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Role of lipid rafts in the regulation of LFA-1 function Marwali, Muhammad Reza Perkasa 2004

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R O L E O F L I P I D R A F T S I N T H E R E G U L A T I O N O F LFA-1  FUNCTION  by M U H A M M A D REZA PERKASA M A R W A L I M.D., University of Indonesia, 1991  A THESIS SUBMITTED IN P A R T I A L F U L F I L L M E N T OF T H E REQUIREMENTS FOR T H E D E G R E E OF DOCTOR OF PHILOSOPHY in T H E F A C U L T Y OF G R A D U A T E S T U D Y Department of Medicine, Experimental Medicine Program  We accept this thesis as conforming to the required standard  T H E UNIVERSITY OF BRITISH C O L U M B I A July, 2004 © Muhammad Reza Marwali, 2004  ABSTRACT  Leukocyte function-associated molecule-1 (LFA-1) plays an essential role in cell-cell interactions in the immune system. LFA-1 may be activated by conformational changes and by clustering. In this study, the role of lipid rafts in the regulation of L F A - 1 was investigated. Lipid rafts are plasma membrane microdomains enriched in cholesterol and ganglioside G M 1 , and they are thought to regulate functions of various cell surface receptors by mediating their clustering. Disruption of rafts on T cells by cholesterol depletion inhibited phorbol-ester-induced LFA-1 binding to its ligand I C A M - 1 , suggesting that rafts regulate L F A - 1 functions. Isolation of rafts by non-ionic detergents followed by sucrose gradient centrifugation as well as sensitivity to cholesterol depletion and co-capping experiments suggested that the rafts of primary resting T cells are heterogeneous and that LFA-1 associates with a subset of lipid rafts that are rich in cholesterol but poor in G M 1 . To further elucidate the role of lipid rafts in LFA-1 function, the formation of the immunological synapse on cytotoxic T lymphocytes (CTL) was examined. Disruption of rafts by cholesterol depletion inhibited C T L cytotoxicity. However, the L F A - 1 on C T L was constitutively active and was not inhibited by cholesterol depletion. Instead, rafts seemed to mediate the association between LFA-1 and the CD3/T cell receptor (TCR) complex on CTL, but not on resting T cells. Deconvolution and confocal microscopy as well as 3-D image reconstruction showed that L F A - 1 , CD3/TCR and GM1 co-distribute in the periphery of the C T L immunological synapse in an antigen-independent manner while cholesterol was distributed more widely. These results suggest that the binding of L F A - 1 on C T L to ICAM-1 on target cells initiates the formation of the synapse on C T L and recruits the CD3/TCR complex to the synapse through their co-distribution in rafts.  ii  The role of LFA-1 in the reorganization of the cytoskeleton following formation of the immunological synapse was also analyzed. LFA-1 ligation induced antigen-independent F-actin accumulation in the synapse, whereas tyrosine phosphorylation in the synapse and translocation of the microtubule organizing center required recognition of specific antigen by the TCR. Thus, LFA-1-mediated signalling is important for the activation of the actin cytoskeleton, but not sufficient for microtubules reorganization in CTL.  iii  T A B L E OF CONTENTS  TITLE P A G E ABSTRACT T A B L E OF CONTENTS LIST OF FIGURES LIST OF A B B R E V I A T I O N S ACKNOWLEDGEMENT  i ii h vi vii xi  CHAPTER 1 INTRODUCTION 1.1. LFA-1 1.1.1. Functions of LFA-1 1.1.2. Ligands 1.1.3. Regulation 1.1.3.1. Structure-function relationship and affinity activation 1.1.3.2. Avidity regulation 1.1.3.3. Inside-out signaling 1.1.3.4. Outside-in signaling 1.2. Lipid rafts 1.2.1. Functions of lipid rafts 1.2.2. Lipid rafts and integrins 1.3. Immunological synapse 1.4. Thesis objectives  ...1 1 2 3 6 9 9 24 25 29 32 36 38 39 45  CHAPTER 2 MATERIALS AND METHODS 2.1. Animals 2.2. Cell culture 2.3. Antibodies and reagents 2.4. Cell adhesion assay 2.5. Generating single cell suspension of splenocytes 2.6. Antibody purification 2.7. Purification of soluble ICAM-1 2.8. Sucrose gradient centrifugation and Western blotting.. 2.9. Cholesterol depletion, sequestration and reconstitution 2.10. Transfection 2.11. Preparation of C T L 2.12. Cytotoxicity assay 2.13. Flow cytometry 2.14. Fluorescence microscopy  =  46 46 47 47 47 49 50 50 50 51 52 52 53 53 54 54  57 CHAPTER 3 M E M B R A N E C H O L E S T E R O L R E G U L A T E S LFA-1 F U N C T I O N A N D L I P I D R A F T HETEROGENEITY 57  iv  3.2. Results 61 3.2.1. M C D disrupts lipid rafts and inhibits LFA-1-mediated adhesion of T cell lines 61 3.2.2. M C D inhibits LFA-1 on primary T cells but does not disrupt Triton X-100-insoluble lipid rafts 65 3.2.3. Specificity of M C D treatment 67 3.2.4. Filipin inhibits LFA-1 activation 69 3.2.5. Lipid rafts of T cell lines and primary T cells are different 71 3.2.6. LFA-1 in primary T cells are found in Brij 35-insoluble and MCD-sensitive lipid rafts 71 3.2.7. LFA-1 and cholesterol on primary T cells co-cap 74 3.3. Discussion 77 CHAPTER 4 85 L I P I D R A F T S M E D I A T E A S S O C I A T I O N O F L F A - 1 A N D CD3 A N D F O R M A T I O N O F THE I M M U N O L O G I C A L SYNAPSE OF C T L 85 4.2. Results 88 4.2.1. Lipid rafts are important for cytotoxicity of C T L 88 4.2.2. LFA-1 and CD3 on C T L localize in MCD-sensitive lipid rafts 90 4.2.3. LFA-1 and CD3 on CTLs, but not resting T cells, co-cap 93 4.2.4. LFA-1 and CD3 co-distribute with cholesterol and GM1 on C T L 95 4.2.5. L F A - 1 , CD3 and lipid rafts form the immunological synapse of C T L 97 4.3. Discussion 103 CHAPTER 5 108 ANTIGEN-INDEPENDENT ACTIN CYTOSKELETON REORGANIZATION AND ANTIGEN-DEPENDENT M T O C T R A N S L O C A T I O N AND C E L L SIGNALING IN T H E CTL I M M U N O L O G I C A L SYNAPSE 108 5.1. Introduction 109 5.2. Results Ill 5.2.1. Actin cytoskeleton accumulates in the IS in an antigen-independent manner Ill 5.2.2. LFA-1 capping does not induce co-capping of F-actin cytoskeleton and they are independent of lipid rafts in C T L 113 5.2.3. Accumulation of phospho-tyrosine in the IS is antigen-dependent 113 5.2.4. Lck is enriched in the IS and co-localizes with GMl-rich rafts in an antigenindependent manner 117 5.2.5. M T O C translocation to the IS is dictated by TCR recognition of target 120 5.3. Discussion 122 CHAPTER 6 S U M M A R Y A N D DISCUSSION  126 126  REFERENCES  135  LIST OF FIGURES Page  Fig. 1.1  Schematic representation of LFA-1 structure  11  Fig. 1.2  Ribbon diagram of fi-propeller structure  12  Fig. 1.3  Crystal structure of I-domain  15  Fig. 1.4.  Schematic figures of headpiece conformations of integrin  20  Fig. 3.1  Disruption of lipid rafts and inhibition of LFA-1 activation by  63-64  M C D treatment Fig. 3.2  Effects of M C D treatment on primary T cells  66  Fig. 3.3  Specificity of M C D treatment  68  Fig. 3.4  Effects of Filipin on LFA-1  70  Fig. 3.5  Comparison of levels of GM1 and cholesterol in the plasma  72  membrane of splenic T cells and T cell lines Fig. 3.6  Detection of LFA-1 in Brij 35-insoluble, M C D - and Filipin-  73  sensitive lipid rafts of primary T cells Fig. 3.7  Confocal microscopic analysis of co-capping of L F A - 1 and  76  cholesterol Fig. 3.8  Intracellular staining caused extraction of membrane GM1  79  Fig. 3.9  A model for lipid rafts heterogeneity in primary T cells  81  Fig. 3.10  Most of LFA-1 on thymocytes is detected in high density fractions  84  when extracted with 1% and 0.05% Triton X-100 Fig. 4.1  Cholesterol depletion inhibits C T L cytotoxicity  89-90  Fig. 4.2  LFA-1 and CD3 are found in MCD-sensitive detergent insoluble  92  fractions Fig. 4.3  LFA-1 and CD3 co-cap on C T L but not on resting T cells  94  Fig. 4.4  LFA-1 co-cap with both GM1 and cholesterol but CD3 and  96  cholesterol do not co-cap with GM1 Fig. 4.5  C T L tightly bind to L cells transfected with ICAM-1 or ICAM-1 and M H C class I D  98  b  Fig. 4.6  L F A - 1 , CD3 and lipid rafts form C T L immunological synapse  100-1  Fig. 4.7  C T L and R M A cells conjugates and IS  102  Fig. 5.1  Actin cytoskeleton accumulates in IS in antigen-independent  112  manner Fig. 5.2  LFA-1 capping did not induce co-capping of F-actin cytoskeleton  114  and they were independent of lipid rafts in C T L Fig. 5.3  Tyrosine-phosphorylated proteins and M T O C accumulate in  116  antigen-specific C T L synapse Fig. 5.4a  Lck is found in lipid rafts isolated by 1% Brij 35 and resistant to  118  M C D treatment in both resting T cells and C T L Fig. 5.4b,c  Lck is enriched in the IS and co-localizes with GMl-rich rafts in  119  antigen-independent manner Fig. 5.5  M T O C translocation to the IS is dictated by TCR recognition of target  vii  121  LIST OF ABBREVIATIONS Ab  Antibody  APC  antigen presenting cell  BSA  bovine serum albumin  CD  cluster of differentiation  Cdc42  cell division cycle protein 42  CHAPS  3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate  Con A  concanavalin A  c-SMAC  central-supramolecular activation cluster  C T L IS  cytotoxic T lymphocyte immunological synapse  CTL  cytotoxic T lymphocyte  CTxB  cholera toxin subunit B  DC  dendritic cell  DIC  differential interference contrast  DMEM  Dulbecco's modified minimal essential medium  DRM  detergent resistant membrane  EDTA  ethylene diamine tetraacetate  EGF  epidermal growth factor  EM  electron microscopy  Erk  extracellular signal-regulated kinase  ERM  ezrin-radixin-moesin  FACS  fluorescence activated cell sorter  FAK  focal adhesion kinase  FCS  fetal calf serum  FITC  fluorescence isothiocyanate  FRET  fluorescence resonance energy transfer  GD3  ganglioside ceramide dihexoside (Cer-Glc-Gal-Sialic acid-Sialic acid)  GFP  green fluorescent protein  GM1  ganglioside ceramide tetrahexoside (Cer-Glc-Gal-Sialic acid-GalNac-Gal)  GM3  ganglioside ceramide dihexoside (Cer-Glc-Gal-Sialic acid)  viii  GPI  glycosyl-phosphatidylinositol  HBSS  Hanks' balanced salt solution  HEPES  N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid  hr  Hour  IAP  integrin associated protein  ICAM  intercellular adhesion molecule  IFN  Interferon  Ig  Immunoglobulin  IL  Interleukin  IS  immunological synapse  JAM  junctional adhesion molecule  kD  Kilodalton  LAD  leukocyte adhesion deficiency  LAT  linker for activation of T cell  LFA-1  leukocyte function-associated antigen-1  LPS  Lipopolysaccharide  mAb  monoclonal antibody  Mac-1  macrophage antigen-1  MAPK  mitogen-activated protein kinase  MCD  methyl-P-cyclodextrin  MHC  major histocompatibility complex  MIDAS  metal ion dependent adhesion site  MIP  macrophage inflammatory protein  MTOC  microtubule organizing center  NMR  nuclear magnetic resonance  PAGE  polyacrylamide gel electrophoresis  PBS  phosphate buffer saline  PdBU  phorbol dibutyrate  PE  Phycoerythrin  PH  plecktrin homology  PI3K  phosphatidylinositol-3-OH kinase  PIP  phosphatidylinositol phosphate  PKC  protein kinase C  PLC  phospholipase C  PMA  phorbol 12-myristate 13-acetate  PMN  Polymorphonuclear  PSI  plexin semaphorin integrin  p-SMAC  peripheral-supra molecular activation cluster  Rag  recombination-activating genes  RGD  Arg-Gly-Asp  RPMI  Roswell Park Memorial Institute  RT  room temperature  SDS  sodium dodecyl sulphate  SH  Src homology  SLAP  SLP-76-associated phosphoprotein  TCR  T cell receptor  Tg  Transgenic  T  melting temperature  TNF-a  tumor necrosis factor-a  VLA  very late antigen  WASP  Wiskott-Aldrich Syndrome Protein  ZAP  zeta-associated protein  ACKNOWLEDGEMENTS  Alhamdulillah. First, I would like to express my utmost gratitude to my parents, who always provide me with support and encouragement. I would like to thank my supervisor, Dr. Fumio Takei for giving me the opportunity to do my graduate study in his laboratory and for his excellent teaching and very stimulating scientific discussions. I am very grateful for his patience and consideration throughout my training. I am also deeply indebted to my supervisory committee Dr. Pauline Johnson, Dr. Michael Gold and Dr. Marcel Bally for their inputs, suggestions and ideas for my research projects. I would like to express thanks to Dr. Jose ReyLadino and Dr. Motoi Maeda for very helpful discussions and suggestions and Lisa Dreolini, Matthew MacLeod, Nastaran Mohammadi and Gayle Thornbury for their help.  xi  CHAPTER 1 INTRODUCTION  i  1.1. LFA-1  Cell-cell interactions are essential for the immune system. A number of cell adhesion molecules that regulate leukocyte interactions with other leukocytes or endothelial cells have been identified. Among them, LFA-1 plays a critical role in almost all immune responses involving cell-cell interactions. Although LFA-1 has been extensively studied and its importance for the immune system well established, the regulation of LFA-1 function is still not well understood. Recent studies have suggested that membrane microdomains, termed lipid rafts, may play an important role in the regulation of many cell surface receptors. Therefore I initiated my research to investigate the relationship between lipid rafts and LFA-1 regulation. LFA-1  (ccLfe;  CDlla/CD18), a member of the integrin family, is expressed on almost all  types of leukocyte except some tissue macrophages (Kurzinger et al., 1981). Integrins mediate cell-cell interactions and belong to the adhesion molecule family. LFA-1 is a non-covalent heterodimer consisting of the a ( C D l l a ) chain of 180 kD and the P2 (CD18) chain of 95 kD L  (Kishimoto et al., 1987b; Larson et al., 1989). Both human and mouse L F A - 1 were initially identified through the generation of monoclonal antibodies (mAbs) that blocked C T L killing and T cell proliferation (Davignon et al., 1981a; Pierres et al., 1982; Sanchez-Madrid et al., 1982). It was subsequently noted that this killing inhibition seemed to be due to inhibition of CTL-target cell conjugate formation rather than inhibition of the killing event itself (Davignon et al., 1981b). LFA-1 is a type I trans-membrane receptor with the N-termini of both chains in the extracellular space. The C-termini have relatively short cytoplasmic domains. The chromosomal location of the human C D l l a gene is in 1 6 p l l . l - p l 3 and the CD18 gene is in 21q22.3 (Corbi et al., 1988). Expression level of LFA-1 on the cell surface is unchanged by inflammatory stimulation.  