis of complement-opsonised particles (Micklem & Sim 1985, Malhotra et al 1986, Myones et al 1988, Bilsland et al 1994, Vorup-Jensen et al 2003, Chen et al 2012). Th e roles of Mac-1/CR3 and CR4 in complement-mediated phagocytosis have raised questions, as the two quite similar molecules are co-expressed on many cell types. In alveolar macrophages Mac-1 is the major iC3b receptor although CR4 is expressed in higher quantities. Diff erent mobility of Mac-1 (more mobile on the membrane, forming patches) and CR4 (less mobile, uniform staining) was reported, and increased mobility was shown to mediate increased CR4-iC3b binding (Ross et al 1992). Th e signifi cance of CR4 in phagocytosis has been verifi ed throughout the last three decades. It is involved in the complement-mediated phagocytosis of Mycobacterium leprae by macrophages (Schlesinger & Horwitz 1991) and of Mycobacterium tuberculosis by alveolar macrophages of the lung (Hirsch et al 1994). Importantly, uptake and removal of apoptotic cells is partly caused by CR4. Phagocytosis of apoptotic cells is essential for the prevention of autoimmune responses, as it enhances anti-infl ammatory cytokine release, blocks infl ammatory cytokine release and induces T cell tolerance (Mevorach et al 1998, Pittoni & Valesini 2002). CR4 is also heavily involved in the erythrophagocytosis, the uptake of senescent red cells by spleen macrophages (Toivanen et al 2008). Other soluble ligands for CR4 are heparin (Diamond et al 1995, Vorup-Jensen et al 2007) and fi brinogen (Loike et al 1991, Ruf & Patscheke 1995, Nham 1999, Lishko et al 2001, Choi & Nham 2002). Fibrinogen expressed by platelets binds CR4 on neutrophils, inducing an oxidative burst (Ruf & Patscheke 1995). Serum albumin has also been reported to be a CR4 ligand, but interestingly only when CR4 is expressed on macrophages, not on monocytes (Brevig et al 2005). Plasminogen (zymogen of plasmin that is an enzyme degrading the fi brin clot) can also interact with CR4 with moderate affi nity (Gang et al 2007). Interaction of CR4 with LPS leads to cell activation, and transcription factor B translocation (Ingalls & Golenbock 1995). CR4 on monocytes also binds type I collagen, which promotes cell adhesion and activation in the initial phase of infl ammation (Garnotel et al 2000). Lately CR4 has been shown to interact with collagen IV through its I domain and the GFOGER motif on collagen, which is traditionally a β1 integrin binding motif. Interestingly, the CR4 I domain binds collagen signifi cantly better than LFA-1 or Mac-1 (Lahti et al 2013). CR4 and Mac-1 (but not LFA-1) on monocytes bind to denatured proteins (Davis 1992). Negatively charged residues in disintegrated proteins probably serve as a pattern recognition motif for CR4. Th ese fi ndings have led to the hypothesis that the pericellular proteolysis at the leading edge of a migrating would produce binding sites for CR4, enabling adhesion needed in migration (Vorup-Jensen et al 2005). It was found already in the beginning of the 1990s that activated endothelial cells bind to CR4 (Stacker & Springer 1991). Since then, CR4 has been reported to interact with several cellular ligands. Th ese include ICAM-1 and more precisely its fourth domain (Diamond et al

1993, Blackford et al 1996, Frick et al 2005). Th e binding residues on the αX I domain to ICAM-1 have also been characterised (Choi 2010). CR4 also binds to ICAM-2 and VCAM-1 (Sadhu et al 2007) but not to ICAM-3 (de Fougerolles et al 1995). Another reported ligand of CR4 is Th y-1,

23 Review of the literature a involved in cell adhesion and signaling regulation in neurons and T cells

(Choi et al 2005). CD23 is an IgE receptor lectin and the binding of CR4 to it induces NO2 and

H2O2 production (Lecoanet-Henchoz et al 1995). Recently, studies on the role of CR4 in hypercholesterolemic mice and its involvement in the development of atherosclerotic plaques have revealed an important role for αXβ2 on activated monocytes and in the formation of atherosclerotic plaques. CR4 binding to VCAM-1 is partly responsible (together with VLA-4) for the capture and transmigration of monocytes to infl amed human aortic endothelial cells. Also delayed-type hypersensitivity reaction induced by SRBC

(sheep RBC) in mice was reduced in the presence of anti-αX antibody (Sadhu et al 2007). Hypercholesterolemic (apoE defi cient) mice display increased CR4 expression on monocytes when fed with a western high-fat diet. CR4 is needed for these cells to arrest on VCAM-1 and E-selectin in shear fl ow, as well as for monocyte/macrophage accumulation in atherosclerotic lesions. Lack of CR4 expression decreases atherosclerosis in apoE depleted mice on high-fat diet

(Wu et al 2009). In humans, the amount of αXβ2 on the monocytes of healthy subjects is increased aft er a fatty meal, simultaneously with lipid particle uptake. Monocyte adhesion to VCAM-1 under shear fl ow depends partly on CR4 (Gower et al 2011) and the engagement of CR4 on monocytes leads to the production of proinfl ammatory cytokine (Lecoanet-Henchoz et al 1995). CR4 is also implicated in fat tissue reactions, as there is less adipose tissue infl ammation induced by the high-fat diet in the αX defi cient than in wt mice (Wu et al 2010). CR4 seems to be heavily involved in the uptake of antigens to dendritic cells (Sadhu et al 2007, Castro et al 2008, Faham & Altin 2011). In helminth infection CR4 is essential for the development of a correct Th 2 response (Phythian-Adams et al 2010). Targeting to CR4 helps in the uptake of antigen also in an artifi cial situation such as vaccination (Reddy et al 2006). Th is fi eld of research has become extremely important, as the DC related vaccines are being developed based on the discoveries of Ralph Steinman (Steinman & Banchereau 2007). Promising results with CR4 targeting have been obtained. T cell responses may be induced by targeting the antigen to the CR4 with a single chain fragment (scFv) recognising CR4 (Ejaz et al 2012). Also peptides binding CR4 have been successfully used in eliciting T cell priming and antibody production in mice vaccinated with antigen-containing liposomes engraft ed with these peptides (Faham & Altin 2011). It also seems that the Mac-1 and CR4 in general are more relevant in the antigen uptake to DCs than in the immune synapse formation or migration (Grabbe et al 2002, Lammermann et al 2008). CR4 integrin has also been implicated in the development of experimental autoimmune encephalomyelitis (EAE), which is a murine model of MS (Bullard et al 2007). Expression of CR4 on T cells and other leukocytes is a prerequisite for the development of the disease, but also other integrins such as Mac-1 are involved. CR4 is also a receptor for some pathogens. Candida albicans hyphae (fi lamentous form, but not the fungal form) has been reported to bind to CR4, and this recognition is important for protection against C. albicans infections through Kupff er cells in the liver and microglia in the brain (Jawhara et al 2012). Rotaviruses use CR4 for entry to the host cell (Graham et al 2003). It seems that Herpes simplex virus 1 (HSV-1) exploits CR4 on dendritic cells to promote its infectivity. In fact, depletion of CR4 leads to decreased HSV-1 replication, increased cytokine production, and increased levels of virus-specifi c CD8+ T cells (Allen et al 2011). One of CR4 ligands is osteopontin, a multifunctional cytokine that is able to opsonize some (Streptococcus and Staphylococcus) bacteria for phagocytosis. Th is phagocytosis requires the presence of CR4 (Schack et al 2009).

24 Review of the literature 4 Regula on of integrin ac vity As discussed earlier, the integrins on leukocytes are responsible for a growing number of reported functions in health and disease. Th us solving the mechanisms of integrin signalling is essential for the understanding of leukocyte behaviour in physiological and clinical conditions. Integrins off er an eff ective target for therapies against various immunological and other malignancies, autoimmune diseases and invading pathogens, which has induced a lot of research and clinical trials (Hilden et al 2006, Millard et al 2011, Marelli et al 2013). Because of the fundamental connection between integrin ligand-binding activity and their conformation, many antibodies recognising the diff erent conformations have been developed (Byron et al 2009). Th ey provide an elegant way to study the implications of e.g. diff erent activation methods or the role of the numerous intracellular signalling and adapter molecules in the signalling cascades leading to cell adhesion. Especially they allow the researchers to study two diff erent paths to enhanced ligand binding: increase in a single molecule’s ability to bind its ligand (affi nity) or increase in the number of integrins at the adhesion site (avidity). Th e only endogenous inhibitor of leukocyte functions reported so far is developmental endothelial locus-1 (Del-1) that inhibits LFA-1 and Mac-1 functions. It binds to β2 integrins and is able to outcompete ICAM-1 from LFA-1 binding and inhibit macrophage phagocytosis of iC3-coated RBC (Choi et al 2008, Mitroulis et al 2014). A “natural”inhibitor for Mac-1 called neutrophil inhibitory factor or NIF has been found in canine hookworm. It selectively inhibits Mac-1 dependent eosinophil transmigration in and thus is promising candidate for treatment of allergies (Moyle et al 1994, Schnyder-Candrian et al 2012).

4.1 Affi nity/avidity Integrin-dependent leukocyte adhesion to ligands can plausibly be regulated at two levels. First, the affi nity of a single integrin may be enhanced so it can bind ligands better (see chapter 3.1). Second, the amount of ligand-binding integrins at the contact site may be raised due to their movement in the plane of the plasma membrane. It has been suggested that rapid inside-out activation of the integrins would change integrin conformation and increase their affi nity, whereas outside-in signalling would cause changes in the actin cytoskeleton, leading to integrin clustering at the ligand binding area and thus reinforcing the adhesion (Dustin et al 2004, Hogg et al 2011). Th is idea has been endorsed by many reports, especially by the fi nding that LFA-1 clustering follows and does not precede ligand binding to integrins (Kim et al 2004). Probably the whole picture is more complicated than that. In addition, in diff erent cell types or cells in various stages of maturation, diff erent activation mechanisms may prevail. It has also been suggested that the inside-out activation and hence the conformational change of the integrin would be transduced by the β chain, and the outside-in signalling, leading to integrin clustering (among other consequences) conveyed through the α chain (Kliche et al

2012). However, for example β2 Th r-Th r-Th r sequence mutation to Ala-Ala-Ala does not change initial binding, but completely abrogates the strengthening of the adhesion (Morrison et al 2013). Mechanical force (shear force experienced by the leukocytes in the blood fl ow) has also been implicated in integrin activation. It stimulates conformational activation (Katsumi et al 2005) as well as clustering (Knies et al 2006) of integrins. In the shear-free environment of lymph nodes the VLA-4 and LFA-1 integrins are not able to support adhesion (Woolf et al 2007, Alon & Dustin 2007).

25 Review of the literature

Clustering of the integrins takes place upon dynamic changes of the cytoskeleton, as well as regulation of integrin cytoplasmic tail binding to the actin network through adapter proteins. Th e release of the integrins from the cytoskeleton increases LFA-1 binding to their ligands due to integrin clustering, not affi nity changes (Kim et al 2004).

4.2 Inside-out ac va on of integrins Inside-out activation of integrins is a series of events where binding of ligand to non-integrin receptors on the plasma membrane initiates an intracellular signalling cascade leading to integrin activation (fi gure 11). Inside-out activation may occur upon activation through the TCR, the B cell receptor (BCR), chemokine receptors, selectins, or other molecules. Aft er the initial signal from non-integrin receptor, the signalling cascade continues in the cytoplasm, recruiting adapter and signalling proteins. Th e signal is further delivered to the intracellular parts of the integrins, which leads to the straightening of the integrin extracellular part from bent to extended conformation and enhancement of ligand binding (Abram & Lowell 2009, Springer & Dustin 2012). Th e intracellular signalling cascades in immunological synapse, extravasation and phagocytosis were discussed to some extent in chapter 1.2. Although the signalling events aft er diff erent activation mechanisms most probably resemble each other in all leukocytes, there are diff erences between cell types, maturation stages, types of activation, integrins in question etc. For example, eff ector T cells are not dependent on chemokine signalling when adhering to the endothelium in the very beginning of extravasation, whereas naïve T cells need chemokines in order to attach properly. In the next steps of extravasation, also eff ector cells are dependent on chemokines (Shulman et al 2011, Lek et al 2013). It is also good to keep in mind that many of the studies presented here have been carried out in an experimental setup where a limited number of cell types, activation methods and integrin outcomes have been utilized.