2  1.1.1. Functions of LFA-1 The importance of LFA-1 has been demonstrated in leukocyte adhesion to endothelial cells in inflammatory tissues, leukocyte trafficking into high endothelial venules in peripheral lymph nodes, T cell activation and C T L and Natural Killer (NK) cell functions. Leukocyte recruitment during inflammation can be considered as a multistep process of not merely ' leukocyte-endothelial cells interactions but also platelet and fibrin formation (Mclntyre et al., 2003). Normally leukocytes circulating in the blood vessels are not adhesive to the endothelial cells. But during inflammation, the endothelium becomes activated due to pathogen products, cytokines, chemokines, traumatic stimuli or oxidants. The activated endothelial cells upregulate the expression of adhesion molecules, such as P-selectin, E-selectin and ICAM-1. The initial steps involved capture and rolling mediated by the interactions of P- and E-selectin on endothelium with PSGL-1 (P-selectin glycoprotein ligand-1) and L-selectin on leukocytes. Rolling is also mediated by pre-activated leukocyte integrins. Chemokines, such as IL-8, P A F (platelet activating factor), SDF-loc (stromal cell derived factor-la), R A N T E S (regulated on activation normal T cell expressed and secreted) or MCP-1 (monocyte chemotactic protein-1), activate leukocyte integrins.after they bind to chemokine receptors, through an inside-out signal (Constantin et al., 2000; Mclntyre et al., 2003). The ensuing activation of leukocyte integrins, such as L F A - 1 , Mac-1 and V L A - 4 (very late antigen-4; a$i) results in firm adhesion and arrest of the leukocyte, which allows the following step of extravasation and emigration to take place. Consequently, in mounting good immunological response in inflammation, the leukocyte integrins have a very important role. Studies with blocking antibodies have demonstrated that LFA-1- and Mac-1, together with V L A - 4 , are essential for leukocyte adhesion and migration through endothelium (van Kooyk et  3  al., 1993; Issekutz and Issekutz, 1995; Shang and Issekutz, 1998; Hentzen et al., 2000). Interestingly, a recent study showed that during monocyte and neutrophil adhesion to human umbilical vein endothelial cells (HUVEC), the endothelial cells form microvilli projections containing ICAM-1 that wrap the leukocytes along their sides (Carman et al., 2003). The role of LFA-1 in this process is evident from studies with LFA-1 deficient mice. Comparing L F A - 1 , Mac-1 and C D 18 deficient mice, Ding et al. reported that both LFA-1 and Mac-1 contribute to neutrophil adhesion to endothelium. However, the role of LFA-1 is more dominant than Mac-1. On the other hand, neutrophil extravasation in response to TNF-oc stimulation is lower in LFA-1" mice than wild type mice whereas it is higher in Mac-1 deficient mice (Ding et al., A  1999). Utilizing L F A - l ' m i c e and blocking mAb, L F A - 1 , but not Mac-1, was demonstrated to be important for rolling and for the firm adhesion of neutrophils. However both are important for leukocyte migration. In addition, V L A - 4 can compensate for LFA-1-deficiency in mediating rolling and adhesion (Henderson et al., 2001). Another study using LFA1" " and M a c - l " mice 7  7  reported that both LFA-1 and Mac-1 are important for the rolling of neutrophils, with LFA-1 being a more important factor in inducing firm adhesion (Dunne et ah, 2002). L F A - 1 " mice are 7  resistant to ConA-induced fulminant hepatitis or LPS-induced shock liver injury (Matsumoto et al., 2002; Emoto et al., 2003). These studies provide strong evidence for the crucial role of L F A 1 in leukocyte adhesion and migration from the blood vessels, a critical step in the normal inflammatory response. The role of LFA-1 in human is evident in human disease where LFA-1 is defective. Leukocyte Adhesion Deficiency (LAD) type I is an autosomal recessive disorder of the immune system characterized by recurrent bacterial infections. L A D type I patients have either defective P2 leukocyte integrin expression level (Kishimoto et al., 1987a) or normal expression level but  4  non-functional P2 integrin (Kuijpers et al, 1997; Hogg et al., 1999; Harris et al, 2001; McDowall et al., 2003; Alon et al., 2003; Kinashi et al., 2004). This defect causes inability of neutrophils to migrate to the inflamed sites. However, lymphocyte adhesion and migration seem normal, probably due to a compensation mechanism involving V L A - 4 (Wehrle-Haller and Imhof, 2003). A n early report using mAb against LFA-1 and T cells from L A D type I patient suggested the involvement of LFA-1 in lymphocyte homing to lymph nodes (Pals et al., 1988). LFA-1 is essential for leukocyte trafficking into the lymph nodes. LFA-1 mediates the firm adhesion of lymphocyte to high endothelial venules in Peyer's patch while L-selectin and a (3 integrin mediate the rolling and tethering steps (Bargatze et al., 1995). LFA-1 also 4  7  mediates similar process in other peripheral lymph nodes, i.e. iliac and para-aortic lymph nodes (Warnock et al., 1998). Using L F A - l " mice and mAbs against other leukocyte integrins, it was 7  determined that LFA-1 is important not only for lymphocyte homing into peripheral lymph nodes but also into bone marrow, which could be compensated for by ot integrins (Andrew et 4  al., 1998; Berlin-Rufenach et al., 1999). Intracellular signaling molecules, Rho A and PKC£, mediate the activation of LFA-1 for lymphocyte homing into Peyer's patch (Giagulli et al, 2004). Both LFA-1 and V L A - 4 were demonstrated to have a principal role in lymphocyte entrance into splenic white pulp (Lo et al., 2003). Together these studies have elucidated the functional role of LFA-1 in leukocyte homing. C T L binding to target cells, as well as T cell activation, is mediated by LFA-1 and L F A 3/CD2 interactions (Davignon et al., 1981a; Pierres et ah, 1982; Kaufmann et ah, 1982; Sanchez-Madrid et al, 1982; Krensky et al, 1983; Krensky et al, 1984; Shaw et al, 1986; Mentzer et al, 1987; Dustin et al, 1987; Selvaraj et al, 1987). There are conflicting reports as to whether LFA-1-deficiency affects C T L killing function. Schmits et al. reported that C T L killing  5  function is not affected in LFA-1" " mice, instead only N K cell killing function is defective 7  (Schmits et al, 1996). The authors argue that the C T L in these LFA-1 deficient mice might have some other compensating mechanism. However, Shier et al. demonstrated that C T L generated from LFA-1" " (T cell receptor) TCR-transgenic (Tg) mice cannot kill target cells and that this 7  inhibition cannot be compensated for by increasing the peptide/MHC density on target cells (Shier et al., 1999). Both reports demonstrated that T cell activation in these knockout mice is defective. Other studies using LFA-1" " mice also showed severely impaired T cell activation 7  (Shier et al, 1996; Bachmann et al, 1997). Thus, LFA-1 is important for C T L killing and T cell activation.  1.1.2. Ligands The ligands for LFA-1 are I C A M (inter-cellular adhesion molecule-)-1, ICAM-2, I C A M 3, ICAM-4 and ICAM-5 and J A M (junctional adhesion molecule)-lor J A M - A . ICAM-1 is a single chain glycoprotein of 90 kD and a member of the immunoglobulin (Ig) superfamily, consisting of 5 Ig-like domains (D1-D5) (Rothlein et al, 1986; Marlin and Springer, 1987; Staunton et al, 1988; Siu et al, 1989; Horley et al, 1989). It was identified in mouse as M A L A 2 (murine activated lymphocyte antigen-2) by a mAb that inhibits mixed lymphocyte reactions (Takei, 1985). ICAM-1 was later confirmed to be the ligand for LFA-1 (Marlin and Springer, 1987; Simmons et al, 1988; Makgoba et al, 1988a; Makgoba et al, 1988b). ICAM-1 is expressed on endothelial cells, epithelial cells, synovial cells, lymphocytes, monocytes and keratinocytes. Its level of expression is upregulated upon inflammatory stimulation (Dustin et al, 1986; Rothlein et al, 1988).  6  Mutagenesis studies have shown that the ICAM-1 interaction with LFA-1 is mediated by the first and second Ig-like domain (D1-D2) of ICAM-1. The affinity and kinetics of this interaction have been determined by surface plasmon resonance (Tominaga et al, 1998). In this study the authors used monomeric soluble LFA-1 (sLFA-1) or membrane-bound LFA-1 (mLFA1) and the D1-D2 or D1-D5 of ICAM-1 fused to the Fc portion of human I g G i . LFA-1 was activated with M g  2 +  and the binding affinity was determined using a BIACore system. Both  sLFA-1 and mLFA-1 had a similar Kd of 500 n M and a dissociation rate of (& ) 0.1 s* . The 1  diss  affinity of monomeric soluble ICAM-1 for LFA-1 was hard to measure by other methods (Miller et al, 1995; Reilly et al, 1995; Casasnovas et al, 1998). It is thought that ICAM-1 exists on the surface of the cell as a dimer, and therefore it increases its binding affinity with L F A - 1 . A recent study using BIACore measurement has shown that the higher LFA-1 binding affinity to dimeric ICAM-1 is simply due to increase in avidity (divalent) and not because the dimer is required for forming a complete binding site area (Jun et al, 2001). LFA-1 also binds ICAM-2 (Staunton et al., 1989; de Fougerolles et al., 1991; X u et al, 1992). ICAM-2 is a single chain protein of 55 kD containing two Ig-like domains (D1-D2), that is constitutively expressed on leukocytes and endothelial cells. Mutagenesis study of ICAM-2 revealed that the binding site for LFA-1 in ICAM-2 is confined to the upper part of the D I (Casasnovas et al., 1999) ICAM-3, which is expressed on leukocytes in a constitutive manner, is another ligand for LFA-1 (van Kooyk et al., 1996). It is a 124 kD protein containing five Ig-like domains. Its interaction with LFA-1 is mediated by first and second domain (D1-D2) (de Fougerolles and Springer, 1992; Fawcett et al., 1992; de Fougerolles et al., 1993; Holness et al, 1995; Klickstein et al, 1996; Bell et al, 1998). However, it was reported that the binding of LFA-1 to ICAM-3 is  7  nine times weaker compared to ICAM-1 binding (Woska, Jr. et al, 1998). Later it was shown that ICAM-3/Fc has a higher affinity for DC-SIGN (dendritic cell-specific ICAM-3 grabbing non-integrin) than for L F A - 1 on the HSB T cells (Geijtenbeek et al, 2000). The DC-SIGNICAM-3 interaction is thought to mediate DC-naive T cell interaction in both antigen dependent or independent interaction. It should be noted that murine ICAM-3 has not been identified and that a murine homologue of human ICAM-3 gene has not been found in the mouse genome. The red cell Landsteiner-Wiener (LW) blood group antigen is a glycoprotein molecule of 42 kD. It was shown to have 30% sequence homology with I C A M - 1 , 2 and 3 and two Ig-like domains. It was therefore designated to be ICAM-4 (Bailly et al, 1994). I C A M - 4 was later demonstrated to bind to L F A - 1 , Mac-1, C D l l c / C D 1 8 ,  cc Pi, oc P3, ctvPs, v  v  a (3i and anhP3 (Bailly 4  et al, 1995; Spring et al, 2001; Hermand et al, 2003). Mutagenesis studies of ICAM-4 suggested that the first Ig-like domain (DI) mediates binding to L F A - 1 (Hermand et al, 2000; Ihanus et al, 2003). ICAM-5, also known as telencephalin (TLN), is a 130 kD glycoprotein expressed exclusively in neurons of the telencephalon of the brain. It has nine Ig-like domains and the first five NH2-terminal Ig-like domains are closely related to those of I C A M - 1 , 2, 3 and 4 (Yoshihara et al, 1994; Mizuno et al, 1997). ICAM-5 is the ligand forLFA-1. It was demonstrated by T cell and B cell adhesion to immobilized ICAM-5, which can be inhibited by antibody against LFA-1 (Tian et al, 1997). However this binding is independent of divalent cations and P M A activation (Mizuno et al, 1997). Using a mutagenesis approach and blocking mAb to the first Ig-like domain, it was demonstrated that this domain is important for binding to L F A - 1 (Tian et al, 2000).  8  By using a yeast two hybrid system to identify ligands for L F A - 1 , Ostermann et al. discovered that JAM-1/JAM-A bound to LFA-1 and has an important role during leukocyte trans-endothelial migration (Ostermann et al., 2002). JAM-1 (40 kD) has two Ig-like domains and is expressed on endothelial and epithelial cells (Martin-Padura et al., 1998; Williams et al., 1999). It is distributed on the apical side of tight junctions (TJ) of endothelial cells.  1.1.3. Regulation The activity of LFA-1 and other integrins binding to their ligands is strictly regulated. Regulation by modifying the expression level on the cell surface is not involved, because LFA-1 is constantly expressed at similar level (Rothlein and Springer, 1986). It has been proposed that regulation can be achieved by changes in affinity or changes in avidity (Carman and Springer, 2003). A n increase in affinity is defined as a measure of equilibrium interaction of monovalent receptor and ligand in solution (Dustin et ah, 2004). A n increase in affinity for ligands occurs through conformational changes. Evidence for this idea came primarily from structure-function studies and various mAbs that can recognize epitopes on activated LFA-1 and mAbs that can activate L F A - 1 .  1.1.3.1. Structure-function relationship and affinity activation The OCL chain has several domains in its extracellular domain, namely the (3-propeller, the I-, the thigh, the calf-1 and the calf-2 domains (Figure 1.1). The P2 chain has the PSI, the hybrid, the I-like, the I-EGF and the (3-tail domains. The three-dimensional structure of LFA-1 has not been resolved. However, structural studies of other integrins have provided structure-function relationship of integrins in general. The (XL-chain extra-cellular domain has seven segments, each  9  containing 60 amino acid residues with weak homology to each other. They were predicted to form a seven bladed beta propeller structure similar to the trimeric G-protein P-subunit (Huang and Springer, 1997). This prediction was corroborated by the X-ray crystallography analysis of crystal structure of integrin aVP3 (Xiong et al, 2001) (Figure 1.2a, top view; 2b, side view). It contains seven P-sheets that produce propeller shape, each P-sheet representing one blade. Every blade consists of four antiparallel P-strands connected by P-hairpin loops. A l l seven blades are arranged in a manner that P-strand 1 in each blade facing a central cavity and P-strand 4 forming the outer rim (Xiong et al., 2001). The P-propeller forms a closed circle by the union of P-strand 3 of the 7 blade . th  with P-strand 1 of the 1 blade. Four C a - binding sites are located in the P-strand 1 and 2 P st  2+  hairpin loops of blades 4-7 at the propeller's bottom. The top portion of the propeller forms an interface with the P chain and together they form the globular head of integrins. Mutagenesis studies of integrin anbP3 showed that ligand binding residues are clustered to one portion of the top and side of the P-propeller on blade 3 (Kamata et al., 2001). In L F A - 1 , this P-propeller structure does not play any direct role in ligand binding activity, as it was shown that its Idomain is sufficient for ligand binding (Lu et al., 2001b; L u et al., 2001c). The PSI domain could not be appreciated very well in X-ray crystallography, but it was thought to interact with the IE G F domain by a disulfide bond (Xiong et al, 2001; Hynes, 2002). In vertebrates there are 19 different a and 8 different P subunits, which non-covalently associate to form heterodimers of at least 25 distinct integrins (Hynes, 2002; Shimaoka et al, 2002). Nine of the 19 a subunit contain a domain of around 200 amino acids, the I-domain or also known as the A domain or von Willebrand factor A domain. These nine integrins are CCL, OC , OCX, (XD, OCE, (XI, C62, OC10 and a n . The I-domain loops out from the 2 M  10  nd  and the 3 P-sheet rd  Figure 1.1. I-domain  a  1  2  3  4  5  (3- propeller  6  7 1  | I-like domain  Thigh  1  Calf-1  2  3  4  Calf-2  p-TD  I-EGF domain  PSI domain Hybrid domain  Takagi J. Irnrn. Rev. 2002 Aug 186:141-63 Xiong TP. Science 2001 Oct 12;294(5541):339-45.  