Integrin

β α CD4 LAT TCR PtdIns(4,5)IP2 Talin Lck PIP5K1γ87 SLP-76 ZAP-70 Kindlin Chemokine receptor PLD

PLCγ PIP2 DAG

IP3 Arp2/3 Abl WAVE

2+ CalDAG CRKL Ca Mst1 -GEF -C3G ADAP Kindlin Mst1 ADAP RIAM SKAP55 Talin RAPL SKAP55 Rap1- Rap1- GDP GTP

SPA1 Intracellular vesicle Figure 11. A simplifi ed scheme of pathways involved in integrin inside-out activation.

26 Review of the literature

Th e fi nal outcome of the inside-out signalling is integrin activation through a conformational change. An essential event for this is the dissociation of integrin α and β cytoplasmic parts. In resting state, they form a salt bridge between each other but aft er cell activation the separation of the tails occurs. Th is has quite unanimously been reported to happen upon talin head binding to the β chain, forcing the two tails to separate, further leading to conformational change of the extracellular domain towards an extended, ligand-binding state (Tadokoro et al 2003, Simonson et al 2006). Before talin binding to the integrin cytoplasmic tail, the signals from several activation routes converge in the formation of multi-protein complexes. Th ese include Ras-related protein 1 (Rap1, or Rap2 in B cells), a small GTPase, that has been shown to be essential for LFA-1 activation, interacting with one of its eff ector proteins: RIAM (Rap1 interacting adapter molecule) or RAPL (regulator for cell adhesion and polarization enriched in lymphoid tissues) and mammalian Ste20-like kinase (Mst1). Other important mediators for at least LFA-1 activation are constitutively interacting adapter proteins ADAP and SKAP55. LFA-1 and Rap1 with its aforementioned interaction partners have been localised to intracellular vesicles in T cells, and the proper activation of the integrin requires Rap1 activation and the vesicle traffi c to the plasma membrane. Th e exact order of the delivery of the vesicles to the plasma membrane and the activation events is not fully elucidated (Sebzda et al 2002, Shimonaka et al 2003, Mor et al 2009, Bivona et al 2004, Katagiri et al 2006, Kliche et al 2012). Th ese signalling events have been extensively characterized, also in the context of platelet integrin activation, and the same molecules (Rap1, RIAM and talin) regulate also αIIbβ3 activity (Calderwood 2004, Han et al 2006). Rap1 activation to a GTP-bound form is induced by two diff erent Rap1 GEFs (guanine- nucleotide exchange factors, activators of Rap1): CalDAG-GEF1 (Ca2+ and diacylglycerol- regulated guanine nucleotide exchange factor 1) and C3G. Following TCR ligation, PLCγ1 (phospholipase C γ1) is activated and Ca2+ and diacylglycerol (DAG) are generated. Th ese in turn are able to activate CalDAG-GEF1 (Katagiri et al 2004, Ghandour et al 2007, Bergmeier et al 2007). C3G is closely associated to CRKL (CRK-like protein) and needs the help of additional actin-binding complex Arp2/3 for the delivery to the plasma membrane (Nolz et al 2008, Mor et al 2009). Interestingly, CalDAG-GEF1 is not needed for VLA-4 activation (Ghandour et al 2007). Th e identity of the Rap1- containing complex in the vesicles has drawn some interest, and at least two diff erent complexes have been described: one with Rap1, RIAM, Mst1 and the ADAP/SKAP55 module, including talin and kindlin3 (the role of talin and kindlin, see below) and another with Rap1, RAPL Mst1 and the ADAP/SKAP55 module. It has been suggested that the fi rst complex is involved in the inside-out activation through β2, whereas the second,

αL-interacting complex would be important in outside-in signalling. RAPL is able to interact directly with the αL chain on Lys1097 and Lys 1099, whereas the role of Mst1 contributes to translocating LFA-1 to the leading edge of a migrating cell (Katagiri et al 2003, Katagiri et al 2006, Kliche et al 2012). Altogether, the importance of Rap1 cannot be denied, but its actions still need clarifi cation. It seems to display slightly diff erent functions in diff erent cell types. In B-cell line BAF, the activation of Rap1 leads to an increase in LFA-1 affi nity (Katagiri et al 2000, Tohyama et al 2003), whereas in mouse thymocytes it induces clustering (Sebzda et al 2002). In platelets the activation of Rap1 and subsequent formation of the RIAM-talin complex also increases the affi nity, not the avidity of αIIbβ3 (Han et al 2006). In the resting state, fi lamin, a large, actin-crosslinking protein, is bound to the cytoplasmic part of the β2 chain, and the binding keeps the integrin in a resting, non-adhesive conformation.

27 Review of the literature

Filamin binds only to the non-phosphorylated β2 and the phosphorylation of the β2 chain on threonine 758 that happens upon cell activation (Valmu & Gahmberg 1995), is a negative regulator of fi lamin binding. Phosphorylation of the β2 does not directly aff ect talin binding as the binding site does not contain any phosphorylatable residues, but fi lamin and talin probably compete for the binding of the integrin cytoplasmic tail (Kiema et al 2006). Especially talin could be important for the early steps in the adhesion when it probably competes out the cytoplasmic moiety of the αL chain from binding to the β2, as it has higher affi nity towards the β2 cytoplasmic tail than αL. Talin binding leads to the separation of the αL and β2 cytoplasmic parts and results in straightening of the integrin conformation. Later, aft er Th r758 phosphorylation, talin itself is probably outcompeted by the 14-3-3 protein, an adapter protein that binds to the phospho- Th r758 with extremely high affi nity (see more in the next chapter about outside-in signalling) (Fagerholm et al 2002, Fagerholm et al 2005, Kiema et al 2006, Takala et al 2008, Bhunia et al 2009). In addition to its role in increasing the integrin affi nity by conformational change, talin also infl uences the clustering of the integrins thus enhancing the avidity of the integrins (Simonson et al 2006). Talin transfer to the plasma membrane and hence close to the integrin cytoplasmic part, is thought to happen upon binding of the plextrin homology (PH) domain of talin to the phosphatidyl inositol 4,5-bisphosphate (PtdIns(4,5)P2) lipid on the plasma membrane (Martel et al 2001, Anthis et al 2009). PtdIns(4,5)P2 is produced by the action of PIP5K1γ87, which is also essential for the high affi nity LFA-1 (Bolomini-Vittori et al 2009, Wang et al 2004). PIP5K1γ87 itself is activated by phosphatidic acid (PA), which is formed by phospholipase D 1 (PLD1) upon TCR or chemokine receptor activation (Mor et al 2009). Talin has been localised in the mid-cell high-affi nity LFA-1 zone (Smith et al 2003), whereas α-actinin, another2 β -binding actin-cytoskeleton-associating protein colocalizes with the intermediate-affi nity LFA-1 to the leading edge (Stanley et al 2008). It has been suggested that the binding of α-actinin is made possible when talin is cleaved by calpain upon cell activation. Th is would release LFA-1 from the cytoskeleton, leaving it to move more freely on the plasma membrane e.g. to the site of adhesion. It also uncovers an α-actinin binding site. α-actinin may then attach the LFA-1 cytoplasmic tail back to the cytoskeleton, supporting formation of integrin clusters (Pavalko & LaRoche 1993, Sampath et al 1998, Stewart et al 1998). Another important molecule associated in integrin activation is kindlin-3 that binds to integrin cytoplasmic tails. Its absence leads to LADIII (leukocyte adhesion defi ciency III), a condition where leukocyte and platelet integrin functions are severely defective (Svensson et al 2009, Malinin et al 2009). Kindlins are a family of three FERM (4.1 protein, ezrin, radixin, moesin) domain- and PH (plextrin homology) domain-containing proteins with distinctive expression patterns. Kindlin-3 is expressed only on cells of haematopoietic origin and it binds to β1, β2 and β3 cytoplasmic tails and, more specifi cally to the membrane-distal NxxF/Y-motif and to a phosphorylatable Th r-Th r-Th r motif on the β2 chain. It also interacts with several other, actin-associated proteins such as ILK (integrin-linked kinase), FAK (focal adhesion kinase) and α-actinin. Kindlin-3 has recently been found to be essential for the activation of the integrins in haematopoietic cells (Moser et al 2009, Morrison et al 2013, Karakose et al 2010). Th e role of kindlin-3 in β2 integrin activity regulation has been studied in detail and, interestingly, it is involved in some but not all processes in β2 integrin activation. A possible mechanism of action is to optimize the association of the LFA-1 tail with talin and possibly other intracellular regulators of integrin activities. Furthermore, kindlin-3 does not leave the crime scene aft er the initial integrin activation, but continues to be active and takes part also in the outside-in

28 Review of the literature signalling events. It has an adhesion-strengthening role, probably due to its interactions with the actin cytoskeleton. In T cells it is needed for homing to lymph nodes but not for affi nity increase

(Morrison et al 2013) and it has also been reported to take part in αMβ2-dependent outside-in regulation of Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 homolog (Cdc42) activities through spleen tyrosine kinase (Syk) and proto-oncogene Vav1 (Xue et al 2013). Most of the data previously cited here has been conducted in the T cell or T cell lines expressing LFA-1 as the only member of β2 family. However, the signalling events have been also studied extensively in the context of Mac-1–dependent phagocytosis. Rap1 is a vital activator of Mac-1 dependent phagocytosis, and its interaction with talin localises both of them in the phagocytic cup. Contrary to LFA-1, Rap1 signalling to Mac-1 has been reported to use only RAPL and not RIAM (Lim et al 2010, Goult et al 2010). Th e inside-out activation and ligand binding of Mac-1 occurs upon talin head binding, but the rod domain is indispensable for phagocytosis (Lim et al 2007). As for VLA-4, also other molecules and processes are involved in the activation cascades, but many of the results presented earlier hold true also for VLA-4 signalling (Imai et al 2010). Th e largest structural diff erence between VLA-4 and the 2β integrins is that VLA-4 does not contain the α chain I domain that is almost exclusively responsible for the ligand binding to β2 integrins. Th e lack of I domain, however, seems to increase VLA-4 fl exibility and ability to bind ligands in various conformations (Chigaev & Sklar 2012). Another factor that diff erentiates VLA-4 is the binding of paxillin to the α4 cytoplasmic part. Paxillin is essential for VLA-4 adhesion under shear but not in no-shear conditions (Alon et al 2005). Paxillin-α4 interactions are strongly regulated by the phosphorylation of α4, so that paxillin only binds to non-phosporylated α4 and is released upon phosphorylation. Th e binding of paxillin decreases cell spreading and migration, whereas its release from the α4 tail enhances spreading. Importantly, a dynamic phosphorylation- dephosphorylation cycle is needed for the proper function of VLA-4 in e.g. cell migration (Han et al 2001, Han et al 2003).

4.3 Outside-in signalling ini ated by integrin ligand binding Outside-in activation of integrins may happen upon ligand binding, activating antibody binding (Campbell & Humphries 2011) or divalent cations (Dransfi eld et al 1992). Specifi c signalling cascades are induced upon ligand binding, leading to increase in cell adhesion, spreading and migration, although the outcome depends on the cell type and function. For example in T cells, outside-in signalling is associated with cell proliferation and interleukin-2 (IL-2) production as well as stabilisation of the immune synapse between T cell and APC. In neutrophils the result is diff erent: enhancement of degranulation and activation of NADPH oxidase that enables ROS production that is further used for cytotoxic eff ects. As for macrophages, the outcome of outside-in signalling consists of cell diff erentiation and IL-1β mRNA stabilisation (Abram & Lowell 2009). As previously discussed, it is occasionally diffi cult to dissect the role of the intracellular signalling and adapter proteins in inside-out and outside-in cascades, also because many molecules are used in both pathways according to the present knowledge. In order to bypass the inside-out route and elicit only outside-in signalling, it is possible to use specifi c activators such as monoclonal antibodies and divalent cations (Mn2+) or integrin ligands. Using these methods,

29 Review of the literature many of the following results have been obtained. A schematic model of some of the pathways involved in integrin outside-in signalling is shown in fi gure 12. In outside-in signalling, a crucial downstream step is the formation of FA or focal adhesion, a large, dynamic complex consisting of about 150 intracellular proteins. Another important consequence of integrin outside-in signalling are the changes in the assembly of the actin cytoskeleton and changes in integrin avidity .

Integrin α β I T A M P Syk ZAP-70 SFK SLP-76 VAV1 Ca2+ P 14-3-3 PLCγ IP3

JAB-1 Cytohesin FAK SLP-76 ADAP PRAM-1

Nucleus Pyk2 C-Cbl C-Jun Erk IL-2 PI3K AP-1 Integrin TIAM1 clustering

Rac-1ac-1 ActinA reorganisationre

Figure 12. Pathways of integrin outside-in signalling.