Figure 1.1. Domains of integrin are shown in different colors. The I-domain is inserted between loops 2 and 3 and I-like domain is inserted in hybrid domain (a). Schematic representation of integrin structure is shown with relevant colors as in a (b). I-domain is shown with interrupted line. Figure c shows the crystal structure of integrin a V 0 3 , which lacks I-domain, in a bent shape and extended shape.  ll  Figure 1.2.  Springer, T.,Proc Natl Acad Sci U S A 1997 Jan 7;94(l):65-72. Figure 1.2. Ribbon diagram of P-propeller structure of a chain is shown from top (a) and side (b) view. Each loop forms a blade and shown in different color and divalent cations are shown as spheres.  12  blades of the P-propeller structure to form a separate tertiary structure. I domains are essential for ligand binding (Michishita et al., 1993; Randi and Hogg, 1994). Evidence for this came primarily from mapping of mAbs that inhibited ligand binding to the I-domain (Diamond et al., 1993; Huang and Springer, 1995), mutagenesis studies (Kamata et al., 1995; Huang and Springer, 1995) and the ability of isolated I-domain to bind with ligand (Michishita et al., 1993; Shimaoka et al., 2003b). Moreover, deletion of the I-domain of OIL results in no binding to ligands (Yalamanchili et al., 2000; Leitinger and Hogg, 2000). In addition, GPI-anchored I-domain of LFA-1 expressed on baby hamster kidney cells demonstrated a rolling interaction with lipid bilayers containing ICAM-1 or ICAM-3 (Knorr and Dustin, 1997). Furthermore, the I-domain of LFA-1 has been shown to mediate the transition from rolling to firm adhesion (Salas et al., 2002). Initially Pytela reported the homology of the alpha chain of Mac-1 to von Willebrand factor (Pytela, 1988) and this vWF type A was later predicted to be similar to human Ras-p21 (Edwards and Perkins, 1995). Crystal structures of I-domain of OCL and a  M  were resolved  subsequently (Qu and Leahy, 1995; Lee et al., 1995b). They showed similarities to trimeric G protein a-subunit. Qu reported 1.8 A crystal structure of the CD1 l a I-domain with a bound manganese ion, which most likely represents the high affinity form. Three-dimensional structure revealed a dinucleotide binding or Rossman fold, which contains seven a-helices and five parallel and one antiparallel P-strands (Figure 1.3a). The a-helices surround the central P-sheets. The divalent cation is found at the top, ligated by five side-chains located in three different loops, which is called metal ion-dependent adhesion site (MIDAS). The first loop, P i - a i , has three important residues D X S X S of MIDAS that mediate divalent cation binding.  13  The I-domain is thought to assume two different conformations, namely open and closed, because the oc I domain was found to crystallize in two different ways depending on which M  divalent cation it binds to (Lee et al, 1995a). The Mg -bound I domain is considered to 2+  represent the active state and the Mn -bound I domain represents the inactive state. Later they 2+  were designated the open and the closed conformation, respectively. However, further studies revealed that the closed conformation could be seen with M g , M n , C d 2 +  2 +  2 +  or absence of  divalent (Qu and Leahy, 1996; Baldwin et al., 1998), while the open conformation could also be observed with M g  2 +  and probably M n  2 +  (Lee et al., 1995b; Emsley et al, 2000).  The major difference between these two structures is that in the open conformation glutamic acid (Glu) residue donated by the ligand interacts with the divalent cation in the MIDAS sequence (Stanley and Hogg, 1998). In the closed conformation a Glu residue from the ligand is replaced by water, accompanied by movements of the surrounding amino acid residues. The other major difference is the wide (10 A) downward movement of C-terminal helix, al (Figure 1.3b). Employing the CBRM1/5 mAb, Oxvig et al. provided evidence to support the physiological relevance of the conformational change seen in these two different forms (Oxvig et al, 1999; Takagi and Springer, 2002). Supporting this idea, Shimaoka et al. demonstrated by computationally designing mutants of 0CM that stabilized either the open or closed conformations, that the two conformations correspond to active (ligand-binding) and inactive conformations, respectively (Shimaoka et al, 2000). The crystal structure of a I-domain appears to exist in closed conformation when L  compared to the conformations of CCM- Its structures in the presence of M n , M g 2 +  2 +  or no metal at  the MIDAS are similar (Qu and Leahy, 1995; Qu and Leahy, 1996; Kallen et al, 1999).  14  Figure 1.3.  I-domain  Takagi J. Imrn. Rev. 2002 Aug 186:141-63  Takagi J. 1mm. Rev. 2002 Aug 186:141-63  Shimaoka M., Cell 2003 Jan 10; 112(1):99-111.  Figure 1.3. Structure of the I-domain is shown as ribbon diagram. Figure a shows the helices in blue and P strands in yellow, M g as green sphere and loops in brown. Side-chains of residues that form primary or secondary coordinations to the metal ion (D140, SI 42, SI44, T209 and D242) are shown with gray bonds and carbon atoms and red oxygen atoms. Coordinating water molecule oxygens are gold, and the oxygen of the ligand-mimetic Glu from another I domain is magenta. Figure b shows the changes observed in the closed or inactive (blue) and open or active (yellow) conformation. Similar backbone regions are in gray. Metal atoms and coordinating side-chain bonds and carbon atoms are in yellow (open or active) and blue (closed or inactive); oxygen atoms are red. The I domains were superimposed. Figure c shows the structure of I-domain (yellow) interacting with ICAM-1 (blue). Divalent cation M g is shown in purple.  15  To investigate the role of conformational change in 0CL, Shimaoka et al. introduced pairs of cysteine residues to form disulfides that could lock the OCL domain in either the open or closed conformation by locking the C-terminal al helix. Locking the I-domain open resulted in a 9,000fold increase in affinity to ICAM-1, which was reversed by disulfide reduction in surface plasmon resonance measurement (Shimaoka et al., 2001). Similar conclusion was demonstrated when these two forms were tested with cell adhesion assay. In addition, it was shown that locking the I-domain in closed conformation made them resistant to activation by M n  2 +  or mAb.  These results showed that by manipulating the P6-a7 loop it is sufficient to regulate the affinity of the ligand-binding site at the MIDAS (Figure 1.3b, yellow ribbons). N M R spectroscopy study showed that the CD 11a interaction with ICAM-1 affects two clusters of residues. First, the cluster around MIDAS is severely perturbed upon ICAM-1 binding. Second, another cluster in the C-terminal cc-helix is also perturbed. This result supports the idea of conformational changes in LFA-1 upon binding to ligand (Huth et al., 2000). The most recent evidence came from crystal structure of a I-domain and its complex with ICAM-1 L  (Shimaoka et al., 2003b) (Figure 1.3c). The complex structure revealed the open conformation. The I-domain M g  2 +  directly coordinates residue Glu-34 of ICAM-1. It was also demonstrated  that C-terminal al helix was pulled down by introducing disulfide bond locking the|36-a7 loop into three different positions in the closed, intermediate and open conformations that also correlates with increase in affinity. By mimicking the effect of inside-out signaling by pulling down the C-terminal al helix with disulfide bonds in the absence of ligand binding, it was shown that allosteric signals could convert the closed conformation to intermediate or open conformations without ligand binding.  16  The C-terminal stalk of the a-chain consists of cysteine-rich EGF-like domains. They are termed thigh, calf-1 and calf-2 domains. In the crystal structure of avP3, the area in the a-chain between the thigh and calf-1 domains, called genu, and in the P-chain between the I-EGF2 and -3 domains were found to be bent some 135" from the linear form (Xiong et al., 2001; Xiong et al., 2003) (Figure 1.1c). The P chain of LFA-1 starts with 50 amino acid residues at the N-terminal that resemble other membrane proteins including plexins, semaphorins and the c-met receptor. It is called the PSI domain for plexins, semaphorins and integrin (Bork et al., 1999). In human 0CxP2, it has been demonstrated that this cysteine-rich region is important in restraining the integrin in the inactive state by making disulfide bond with another C-terminal cysteine-rich region in the P-chain (Zang and Springer, 2001). In oc P3 crystal structure, this domain is located between the hybrid domain v  and the EGF-1 domain of the P-chain. However, the electron density in this domain is weak and its C a chain cannot be traced with certainty (Xiong et al., 2001). The globular head of the P-chain is formed by its I-like (PA) domain and hybrid domain. It is termed I-like domain, because it shares homology with the a-chain I-domain and it contains the MIDAS sequence motif (DXSXS). The I-like domain is inserted in the hybrid domain, and on crystal structure of avP3 it assumes the nucleotide-binding (Rossman) fold found in GP and a-chain's I-domain (Xiong et al., 2001). It consists of a central six-stranded P strands surrounded by eight helices. Its M I D A S motif (DXSXS) is located at the top. In avP3 crystal structure, the presence of metal was not clearly appreciated. Adjacent to M I D A S lies a metal ion-binding site termed A D M I D A S . From the crystal structure of unliganded avP3, the I-like domain seems to assume the open conformation (Xiong et al., 2003).  17  In integrins lacking I-domain in a chain, such as otvP3, this I-like domain has been demonstrated by X-ray crystallography to bind to ligand (cyclic RGD). Upon ligand binding the open conformation takes shape and is accompanied by conformational changes in the MIDAS motif. A M n  2 +  ion is recruited to MIDAS and the ligand interaction site (Xiong et al, 2002).  However, in integrins with an I-domain in a chain, e.g. LFA-1 and Mac-1, the I-like domain does not interact directly with the ligands. It is thought to play a role as regulator of ligandbinding whereas the I-domain takes over the function of mediating direct binding to the ligands (Carman and Springer, 2003; Shimaoka et al, 2003b). Allosteric I-like domain antibodies (Lu et al, 2001b; L u et al, 2001c), I-like domain MIDAS mutation (Hogg et al, 1999) and small molecule inhibitors of the I-like domain (Welzenbach et al, 2002; Shimaoka et al, 2003a) inhibit ligand binding by the I-domain. The isolated I-domain of « L locked in open conformation binds to immobilized-ICAM-1 with similar affinity to that of activated L F A - 1 . When expressed on the cell surface, it also mediates similar cell adhesion as the intact L F A - 1 molecule. The binding of LFA-1 containing a locked I-domain is resistant to inhibition by mAbs to the P2 I-like domain that inhibit ligand binding by activated wild type L F A - 1 . Treatment with dithiothreitol (DTT), which disrupts disulfide bonds, restores the susceptibility to inhibition by mAbs, suggesting that the locked I-domain overrides the regulation of the I-like domain (Lu et al, 2001c). Divalent cations are widely known to play an important role in LFA-1 binding to I C A M 1. M g  2 +  and M n  2 +  can activate LFA-1 whereas the presence of high concentration of C a  2+  can  inhibit it (Dransfield et al, 1992; Griggs et al, 1998; Labadia et al, 1998). This effect might be mediated by the MIDAS and the A D M I D A S motifs in the I-like domain of P-chain, acting as regulators of the I-domain in the a-chain, because binding of isolated CCL I-domain to its ligand is not inhibited by high concentration of C a  2+  (Shimaoka et al, 2002).  18  The hybrid domain is similar to Ig domains. It makes an extensive contact with the I-like domain, which suggests a minimal inter-domain movement between the I-like-hybrid domains (Xiong et al, 2001). However, in a study with high-resolution electron microscopy (EM), the hybrid domain of oc P3 and anbP3 swings outward by -80° into an "open orientation" upon v  ligand binding (Takagi et al., 2002). In integrins lacking I-domain, it was proposed that this wide swing would pull down the C-terminal al of I-like domain and open its ligand-binding site. This would allow the I-like domain binding site to interact with P-propeller in the a chain and resulted in downward pulling of C-terminal al of the I-domain. The latter conformation of Idomain represents the open conformation or active state (Figure 1.4a, upper panel) (Alonso et al, 2002; Carman and Springer, 2003). The C-terminal stalk of the p-chain contains four tandem cysteine-rich repeats that each assumes the structure of an E G F fold (Xiong et al, 2001). They are termed the integrin-EGF (IEGF) domains (Takagi et al, 2001a; Beglova et al, 2002). Many activating mAb, K I M 127, MEM48 and C B R LFA-1/2 mAbs bind to the I-EGF2 and I-EGF3 regions (Lu et al, 2001a). N M R and a mAb study suggested that this area is buried in an inactive integrin and becomes exposed upon integrin activation. In interpreting this data, the authors proposed the switchbladelike model, where an opening motion extends the integrin into its active conformation (Beglova et al, 2002). The P-Tail Domain (PTD) consists of four-stranded P-sheets that contain antiparallel and parallel strands (Xiong et al, 2001). Only two weak hydrophobic contacts are found between PTD and EGF-4 from a chain, suggesting flexibility at this interface. The function of this domain is still unclear.  