Th e fi rst steps in outside-in signalling are the phosphorylation events of Src family kinases (SFK). In myeloid cells the SFK involved are called Hck, Fgr and Lyn (Giagulli et al 2006, Baruzzi et al 2008) and in T cells Lck and Fyn (Suzuki et al 2007). SFKs then phosphorylate ITAMs on DAP12 and FcγR, which off er docking sites for the next players in the sequence, the Syk family kinases Syk in myeloid cells or ZAP-70 in T and NK cells (Mocsai et al 2002, Mocsai et al 2003, Epler et al 2000). Th e reports of the importance of Src and Syk family kinases in migration are controversial and it seems to be dependent on cell type (monocyte / macrophage / neutrophil / DC) and the infl ammation site (peritoneum / lung / cremaster muscle) (Baruzzi et al 2008, Lammermann et al 2008).

Talin is needed to obtain the high-affi nity integrin. It shows diff erent stages in β2 tail binding (initial binding in the inside-out signalling and further binding to both NxxF/Y sites and to a membrane-proximal, additional site), leading to a stable, high-affi nity conformation (Wegener et al 2007). In VLA-4 outside-in signalling, the role of Zap-70 is to deliver talin to the plasma membrane and to phosphorylate Vav1, which dissociates it from talin. Th is makes it possible for talin to occupy the β tail and the integrin adopts a full high-affi nity conformation (Garcia-Bernal et al 2009). Rap1 and RAPL play critical roles also in the adhesion strengthening phase of transendothelial migration. RAPL binds to two membrane-proximal lysines (Lys1097 and

Lys1099) in the αL chain, and it may participate in the stabilisation of the conformation by keeping the intracellular tails separated. RAPL is suggested to be dispensable for the inside-out

30 Review of the literature affi nity change aft er chemokine activation, whereas Rap1 is needed also in inside-out activation (Katagiri et al 2003, Hogg et al 2011, Ebisuno et al 2010). Very important players in the outside-in cascade are the tyrosine kinases FAK and Pyk2 that are situated downstream of SFKs. Upon ligand binding, Src phosphorylates and hence activates FAK. FAK and Pyk2 functions in myeloid cells have been studied and the FAK or Pyk2 defi cient cells show similar kind of defects in lamellipodia stabilisation, directed , migration in vivo and cell polarisation (Owen et al 2007, Okigaki et al 2003, Han et al 2003). Close localisation is a prerequisite for such an activation cascade to take place. In that sense, it is important to decipher how the above-mentioned kinases and the integrins initiating the signalling are brought together. Even though the integrin cytoplasmic parts lack traditional SH2 or SH3 domains needed for Src and Syk kinases to bind, it has been reported that they are attached to the integrins, even in resting cells (Arias-Salgado et al 2003, de Virgilio et al 2004). Th e ITAM-phosphorylation and subsequent activation steps are partly similar in outside-in signalling that they are in the TCR- or BCR-mediated inside-out signalling. Th e integrin tails do not themselves contain ITAM-sequences, but they forward the signal through ITAMs of other cytoplasmic proteins. Further separation of α and β tails is needed for proper outside-in signalling and this might bring the integrin tails to close proximity of ITAM sequences (Zhu et al 2007). Another way to bring integrins close to the ITAMs could be the association of the two molecules in lipid raft s that have been implicated in LFA-1 and VLA-4 signalling (Marwali et al 2003, Krauss & Altevogt 1999, Solomkin et al 2007). Downstream of SFK and Syk family kinases is the important scaff old protein called SH2 domain-containing leukocyte phosphoprotein of 76kDa (SLP76). Its expression has been detected in neutrophils, DCs and platelets (Bezman & Koretzky 2007), and it is probably also involved in immunoreceptor (TCR, BCR, FcR) signalling (Abtahian et al 2006). Also ADAP (T cells) and PRAM-1 (myeloid) are molecules that are considered important in outside-in signalling in neutrophils (Clemens et al 2004, Bezman & Koretzky 2007). Th e SLP76 and ADAP may be contributing to inside-out signalling in T cells, whereas in myeloid cells they are involved in outside-in signalling. Yet another protein probably involved in outside-in signalling is cytohesin-1 that becomes phosphorylated aft er LFA-1 ligation, although it functions in inside-out signalling without being phosphorylated upon TCR activation. It is involved in ERK activation and IL-2 production (Perez et al 2003). Th e transcription factor JAB-1 (Jun-activating binding protein-1) has also an important role in LFA-1 outside-in activation. When LFA-1 binds to its ligand, JAB-1 is released from the LFA-1 cytoplasmic tail and transferred to the nucleus, where it co-activates the c-Jun transcription factor, activating activator protein-1 (AP-1) and resulting in responses such as IL-2 production (Perez et al 2003). In neutrophils, Mac-1 outside-in signalling leads to IL-8 production (Walzog et al 1999) and in monocytes the transcription repressor forkhead fox protein P1 (Foxp1) is downregulated, leading to M-CSF receptor expression upon Mac-1 stimulation (Shi et al 2001, Shi et al 2004). In myeloid cells, casitas B-lineage lymphoma protein (c-Cbl) is involved in integrin outside-in signalling, in addition to its other functions as adapter and ubiquitin ligase protein (Caveggion et al 2003). It transfers cytoplasmic phosphoinositide 3-kinase (PI3K) to the plasma membrane (Meng & Lowell 1998). PI3K is needed for fi rm arrest of VLA-4 and LFA-1 (Hyduk & Cybulsky 2009). Th e involvement of the actin cytoskeleton in outside-in signalling is transmitted through modulation of Rho GTPases that are regulated by Vav family of Rho-GEFs. In neutrophils,

31 Review of the literature the lack of Vav leads to the inability to support binding in shear fl ow even though the initial attachment is not aff ected (Graham et al 2007). Vavs regulate also PLCγ and Ca2+ signalling from TCR and BCR (Swat & Fujikawa 2005) and from integrins in neutrophils (Graham et al 2007). Th e most extensively studied Rho GTPases are Rac, Rho and Cdc42, that are needed for polarisation and cellular migration (Ivetic & Ridley 2004). A specifi c signalling pathway initiated by LFA-1 activation and leading to Rac1 activation and actin cytoskeleton rearrangements has been recently solved. Activation of LFA-1 induces the phosphorylation of the β2 chain on threonine 758 (Valmu & Gahmberg 1995, Hilden et al 2003). Th is is an important switch in the binding of some cytoplasmic signalling and adapter proteins

(Takala et al 2008). Th e affi nity of 14-3-3 proteins is high towards T758-phosphorylated β2, and it is able to outcompete fi lamin from the binding site. Recruitment of 14-3-3 leads to activation of T-cell lymphoma invasion and metastasis-inducing protein 1 (Tiam1) and further to Rac1 activation and actin cytoskeleton rearrangements (Fagerholm et al 2005, Nurmi et al 2007,

Gronholm et al 2011). Similar results have been obtained for β1 integrins (O’Toole et al 2011). Most recent reports have focused on kindlin action in the inside-out signalling of integrins, but recently, more evidence on their role in the outside-in signalling has been gathered. Talin binds to integrin β chains through the membrane-proximal Nxx(Y/F) sequence, causing the separation of α and β cytoplasmic tails, whereas kindlin F3 domain binds to the membrane- distal Nxx(Y/F) (Manevich-Mendelson et al 2009). Kindlin-3 is involved in outside-in signalling of LFA-1 and αIIbβ3 by inducing spreading. It also induces microclustering of the integrin using its PH and F3 domains (Feng et al 2012). Kindlin-3 is also required for αMβ2 mediated fi rm adhesion (Xue et al 2013). Atomic force microscopy studies revealed that the kindlin-3/β2 tail TTT –motif interaction is not essential for the initial ligand binding, but is defi nitely required for the strengthening of the adhesion as well as lymphocyte homing in shear fl ow, whereas in shear- free environment this interaction is not necessary (Morrison et al 2013). In phagocytosis, the diff erent phagocytic receptors may induce separate signalling pathways, leading to slightly varied modes of phagocytosis. Fc-receptors use Rac1/Cdc42 to induce “reaching phagocytosis” and complement receptors produce ”sinking phagocytosis” through Ras homolog gene family, member A (RhoA) (Caron & Hall 1998, Dupuy & Caron 2008). RhoG binding to the Th r-Th r-Th r motif is also involved (Tzircotis et al 2011). Also Rap1 and RhoA induce outside-in activated phagocytosis (Kim et al 2012). One aspect of signalling is the diff erence between the various activation methods. In fact, it seems that the TCR-mediated integrin activation that takes place in the static conditions of the lymph nodes or tissues and the chemokine- or selectin-mediated activation in the shear fl ow environment lead to diff erent outcomes of integrin activation. According to some reports, the affi nity of the integrins does not necessarily change aft er TCR activation (Stewart & Hogg 1996, Burbach et al 2007) and TCR ligation has been suggested to, in fact, prime the integrins for activation, not directly extend them (and leading to ligand binding even by bent form) (Feigelson et al 2010). Th e LFA-1 molecules on eff ector cells diff er from the naïve T cells in the leukocyte integrin activation status and mechanisms. In eff ector cells, there are more LFA-1 integrins expressed than in naïve T cells and, importantly, it shows spontaneous binding to ICAM-1, as opposed to the situation in the naïve cell LFA-1. Th e strengthening of the adhesion is extremely rapid. Th e binding of the eff ector cells to the endothelium is independent of chemokine activation, whereas they do need chemokine signals for transendothelial migration (Shulman et al 2011, Lek et al 2013).

32 Review of the literature 4.4 Phosphoryla on As seen in the previous chapters, the binding of the cytoplasmic adapter and other proteins is essential for the accurate regulation of integrin functions. All the proteins able to bind to the short cytoplasmic integrin tails cannot be present at the same time due to spatial limitations, so there has to be some factors that determine which protein(s) can bind under certain conditions. Th e cellular localisation of the regulators and the diff erences between binding affi nities are important mechanisms to direct the protein-protein interactions. Another way to regulate the adaptor binding to integrin tails is phosphorylation of the integrin cytoplasmic tails, which may increase or decrease the binding of certain molecules. Most α and β chains of the integrins have been reported to undergo phosphorylation either constitutively or upon cell activation (fi gure 9) (Fagerholm et al 2004, Gahmberg et al 2009).

4.4.1 β2 integrins Integrin phosphorylation has been reported already almost 30 years ago, and more specifi c studies on β2 family phosphorylation showed that the β2 chain is phosphorylated upon cell activation, whereas the α chains (αL, αM, αX) are constitutively phosphorylated. In the initial studies, serine was found to be the predominant phosphorylation target in all polypeptide chains studied. Weak threonine phosphorylation was detected in αX and β2, and β2 showed also tyrosine phosphorylation (Chatila & Geha 1988, Chatila et al 1989, Buyon et al 1990). Later, strong threonine phosphorylation has been detected using phosphatase inhibitors (Valmu & Gahmberg 1995).

Th e following phosphorylation sites on β2 (CD18) chain have been reported. Tyrosine 735 is phosphorylated aft er IL-2 treatment in NK cells (Umehara et al 1993), and aft er collagen binding in neutrophils (Garnotel et al 1995). Th is phosphorylation is reported to be involved the internalisation of LFA-1 at the rear end of a migrating cell (Tohyama et al 2003). Serine 745 is phosphorylated aft er phorbol ester treatment that is mimicking PKC activity in the cells (Fagerholm et al 2002). An interesting function of Ser745 phosphorylation is that it releases JAB-1 from LFA-1, leading to transcription of certain genes (see previous chapter) (Perez et al 2003). Serine 756 was initially found to be the major phosphorylation site aft er activation with phorbol esters but it was reported not to be essential for integrin adhesion to its ligands (Hibbs et al 1991, Fagerholm et al 2002). Later Ser756 has been shown to be essential for Mac-1 dependent phagocytosis through Rap1 and talin recruitement to β2. Interestingly, the talin head alone is able to activate the phosphorylation mutant Mac-1 bearing the Ser756Met mutation. Th e kinase involved is calmodulin-associated molecule CaMKII (Lim et al 2011). Phosphorylation of the threonine triplet 758-760 has been under profound investigation and indeed has proven to be essential in leukocyte integrin functions. Th e Th r-Th r-Th r sequence was early shown to be important for LFA-1 mediated adhesion (Hibbs et al 1991, Peter & O’Toole 1995) but the threonine phosphorylation was ignored as it was so labile that it could not be seen unless phosphatase inhibitors were used (Valmu & Gahmberg 1995). Th e triplet is phosphorylated aft er TCR engagement or phorbol ester treatment, but in slightly distinct patterns, and all three threonines cannot be phosphorylated at the same time (Valmu et al 1999, Hilden et al 2003). Th is Th r-Th r-Th r sequence is associated with actin reorganisation, “postreceptor events” (avidity/ clustering), but not in affi nity regulation (Peter & O’Toole 1995).

Kinases phosphorylating the β2 chain have been described. Th e main responsible kinases for Ser745 and Th r758 are PKCδ and PKCβI/II. Also PKCα and PKCη may phosphorylate these

33 Review of the literature residues and PKCα is also able to phosphorylate Th r-760. Ser-756 is not directly phosphorylated by any PKC isoform, but is still phosphorylated upon phorbol ester treatment, indicating the involvement, but perhaps indirect, of a PKC (Fagerholm et al 2002).

Th e cytoplasmic domains of the four α chains of β2 integrins vary in length and sequence, enabling specifi city for cytoplasmic interactions and signal transduction. Constitutive phosphorylation of β2 integrin α chains was reported several years ago (Chatila et al 1989, Buyon et al 1990, Valmu et al 1991) and the specifi c phosphorylation sites and the functions of the phosphorylation of αL and αM have been elucidated (Fagerholm et al 2005, Fagerholm et al 2006).

Phosphorylation of αX is dealt in chapter 8.2. αDβ2 phosphorylation has not been reported.

Th e αL chain is phosphorylated on Ser1140 and the phosphorylation aff ects LFA-1 ligand binding and activation epitope expression. Approximately 40 % of the total αL chain molecules (in heterodimers) are phosphorylated. TCR engagement and phorbol esters are able to activate 2+ also the αL-Ser1140Ala mutant, but when activated with activating antibody MEM83 or Mg , the phosphorylation mutant binds ICAM-1 less effi ciently. Phosphorylation of the α tail also infl uences the overall conformation of the extracellular region, which can be studied with antibodies recognising diff erent (extended or extended-open, see chapter 3.2) conformations. Th e Ser1140Ala-mutated LFA-1 cannot adopt the high-affi nity conformation detected with antibody mAb24, an antibody recognising the open, extended conformation (Kamata et al 2002), if the cells are activated with cytokine stromal-cell derived factor-1α (SDF-1α) or ICAM-2.

Anyway, talin head domain is able to activate both β2 Th r758Ala and αL Ser1140Ala (Fagerholm et al 2005).

Th e αM chain has only one cytoplasmic serine (Ser1126), which is constitutively phosphorylated. Phorbol esters or extracellular Mg2+/EGTA treatment are not able to activate the Ser1126Ala mutant to bind to ICAM-1 or ICAM-2. However, the binding to other ligands, such as iC3b and denatured bovine serum albumin (BSA), is not aff ected. Expression of the conformation-specifi c activation epitopes recognised by antibodies mAb24 and KIM127 (that binds to extended integrin, (Lu et al 2001) is decreased and the binding to soluble ligands is downregulated by the mutation, showing impaired affi nity. Importantly, migration through the endothelium and homing of lymphocytes in vivo are also severely aff ected in the cells where αM phosphorylation is not possible (Fagerholm et al 2006).

4.4.2 VLA-4

Th e α4 chain of VLA-4 is one of the integrin α chains whose phosphorylation has been studied quite extensively. Th e phosphorylation of serine 988 by PKA regulates paxillin binding to the α4 cytoplasmic tail and this in turn regulates the functions of VLA-4. Paxillin does not bind to the

α4 tail phosphorylated on serine 988. Interestingly, in primary cells and diff erent cell lines the α4 phosphorylation varies a lot (from 0% of some cells to 60% of α4 chains being phosphorylated in

Jurkat cell line). Point mutations made on α4 chain revealed very interesting consequences of the

α4 phosphorylation. Ser988Asp, which mimics constant phosphorylation, accelerates spreading and inhibits migration, whereas Ser988Ala (incapable of phosphorylation) inhibits spreading and reduces migration. Both mutations are able to bind VCAM and both wt and Ser988Ala are able to undergo outside-in signalling upon VCAM-1 binding, whereas Ser988Asp is not able to induce outside-in signalling. Especially cell migration requires a dynamic phosphorylation – dephosphorylation cycle (Han et al 2001, Han et al 2003). α4 interaction with paxillin is needed for the VLA-4 –VCAM-1 strengthening under fl ow and for leukocyte recruitment to the sites of infl ammation (Alon et al 2005, Feral et al 2006). 34 Review of the literature

Also the spatial regulation of integrin activities may be controlled by phosphorylation. In migrating cells, phospho-α4 is accumulated at the leading edge, and if α4 phosphorylation is blocked (Ser988Ala mutation), migration as well as lamellipodia stability are decreased, even though there are no changes in ligand binding capacity. In Ser988Ala cells that are not able to spread, paxillin and α4 co-cluster at the membrane. On the contrary, in Ser988Asp cells paxillin is located in all parts of the cell and there is no colocalisation with α4 (Goldfi nger et al 2003).

α4 phosphorylation on another serine, Ser978, induces 14-3-3ζ binding and the formation of a complex consisting of the α4 chain, 14-3-3ζ and paxillin, which is needed for Cdc42 (Rho GTPase) activity on the leading edge and lamellipodia of a migrating cell, and for directed cell movement. Interestingly, α4 interaction with paxillin only is suffi cient for the regulation of another Rho GTPase, Rac1 (Deakin et al 2009).

Phosphorylation of the β1 chain has also been studied with point mutations in the cytoplasmic moiety. Th e phosphorylation of Tyr783 and Tyr795 do not aff ect integrin functions (Wennerberg et al 1998), but phosphorylation of Ser785 induces integrin localisation to FAs, enhances attachment and inhibits spreading and migration (Barreuther & Grabel 1996, Mulrooney et al 2001). Phosphorylation of threonines 788 and 789 (corresponding to the Th r-Th r-Th r motif in the β2 tail) is needed for attachment to Fn and activation epitope expression (Wennerberg et al 1998). Th reonine 788 mutation to aspartic acid, which mimics phosphorylation, induces a constitutively active integrin and increases cell adhesion through an inside-out activation pathway (Nilsson et al 2006). Phosphorylation of threonines 788 and 789 is induced with elevated pressure, which increases β1 affi nity towards extracellular matrix components such as collagen, Fn and laminin, whereas serine 785 phosphorylation is not needed for pressure-controlled adhesion (Craig et al 2009). Also tyrosine phosphorylation of β1 is required for migration (Sakai et al 1998b, Sakai et al 1998a).

4.5 Transdominant regula on of integrins 4.5.1 Leukocyte integrins Integrin transdominant regulation or cross-talk is a phenomenon where the activation of one integrin regulates the functions of another in the very same cell. Th e fi rst report of mutual regulation of diff erent integrins expressed in the same cells dates back to two decades ago, when van Kooyk et al. noticed that in native T cells and diff erent T cell lines there are large diff erences in the proportions of LFA-1 and VLA-4 mediated adhesion to activated endothelium. For example, in freshly isolated T cells the binding was equally LFA-1 and VLA-4 dependent, but when further cultured, the equilibrium moved towards LFA-1 dependency. Some of the T cell clones were LFA-1 dependent, some VLA-4, even though both integrins are expressed on all lines examined. Usually LFA-1 is used for adhesion whenever it is available, i.e. expressed, functional and activated, and VLA-4 binding to VCAM-1 only takes place when LFA-1 cannot be activated (van Kooyk et al 1993). Next, Porter & Hogg reported that when LFA-1 is activated, VLA-4 (and VLA-5, to some extent) are inhibited to bind their ligands VCAM-1 and Fn. It is the occupancy of LFA-1 that induces the cross talk (Porter & Hogg 1997). A somewhat controversial but intriguing aspect was detected when Leitinger and Hogg studied the functions of LFA-1 integrin lacking the I domain, using the Jβ2.7 T cell line. Th ey reported that removal of the I domain induced the expression of the activation epitopes and very effi cient activation of VLA-4 and VLA-5. Th e changes in VLA-4 were not affi nity-based, but the

35 Review of the literature localisation of VLA-4 on the plasma membrane was clearly clustered. Th e increased adhesion was due to actin rearrangements (Leitinger & Hogg 2000). Interestingly, a similar of phenomenon, but vice versa (VLA-4 activation increasing LFA-1 activation) was observed in a study by Chan et al. When VLA-4 bound to VCAM-1, LFA-1 binding to ICAM-1 was also enhanced, probably due to clustering of LFA-1 on the cell surface. Th e activation status of LFA-1 detected by mAb24, did not change and cytochalasin D abolished the increase in binding but did not, however, change the basal binding to ICAM-1. Th ese fi ndings suggest that the VLA-4 activation-dependent increase in LFA-1 ligand binding is dependent on the cytoskeleton and increase in LFA-1 avidity rather than affi nity (Chan et al

2000). Interestingly, the phosphorylation of the α4 chain regulates this transdominant activation eff ect of VLA-4, because the Ser988Asp mutant (“constant phosphorylation”) that is unable to bind paxillin, is also not capable of signalling to increase the LFA-1/ICAM-1 interactions (Han et al 2003). Mac-1 has also been shown to be involved in the transdominant regulation of integrins. In neutrophils, the activation of β1 with an activating antibody leads to activation epitope expression and increased Mac-1-dependent binding to Fn (van den Berg et al 2001). αVβ3 has also been reported to modulate αMβ2 ligand binding (Van Strijp et al 1993, Ishibashi et al 1994). Th e role of Mac-1 in negative regulation of immune responses was dealt with in chapter 3.3. Also pathogens may use the cross-talk to conquer the host cells. In Bordetella pertussis (whooping cough) pathogenesis, the bacterium adheres to monocytes/macrophages through a Mac-1 dependent mechanism, but the adhesion seems to be enhanced through a β1 integrin signalling pathway (Ishibashi et al 1994). CR4 has also been associated with cross-talk in monocytes. As previously mentioned, CR4 expresssion is increased on monocytes aft er a high-fat meal, which enhances, together with VLA-4, monocyte adhesion to aortic endothelium VCAM-1. It has been suggested that the role of CR4 in these monocytes is not necessarily only to bind VCAM-1, but to support the VLA-4/ VCAM-1 interactions through cross talk (Gower et al 2011).

4.5.2 Other integrins in cross talk In Glanzmann’s thrombastenia, a mechanism involving integrin cross talk has been suggested. In this disease the platelet aggregation is defective and severe bleeding is detected. In fact, there are two reported pathways that may aff ect platelet functions. In both models, a mutation (Ser752Pro) in the β3 chain, that inhibits correct αIIbβ3 inside-out activation, aff ects α2β1 integrin binding to collagen. In the model proposed by Riederer and colleagues, binding of fi brinogen to and thus activation of αIIbβ3 leads to inhibition of α2β1-mediated collagen binding. van de Walle et al on the other hand have reported a positive regulation initiated by ligand binding to αIIbβ3 and resulting in enhanced collagen binding by α2β1 (Riederer et al 2002, Van de Walle et al 2007, Gonzalez et al 2010).

Early reports indicated that also the ligation of αVβ3 is able to regulate α5β1 functions.

Initially, it was shown that when the phagocytosis through αVβ3 was blocked, α5β1-mediated phagocytosis was also inhibited. Th e mechanism of this cross talk has been studied quite thoroughly and the suppression was fi rst reported to take place through PKC, and later CamKII was shown to be the major signal mediator between the two integrins (Calderwood et al 2004). It seems that CamKII, that is essential for α5β1-dependent phagocytosis and migration in a normal situation, is inhibited upon αVβ3 ligation (Blystone et al 1994, Blystone et al 1995, Blystone et al

36 Review of the literature

1999). αVβ3 integrin ligation has been shown to enhance VLA-4 (α4β1) dependent migration on VCAM-1, followed by rapid extravasation and migration under infl ammatory conditions (Imhof et al 1997). Also LFA-1 binding to ICAM-1 has been reported to be downregulated upon αVβ3 activation (Weerasinghe et al 1998). Yet another situation where cross-talk has been reported is the fi bronectin binding in cells expressing αIIbβ3 and α5β1. When αIIbβ3 is not activated, binding takes place through α5β1. But when αIIbβ3 is ligated and its conformation changes, Fn binding is blocked and spreading on Fn is reduced (Diaz-Gonzalez et al 1996).