19  Carman C V , Curr Opin Cell Biol. 2003 Oct;15(5):547-56  Figure 1.4. Schematic figure of headpiece conformations of integrins lacking an I-domain (Figure 4a, upper panel) and integrins containing an I-domain (Figure 4a, lower panel) are shown. Inside out signal will relay conformational changes that results in 80° outward swing of hybrid domain that pulls the C terminal helix downward and expose the binding sites in Idomain or I-like domain. This allows the external or internal ligands to bind to I-like domain and I-domain. Figure 4b shows the model of integrin activation by extension of extracellular domain, (i) inactive; (ii) intermediate; (iii) activated.  20  The stalk region of integrins is thought to play an important role in relaying the activating signal from intracellular area to the headpiece. It appears that disruption of the association of the a- and p-chains at the stalk region is the first step of inside-out signaling. Replacement of the cytoplasmic domains of CD1 l a and CD 18 with complementary a helices that formed a heterodimeric a helical coiled-coil keeps the receptor in low affinity. In contrast, replacing it with noncomplementary a helices results in constitutive activation of LFA-1 (Lu et al., 2001d). More recently, it has been demonstrated using Fluorescence Resonance Energy Transfer (FRET) that upon inside-out activation or ligand binding (outside-in activation) the 0CL and P2 cytoplasmic domains are separated, suggesting the bi-directional transmembrane signaling occurs in the stalk regions of LFA-1 (Kim et al., 2003). Further experimental evidence comes from mAbs that recognize activation epitopes, which become exposed upon receptor activation and are mapped to the stalk region in the I-EGF domains 2-4 of the P-chain (Stephens et al., 1995; Zang et ah, 2000; La etal., 2001a). Xiong et al. suggested that the bent form of integrin  0CvP3 in  X-ray crystallography  represents the active conformation (Xiong et al., 2001). The bent structure is made from the extracellular domain lacking the cytoplasmic and the transmembrane domains, which has been demonstrated to be in constitutively active state (Dana et al, 1991; Hughes et al., 1996; Mehta et al., 1998). In addition, this structure is preserved upon ligand (cyclic RGD) binding (Xiong et al., 2002). When comparing the conformation of ligand-bound and non-ligand-bound I-like domain, this structure seems to show the open confirmation in both conditions. B y mAbs epitope mapping of  0CnbP3,  Calzada et al. demonstrated that the bent conformation represent both active  and inactive integrin (Calzada et al., 2002). Xiong et al. proposed that the inside-out activation of integrins occur through the sliding movement of the transmembrane domain, which unlocks  21  the "deadbolt" contact of PTD and P chain I-like domain (Xiong et ah, 2003). However, studies on integrins OvP3 and anbP3 by electron microscopy (EM) suggested that the bent form conforms more to the inactive state than active state. The addition of divalent cation M n  2 +  or a ligand  mimetic peptide results in a switchblade-like opening to an extended structure. The studies also showed that a breakage of a C-terminal clasp between the a- and P-chains enhances the M n 2 +  induced unbending (Du et al., 1993; Takagi et al., 2002). Locking the stalk regions of the a- and P-chains by introducing a disulfide bond in this region made the two chains close to each other, which made avp3 and anbP3 integrins inactive and resistant to activation by M n o r activating 2+  mAbs. A n addition of the disulfide bond-reducing agent dithiothreitol (DTT) allowed the activation to ensue again. a P3 crystallized with C a v  2+  and no M g  2 +  had the stalk regions of both  a and P chains closed together, suggesting that the bent form is not favourable for ligand binding and it represents the inactive form (Shimaoka et al., 2002). Furthermore, the activation epitopes defined by the mAbs K I M 127 (anti-p I-EGF2), CBR L F A 1/2 and M E M 4 8 (anti-I-EGF3) that 2  recognize activated integrins were mapped to the stalk regions buried in the bent form (Lu et al., 2001a). According to this argument, relaying signals from the intracellular domain would only require destabilization of the headpiece-tailpiece interface from the bent form, which results in a release of the headpiece from the tailpiece and allows the integrin to assume the extended form by swinging in the genu area (Figure 1.4b). This global conformational change would also cause the stalk regions to become more separated and also the hybrid domain to swing outward and propagate the allosteric changes to the pulling down of the C-terminal al in I-like domain of P chain (Figure 1.4a, upper panel for integrin lacking I-domain and lower panel for I-domain containing integrin). This would cause the MIDAS in the I-like domain to be exposed and  22  interact with the linker residue in P-propeller in the a-chain, which will pull down the C-terminal al of a chain I-domain. Consequently, the M I D A S motif would be exposed and interact with appropriate ligands (Takagi and Springer, 2002). However, it is also possible that an intermediate conformation may exist as shown in E M study (Takagi et al, 2002). Moreover, the crystal structure of the I-domain-ICAM-1 complex showed the existence of an intermediate state (Shimaoka et al., 2003b). Springer suggested that the regulation of conformational change occurs as a shifting equilibrium between closed, intermediate and open conformations, rather than the flipping of a switch. In addition, the interaction with ligand might favour the shift toward the open conformation, because M n  2 +  induces a mixture of open, intermediate and closed  conformations, and the addition of saturating ligand produces almost entirely open conformation (Takagi et al, 2002; Carman and Springer, 2003).  23  1.1.3.2. Avidity regulation Avidity' is a broad term that describes a multivalent interaction. Affinity is one component of avidity. The term avidity tends to be physiological or functional measures of relative adhesive strength. Two-dimensional affinity, two-dimensional kinetic rates, receptor lateral mobility, receptor clustering, membrane deformability and the effects of force are included in avidity (Dustin et al., 2004). A n increase in LFA-1 adhesiveness to ligands can also occur through clustering. In this thesis work, avidity is used interchangeably with increase in clustering. The NKI-L16 mAb that recognizes a Ca -dependent activation epitope on human 2+  LFA-1 only binds to clustered LFA-1 (van Kooyk et al, 1991; van Kooyk et al., 1994). P M A activated LFA-1 has increased mobility as detected by single particle tracking, suggesting the involvement of cytoskeletal restraint. Cytochalasin D, which disrupts the actin cytoskeleton, increases LFA-1 mobility (Kucik et al., 1996). In human peripheral blood T lymphocytes, the actin cytoskeleton has a dual effect on L F A - 1 . In resting T cells, disruption of actin increases LFA-1 binding to ICAM-1 whereas it has an inhibitory effect on IL-2/PMA-activated T cells (Lub et al., 1997). Stewart et al. showed that treatment of T cells with ionomycin to increase intracellular C a  2+  would activate calpain, which will release LFA-1 from cytoskeletal constraint  and activate LFA-1 by increasing its clustering (Stewart et al., 1998). L F A - 1 on human neutrophils binds to talin during the resting state and upon activation its cytoplasmic domain interacts with a-actinin, suggesting a release and re-association with the cytoskeleton upon activation (Sampath et ai., 1998). Deletion of the CD18 cytoplasmic domain or the conserved K V G F F K R sequence in C D l l a results in constitutive binding of LFA-1 to ICAM-1. However, this activation does not result in binding of soluble ICAM-1, suggesting that the increase in L F A 1 binding is due to avidity enhancement rather than conformational changes. Upon cytoskeletal  24  disruption with cytochalasin D, there is no inhibition of L F A - 1 , indicating that deletion of the cytoplasmic domain causes release of LFA-1 from the cytoskeleton and its activation (van Kooyk et al, 1999). PMA-activated LFA-1 binds to ICAM-1-coated microspheres but not soluble ICAM-1, which suggest the importance of avidity (Welder et al., 1993; Pyszniak et al., 1994). Several signaling molecules that mediate LFA-1 clustering induced by the TCR have been identified. The small GTPase, Rapl, through its effector, R A P L (regulator for cell adhesion and polarization enriched in lymphoid tissues), and also V a v l become activated upon TCR stimulation (Katagiri et al., 2000; Sebzda et al., 2002; Katagiri et al., 2003). Phosphorylated SKAP-55 (Src kinase-associated phosphoprotein of 55 kD) binds to adaptor protein A D A P (SLAP-130/Fyb) and activates LFA-1 through an increase in clustering upon TCR stimulation (Griffiths et al, 2001; Peterson et al, 2001; Wang et al, 2003).  1.1.3.3. Inside-out signaling LFA-1 can be activated by stimulating various surface receptors including the TCR (Dustin and Springer, 1989), G protein-coupled receptors, chemokine receptors (Laudanna et al, 2002), CD2 (van Kooyk et al, 1989a), L-selectin (Gopalan et al, 1997) and the leukocyte integrin a$\ (integrin cross-talk) through a signaling process termed inside-out signal (Porter and Hogg, 1997; Chan et al, 2000). Similar activation pathways can be induced by phorbol esters (PMA, PdBU) through protein kinase C (Rothlein and Springer, 1986) and ionomycin through increases in intracellular C a  2+  (Stewart et al, 1998). Activation of LFA-1 by TCR  stimulation is essential for T cell interaction with antigen presenting cell (APC). The initial interaction of naive T cell with A P C is thought to be mediated by antigen-independent  25  interactions of DC-SIGN of dendritic cells (DC) with ICAM-3 on T cells (Geijtenbeek et al, 2000). Upon TCR engagement with cognate peptide-MHC complexes, an activation signal through an inside-out pathway will activate L F A - 1 , which allows longer and more sustained stimulation of naive T cells. In human peripheral blood leukocytes, it was demonstrated that upon TCR crosslinking or P M A stimulation LFA-1 associates with the cytoskeleton, as detected in the pellet after cell lysis and by fluorescence microscopy. In addition, LFA-1 was found to colocalize with F-actin only after TCR stimulation (Pardi et al., 1992). Overexpression of S K A P 55 increases LFA-1 clustering which results in increased binding of T cell to A P C . This effect could also be observed by overexpression of A D A P (adhesion and degranulation-promoting v  adaptor protein), also known as Fyb (Fyn binding protein) and S L A P (SLP-76 associated protein) (Wang et al, 2003). Fyb/SLAP (ADAP) was shown to couple T C R signaling to LFA-1 activation through clustering, as shown using Fyb/SLAP knockout mice (Griffiths et al., 2001; Peterson et al, 2001). In a study using constitutively active R a p l A Tg mouse, Rap 1A activates LFA-1 through an increase in LFA-1 avidity. In addition, TCR stimulation activates Rap 1A (Sebzda et al, 2002). This finding confirmed earlier reports about the role of Rap 1 in mediating TCR-induced LFA-1 activation (de Bruyn et al, 2002; Tohyama et al, 2003), which is distinct from the P K C and phosphatidylinositol-3-OH kinase (PI3K) pathways (Katagiri et al, 2000), yet dependent on P L C - y l (Katagiri et al, 2004). Preventing activation of Rap by RapGAPII (Rap-specific GTPase-activating protein II) significantly decreases LFA-1 binding activity in B lymphocytes (McLeod et al, 2004). Using the yeast two-hybrid system, Katagiri et al. identified RapL as an effector of Rap, which interacts only with active GTP-Rapl. RapL mediates both TCR and chemokine (SDF-1) induced LFA-1 activation and this activation involves an increase in affinity  26  and avidity. RapL was shown to bind to LFA-1 upon TCR crosslinking, and this interaction is increased after expression of coristitutively active Rap 1 (Rap 1V12) (Katagiri et al., 2003).The guanine nucleotide exchange factor (GEF) V a v l is also a central signaling molecule that the TCR uses for inside-out activation of LFA-1 (Krawczyk et al., 2002). During leukocyte adhesion and extravasation from blood vessels to a site of inflammation, chemokines induce activation of leukocyte integrins, resulting in firm adhesion and arrest. The chemokines SDF-lrx (CXCL12), E L C (CCL19), and SLC (CCL21) induce rapid and transient (1-2 minutes) increases in the affinity of L F A - 1 , as measured by binding to soluble ICAM-1. These chemokines also increase the lateral mobility and clustering of L F A - 1 , which is dependent on PI3K (Constantin et al., 2000). This inside-out activation is mediated by RhoA and PKC£ (Giagulli et al., 2004). In an in vivo model using Tg mice with GFP-expressing naive T cells, TCA-4 (thymus-derived chemotactic agent-4) causes LFA-1 mediated homing of T cells to mouse peripheral lymph nodes (Stein et al., 2000). Rap 1 has important role in the activation of LFA-1 by SLC and SDF-1 (Shimonaka et al., 2003). SDF-1-induced leukocyte migration also requires Rho A , myosin, G i and Cdc 42 (Soede et al., 2001). Moreover, M l P - l a and P activate LFA-1 through H-Ras and F-actin polymerization (Tanaka et al., 1999). Thus, chemokines are potent LFA-1 activators through inside-out signaling pathways. Cytohesin-1, a P H domain containing signaling molecule, was isolated using the yeast two hybrid system and shown to interact with the CD18 cytoplasmic domain. Overexpression of cytohesin-1 results in increased LFA-1 binding to ICAM-1 in Jurkat cells (Kolanus et al., 1996). The effect of cytohesin 1 on LFA-1 occurs through direct interaction with the p chain cytoplasmic domain and its GEF activity acting on A D P ribosylation factors (ARF) (Geiger et al., 2000). Overexpressed cytohesin-1 can increase LFA-1 mediated leukocyte arrest and  27  transmigration upon chemokine stimulation (Weber et al, 2001). Talin (Kim et al., 2003), PI3K (Nagel et al., 1998) and Cbl, a ubiquitin ligase adaptor protein, that consists of an amino-terminal SH2-like> domain, a RING finger, and carboxyl-terminal proline-rich sequences (Zhang et al., 2003) have all been demonstrated to have important roles in the inside-out signaling that activates L F A - 1 . The cytoplasmic domains of integrins play a crucial role in relaying the inside-out signal to the extracellular domain, which will require propagation of long-range conformational changes. So far this type of activation has never been observed in any other cell surface receptor. The most compelling evidence came from N M R study on integrin anbP3 cytoplasmic domain (Vinogradova et al., 2002). In this study, an P3 cytoplasmic domain in aqueous solution was b  shown to form a weak clasp or handshake from hydrophobic and electrostatic interactions mediated by both a helices in membrane proximal a and (3 chains. Mutations introduced into these areas perturb the weak interaction. In a more physiological situation, talin head was shown to also perturb the interaction. Deletion and protease digestion experiments demonstrated that talin-head binds to the p tail with high affinity (Kd -100 nM) (Calderwood et al, 1999; Yan et 3  al, 2001; Tadokoro et al, 2003). This is further supported by the analysis of  OC5P1.  