Recently αV integrins have been shown to control each other in the specifi c situation of glaucoma that was studied in human trabecular meshwork cells (HTM). Researchers studied phagocytosis mediated by αVβ5 and FAK, that is critical for the maintenance of the physiological conditions in the eye. Activation of αVβ3 that may happen upon steroid treatments inhibits this phagocytosis and may lead to glaucoma (Gagen et al 2013). Th e molecular mechanisms of these cross-talk events have been studied to some extent, but no common factor has been found. For example in some cases the affi nity of the target integrin is changed, whereas in others, the enhancement of adhesion is due to avidity changes, i.e. integrin clustering on the cell surface. Th e signalling cascades leading to inhibition or activation of another integrin have been reported in only some cases. Talin sequestration by the inhibiting integrin has been suggested to be the mechanism of cross talk control in the case of αIIbβ3 and

α5β1 integrins (Calderwood et al 2004). In another situation, when β1 controls laminin binding through αVβ3, talin doesn’t seem to play a restrictive role. Instead, a signalling cascade initiated by PKA activation and leading to inhibitor-1 phosphorylation, inhibition of protein phosphatase-1 and sustained phosphorylation of β3, has been reported to control the cross-talk (Gonzalez et al 2008).

37 Summary of the study SUMMARY OF THE STUDY 5 Aims of the study Adhesion events of blood cells are essential for their function and require constant regulation. Th e purpose of these studies was to defi ne mechanisms behind the multi-step adhesion events of leukocytes and red blood cells. Th e specifi c aims were:

1. To analyse the interactions between erythrocyte ICAM-4 and leukocyte integrin CR4

(αXβ2) and to characterise the binding between ICAM-4 and LFA-1 (αLβ2), Mac-1 (αMβ2)

and CR4 (αXβ2).

2. To identify the phosphorylation site of CR4 and characterise the role of the phosphorylation in the functions of the integrin.

3. To determine the molecular mechanisms leading to cross-talk or transdominant

regulation between β2 integrins and VLA-4.

38 Summary of the study 6 Experimental procedures

Materials and methods used in this study have been described in detail in the original publications.

Experimental procedure Original publication Antibodies I II III IV Cell adhesion assays (shear fl ow) IV Cell adhesion assays (static) I II III IV Cell lines and cell culture I II III IV Co-immunoprecipitation IV Erythrophagocytosis assay II Flow cytometry I II III IV Immunofl uorescence microscopy IV Immunoprecipitation III IV Migration assays IV Pep-spot assay II Peptide transfections IV Phagocytosis assay III Production of point mutations III Protein production and purifi cation I II Protein purifi cation from buff y coat cells I II Radioactive labelling of cells III Solid-phase ELISA assays I II Stable cell line production III IV Th ree-dimensional protein modelling II Transfection III

39 Summary of the study 7 Results and discussion 7.1 Interac ons of ICAM-4 with leukocyte integrins (I & II) 7.1.1 CR4 is a new ICAM-4 binding partner (II)

β2 integrins LFA-1 and Mac-1 had previously been reported to bind to erythrocyte ICAM-4, but the results concerning the interaction between CR4 and ICAM-4 were controversial. In a previous report, the adhesion of various types of leukocytes (B cells, NK cells and monocytes) to purifi ed LW/ICAM-4 was indeed shown to be partly CR4-dependent, but in the same paper, red cells failed to adhere to purifi ed CR4 integrin (Bailly et al 1995). We set out to study the interaction. In the present paper (II), we used a diff erent purifi cation method for CR4, one that has been shown to yield functionally active CR4 protein (Stacker & Springer 1991). It is possible that the purifi cation procedure used previously was not suitable for preserving integrin activity. Th is is especially important as CR4 has been shown to be resistant to activation and to preferably stay in the inactive state (Bilsland et al 1994, Zang & Springer 2001). Here, we found that CR4, when purifi ed according to the protocol of Stacker & Springer, or expressed in L cell transfectants, was able to bind to ICAM-4. Th e interaction was characterized in a variety of experiments. First, adhesion assays with red cells binding to purifi ed CR4 coated on plastic were performed in 96-well plates. Th is adhesion was ICAM-4 and CR4 specifi c, as shown with inhibiting antibodies against ICAM-4, αX and β2. Next, L cells stably transfected with ICAMs were allowed to adhere to purifi ed integrins and also the interaction between ICAM-4 transfectants and CR4 was specifi c (II fi g 1). Th ird, the interactions were analyzed in a solid-phase ELISA assay, where purifi ed proteins were allowed to interact on a 96-well plate. ICAM-4 was found to directly bind to LFA-1, Mac-1 and CR4 in a dose-dependent fashion. Again, the specifi city of the interaction was verifi ed with antibody inhibition (II fi g 2). Fourth, the adhesion of L cells stably transfected with CR4- to purifi ed ICAM-4 was studied and found to be specifi c (II fi g 3a-b). Th e fi nding that ICAM-4 binds to leukocyte integrin CR4, was in a way a return back to the primary fi ndings (Bailly et al 1995). Th e fact that CR4 is a ligand for ICAM-4 is not surprising as such, as it shares many ligands with Mac-1 and LFA-1. Anyhow, interesting diff erences between the binding properties could be discerned when comparing the binding of ICAM-4 to the three

β2 integrins, as will be discussed in the following section.

7.1.2 Characterisa on of the interac ons between ICAM-4 and CR4 (II) Th e Ig domains and amino acid residues on ICAM-4 responsible for LFA-1 and Mac-1 binding as well as those needed for interactions with αIIbβ3 and αV-integrins have been determined (Hermand et al 2000, Mankelow et al 2004, Hermand et al 2004). We continued this work and characterised the ICAM-4 domains and amino acid residues responsible for CR4/ICAM-4 interactions. Th is was studied in an adhesion assay, where CR4-transfectants were allowed to adhere to purifi ed wild type, truncated or point mutated ICAM-4-Fc protein. Th e results show that CR4 needs both Ig domains (the outermost D1- and the membrane proximal D2-domain) of ICAM-4 for maximal binding, even though domain 2 alone binds CR4 to a lesser extent (II fi g 4a). Th is was also supported by mutation studies, where the amino acids needed for binding were shown to be located in both domains as expected. Th e following amino acid were found to be essential for binding (see three dimensional construction of ICAM-4 in chapter 2.2.2): Trp19 on the A strand of D1, Arg52 on the C strand of D1, Trp77 on the EF loop of D1 and Th r91,

40 Summary of the study

Trp93 and Arg97 on the G strand of D1. On D2, Glu166 on strand E was needed for effi cient binding (II fi g 4b). Th e domain deletion and amino acid substitution studies were further confi rmed using synthetic peptides derived from domains 1 and 2 (P-D1 and P-D2, respectively). Th e specifi c peptide sequences were determined based on a PepSpot assay, where purifi ed CR4 integrin selectively bound to two sequences, one from each domain (II fi g 6a). Both peptides were 13-mers, and P-D1 contained the Arg52 and P-D2 included the Glu166 that were both found to be essential for the binding in the previous assay. Both peptides bound to CR4-transfected cells and were able to inhibit CR4-transfectant binding to ICAM-4-Fc, although they yielded the maximal inhibition only when used together (II fi g 6B-C). Th e peptides have been illustrated in the model of ICAM-4 fi gure 13.

Figure 13. Model of ICAM-4 based on ICAM-2 three-dimensional structure. Th e peptides P-D1 (green) and P-D2 (red) binding to CR4 are shown. From Ihanus et al 2007.

Th e results with amino acid point mutations in the ICAM-4 extracellular part continue the set of reports where the exact binding sites of diff erent integrins on ICAM-4 have been elucidated (Hermand et al 2000, Mankelow et al 2004, Hermand et al 2004). Th e domains and residues involved in the binding (table 1) show interesting diff erences between the diff erent integrins. A model of ICAM-4 is shown in fi gure 13. All integrins studied, except LFA-1, need both domains of ICAM-4 for maximal binding. All the β2 integrins seem to bind to the CFG-face of the fi rst domain, whereas the binding site of αIIbβ3 is on a spatially distinct location, in the AB-face of the domain 1. αVβ3 binds to the BE-strands of D1, to an area that is adjacent to, but not much overlapping with the αIIbβ3 binding site. αVβ1 and αVβ5 integrin binding sites encompass the AG-face of domain 1 with an input from the B strand. When looking at D2, most binding sites reside in the proximity of D1. Th e C’E loop of domain 2 is used by Mac-1, αIIbβ3 and αVβ3. αVβ1

41 Summary of the study and αVβ5 need Lys118 on the B strand and CR4 needs the Glu166 that resides in the bottom of the E strand, fairly distant from D1.

Table 1. Binding sites of reported ICAM-4 binding integrins on domains 1 and 2 of ICAM-4. (Adapted from Toivanen et al 2008).

Integrin

ICAM-4 αVβ1 & ICAM-4 domain αLβ2 αMβ2 αXβ2 αIIbβ3 αVβ3 αVβ5 strand/loop F18A W19A W19A W19A W19A A V20T Q30A A-B G32A K33A K33A B Q36A R52A/E R52A/E R52A/E C D W66A W66A D1 Y69A E D73A W77A/F W77A/F W77A/F W77A/F W77A/F E-F L80G/F L80G/F L80G/F F R92E W93A W93A W93A A94L G T95L S96A R97A/E R97A/E R97A/E R97A R97E K118E B E151A/K E151A E151A/K D2 C’-E T154V T154V E166A E

Especially the CR4 binding site on ICAM-4 encompasses almost the whole length of the molecule, which suggests a reasonably large area of integrin making contact with ICAM-4. Th is is further confi rmed by adhesion assays in the presence of CR4 I domain specifi c adhesion- blocking antibody 3.9. Th is antibody effi ciently inhibits CR4 binding to D1 of ICAM-4, but it is not as effi cient in inhibiting adhesion to D2, suggesting CR4 I domain interaction with D1 of ICAM-4 and another integrin part binding to D2. Another result strengthening this idea is that the peptides from both ICAM-4 domains (P-D1 and P-D2) were needed to obtain maximal inhibition between ICAM-4 and CR4, suggesting two distinct binding sites for ICAM-4 on CR4. Co-crystallisation studies with CR4 and ICAM-4 would be required to confi rm this data. Th e integrin-binding Ig-domains of ICAM family members share approximately 30 % of amino acid homology. When comparing the amino acids and domains responsible for ligand binding in ICAM-4 to the other ICAMs, some interesting observations can be made. First, all the other ICAM family members have a glutamate and a glutamine in positions that are highly important for ligand binding (Glu34 and Gln73 in ICAM-1). However, ICAM-4 has an arginine

42 Summary of the study and a threonine in these positions (Arg52 and Th r91), and mutating them “back” to glutamate and glutamine, respectively, does not improve ligand binding (Hermand 2000). It is also interesting to note that these amino acids are not used by all ICAM-4-binding integrins. Second, the integrin binding domains on other ICAMs do not encompass more than one domain, the outermost Ig-domain being the most popular. Only Mac-1 makes an exception as it binds to the third domain of ICAM-3 (Diamond 1991).

7.1.3 ICAM-4 binds to β2 integrin I domains (I & II) Th e I domain is the major binding site of ligands in all α chain I domain containing integrins. In I-less integrins the β chain I-like domain is critical for ligand binding (see chapter 3.2). ICAM-4 has been reported to bind both to integrins containing the α chain I domain (LFA-1, Mac-1 and

CR4) (Hermand et al 2000) and to integrins without I domain (αV integrins and αIIbβ3) (Hermand et al 2004, Mankelow et al 2004). In original articles I and II the binding of ICAM-4 to I domains of LFA-1, Mac-1 and CR4 was confi rmed. Th e ICAM-4 binding to LFA-1 and Mac-1 was studied in detail with red cells (I fi g 1 and 2), ICAM-transfectants (I fi g 4-7) and in a solid phase assay with purifi ed proteins (I fi g 10). Th e binding was shown to be specifi c, as shown with antibodies against ICAM-4 and the I domains (I fi gs 1B, 1D, 6 and 7) and by inhibition of the binding with purifi ed proteins (I fi gs 2, 4 and 11). According to the results obtained in a solid phase assay, the αL I domain is not able to block αM I domain binding to ICAM-4, even though soluble αM I domain inhibitis ICAM-4/αL I domain binding to some extent (I fi g 11). Th is suggests that the binding sites on these two integrins are partly overlapping but not identical. ICAM-1 and ICAM-2 transfectants were also used in the cell adhesion assays, and their binding to LFA-1 and Mac-1 was characterised in a similar way as that of ICAM-4 (I fi gs 4-7). When comparing the interactions between ICAM-1, ICAM-2 and ICAM-4 to the I domains of LFA-1 and Mac-1, certain distinctions could be made. Th ese diff erences were studied in adhesion assays performed in the presence of a variety of antibodies against these three ICAMs as well as the I domains of αL and αM. We noticed that the I domains of the two integrins do not necessarily bind to the same region in a certain ICAM molecule, and, similarly, the three ICAMs studied here do not bind to exactly the same site on the I domains (I fi g 6 & 7). Th ese results could be explained by relatively low homology between αL and αM sequences (35%, also in I domain area) and between diff erent ICAMs (approximately 30 %) (Harris et al 2000, Gahmberg et al 1997). Th e role of the CR4 I domain in binding was verifi ed in an adhesion assay where the active CR4 I domain, containing the activating mutation Ile314Gly effi ciently inhibited CR4/ICAM-4 interaction (II fi g 3D).

7.1.4 Interac ons between ICAM-4 and leukocyte integrins require divalent ca ons (I & II) As integrin adhesion is cation-dependent (see chapter 3.2), the requirements for cations in integrin-ICAM-4 interactions were studied next. LFA-1 and Mac-1 adhesion to ICAM-4 has previously been shown to be dependent on magnesium and calcium, and to be inhibited with removal of Ca2+ by EGTA (Hermand et al 2000). Th e same cations were shown to be partly responsible for CR4 binding as well, but maximal binding was obtained in the presence of Mg2+, Ca2+ and Mn2+. Even Mn2+ alone showed higher binding than the combination of Mg2+ and Ca2+ (II fi g 3C). Th e binding of ICAM-4 transfectants to purifi ed LFA-1 and Mac-1 I domains also

43 Summary of the study required Mn2+ for maximal binding, (I fi g 8), whereas RBC show high levels of adhesion with a combination of Mg2+ and Ca2+ (I fi g 9) and also with Mn2+ (data not shown). Th ese results are in accordance with several previous reports of integrins requiring divalent cations for binding to their ligands. But contrary to the report of Dransfi eld et al, who showed that Mg2+ or Mn2+ are needed for effi cient LFA-1/ICAM-1 interaction whereas Ca2+ inhibited the interaction (Dransfi eld et al 1992), in our experiments calcium did not show total inhibition of the adhesion. One has to keep in mind that there are diff erences between the experimental design of these reports. Dransfi eld and colleagues studied LFA-1 in T cells, whereas Hermand et al and us (Dransfi eld et al 1992, Hermand et al 2000) performed the experiments with transfected L cells. As for the I domains, the cation-dependency experiments were performed with ICAM-4 transfected cells adhering to purifi ed I domains coated on plastic (I). It is also important to note that the results of the activating eff ect of Mn2+ by us and others have been obtained in the presence of more than a thousand-fold concentration of Mn2+ compared to the normal levels in human blood (Milne et al 1990).

7.1.5 The role of ICAM-4 in erythrophagocytosis (II) Th e in vivo function of the interactions between ICAM-4 and leukocyte integrins was studied in an erythrophagocytosis assay, where monocytes were diff erentiated into macrophages, incubated with RBC, and phagocytosis of RBC was detected. Antibodies against LFA-1, Mac-1 and CR4 as well as anti-ICAM-4 effi ciently blocked erythrophagocytosis (II fi g 7). Similar studies have later been performed with erythrocytes of diff erent ages. As expected, the oldest red cells were the most prone to be phagocytosed and, in agreement with our results (II), erythrophagocytosis could be inhibited by antibodies against β2 integrins. Interestingly, diff erent β2 integrins seemed to be responsible for erythrophagocytosis of RBC of diff erent ages, most effi cient inhibitor of senescent RBC phagocytosis being CR4. Younger cells were phagocytosed most effi ciently by Mac-1 (Toivanen et al 2008).

7.1.6 Possible roles of ICAM-4/integrin interac ons in red cell life cycle Leukocyte integrins LFA-1, Mac-1 and CR4 are clearly important ligands for ICAM-4 and the results from these studies (I & II) and those published later (Toivanen et al 2008) suggest that one of the signals leading to senescent RBC removal by spleen macrophages could be ICAM-4 interaction with CR4 and other β2 integrins. Even though erythrophagocytosis has been extensively studied (see chapter 1.1.2), a lot of controversy still remains. Perhaps there is no single factor causing it, but rather the recognition and engulfment of senescent RBC is a multi-factor sum of diff erent changes in cell composition. One plausible mechanism could be that the loss of sialic acids from cell surface glycoproteins and thus reduction of the overall negative charge of the RBC could enable the senescent cells to come to a closer contact with the macrophages. Th is would then further allow more intimate interactions between phagocytosis receptors like CR4 and their ligands on RBC. Further studies on the mechanisms of erythrophagocytosis are required to show the importance of ICAM-4 and leukocyte integrins in these events.

Th e role of other than β2-family binding partners of ICAM-4 should not be omitted when looking for the candidates responsible for ICAM-4 mediated erythrophagocytosis. However, to our knowledge, most of them have not been implicated in phagocytosis. αVβ5 has been shown capable of phagocytosis in the context of eye, but its function there is highly specifi c, maintaining

44 Summary of the study homeostasis of trabecular meshwork in order to avoid glaucoma (Gagen et al 2013, Dupuy & Caron 2008).

7.2 Phosporyla on of αX chain is important for CR4 func ons 7.2.1 CR4 α-chain is phosphorylated on serine 1158 Phosphorylation of leukocyte integrin alpha chains, although not induced by cell activation, has been shown to be highly important in the functions of integrins LFA-1 and Mac-1 (Fagerholm et al 2005, Fagerholm et al 2006). CR4 has also been shown to be phosphorylated on serine (Chatila et al 1989, Buyon et al 1990), but the exact phosphorylation site has not been identifi ed. In order to specify the site(s) of serine(s) to be phosphorylated, we created serine to alanine point mutations in the two serines of the αX chain cytoplasmic part. Th ese constructs were transfected into COS-1 cells and the cells were labelled with radioactive 32P. Integrins were immunoprecipitated and phosphorylation of the αX chain was detected. Th e primary phosphorylatable serine revealed to be Ser1158, although possibly some trace phosphorylation could be seen also when Ser1158 was mutated to alanine (III fi g 1A). Based on that fi nding, we made stable transfectants of K562 cells expressing wild type (wt) αXβ2 or the phosphorylation mutant Ser1158Ala-αXβ2 (III fi g 1C). Th e sequences and reported phosphorylation sites of diff erent integrin α chains are shown in fi gure 9 and, as already mentioned, they are very heterologous in sequence and in structure, especially in the areas of phosphorylation.

7.2.2 αX chain phosphoryla on is vital for ligand binding

To study the eff ect of αX chain phosphorylation on cell adhesion, we performed adhesion assays where K562 cells transfected with wt CR4 or Ser1158Ala-CR4 were allowed to adhere to complement component iC3b coated on plastic. Th ese experiments showed that the Ser1158Ala mutation reduced the adhesion of CR4 transfectants to the background level. Adhesion was

CR4-specifi c as it could be inhibited with antibodies against αX and β2 (II fi g 2a). To verify the importance of Ser1158 over the other possible phosphorylation sites, we performed adhesion assays also with HEK293T cells transiently transfected with the Ser1161Ala-mutated CR4. We found that the Ser1161Ala mutation does not disturb adhesion, so Ser1158 seems to be the only or at least the most relevant serine controlling integrin adhesion (III fi g 2B). Similar assays were performed to show that phosphorylation could be mimicked with negatively charged aspartate, using Ser1158Asp-CR4 (III fi g 2B). β2 chain phosphorylation in LFA-1 and Mac-1 has been studied (see chapter 4.4.1), but not much is known about β2 chain phosphorylation in CR4. Th erefore, we performed adhesion assays also with CR4 containing Th r758Ala mutation in the

β2 chain. Th is mutation abolished CR4-dependent adhesion to iC3b to the background level, showing that the phosphorylation of β2 chain on Th r758 is needed for the adhesion also in CR4 (III fi g 2B).

Th e αL and αM chains have been shown to be phosphorylated on Ser1140 and Ser1126, respectively (Fagerholm et al 2005, Fagerholm et al 2006). Phosphorylation of these α chains is critical for the functions of LFA-1 and Mac-1, but also diff erences between the three α chains reported so far can be discerned. In LFA-1, the αL chain phosphorylation aff ects ligand binding when the cells are activated with activating antibody MEM83 or Mg2+, but not when activated with TCR ligation or phorbol esters. Th e role of diff erent activation methods will be further discussed in next chapter. Also binding of Mac-1 to ICAM-1 or ICAM-2 is inhibited if the phosphorylation site is mutated to alanine (Ser1126Ala) but it is still able to bind some of its

45 Summary of the study ligands, such as iC3b and dBSA even without a phosphorylatable serine. Interestingly, binding to these two ligands was totally abolished in cells expressing the phosphorylation-mutant CR4 (III fi g 2a). iC3b binding to integrins has been characterised reasonably well, and a very detailed picture of iC3b/CR4 binding has been received with negative-stain electron microscopy (Chen et al 2012). I domain is the major binding site of iC3b on CR4 as it has been reported to be on Mac-1 as well (Diamond et al 1993). However, other regions on Mac-1 may contribute (Xiong et al 2002, Li & Zhang 2003). Th ese fi ndings suggest that Mac-1 and CR4 use diff erent, α-chain phosphorylation-independent and -dependent mechanisms for iC3b binding regulation, respectively. Th is kind of regulation could enable the cell to use these phagocytosis receptors in diff erent conditions, depending for example on cell activation status. Also the replacement of the phosphorylatable serine with a negatively charged aspartate results in diff erent outcomes in LFA-1 and CR4. Th e Ser1140Asp mutation on αL chain inhibits adhesion in a similar fashion as the Ser1140Ala-mutation, whereas in the αX chain the aspartate seems to mimick phosphorylation and does not aff ect ligand binding (Fagerholm et al 2005). We also verifi ed the importance of Th r758 of the β2 in CR4 functions, as β-chain phosphorylation in CR4 had not been reported. Phosphorylation of Th r758 (along other phosphorylation sites) has been shown to be crucial for LFA-1 functions (Valmu & Gahmberg 1995), but Ser756 is essential for Mac-1 dependent phagocytosis (Lim et al 2011). And at least Th r758 is needed for CR4 functions. When comparing the diff erences, it is worth noting that the studies of LFA-1 and Mac-1 have been made in leukemic T cell line Jurkat or its derivative Jβ2.7 (Weber et al 1997) and the CR4 phosphorylation was studied in K562 cells that are quite primitive erythroid/myelomonocytic precursor cells (Andersson et al 1979).