Soluble  recombinant protein of the extracellular domain of this integrin was inactivated when artificial clasp was introduced by mutation in the membrane proximal area. After cleavage with protease, it became activated in binding to ligand fibrinogen (Takagi et al, 2001b). In L F A - 1 , the separation of both chain cytoplasmic domains was demonstrated to be important by using FRET technique and this separation could occur upon talin-head binding (Kim et al, 2003). L u et. al. showed that forced association of membrane proximal region of cytoplasmic domains of both chains constrain LFA-1 in inactive state (Lu et al, 2001d).  28  LFA-1 cytoplasmic domains are short and do not seem to have any enzymatic activity. Phosphorylation of the cytoplasmic domain of L F A - 1 has never been proven to be important for inside-out signaling (Hibbs et al, 1991a). Two possible phosphorylation sites are S745 and S756, while the important tyrosine phosphorylation sites in other integrins, the N P X Y motifs, are missing in L F A - 1 , which has N P L F sequence instead (Calderwood, 2004). The a-chain contains the conserved G F F K R sequence. Deletion of the G F F K R sequence or the whole CCL cytoplasmic domain results in constitutively active LFA-1 (Pardi et al., 1995; L u and Springer, 1997). Deletion of the whole (3 cytoplasmic domains or three threonine residues (TTT 758-760) or Phe 2  766 inhibits binding to ICAM-1 (Hibbs et al, 1991a; Hibbs et al, 1991b; Peter and OToole, 1995). Overexpression of membrane-bound CD18 cytoplasmic domain inhibits LFA-1 (ReyLadino et al, 1998). These studies suggest that it is possible that L F A - 1 cytoplasmic domains are connected to the cytoskeleton, mediated by the cytoskeleton-associated molecule, like talin and paxilin that binds directly to these amino acid sequences.  1.1.3.4. Outside-in signaling E M studies of 0CvP3 and  ccirbP3  structures showed that their conformations change upon  ligand (cyclic RGD) binding (Takagi et al, 2002). Separation of the a- and the (3-chains of L F A 1 upon ligand binding has also been detected by FRET (Kim et al, 2003). Therefore, ligand binding seems to induce outside-in signal to further enhance integrin functions. LFA-1-mediated outside-in signals include activation of P L C - y l (Kanner et al, 1993). During LFA-1-induced T cell locomotion, PKC-P(I) translocates to the microtubule cytoskeleton. Upon crosslinking with immobilized mAb, LFA-1 forms a complex with tubulin and PKC-P(I), as demonstrated by in situ immunoprecipitation. In this method cells were  29  allowed to bind to immobilized ICAM-1 for 3 hours at 37°C then lyzed in microtubulestabilizing buffer. The remaining LFA-1-cytoskeleton complexes were extracted from the surface by scraping into the SDS-containing buffer then separated by SDS-PAGE (Volkov et al., 1998; Volkov et al, 2001). Furthermore, LFA-1 binding to ICAM-1 induces M L C K (myosin light chain kinase)-mediated attachment of the leading edge and ROCK-mediated detachment of the trailing edge during T cell migration on immobilized I C A M - l / F c (Smith et al, 2003). LFA-1 crosslinking by antibody has long been known to induce activation of other signaling molecules. Rho, R O C K and Pyk2 (Rodriguez-Fernandez et al, 2001), Paxillin and Pyk2 (Rose et al, 2003), oc-actinin (Pavalko and LaRoche, 1993), filamin (Sharma et al, 1995), ZAP-70 (Soede et al, 1998) and F A K tyrosine phosphatase (Giannoni et al, 2003), all have been shown to be involved in the LFA-1 outside-in signalling. LFA-1 engagement has been demonstrated to provide a co-stimulatory signal in T cells (Wacholtz et al, 1989; Van Seventer et al, 1990; Vyth-Dreese et al, 1993; N i et al, 1999; N i et al, 2001) as well as T h l polarization (Smits et al, 2002) and regulation of Th2 cytokine production (Salomon and Bluestone, 1998). LFA-1 on macrophages was reported to induce MIP (macrophage inflammatory protein)-a and P secretion (Murphy et al, 2000). In P M N , LFA-1 crosslinking induces respiratory burst (Menegazzi et al, 1999; Decleva et al, 2002) and protein tyrosine phosphorylation (Walzog et al, 1996). LFA-1 induces F A K and Pyk 2 activation and microtubular cytoskeleton rearrangement and C a  2+  signaling as well (Rodriguez-Fernandez et al,  1999; Rodriguez-Fernandez et al, 2002). In the B-lymphoblastoid cell line JY, it also induces tyrosine phosphorylation of Cas (Crk-associated substrate) and its binding to c-Crkll (Petruzzelli etal, 1996).  30  More recently it was demonstrated that L F A - l - I C A M - 1 interaction results in phosphorylation of V a v l in N K cells, which is upstream of actin cytoskeleton and lipid rafts reorganization (Riteau et al, 2003). Supporting this finding in T cells, it was revealed that L F A 1 stimulation induces transient activation of Rac-1 (Sanchez-Martin et ah, 2004). JAB1 (Jun activation domain-binding protein 1), a co-activator of the c-Jun transcription factor, was found to interact with LFA-1 cytoplasmic domain by yeast-two hybrid method and was shown to colocalize with L F A - 1 , and upon LFA-1 binding is translocated to nucleus to induce gene transcription (Bianchi et al., 2000). This event was also shown to lower T cell activation threshold for IL-2 production and T h l differentiation and to activate Erk 1/2 through cytohesin-1 (Perez et al, 2003). Lateral association of LFA-1 on the plasma membrane has been noted to play an important role for outside-in signaling as well. CD226 (DNAM) is an adhesion molecule found on C T L and N K cell and its ligation can trigger killing function. D N A M was found to physically associate with L F A - 1 in N K cell or CD3 stimulated T cell. Tyrosine phosphorylation of D N A M is induced upon crosslinking of LFA-1 (Shibuya et al, 1999). This interaction was demonstrated to have an important role to provide co-stimulatory signal for T cell differentiation and proliferation (Shibuya et al, 2003). In a search for 0 2 cytoplasmic domain interacting protein using yeast two-hybrid system, RanBPM was identified. It is a membrane-bound protein capable of binding to small GTPase Ran. Expression of RanBPM potentiates L F A - 1 dependent activation of an AP-1 promoter (Denti et al, 2004). Tetraspanins are membrane protein that has been shown to interact with integrins. CD63, a member of tetraspan family, was found to associate by immunoprecipitation with Mac-1 in P M N but it is unclear whether it is also important for LFA-1 (Skubitz et al, 1996; Skubitz et al, 2000).  31  1.2. Lipid rafts The fluid-mosaic model (Singer and Nicolson, 1972) states that the plasma membrane arranges itself as a uniform lipid bilayer where all the membrane-associated surface receptors are distributed randomly. However, in recent years evidence has accumulated pointing out that plasma membrane is not a uniform lipid bilayer but rather it contains sphingolipid- and cholesterol-rich microdomains, named lipid rafts (Simons and Dconen, 1997). Lipid rafts are thought to play a role in a number of signaling processes involving receptors expressed by a variety of cell types. A l l cell membranes contain glycerophospholipids, which consist of a glycerol backbone and unsaturated acyl chains. Another group of lipid is sphingolipid, with ceramide backbone combined with either a phosphocholine head group (sphingomyelin) or carbohydrate (glycosphingolipid). The acyl chains in sphingolipids are usually saturated. The last group of lipid in eukaryotic cells is sterol, consists of rigid four-ring structure. The principal sterol in vertebrate is cholesterol. Cholesterol and sphingolipids are mainly found in the plasma membrane and endosome and they are very low in internal membranes, since cholesterol is synthesized in the endoplasmic reticulum (ER) and sphingolipids in Golgi. There are also differences in the outer leaflet and the inner leaflet of plasma membrane. Most, if not all, of the sphingolipids are found in the outer leaflet, while some of the glycerophospholipids (phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine) are localized in the inner leaflet (Munro, 2003). Cholesterol preferentially interacts with sphingolipids (Ramstedt and Slotte, 2002). The outer leaflet of lipid rafts is composed of both sphingolipids with saturated acyl chains that arrange tightly into gel-like microdomains and cholesterol. Sphingolipids differ from most biological phospholipids in their long, largely saturated acyl chains. This allows them to  32  readily pack tightly together, a property that gives sphingolipids much higher melting temperatures (T ) than membrane glycerophospholipids, which are rich in kinked unsaturated m  acyl chains. The interaction of small-sized cholesterol with the straight-chained sphingolipids promotes formation of a liquid ordered phase (Schroeder et al., 1998). The sphingolipids and cholesterol exclude themselves out of the surrounding glycerophospholipid bilayer, which exists in a liquid disordered phase due to the unsaturated, kinked acyl chains of glycerophospholipids. Therefore lipid rafts are relatively ordered domains that float in the disordered glycerophospholipid bilayer. The inner leaflet of lipid rafts is less well characterized but is probably composed of saturated phospholipids with saturated fatty acids and cholesterol (Fridriksson et al., 1999). The inner and outer leaflets are coupled, although the nature of the coupling is unclear. One possible mechanism is that long fatty acids of sphingolipids in the outer leaflet couple the outer and inner leaflets by interdigitation. Another possibility is that transmembrane proteins could also stabilize this coupling (Simons and Ikonen, 1997). The evidence for the existence of lipid rafts has been gathered by lipid model membrane studies, which showed that liquid ordered phase and liquid disordered phase domains could coexist. The phase separation could occur in mixtures of cholesterol with two phospholipids (or a phospholipid and a sphingolipid) that have different T and therefore different tendencies to form m  an ordered phase (Silvius et al., 1996; Ahmed et al., 1997; Wang and Silvius, 2003). In these mixtures, liquid ordered phase domains enriched in the high T lipid separate from liquid m  disordered phase domains enriched in the low T lipid. It was also demonstrated that in m  supported planar lipid monolayer glycosylphosphatidylinositol (GPI)-anchored protein, Thy-1, partitioned into glycosphingolipid GM1 rich domain (Dietrich et al., 2001b). The presence of lipid raft-like domains was subsequently observed in giant unilamellar vesicles (GUV) (Dietrich  33  et al, 2001a; Baumgart et al., 2003). However, a study on membrane model with lipid composition mimicking the inner leaflet of plasma membrane found that cholesterol does not induce a liquid ordered domain (Wang and Silvius, 2001). It is still possible that the outer leaflet may dictate the structure/phase of inner leaflet domain (Munro, 2003). In living cell membrane, the presence of lipid rafts microdomains was demonstrated most convincingly by measuring the FRET and fluorescence anisotropy amongst GPI-anchored molecules coupled with one type fluorochrome. This method combined with mathematical modeling concluded that, in fact GPIanchored. molecules occurred in cluster on living cell membrane and the size of this cluster is estimated in the nanoscale range (5 nm) and these clusters were actually sensitive to cholesterol disruption (Varma and Mayor, 1998; Pralle et al., 2000; Sharma et al., 2004). Studies on living membrane cells using FRET technique on M D C K (Madin-Darby canine kidney) cells of Aequorea fluorescent proteins with modified lipid anchors showed that acyl modification promotes clustering in rafts (Zacharias et al., 2002). On the other hand, other studies using simple FRET methods did not detect any clustering of GPI-anchored molecules (Kenworthy and Edidin, 1998; Kenworthy et al., 2000; Glebov and Nichols, 2004). Crosslinking of surface receptors to induce patching has been used to evaluate rafts (Harder et al., 1998; Janes et al., 1999). Figdor et al. demonstrated DC-SIGN to be localized in rafts with electron microscopy (Cambi etal, 2004). Lipid rafts are most commonly isolated from the plasma membrane by sucrose gradient centrifugation method. Lipid rafts domains are insoluble in certain nonionic detergents at 4° C and can be separated from the soluble membranes based on their low buoyant density (Simons and Toomre, 2000). Therefore, the identification of a protein association with rafts is operational and dependent on the type of detergent and their concentration. A number of issues arise from  34  the use of detergent solubility to define the microenvironment of a protein on the plasma membrane. However, evidence is rapidly accumulating that supports the existence of rafts in the membranes of living cells, as mentioned above (Dykstra et al., 2003). Munro advised caution in interpreting the results of lipid raft isolation by this method alone (Munro, 2003). The presence of heterogeneity in lipid rafts has been suggested by several studies. In migrating cells, ganglioside GM3 and GM1 are differentially distributed in the leading edge and the uropod, respectively (Manes et al., 1999; Gomez-Mouton et al., 2001). High resolution E M studies of intact 2-D sheets of apical plasma membrane, ripped off from adherent cells, demonstrated that H-ras and K-ras are clustered in different type of microdomains, as defined by their sensitivity to cyclodextrin treatment (Prior et al., 2003). In human peripheral blood T lymphoblasts, Lck and L A T are distributed differently, supporting the idea of lipid raft heterogeneity (Schade and Levine, 2002). Membrane caveolae containing caveolin is thought to form a microdomain separate from lipid rafts; eventhough caveolae itself is rich in cholesterol and ganglioside (Kurzchalia and Parton, 1999). Isolated caveolin-containing plasma membrane of the apical surface of rat lung endothelial cells do not contain GPI-anchored proteins (Schnitzer et al., 1995). However, fibroblast derived from caveolin-r mice showed intracellular retention A  of GPI-anchored proteins (Sotgia et al., 2002), whereas lipid rafts were demonstrated to have an important function in sorting of GPI-anchored proteins to the apical cell surface (Brown and Rose, 1992). Thus, the relationship between GPI-linked proteins and caveolin remains controversial. Another type of membrane microdomain is tetraspanin microdomain, which is also thought to be distinct from lipid rafts, because they do not contain GPI- anchored molecule and caveolin (Kropshofer et al., 2002; Hemler, 2003). Tetraspanins are membrane receptors,  35  which have four transmembrane domains, like CD9 or CD81. They are important for B cell activation and cell fusion.  