7.2.3 Eff ects of αX chain phosphoryla on on inside-out and outside-in signalling To study the eff ects of Ser1158 phosphorylation on inside-out signalling, we transfected the cells with constitutively active Rap1 (Rap1V12, where amino acid in position 12 has been replaced with a valine) that has been shown to activate LFA-1 (Fagerholm et al 2005). Phosphorylation of Ser1158 was found to be needed for proper inside-out activation of the integrin, as the Rap1V12 did not increase Ser1158Ala adhesion to iC3b (III fi g 2D). On the contrary, when studying outside-in signalling, we did not fi nd diff erences between wild type and phosphorylation- mutant CR4. Th is was observed in two experiments, fi rst in an adhesion assay where activation was induced with a β2-binding activating antibody. In the second assay Syk phosphorylation, which is known to take place downstream of outside-in activation of integrins, was studied by immunoprecipitation. When phosphorylated Syk was detected, there were no diff erences between wild type and Ser1158Ala-mutated CR4. So we conclude that the αX chain phosphorylation does not aff ect outside-in signalling from CR4 (III fi g 2C & 2F). Th e inside-out signalling of LFA-1, when studied with Rap1V12 transfection, showed similar dependence on α-chain phosphorylation as CR4 (Fagerholm et al 2005). Corresponding studies have not been made with Mac-1, nor has the α-chain phosphorylation in signalling events been studied in LFA-1 or Mac-1. One important tool in integrin activation studies are the activating or activation epitope recognising antibodies. When activated with monoclonal antibody MEM83 (or with Mg2+),

LFA-1 binding to ICAM-1 is dependent on αL chain phosphorylation, whereas induction of

46 Summary of the study ligand binding with TCR ligation or phorbol esters, that mimick the functions of PKC, is not

αL chain phosphorylation-dependent (Fagerholm et al 2005). Interestingly, the activating antibody CBR LFA-1/2 was able to activate CR4 despite of the phosphorylation status (III fi g 2C). Antibodies detecting the integrin activation status used in these studies were mAb24 that recognises the extended, open conformation and KIM127 that binds to extended integrin. LFA-1

αL chain phosphorylation mutant is not able to adopt the fully active conformation detected with mAb24 when activated with ICAM-2 (soluble LFA-1 ligand) or SDF-1α (chemokine) (Fagerholm et al 2005). Phosphorylation mutant Mac-1 (αM-Ser1126Ala) is not able to support maximal binding of either of the two antibodies (Fagerholm et al 2006). Interestingly, CR4 binding to activation epitope recognising antibodies was not greatly aff ected upon phosphorylation site mutation (data not shown). On the basis of these and other results, discussed in chapter 4 (Regulation of integrin activity), it is intriguing to search for a molecular mechanism that would explain the roles of α- and β-chain phosphorylations in integrin activation and signalling. Diff erent models for the specifi c functions of α and β chains and their phosphorylations in the integrin signalling have been proposed. According to Fagerholm et al (Fagerholm et al 2005), the role of αL phosphorylation would be needed for the affi nity change and β2 would function as an avidity regulator. On the other hand, it has been suggested that the role of αL-interacting -module would regulate the outside-in activation of the integrin, whereas the ADAP/SKAP55/RIAM-module, interacting with β2 chain, would be needed in the inside-out activation (Kliche et al 2012). When keeping in mind that the affi nity changes are thought to happen as a result of inside-out signalling, and that clustering and changes in avidity have been reported to occur aft er ligand binding (and thus being the outcome of outside-in signalling) (Kim et al 2004) it is clear that more studies are needed to specify the role of intracellular phosphorylations in these events. It is also likely that the phosphorylation and other regulation events of inside-out and outside-in are intertwining, and it may be next to impossible to dissect the activation cascades with this kind of classifi cation.

7.2.4 Eff ect on αX phosphoryla on on phagocytosis Finally, we set up a phagocytosis assay with PMA-activated K562 cells to elucidate the role of

α-chain phosphorylation on phagocytosis. Th e experiments show that αX chain phosphorylation on Ser1158 is indispensable in CR4-mediated phagocytosis (III fi g 2E). As a complement receptor, CR4 has been indicated in pathogen phagocytosis as well as apoptotic cell removal (Schlesinger & Horwitz 1991, Hirsch et al 1994, Mevorach et al 1998). However, the molecular mechanisms involved in CR4 mediated phagocytosis have not been studied in detail. Mac-1 signalling mechanisms have been thoroughly studied and reports (Lim & Hotchin 2012) show similarities but also discrepancies, when compared to LFA-1 signalling cascades, as discussed in chapters 3.4 and 4. Some examples of the diff erences are the immunosuppressive role of Mac-1, the importance of phosphorylation of β2 chain Ser756, and the independence of RIAM signalling. Instead, Mac-1 uses only RAPL for Rap1-mediated activation. Along these lines, further studies on CR4 signalling mechanisms probably reveal interesting distinctions between the signalling routes used by the Mac-1 and CR4.

47 Summary of the study 7.2.5 Why is this important? For a long time, CR4 has been considered a marker of DC, but recent studies have shown its role in the adhesion of monocytes to the endothelium aft er a high-fat meal (Gower et al 2011), in adipose tissue infl ammation associated with obesity (Wu et al 2010) and in antigen uptake by DC (Ejaz et al 2012). Th ese functions are involved in atherosclerosis, insulin resistance and, on the other hand, in the exciting development of new therapies against cancer. Th ese are probably some of the major health problems of the modern civilisation. Th erefore, it is of high importance to learn the molecular mechanisms involved in the regulation of these events, in order to develop specifi c, targeted cures against these diseases. Perhaps the highly specifi c nature of phosphorylation of the leukocyte integrin α-chains could serve as a target for future therapies aimed to activate or inhibit specifi c leukocyte functions.

7.3 Mechanism of trans-dominant inhibi on between β2 integrins and VLA-4 (IV) Already when studying CR4 phosphorylation we noticed that, instead of inducing adhesion, the expression of functional CR4 on K562 cells inhibited binding to VCAM-1. Interestingly, cells expressing the αX chain phosphorylation mutant (CR αX-S/A) bound strongly to VCAM-1, just like cells not transfected with CR4 (IV fi g 3D). Th is lead to further studies on transdominant inhibition of integrins and we observed that Jβ2.7 cells expressing LFA-1 bound less to VCAM-1 (that is not an LFA-1 ligand) than the cells that did not have LFA-1. Binding of Jβ2.7 cells to VCAM-1 was found to be VLA-4 and VCAM-1 dependent and VLA-4 is the only VCAM-1 receptor in Jβ2.7. Jurkat (of which the Jβ2.7 cells are derived) is a well-known cell line used widely in T cell and leukocyte integrin studies and their signalling cascades and activation methods have been extensively reported. Jβ2.7 are also more prone to adhere, migrate and spread than K562, which makes it easier to detect the outcome of specifi c activations or inhibitions. All these facts led us to study the transdominant inhibition in Jβ2.7 cells. Trans-dominant inhibition of VLA-4/ VCAM-1 binding by LFA-1 activation in T cells was reported already almost twenty years ago (Porter & Hogg 1997), but the mechanism of this regulation have not been elucidated. In the original article IV we propose a mechanism of this regulation.

7.3.1 VLA-4 binding to VCAM-1 is blocked by ac vated β2 integrins

Jβ2.7, which is an αL chain-defi cient derivative of the parental Jurkat T cell line and lacks LFA-1, adhere strongly to VCAM-1 through VLA-4 integrin. Th is adhesion was inhibited when LFA-1 was expressed in the cells and even more effi ciently so, when LFA-1 was activated with chemokine SDF-1α. Th e inhibition was seen in static adhesion assays as well as in fl ow adhesion on purifi ed VCAM-1 and on activated endothelium (IV fi g 1). Jurkat cells have been reported to adhere effi ciently to VCAM-1 through VLA-4 (Manevich et al 2007) and this strong adhesion was also seen in the migration assays we performed. Without treatment, the cells lacking LFA-1 were not able to transmigrate in a Transwell assay, but stayed bound on the fi lter coated with VCAM-1. Th e strong adhesion was released with antibodies against α4 chain of VLA-4 and with the expression of functional LFA-1. Also the retraction of the uropod at the rear of the migrating cell was impaired (IV fi g 2).

48 Summary of the study 7.3.2 LFA-1/CR4 phosphoryla on is essen al for transdominant inhibi on To begin the elucidation of the signalling mechanisms behind this phenomenon, we studied how the α-chain phosphorylation mutant of LFA-1 regulated the VLA-4 adhesion. Th e cells bearing the Ser1140Ala-mutation in the LFA-1 chain were not able to detach the uropod but left long extension behind them while migrating on VCAM-1, just like the cells lacking LFA-1. In a similar way, the cells expressing the Ser1140Ala-mutant adhered tightly to VCAM-1 thus inihibiting the migration (IV fi g 3A-C). As already mentioned, the same phenomenon was seen in CR4-K562 transfectants, where the expression of wt CR4, but not that of α-chain phosphorylation mutant Ser1158Ala-CR4, inhibited the VLA-4-mediated adhesion to VCAM-1 (IV fi g 3D).

Interestingly, the eff ect of αL chain phosphorylation is dependent on the activation method. In cells activated with SDF-1α, the phosphorylation of the αL chain is essential for the transdominant inhibition to occur, whereas activation through TCR and outside-in activation with ligand binding or activating antibody are independent of α-chain phosphorylation (IV fi g 4A-B). To fi nd out if the transdominant inhibition is related to the conformation of LFA-1, we studied the expression of activation epitopes with antibodies KIM127 (that recognises the extended integrin) and mAb24 (extended, open conformation). It was shown that the KIM127 epitope was constitutively expressed on cells regardless of phosphorylation or activation, whereas mAb24 only recognised wt LFA-1 (and not Ser1140Ala-mutant) and only when activated with SDF-1α , anti-CD3 or ligand binding (IV fi g 4C-D). Th ese results are in accordance with the previously reported αL phosphorylation studies, where SDF-1α could not induce full activation of

αL chain phosphorylation mutant LFA-1 but TCR ligation could. On the other hand, Fagerholm et al did not see mAb24 epitope expression following soluble ICAM-2 activation in the Ser1140Ala mutants, whereas our results show that the Ser1140Ala mutant is both expressing the mAb24 epitope and capable of ICAM-induced transdominant inhibition (Fagerholm et al 2005). Th e phosphorylation of the β2 chain is induced when the cells are activated with SDF-1α or through

TCR ligation, indicating that β2 phosphorylation may be needed for this signalling (IV fi g 4F).

Next, we went on to study the eff ects of β2 chain phosphorylation on transdominant inhibition. Th e Jβ2.7 cells lacking LFA-1 were transfected with β2-chain cytoplasmic peptide where Th r758 is phosphorylated (pβ2). Th is transfection was enough to induce transdominant inhibition, i.e. decrease in VCAM-1 binding (IV fi g 5A). Th is clearly shows that, even if the α-chain is needed for the activation of the integrin with some activation methods, the phosphorylated β2 is needed for signalling to inhibit VLA-4/VCAM-1 interaction.

7.3.3 Signalling through 14-3-3, Tiam1 and Rac-1 leads to VLA-4 inhibi on An important signalling cascade, involving 14-3-3 protein binding to the phosphorylated

β2 tail and subsequent activation of Tiam1 leading to Rac-1 activation and actin cytoskeleton rearrangements has been reported (Gronholm et al 2011). To test if this pathway was involved in transdominant inhibition of VLA4, we treated the cells with 14-3-3 blocking peptide or

Tiam1-inhibitor prior to fl ow adhesion assays. Indeed, pβ2-induced transdominant inhibition was overridden with these inhibitors, suggesting that 14-3-3 and Tiam1 are essential for the transdominant inhibition to occur (IV fi g 5B-C). Th e interaction of 14-3-3 with β2 tail upon cell activation was also studied and, in consensus with previous reports (Gronholm et al

2011) and results presented above, the 14-3-3 binding to β2 chain is attenuated when LFA-1 is not fully activated, as in the case of the α-chain phosphorylation mutant aft er activation with SDF-1α (IV fi g 5D-F). As discussed in chapter 4.3, Rac-1 is an important GTPase regulating

49 Summary of the study cytoskeletal rearrangements, which invites to hypothesize on the role of the actin cytoskeleton in the regulation of VLA-4 by LFA-1. Indeed Rac-1 inhibitor abolished cell adhesion and migration of all three cell lines (Jβ2.7 without LFA-1, with wt LFA-1 and with LFA-1 S/A), so the input of Rac-1, specifi cally downstream of LFA-1 in the transdominant inhibition could not be studied.

7.3.4 LFA signalling leads to changes in VLA4 phosphoryla on and complex forma on To fi gure out what the consequences of these signalling events are on VLA-4, we elucidated the phosphorylation status of both α4 and β1 chains of VLA-4 aft er LFA-1 activation. Interestingly, phosphorylation of the α4 chain was similar in all three cell lines (Jβ2.7 lacking LFA-1, expressing wt LFA-1 and expressing LFA-1 Ser1140Ala), even though Ser988 phosphorylation has been shown to be essential in dynamic adhesion events through the regulation of paxillin binding

(Han et al 2001). On the other hand, β1 phosphorylation was inhibited in the cells expressing wt LFA-1, whereas in Jβ2.7 or LFA-1 S/A cells, β1 was clearly phosphorylated on Th r788/789. Filamin, a negative regulator of integrin activation (Takala et al 2008), only bound to VLA-4 when wt LFA-1 was present and activated, whereas 14-3-3ζ binding to the α4 chain was decreased in LFA-1 expressing cells (IV fi g 6A). And, on the contrary, when LFA-1 was activated, it bound 14-3-3 whereas in the resting state it interacted with fi lamin (IV fi g 6B). Based on these results, we propose that fi lamin and 14-3-3ζ alternate in binding to LFA-1 and VLA-4, and regulate the activation state of the integrins in this way. 14-3-3ζ is known to bind effi ciently to the phosphorylated β2 tail and to regulate the subsequent signalling events through Tiam1 and

Rac1 (Nurmi et al 2007, Gronholm et al 2011). 14-3-3ζ also binds to VLA-4 α4 chain when it is phosphorylated on Ser978. When bound, it participates in α4 – 14-3-3ζ –paxillin complex formation that activates Cdc42 at the leading edge of a migrating leukocyte (Deakin et al 2009).

Recently the affi nity of phosphorylated β2 towards 14-3-3ζ has been found to be higher than that of phospho-α4. Th e β1 chain was also reported to bind 14-3-3ζ, but this interaction does not require phosphorylation and is of lower affi nity than phospho-α4 or phospho-β2 binding (Bonet et al 2013). With this information available, we propose that activation of LFA-1 and thus phosphorylation of the β2 chain, may sequester the available 14-3-3ζ from the surroundings (and from α4), allowing space for fi lamin to bind to VLA-4 and lead to the inhibition of VCAM-1 binding (fi gure 14).

50 Summary of the study

LFA-1 VLA-4 LFA-1 VLA-4 LFA-1 VLA-4

VCAM-1 ICAM-1/2 VCAM-1 ICAM-1/2 T CELL RECEPTOR CHEMOKINE αβ αβ RECEPTOR

P P P P P P P P P 14-3-3 PKC 14-3-3 14-3-3 P

FILAMIN TIAM1 FILAMIN

ACTIN N FILAMIN RAC

Figure 14. Schematic model of transdominant inhibition of VLA-4 by LFA-1. Phosphorylation of integrin cytoplasmic tails and 14-3-3 protein is indicated by yellow stars. Th e fi gure was kindly provided by Dr. Mikaela Grönholm.

7.3.5 Transdominant inhibi on could be u lised for effi cient therapies In addition to mediating adhesion and signalling, integrins need to be able to release their ligands in order to fulfi l their mission. Th is is evident in transendothelial migration, but also immune synapses need to unwind so that the cells can continue patrolling the circulation. In the context of extravasation, the fundamental steps are selectin-mediated rolling, slow integrin- mediated rolling, tight adhesion and migration along the endothelium and then transmigration towards the infl amed area (the whole process is explained in more detail in section 1.2.2). Diff erent adhesion molecules are involved in these diff erent steps, including selectins and their ligands, VLA-4 and VCAM-1 and β2 integrins, especially LFA-1 and ICAMs. Th e roles of these (and many other) molecules have been attributed to distinct steps of the extravasation, and in order to continue to the next phase of extravasation, the previous receptor-ligand interactions need to be released. Th is is especially true for the migration, as the cell is constantly building up new adhesions at the lamellipodia, strengthening them in FAs and releasing them in the uropod area. Th e regulation of all these steps, adhesion and release in space and time, is of primary importance for many immune functions, but little has been known about the mutual control of the diff erent adhesion molecules involved. Transdominant inhibiton is probably one of the main mechanisms orchestrating the continuum of the steps of extravasation as it enables inactivation of previous adhesion steps simultaneously to the activation of the next adhesion molecule. Another point of view related to transdominant control of diff erent integrins is the therapeutical aspect, as integrins have been targets for drug development almost since they were discovered (Hilden et al 2006, Millard et al 2011). However, the anti-integrin therapies in the market are not numerous although a few successful drugs have been designed. One of the anti-integrin drugs on the market at the moment is or Raptiva, an antibody against LFA-1 used in treatment of psoriasis (Lebwohl et al 2003). On the contrary, a promising therapy against multiple sclerosis and Crohn’s disease ( or Tysabri) was developed based on an antibody against VLA-4 but unfortunately, severe side eff ects causing progressive multifocal leukoencephalopathy (PML) led to withdrawal from the market (Linda et al 2009).

51 Summary of the study

However, the withdrawal was only temporary, as Natalizumab is at the moment widely used in the treatment of severe MS. One possible explanation for unexpected side eff ects or ineffi cient function of a therapeutic agent may be the consequences controlled by transdominant inhibition (or activation) that may induce unwanted cellular events, since this crosstalk appears to be a fairly common form of integrin regulation. However, transdominant inhibition could also be used as a tool to a combined inhibition of for example LFA-1 and VLA-4, if a suitable antibody or other reagent activating the transdominant inhibition of VLA-4 and simultaneously inhibiting LFA-1 adhesion could be developed.

7.4 Regula on of leukocyte integrin binding to Ig-family ligands In the experimental part I have characterized integrin activities from diff erent angles in order to pinpoint some of the regulatory mechanisms involved in blood cell adhesion. On the basis of my research, and with help from the literature I have summarized the most relevant ways to regulate leukocyte integrin functions and signalling. Th ey are as follows. 1. Th e properties of the ligand are naturally the main factor in the specifi city of the integrins. Interesting observations were made when comparing the binding of ICAM-4 to the three members of leukocyte integrins that are close homologues, showing that distinct, although

overlapping regions in both ICAM-4 and on β2 integrins are responsible for the binding. 2. Th e environment of the adhesion, especially the presence of divalent cations Mg2+, Ca2+ and Mn2+ directly aff ects the ligand binding capacity of the integrins. 3. Phosphorylation of the integrin α and β chain cytoplasmic parts is thought to take part in the regulation of the integrins extracellular conformation and transmission of signals across the plasma membrane. Phosphorylation is a specifi c and rapid way to control integrin activities in inside-out activation and outside-in signalling. 4. Binding of intracellular signaling molecules to the cytoplasmic parts of integrins may precede or follow phosphorylation and cascades of signalling proteins may convey signals that are also spatially distant. Th ese oft en integrin-specifi c interactions are able to induce active conformation by separating the cytoplasmic tails, keep the integrin in the resting state, or initiate signalling cascades downstream of integrins. 5. Th e conformation of the extracellular part of the integrin is the ultimate outcome of diff erent activation steps, and it is the limiting factor in ligand binding. However, the active conformation may also be induced from outside with an activating antibody or ligand binding. Strengthening of the conformation is vital for leukocyte adhesion in fl ow. 6. Transdominant inhibition may regulate and coordinate many leukocyte functions where adhesion and release of diff erent integrins are involved.

52 Concluding remarks and future perspectives CONCLUDING REMARKS AND FUTURE PERSPECTIVES

Integrins, and the adhesion and signalling events they mediate, are essential for the proper function of probably all tissues. Especially in blood cells (red cells, leukocytes and platelets), the role of concerted regulation of the adhesion events is the cornerstone of all functions, including haematopoiesis, immune functions, haemostasis and the delivery of oxygen to the tissues. Leukocyte integrins control these adhesions by binding to th’gands, of which the most important group is formed by Ig-superfamily of adhesion proteins. As important as adhesion is the ability to detach from the ligands, when the cells need to move on to the tissues, to the lymph nodes or into circulation. In the current work I have analysed the properties of leukocyte integrins and their ligands as well as the regulation of their interactions. I have observed that the red cell adhesion molecule ICAM-4 can bind to CR4, a leukocyte integrin expressed on monocytes and macrophages, and that the I domain is the ICAM-4 binding site on leukocyte integrins (LFA-1, Mac-1 and CR4). We have also characterised the phosphorylation of the cytoplasmic tail of CR4, and found that αX chain is phosphorylated on Ser1158, and that this phosphorylation is essential for CR4 inside-out activation, adhesion and phagocytosis but not for outside-in signalling initiated by CR4. Finally we analysed the regulation of VLA-4 mediated adhesion to VCAM-1 that is controlled by the β2 integrins. Th e fi ndings of my studies show how leukocyte integrins are involved in numerous blood cell functions and that their functions are tightly regulated. Due to their multifold roles, they also off er attractive targets for therapeutical use. Th e specifi city of phosphorylations or ligands may serve as distinctive factors between diff erent integrins, even members of the same family. Even though the studies presented here may have revealed some of the regulation mechanisms of the leukocyte integrin activities, there remains certainly a large number of questions left unanswered or new ones coming up, which require more experiments and structural studies. One of them is the role of ICAM-4 in erythrophagocytosis, as the results with anti-integrin and anti-ICAM-4 antibodies indicate an important role for these molecules. However, more in vivo studies in the context of spleen are required to answer this question. ICAM-4 also interacts with the platelet integrin αIIbβ3, which suggests red cell adhesion involvement in haemostasis and/or thrombosis, but more work with platelets is needed to solve the role of this interaction. As for regulation of CR4 activities, several aspects may be envisioned. Could atherogenesis be inhibited by decreasing CR4-dependent monocyte adhesion to endothelium and could this inhibition be reinforced by transdominant inhibition between CR4 and VLA-4? Could it be possible to activate the uptake of antigen into DCs and hence strengthen DC-based vaccine effi ciency by manipulating the CR4 activation? Another interesting application for transdominant inhibition between β2 integrins and VLA-4 would be targeted inhibition of autoimmune reactions in diseases like MS.

53 Acknowledgements ACKNOWLEDGEMENTS

Th is study was carried out at the Division of Biochemistry and Biotechnology, Department of Biosciences, Faculty of Biological and Environmental Sciences, University of Helsinki, under the supervision of professor Carl Gahmberg. Th e work has been fi nancially supported by the Magnus Ehrnrooth foundation and the Helsinki Graduate Program in Biotechnology and Molecular Biology. I am deeply grateful to my supervisor Calle for patiently guiding me on my long and winding road towards being a scientist. Especially I appreciate the freedom you have given me to follow my own path and to independently carry out the weirdest experiments I came up with. You have succeeded in creating a welcoming atmosphere of scientifi c wonder in the lab, extending far beyond the world of integrins and adhesion, and shown that scientists should not lock themselves in the lab, but be active members of the society. I also want to thank professor Kari Keinänen, head of the division, for excellent research environment. I want to express my gratitude to Eveliina Ihanus and Mikaela Grönholm for showing me the way. Both of you have taught me important lessons of science in practice. Eve told me all there is to know about cell adhesion in the very beginning of my PhD project, and Miku has shown, amongst other things, how fruitful a good teamwork can be. Th ank you for your support and friendship! I am privileged to have professor Francisco Sánchez-Madrid, who is one of the godfathers of the integrins, as my opponent. I am thankful to professor Jorma Keski-Oja and professor Manuel Patarroyo for the expert review of my thesis, and for providing me valuable feedback and suggestions. I want to thank professor Jukka Finne for serving as my custos. My follow-up group, Tiina Öhman and Pia Siljander did not gather that oft en (that was all my fault), but thank you anyway for your feedback and encouragement. Calle’s lab has, besides excellent facilities, a superb atmosphere. I’m sure that the biochemistry coff ee room is the place where all the problems, scientifi c or wordly, are solved in theory. I want to thank all of you Calle’s lab members for enduring the good and the bad days with me: Esa, Farhana, Lin, Sonja, Kate, Erkki and Miku. A special kiitos goes to Leena Kuoppasalmi for the complete care of the lab (and its members), for fulfi lling our weirdest wishes, and for the patience. Kuoppis has been an important guide on my way, and especially a good friend. Many other people at the biochemistry division are also worth thanking, especially Pirjo Nikula-Ijäs for the expert study counselling and Pia again for taking the immunochemistry course to the next level. Both are also thanked for always being supportive and encouraging. As I have spent quite a few years in the lab, there is also a bunch of former members of the group and the division who I want to thank for sharing the joy of science with me: Anne, Suvi, Maria A., Maarit, Tiina, Suski, Susanna, Minna, Pauli, Suneeta, Emiliano, Kaj, Matti and others I forgot to mention. I have also been lucky enough to be able to dive into the sea of fl ow cytometry with Marias Semenova and Aatonen, thank you for sharing that world with me. Important people from the second fl oor are acknowledged for taking care of the facilities: Esa, Lefa, Paula, Tuukka and Olavi. I want to thank Yvonne, Katarina, Lea and Heidi for doing the paperwork and much more. All the other people at the Division of Biochemistry and Biotechnology are thanked for occasionally laughing at my bad jokes and keeping me scientifi c company in the coff ee room.

54 Acknowledgements

Teaching has been and will hopefully remain an important part of my life and I am very grateful to all the students I have taught and supervised throughout the years. I’m sure I’ve learnt more than you! I want to thank my dear friends Inka, Nelli, Hanna, Ella, Liisa K., Jussi, Lotta, Ankka, Sini and all of you others for the discussions, dinners, drinks, dreams – and giving me better things to think of than cells and adhesion. I am deeply grateful to my parents Helinä (& Heikki) and Tapsa (& Pirjo) for your endless support and belief in me, for not asking the “when is it ready?“ too oft en and for your help in all the areas of life. I want to thank also my in-laws Katri and Voitto for their help and friendship. Heikki, thank you for being there all the time and helping me through the hard times with this. I appreciate your support more than you can ever imagine. Kerttu, without you this or anything else wouldn’t make any sense. Never stop asking why!

Helsinki, October 2014

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