1.2.1. Functions of lipid rafts Lipid rafts have an important role in membrane trafficking (Simons and Ikonen, 1997). Lipid rafts on polarized epithelial cells accumulate in the apical plasma membrane. Basolateral membrane also contains rafts but in smaller amounts, whereas caveolae is present mainly in basolateral side (Vogel et al., 1998). Lipid rafts have been demonstrated to be important both for exocytic and endocytic traffic (Fiedler et al., 1994; Danielsen, 1995; Stoddart et al., 2002). Lipid rafts also play a major role in signaling in the immune system. GPI-anchored proteins preferentially associate with lipid rafts (Ilangumaran et al., 2000). Examples of raftassociated GPI-linked immune receptors include CD 14, the receptor for the bacterial mitogen LPS; CD16, an Fc receptor; and CD48 and CD58, adhesion/co-stimulatory molecules, Thy-1, CD59 and CD55. Although they lack transmembrane or cytoplasmic domains, many GPI-linked proteins have been shown to transduce signals upon crosslinking (Stefanova and Horejsi, 1991; Stefanova et al., 1991). CD48 is thought to act as a co-stimulatory molecule for T cells through lipid rafts association (Moran and Miceli, 1998). Signaling molecules interact with the inner leaflet of lipid rafts through acylation. Molecules that are dually acylated by saturated fatty acids (N-myristoylation and S-palmitoylation) partition into rafts, whereas proteins modified by unsaturated fatty acids or prenyl groups are excluded (Melkonian et ah, 1999). The GTPase H ras, which is palmitoylated and farnesylated, is localized in lipid rafts. In contrast, K-ras, which is farnesylated but not palmitoylated, does not associate with lipid rafts (Prior et al., 2001).  36  Another report showed that heterogeneous fatty acylation functions to regulate signal transduction by membrane-bound proteins (Liang et al., 2001). The role of rafts in T cell activation was initially demonstrated in Jurkat T cell line and mouse thymocyte upon CD3 antibody crosslinking (Xavier et al., 1998; Montixi et al., 1998). TCR stimulation induces TCR/CD3 recruitment into detergent resistant membrane (DRM) fractions followed by an increase in tyrosine phosphorylation and Lck activity in D R M . In addition, Viola et al. established that beads coated with anti CD3 and anti CD28 Ab, but not with anti CD3 alone, cause clustering of lipid rafts toward bead-binding sites (Viola et al., 1999b). The TCR/CD3 complex, Lck, ZAP-70 and CD4 seem to be pre-assembled and constitutively associate with lipid rafts in thymocyte (Drevot et al., 2002). Filipp et al. demonstrated that coaggregation of TCR and CD4 induce subsequent activation of Lck outside lipid rafts followed by its recruitment into the microdomain, which activates co-localized Fyn (Filipp et al., 2003). The linker molecule, L A T requires association with D R M through its palmitoylation in order to become phosphorylated (Zhang et al., 1998). Upon crosslinking by the natural or surrogate ligand (antibody), TCR merge with membrane rafts and ITAMs (immune receptor-based tyrosine activation motifs) present in the cytoplasmic tails of the signaling chain become exposed to the Src-kinases present in the rafts (Horejsi, 2003). In addition, CD4 association with lipid rafts through its palmitoylated cysteine residues is important to provide co-stimulation signal (Fragoso et al., 2003). Other co-stimulation molecule such as CD5 enhances the association of TCR/CD3 with D R M (Yashiro-Ohtani et al., 2000). Similarly in B cell activation, evidence is accumulating of the importance of lipid rafts (Weintraub et al., 2000; Cherukuri et al., 2001; Cheng et al., 2001).  37  1.2.2. Lipid rafts and integrins  Several reports have suggested that lipid rafts play a role in integrin signaling. Krauss et al. reported that LFA-1-mediated adhesion of mouse thymocytes to ICAM-1 is activated by crosslinking ganglioside GM1 with Cholera toxin subunit B (CTxB) or GPI-anchored CD24 with antibody. The activation of LFA-1 on thymocytes is inhibited by rafts disruption with methyl-p 1  cyclodextrin (MCD) (Krauss and Altevogt, 1999). However, Shamri et al. reported that the integrins V L A - 4 and oc p as well as LFA-1 are excluded from rafts of human peripheral blood 4  7  lymphocytes. Disruption of cholesterol rafts with the chelator, M C D , did not affect the ability of these lymphocyte integrins to generate high avidity binding to their respective endothelial ligands and to promote lymphocyte rolling and arrest on inflamed endothelium under shear flow (Shamri et al., 2002). Adding to the controversy, results from Leitinger et al. concluded that LFA-1 on Jurkat T cells does not associate with lipid rafts unless it is activated with manganese or its I-domain is deleted (Leitinger and Hogg, 2002). The I-domain-deleted LFA-1 is thought to mimic activated L F A - 1 , based on the assessment by mAb that recognizes an activation epitope, eventhough this mutant LFA-1 is not capable of binding to ICAM-1 due to the lack of the Idomain. The platelet integrin  anbP3  is found in lipid raft fractions when cells are lyzed with  C H A P S (Mairhofer et al., 2002). Integrin o^Pi is also present in the raft compartment of the newly formed oligodendrocytes, whereas the ccy integrins, also expressed on oligodendrocytes, are almost entirely in the non-raft compartment (Baron et al., 2003). Integrin avP3 was reported to be in lipid rafts in the human ovarian carcinoma OV10 cells when it forms complex with integrin associated protein (IAP) and G-protein (Green et al., 1999). Ligand-coated microspheres induced recruitment of integrins not disrupt intracellular C a  2 +  CC2P1  and a P i into rafts in Jurkat T cells and M C D treatment do 4  storage (Holleran et al., 2003). Part of integrin OeP was shown to 4  38  associate with lipid rafts and this association was important for its signaling to Src family kinase and promotion of EGF-dependent mitogenesis (Gagnoux-Palacios et al, 2003). Both insulin-like growth factor-1 (IGF-1) and integrin Pi are recruited into lipid rafts after IGF-1 stimulation in human multiple myeloma cells (Tai et al., 2003). In human osteosarcoma cells, CX2P1 integrin remains associated with rafts but it moves to caveolae to be internalized upon antibody crosslinking (Upla et al., 2004). CD36 on a melanoma cell line was shown to induce integrins (X3P1 and o^Pi sequestration into lipid rafts and to promote cell migration (Thorne et al., 2000). The complex consisting of the transmembrane 4 super-family (TM4SF) molecule CD9 or CD81 and  CC3P1  is localized in lipid  raft-like microdomain. However, their association is not mediated by cholesterol since M C D treatment has no effect on the complex formation (Claas et al., 2001). Upon CD14 stimulation with LPS (lipopolysaccharide), ceramide recruites Mac-1 into lipid rafts (Pfeiffer et al., 2001). The protein tyrosine phosphatase, SHP-2, when made associated with rafts, induces Pi integrin signaling, mimicking ligand binding (Lacalle et al., 2002). Recent studies demonstrated that integrin on NTH 3T3 fibroblasts regulates the internalization of G M 1 rafts through Rac, F A K and mDia, which in turn regulates the stabilization of microtubule cytoskeleton (Palazzo et al., 2004; del Pozo et al, 2004).  1.3. Immunological synapse The immunological synapse (IS) is a term used to describe the distinct organization of T/NK cell surface receptors involved in antigen recognition. It was first reported by Kupfer et al. using deconvolution microscopy and 3-D reconstruction software. When fixed T cells interact with A P C , its receptors are distributed in a particular manner (Monks et al, 1998). LFA-1 and  39  talin are distributed in the periphery, later called peripheral-supra-molecular activation cluster (pS M A C ) , while the TCR/CD3 complex and signaling molecules, such as protein kinase C 0 (PKC9), Fyn and Lck are clustered in the central-supra-molecular activating cluster (c-SMAC). The dynamics and significance of these structures were studied by using live cells interacting with fluorescent tagged ICAM-1 and MHC-peptide in planar supported bilayer system (Grakoui et al., 1999). Initially TCR-MHC/peptide are localized in the periphery of IS whereas L F A - 1 ICAM-1 are in the center. In the next five minutes, the positions are reversed, where LFA-1 goes to the periphery and the TCR occupies the center, forming a mature IS. This highly organized structure is important for signaling as detected by intracellular C a  2 +  level. Mature IS is formed  when the TCR engages M H C loaded with strong or weak agonist peptides, but not with antagonist or null peptides. This study provided, for the first time, the evidence for the functional importance of IS in T cell activation. This study also demonstrated that CD4 plays a role as a costimulatory molecule for T cell arrest from migration and for mature IS formation. The mature IS configuration is thought to allow the interaction between LFA-1 and I C A M - 1 , which have large sizes, to occur in the p-SMAC, whereas the interaction between the TCR and MHC/peptide, having smaller molecular sizes, to take place in the c-SMAC. C D 45 is larger in size and is excluded from the IS (Shaw and Dustin, 1997; Sims and Dustin, 2002). Similar IS formation was demonstrated on CD8 T cells activated with high concentration of agonist peptide (Potter et al., 2001). Phosphorylation of Lck occurs in the p-SMAC in the first 2-15 minutes and later disappeares from c-SMAC, while phosphorylation of ZAP70 starts in p - S M A C two minutes after cell-cell conjugation and continues for 15 minutes and moved to c-SMAC after 30 minutes. This finding suggested that initial signaling events take place in p - S M A C . In order to induce T cell  40  proliferation, a minimum 2 hours of T cell-APC contact is required, and with longer (over 6 hours) cell contact there is more T cell proliferation (Lee et al, 2002a). Studies with Lck and CD3£ fused with green fluorescent protein (GFP) showed that Lck initially accumulates in pS M A C and translocates to the mature IS while CD3£ is in c - S M A C (Ehrlich et al, 2002). T cells from CD2AP " mice, which cannot down-regulate the TCR and are hypersensitive to antigens, _/  has sustained phosphorylation of Lck in c-SMAC whereas in wild type T cells the phosphorylation of Lck starts in p-SMAC but later continues to c-SMAC and disappears (Lee et al, 2003). The authors suggested that mature IS functions as an adaptive controller to attenuate strong signals and to amplify weak signals from the TCR. Huppa et al. provided evidence which supported the idea that continuous TCR signaling, as detected by intracellular C a  2 +  staining and  PI3K accumulation for hours (~ 24 hours), is required for mature IS and full activation of T cells (Huppa et al, 2003). However, Faroudi et al. demonstrated by using inhibitors of Src-kinases that interrupted signaling and IS formation can also activate EFN-y production of T cells (Faroudi et al, 2003b). Supporting the idea that IS is physiologically relevant, a study of thymocytes showed that during negative selection, CD3£ and Lck are both found to localize in p-SMAC (Richie et al, 2002). CD45 transiently localizes to c-SMAC and later move to outside of the IS (Freiberg et al, 2002), while CD4 is found to localize in the inner side of p - S M A C (Ehrlich et al, 2002). Wulfing et al. demonstrated that co-stimulation molecules (B7.1, B7.2, L F A - 1 , CD2) and endogenous null peptide-presenting M H C are required to maintain mature IS and T cell activation (Wulfing et al, 2002; Wetzel et al, 2002; Zaru et al, 2002). CD4 and CD28 were shown to cooperate to induce auto-phosphorylation of Lck in the IS. CD4 recruits Lck to the IS and CD28 sustains the activation of Lck (Holdorf et al, 2002). As detected by FRET, CD4 and  41  CD3c^ on T cell hybridomas co-localize in the IS upon antigen recognition. However, this interaction does not occur if antagonist ligand is used although the recruitment of both to the IS is not affected (Zai et al., 2002). This supports earlier report that the IS can occur without agonist peptide. In this study, functional IS that results in T cell signaling and weak proliferation can be detected upon naive T cell interaction with D C (Revy et al., 2001). CD43 (sialophorin, leukosialin) is a large, negatively charged transmembrane protein. Due to its abundant glycosylation, CD43 has been found to extend -45 nm, making it one of the largest molecules on the T cell surface. During IS formation, CD43 was found to be excluded and accumulate in the opposite side of cell-cell contact and this antipodal movement is mediated by moesin (Allenspach et al, 2001; Delon et al, 2001). In vivo studies on lymph nodes also demonstrated the formation of the IS on T cells (McGavern et al, 2002; Stoll et al, 2002). Fluorescent labeled naive T cells and DCs were injected subcutaneously and were detected in the regional lymph nodes of antigen-stimulated mice by multiphoton confocal microscopy (Stoll et al, 2002). CD43-GFP fusion protein retrovirally transduced into T cells was found to be excluded from the IS formed on T cell-DC conjugates. McGavern et al. studied the IS in activated CD8 effector T cells or CTLs against lymphocytic choriomeningitis virus (LCMV)-infected target cells in the central nervous system (McGavern et al, 2002). LFA-1 accumulates in the p - S M A C while Lck is more widely distributed but somewhat aggregated in the IS. Lipid rafts are thought to have an essential role for the formation of IS during naive T cell activation (Burack et al, 2002; Jordan and Rodgers, 2003). However, a recent study using FRET to identify the clustering of GPI-anchored fluorochrome has suggested that lipid rafts are not essential in the IS formed between T cells and beads coated with anti-CD3 and anti-CD28 mAbs  42  (Glebov and Nichols, 2004). In this report only GPI-anchored receptor was investigated, and the possibility of heterogeneity was not explored. In addition, FRET is not sensitive enough to detect very close (<10 nm) molecular interactions. Sharma et al. used the more sensitive method of homo-FRET combined with anisotropy and detected clustering of GPI-anchored molecules, which was sensitive to M C D treatment (Sharma et ah, 2004). The regulation of lipid rafts in the IS was shown to involve W A S P (Wiskott-Aldrich syndrome protein), an important signaling molecule that regulates the actin cytoskeleton (Dupre et al., 2002). The signaling pathway between the TCR and W A S P became clearer in a later study, which revealed the involvement of ZAP70-CrkL-WIP (WASP Interacting-protein)-WASP complex and P K C 0 (Sasahara et al, 2002). Another molecule that regulates actin, D O C K 2 , also was found to be important for TCR and lipid rafts translocation to T cell-APC interface (Sanui et al., 2003). P K C 0 localization into lipid rafts followed by accumulation into the IS seems important for T cell activation (Bi et al., 2001) . PIP3 accumulation in the IS area is significant for T cell stimulation (Costello et ah, 2002) . Agrin, an aggregating factor of acetylcholine receptor in neurons, plays a role in aggregating lipid rafts in the IS (Khan et al., 2001). More recently, lipid rafts and the actin cytoskeleton of A P C have been shown to be important in the formation of IS (Gordy et al., 2004) The complex rearrangement of surface receptors involved in the formation of the IS seems to be mainly mediated by the actin cytoskeleton and to certain extent myosin II (Dustin and Cooper, 2000). Using streptavidin beads bound to biotinylated T cell surface receptors, Wulfing and Davis demonstrated that A P C engagement through LFA-1 or CD28 causes active transport of beads to the cell-cell interface, which is mediated by myosin II (Wulfing and Davis, 1998). In addition, LFA-1 transfected into K562 cell line associates with talin (Kim et al, 2003). LFA-1 on human peripheral blood T cells also co-clusters with polymerized actin cytoskeleton,  43  as assessed by fluorescence microscopy, and was detected in the pellet of cell lysate by immunoblot, indicating that LFA-1 associates with the actin cytoskeleton (Pardi et al., 1992). The TCPJCD3 complex may also be associated with actin cytoskeleton. In primary T cell, TCR ligation induces association of tyrosine-phosphorylated CD3£ with cytoskeleton-associated detergent-insoluble pellet fraction (Rozdzial et al., 1995; Caplan et al., 1995). Cytochalasin D was found to block the activation of T cell IFNy production (Valitutti et al., 1995). A recent study with actin-GFP fusion protein revealed that sustained actin dynamics were required for TCR accumulation in the c-SMAC and for efficient T cell proliferation (Tskvitaria-Fuller et al., 2003). Wiskott-Aldrich syndrome patients and WASP" " mice have defective T cell activation and 7  actin polymerization (Snapper et al., 1998; Snapper and Rosen, 1999). V a v l , a regulator of WASP, was shown in a study using V a v l knockout mice to have defective IS formation and T cell activation (Krawczyk et al., 2002). During naive T cell activation, actin polymerization is dictated by LFA-1 whereas microtubule organizing center (MTOC) translocation is determined by TCR engagement (Sedwick et al., 1999). Ezrin, a linker of cytoskeleton with membrane, was found to localize in the p - S M A C together with F-actin after T C R stimulation (Roumier et al., 2001). Das et al. proposed a model that TCR engagement induced accumulation of F-actin and ezrin in the p - S M A C during formation of the IS by T cell-APC interaction (Das et al., 2002). Furthermore ezrin localizes in lipid rafts (Tomas et al., 2002).  44  1.4. Thesis objectives This introduction describes the current pertinent knowledge of LFA-1 regulation, lipid raft microdomains and the IS. At the commencement of this thesis work, the regulation of LFA-1 activation was proposed to involve increases of affinity and avidity. The evidence for the involvement of affinity changes that may occur through conformational modulation came mainly from mAb and mutagenesis studies. Some evidence supporting avidity changes came from mAb that possibly recognized clustered L F A - 1 . Since clustering of GM1 induces LFA-1 activation, lipid rafts may be involved in LFA-1 clustering. However, the concept of lipid rafts and their role in the regulation of LFA-1 was controversial, as discussed above. I set out to investigate the involvement of lipid rafts in the regulation of LFA-1.1 hypothesized that lipid rafts are important for LFA-1 functions. To test this hypothesis, the effects of raft disruption on LFA-1-mediated adhesion of T cell lines and primary splenic T cells are studied in Chapter 3. Chapter 4 describes the physiological significance of lipid rafts and their heterogeneity in the formation of the C T L IS. In chapter 5, the differential role of TCR and LFA-1 in determining the rearrangement of polymerized actin and M T O C in the C T L IS is examined.  45  CHAPTER 2 MATERIALS AND METHODS  46  Materials and Methods 2.1. Animals C57BL/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME) and H Y TCR Tg Rag2 " C57 BL/10 mice were obtained from Taconic Farms (Tarrytown, N Y ) . These mice _/  were bred in the Joint Animal Facility of the B C Cancer Research Centre.  2.2. Cell culture The murine T cell leukemia line EL4, the murine lymphoma line R M A , murine fibroblast L cells were obtained from the American Type Culture Collection (ATCC, Manassas, V A ) . The murine T cell hybridoma line T28 has been described (Takei, 1983). A l l cell lines were maintained in D M E M plus 5% FCS and penicillin/streptomycin (StemCell Technologies Inc., Vancouver, Canada). Splenocytes were harvested from 6-8 week old C57BL/6 mice. Splenic T cells were isolated using a murine T cell enrichment kit, Spin Sep™ (Stem Cell Technologies Inc, Vancouver, Canada). This resulted in 98-99% pure T cells, as assessed by F A C S analysis using a FITC-conjugated anti-CD3 mAb (BD Pharmingen, Mississauga, Canada).  2.3. Antibodies and reagents Rat anti-murine CD18 hybridomas (TIB213 and TIB218) and the mouse anti-D  b  hybridoma (28-8-6S) were from the A T C C . TIB 218 was used in western blots to detect LFA-1 since T cells do not express other 0 2 integrins. The hamster anti-CD 18 (2E6) hybridoma and the mouse anti-rat IgGK hybridoma (TIB 169) were from the A T C C . Rat anti-Thy-1 mAb (clone 3.2) was generated in our laboratory. Its specificity for Thy-1 was confirmed by the binding to the  47  murine lymphoma cell line S49.1 (ATCC TTB28) but not its Thy-1-negative variant S49 (Thy-1a) (ATCC TIB36). Rat anti-CD45 (YE 1/21.2.1) was described previously (Takei, 1983). Purification of these mAbs has been described (Pyszniak et al., 1994). Mouse anti-Lck (3A5) was from Santa Cruz Biotechnology (Santa Cruz, CA) and anti-extracellular signal regulated kinase (Erk) 1/2 (p44/42 M A P Kinase) and anti-phospho-Erkl/2 (phospho-p44/42 M A P Kinase, Thr 202 Tyr 204) were from Cell Signaling Technology (Beverly, M A ) . FITC-conjugated antiCD3e and PE-conjugated anti-CDllc Abs were purchased from B D Biosciences (San Jose, CA). The YN1/1 mAb specific for murine ICAM-1 has been described (Takei, 1985). Mouse antiCD3£ (6B10.2) mAbs were from Santa Cruz Biotechnology. Rabbit polyclonal anti-mouse Lck was a kind gift from Dr. P. Johnson (University of British Columbia, Vancouver, Canada). Antiphosphotyrosine mAb (clone 4G10) was from Upstate, Inc. (Lake Placid, N Y ) and mouse anti-p tubulin mAb was from Chemicon (Temecula, CA). Horseradish peroxidase-conjugated Cholera toxin B subunit (CtxB), FITC-conjugated CtxB, biotin-conjugated CtxB, methyl-|3-cyclodextrin (MCD), filipin TH, water-soluble cholesterol (MCD-cholesterol complex), bovine serum albumin (BSA) fragment V , Triton X 100, Brij 99, Brij 58, Brij 56, Brij 35, CHAPS and protease inhibitors (leupeptin, phenylmethyl-sulfonyl fluoride, aprotinin and pepstatin A) and NaVCU and NaMoCv were from SigmaAldrich (St. Louis, MO). Saponin was obtained from Calbiochem (La Jolla, CA). Murine recombinant soluble ICAM-1 has been described (Welder et al., 1993). Calcein-AM, rhodaminephalloidin, Alexa Fluor 568-, Alexa Fluor 647- and Alexa Fluor 488-conjugated goat anti-rat Ig secondary Ab, Alexa Fluor 568-conjugated streptavidin, Alexa Fluor 568- and Alexa Fluor 488conjugated goat anti-mouse were from Molecular Probes (Eugene, OR). H Y peptide  48  ( K C S R N R Q Y L ) and control randomized H Y peptide ( N Y Q R S L C K R ) were generated by the NAPS facility of the University of British Columbia.  2.4. Cell adhesion assay LFA-1-mediated cell adhesion to immobilized soluble ICAM-1 was assayed as described (Welder et al, 1993; Marwali et al, 2003). Splenic primary T cells, CTLs, EL4 cells or T28 cells were washed with serum-free HBSS and labeled with 1 pg/ml Calcein-AM in HBSS for 10 minutes at 37°C. Labeled cells were then washed with serum-free HBSS and resuspended in HBSS 2% FCS. Flat-bottom microtitre wells (Nalgene Nunc International, Rochester, N Y ) were coated with purified soluble ICAM-1 (10-20 pg/ml in Q.1M sodium bicarbonate buffer, p H 8) by incubating at room temperature (RT) for 60-90 minutes. Unbound soluble ICAM-1 was removed by washing the wells with 100 ul PBS using a multi-channel pipet. Nonspecific sites were blocked using 50 pi of 0.5 mg/ml heat-inactivated (65°C for 30 minutes) B S A in PBS for 30 minutes at RT. Soluble ICAM-1-coated plates were then washed three times with PBS. Labeled cells in HBSS 2% FCS were dispensed to the ICAM-1-coated wells and incubated at 37°C for 30 minutes. Afterwards, non-adherent cells were removed by washing five times with pre-warmed HBSS 2% FCS. The remaining bound cells were measured using a CytoFluor 2300 fluorimeter (Millipore, Bedford, M A ) . The percentages of cell adhesion were determined by the ratio of the fluorescence values of post-wash over pre-wash after subtracting background fluorescence values. For stimulation of L F A - 1 , cells were pre-incubated with 50 ng/ml P M A for 30 min at 37°C prior to binding to immobilized soluble ICAM-1. As a specificity control, 50 pi of 30 pg/ml anti-LFA-1 mAb (TIB 213) was also added together with P M A . For cholesterol depletion,  49  various amount of M C D was added to cells in HBSS 50 m M HEPES at the same time that they were treated with P M A .  2.5. Generating single cell suspension of splenocytes Spleens were isolated from mice under sterile condition. On a 100 mm petri dish, the spleen was crushed with the plunger of 3 ml syringe. Cells were recovered and were resuspended in D M E M 5% FCS and passed through a #21 G needle. Cells were centrifuged and the pellet was resuspended in 0.8% Ammonium Chloride 0.1 m M E D T A to lyse red blood cells. Splenocytes were then washed and resuspended in medium.  2.6. Antibody purification TIB 213 hybridoma was grown in D M E M 5% FCS and the supernatant was passed through a 5 ml Hi Trap affinity protein G column (Amersham Biosciences, Baie d'Urfe, Quebec, Canada). The first column was washed with three column volumes of start buffer (20 m M Naphosphate, pH 7.0). The column was equilibrated with at least two column volumes start buffer. Subsequently the hybridoma supernatant was run through the column using a P-lperistaltic pump (Amersham Biosciences, Baie d'Urfe, Canada) at 1 ml/minute. Next the column was washed with five column volumes of start buffer. The mAb was eluted with three column volumes of elution buffer (0.1 M glycine-HCl, pH 2.7) and collected into tubes containing few drops of 1 M Tris-HCl, pH 9.0. The column was later washed with 20% ethanol to preserve the column.  2.7. Purification of soluble ICAM-1 50  Soluble ICAM-1 was purified from the supernatant of NS-1 cells transfected with c D N A encoding soluble recombinant ICAM-1 (Welder et ah, 1993) by using anti-ICAM-1 mAb (YN1/1.7.4) coupled to Affi-gel 10 (BioRad, Hercules, CA). The supernatant was passed through an affinity column containing YN1/1.7.4 coupled to Affi-gel 10. After washing with PBS, soluble ICAM-1 was eluted with 0.1 M Na-carbonate buffer (pH 11.4) containing 0.8% NaCI. Then fractions were collected into tubes containing 0.5 M sodium phosphate buffer (pH 7.5).  2.8. Sucrose gradient centrifugation and Western blotting Cells (5 x 10 ) were washed twice with PBS and lysed in 1 ml ice-cold lysis buffer 7  containing 20 m M tris (hydroxymethyl) aminomethane (Tris)-HCl (pH 7.2), 150 m M K C l , various concentrations of non-ionic detergents and protease inhibitors. The cell lysates were sheared by five successive passages through #26 gauge hypodermic needles, then mixed with an equal volume of 80% sucrose (w/v) in ice-cold lysis buffer without detergent and transferred to SW41 centrifuge tubes. The samples were then overlaid with 6 ml of 30% sucrose and 3.5 ml of 5% sucrose and centrifuged (Beckman, Palo Alto, CA) at 200,000 x g for 18 hr. Protease inhibitors, 0.1 M NaVCXt and 1 M N a M o 0 were added to the gradients. A l l the procedures were 4  done at 4°C. Following centrifugation, 8 fractions of 1.5 ml each were collected, starting at the top of the gradient. Fractions 2 and 3 corresponding to the 5-30% sucrose interface were referred to as low-density fractions. Fractions 7 and 8 were referred to as high-density fraction. The materials at the bottom of the tube were referred to as the pellet. Aliquots of each fraction were boiled in SDS-PAGE sample buffer (non-reducing conditions), loaded onto SDS-PAGE and transferred to Polyvinylidene Fluoride transfer membrane (Pall Gelman Lab, Ann Arbor MI). Proteins on the blots were detected by specific Abs and visualized by chemiluminescence using  51  an ECL-system (Amersham Biosciences, Piscatway, NJ) according to the manufacturer's protocols. For mitogen activated protein (MAP), kinase analysis, 10 cells were treated with or 6  without 50 ng/ml P M A and with or without 10 m M M C D for 30 min at 37 C, and then subjected to SDS-PAGE and Western blotting.  2.9. Cholesterol depletion, sequestration and reconstitution For cholesterol depletion, cells were treated with various concentrations of M C D in HBSS containing 50 m M N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid (HEPES) for 30 min at 37°C. For cholesterol sequestration, cells were treated with various concentrations of filipin III in HBSS containing 0.2% B S A and incubated at 37°C for 1 hour. To reconstitute the cholesterol of MCD-treated cells, 60 pg/ml water soluble cholesterol in HBSS containing 0.2% B S A was added to the cells and incubated for 30 min at 37°C.  2.10. Transfection L cells were transfected with mouse ICAM-1 c D N A in the p B C M G S expression vector and selected with G418 (Lian et al., 1999). L cells were also co-transfected with a genomic D N A fragment encoding M H C class I D (gift from Dr. Wilfred Jefferies, University of British b  Columbia) and the pRC42 plasmid (gift from Dr. Robert Kay, Terry Fox Lab., Vancouver, Canada) carrying hygromycin-resistance gene and selected with hygromycin. For co-expression of D and ICAM-1, D -transfected L cells were further transfected with ICAM-1 and selected b  b  with both G418 and hygromycin. A l l the transfections were done using Lipofectamine Plus (Invitrogen, Burlington, ON, Canada) according to the manufacturer's instructions. The  52  transfected cells were stained with appropriate Abs and sorted to establish L cell lines expressing high levels of D and/or ICAM-1. b  2.11. Preparation of C T L Dendritic cells were isolated from the spleens of male C57 BL/6 ( 6 - 8 weeks old) mice using anti-CD Mc-PE as the primary Ab then separated using the Easy Sep™ P E selection kit from StemCell Technologies (Vancouver, Canada). Splenocytes and lymph node cells (6 x 10 /ml) from female H Y TCR transgenic Rag 2 " C57 BL/10 mice were stimulated with 6  7  irradiated (30 Gy) dendritic cells (6xl0 /ml) in 2.5 ml media in the presence of 20 U/ml IL-2 in 5  RPMI media supplemented with 10 % fetal calf serum, 2 m M L-glutamine, 5 x 10" M p5  mercaptoethanol and Penicillin/Streptomycin for 5-6 days in 12 well culture plates.  2.12. Cytotoxicity assay R M A cells were pulsed with 1 p M H Y peptide at 37°C for 2-3 hrs, washed and incubated with 100 uCi o f  51  Cr-sodium chromate (NEN, Boston, M A ) for 1 hr at 37°C. C T L were either  treated with M C D or not, or treated with M C D and replenished with cholesterol. After washing twice, the radiolabeled target cells and effector cells were mixed at the indicated E:T ratio in RPMI 0.2% B S A in a final volume of 200 pi in U-bottomed 96-well plates and incubated for 4 hours at 37°C. Culture supernatants (100 pi) were collected and C r release was measured using 5 1  gamma scintillation counter. The percentages of specific lysis was calculated as % lysis = ((cpm sample)-(cpm spontaneous release))/((cpm total)-(cpm spontaneous release)) x 100. Each cytotoxicity assay was done in triplicate.  53  2.13. Flow cytometry Cells were directly stained with FITC-conjugated anti-CD18 mAb 2E6 (10 ng/ml), FITCCTxB (15 u.g/ml) or FITC-conjugated anti-CD3e (5 pg/ml) for 30 min on ice. For the staining of CD45 and Thy-1, cells were incubated with the appropriate hybridoma supernatants for 30 min on ice, washed twice, then stained with 5 pg/ml FITC-conjugated anti-rat IgK (TLB 169) mAb. Stained cells were washed with HBSS containing 2% FCS and 0.1% sodium azide and analyzed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA). For filipin staining (Muller et ah, 1984), cells were treated with 12.5 pg/ml of freshly dissolved filipin in HBSS at 4°C in the dark for 2 h and analyzed using a FACStar Plus (Becton Dickinson) equipped with a 360-nm Coherent Enterprise Argon laser. Emissions were collected via a 640-nm dichroic long pass filter with 424/44-nm band pass filter (Hassall, 1992; Hassall, 1995). Single cell suspensions of mouse fibroblast L cells were prepared by incubation of monolayer cultures with 5 m M E D T A in HBSS and stained with rat anti-mouse ICAM-1 mAb (10 u.g/ml), mouse anti-D mAb (10 u,g/ml) or both for 30 min on ice. After washing, FITCb  conjugated anti mouse-IgG and Alexa Fluor 647-conjugated anti rat-IgG secondary Abs were added and incubated for an additional 30 min. Stained cells were washed and analyzed by a FACSCalibur flow cytometer (Becton Dickinson). The purity of C T L generated in vitro was assessed by staining with FITC-conjugated anti-CD3e Ab.  2.14. Fluorescence microscopy T 28 cell line and mouse primary T cells were stained at 4°C with anti-LFA-1 mAb (TIB 213), anti-Thy-1 Ab, 15 u.g/ml FITC-CTxB and 12.5 fig/ml filipin where indicated. The  54  secondary Ab staining for L F A - 1 , Thy-1 and CD45 were with Alexa Fluor 568-conjugated goat anti-rat Ig. Capping was induced by incubation at 37 C for 30 min, followed by fixation with 4% formaldehyde. Cells were cytospun onto poly-D-lysine coated glass cover slips. Then samples were analyzed by confocal microscopy (BioRad Radiance 2000 Multiphoton, Hercules, C A ) with a 60x objective lens. The lasers used were K r and Mai Tai T i Sapphire. Filipin UI was excited with a multiphoton laser at 779 nm and the emission filter was H Q 450/80 with a B G G 22 blocking filter. FITC was excited by 488 nm and the emission filter was HQ 515/30. Alexa 568 was excited by 568 nm and the emission filter was HQ 600/50. Signals were collected sequentially to avoid bleed through. For co-capping experiments of CTL, cells were incubated at 4°C with 10 pg/ml antiLFA-1 mAb (TIB 213), 10 pg/ml FITC-conjugated anti CD3e Ab, 15 pg/ml FITC-CtxB or 125 pg/ml filipin where indicated. After washing, cells were then incubated with Alexa Fluor 568conjugated, Alexa Fluor 647- or Alexa Fluor 488-conjugated goat anti-rat Ig for.LFA-1 staining, and capping was induced by incubation at 37°C for 30 min, followed by fixation with 4% formaldehyde. For capping of G M 1 , cells were incubated with CtxB-biotin, washed and stained with Alexa Fluor 568-conjugated streptavidin. The stained cells were crosslinked with rabbit anti-cholera toxin sera (Sigma Aldrich, St. Louis, MO) and incubating at 37°C for 30 min. Cells were cytospun onto poly-L-lysine-coated glass cover slips and analyzed by confocal microscopy as above. For immunological synapse experiments, L cells were grown on gelatin coated coverslips overnight. The following day, C T L were added to the L cells after pulsing with 1 p M H Y peptide and incubated at 37°C for 5, 15 and 20 min at 37°C after brief centrifugation. The cells were then fixed with 4% paraformaldehyde. After washing, the cells were blocked with PBS  55  containing 0.5 mg/ml B S A . The fixed cells were stained with various combinations of FITCconjugated CtxB or biotin-conjugated CtxB, anti-LFA-1, FITC-conjugated anti-CD3e Abs and incubated at room temperature for 1 hr. After washing twice, Alexa Fluor 568-conjugated antirat Ab or Alexa Fluor 568-conjugated streptavidin were added and incubated for 1 hr at RT. The cells were washed twice with HBSS and incubated with 125 pg/ml filipin UI on ice for 1 hr. After washing, the stained cells were mounted on Vectashield (Vector, Burlingame, CA). Conjugates were examined with deconvolution microscope (Deltavision, Seattle, W A ) with 0.1pm z steps and 10 iterations for filipin III samples or multiphoton confocal microscopy for samples stained without filipin III (BioRad). Images from deconvolution microscopy were processed using Softworx software program (Deltavision, Seattle, W A ) . Stacks were 3-D reconstructed, and the C T L - L cell interphase was cropped and rotated to obtain a view from the C T L side using the Volocity software (Improvision, Lexington, M A ) . For intracellular staining, CTL-target cell conjugates were permeabilized with 0.5% saponin HBSS 2% FCS, then stained with anti-P-tubulin for M T O C and rhodamine-phalloidin (Molecular Probes, Portland, OR) for F-actin and anti phospho-tyrosine mAb (4G10) for phospho-tyrosine staining.  56  CHAPTER 3 MEMBRANE CHOLESTEROL REGULATES LFA-1 FUNCTION AND LIPID RAFT HETEROGENEITY  The materia] presented in this chapter is essentially as reported in: Marwali, M.R., Rey-Ladino, J., Dreolini, L . , Shaw, D., and Takei, F. (2003). Membrane cholesterol regulates L F A - 1 function and lipid raft heterogeneity. Blood 102, 215-222.  57  3.1. Introduction LFA-1 plays a critical role in the inflammation process. Chemokines released during inflammation activate LFA-1 so that it can mediate firm adhesion of leukocytes to endothelial cells and induce their migration and extravasation from the blood vessels into the tissues or lymphoid organs (Springer, 1994). The binding of LFA-1 on T cells to its ligand ICAM-1 has been shown to provide a second signal for T cell activation (Chirathaworn et al, 2002). LFA-1 also participates in immune responses by forming IS together with the T C R and other costimulatory molecules when T cells interact with A P C (Monks et al., 1998; Grakoui et al., 1999; Dustin and Cooper, 2000). Although resting leukocytes constitutively express L F A - 1 , they do not readily adhere to cells expressing its ligands as the adhesive functions of LFA-1 are regulated by cell activation. Through the process of inside-out signaling, intracellular activation signals convert low-avidity LFA-1 on resting leukocytes into an active form capable of mediating cell adhesion. This conversion does not require an increase in the cell surface expression of L F A - 1 (Dustin and Springer, 1989; Larson and Springer, 1990). The prevailing theories to explain this phenomenon are that LFA-1 undergoes a conformational change upon cell activation or that LFA-1 is redistributed at the cell surface (van Kooyk et al., 1989b; Diamond and Springer, 1994). Evidence for the former comes from the existence of Abs that recognize epitopes on activated LFA-1 (Keizer et al, 1988; Binnerts and van Kooyk, 1999; Beals et al, 2001; L u et al, 2001a). Other studies suggest that clustering of LFA-1 leading to multivalent interaction between LFA-1 and ICAM-1 may be important (van Kooyk et al, 1999; van Kooyk and Figdor, 2000). Lipid rafts are highly organized microdomains of the plasma membrane. They have high content of cholesterol, gangliosides, sphingolipids and phospholipids with long saturated fatty  58  acyl chains and are resistant to extraction with cold non-ionic detergents. Lipid rafts are isolated as detergent-insoluble glycolipid-enriched membranes in low-density fractions of sucrose gradient centrifugation (Simons and Toomre, 2000). Molecules such as GPI-anchored proteins and acylated proteins are known to partition into lipid rafts. Cholesterol plays an important role in maintaining lipid rafts in a liquid ordered phase, whereas the rest of the membrane, which contains phosphatidyl ethanolamine and phosphatidyl choline combined with lesser amount of cholesterol, exists in a liquid disordered phase. Depletion of cholesterol by M C D or sequestering cholesterol out of lipid rafts with filipin is commonly used to disrupt lipid rafts (Cherukuri et al., 2001). Many cell surface receptors and intracellular signaling proteins are thought to localize in lipid rafts. However, direct visualization of these molecules in lipid rafts is difficult, because rafts are thought to be only 50-100 nm in diameter, well below the resolution of optical microscopes (Jacobson and Dietrich, 1999; Brown, 2001). Therefore, the detection of receptors and signaling molecules in the low-density fractions of sucrose gradient centrifugation, combined with raft-disrupting reagents such as M C D and filipin, are often used to determine the localization of receptors in lipid rafts. Crosslinking of various cell surface receptors can induce the fusion of lipid rafts to form clusters large enough to be visible by fluorescence microscope upon staining with fluorescence-conjugated CTxB, which binds to ganglioside G M 1 . Colocalization of molecules with GM1 may be used as an indication that the molecules associate with lipid rafts. However, recent studies have suggested that there is heterogeneity among lipid rafts. For example, in human peripheral T lymphoblasts, cholesterol extraction disrupts lipid rafts that contain Lck, CD4 and T C R ^ but not those containing L A T (Gomez-Mouton et al., 2001;  59  Schade and Levine, 2002). Upon TCR crosslinking these different rafts are thought to coalesce to facilitate T cell activation signaling. The involvement of lipid rafts in LFA-1 regulation is still controversial. LFA-1 on mouse thymocytes was shown to associate with lipid rafts isolated with 1% Triton X-100, and crosslinking of ganglioside GM1 with CTx induces the activation of L F A - 1 (Krauss and Altevogt, 1999). In contrast, confocal microscopic analysis of transfected L F A - 1 expressed in the human T cell line Jurkat showed that LFA-1 does not associate with rafts unless it is activated by M n  2 +  or if the I-domain is deleted from LFA-1 (Leitinger and Hogg, 2002). Shamri  et al. also reported that cholesterol extraction by M C D does not inhibit LFA-1-mediated adhesion of human peripheral blood T cells to ICAM-1 (Shamri et al., 2002). Thus, studies with different cells and techniques have resulted in conflicting results regarding the association of LFA-1 with lipid rafts and its functional significance. Here we show that cholesterol depletion or sequestration strongly inhibits LFA-1-mediated adhesion of murine T cells. Although cholesterol is thought to be an essential component of lipid rafts in general, our results suggest that lipid rafts of primary T cells are heterogeneous and that some lipid rafts seem to contain little cholesterol. LFA-1 is detected in a subset of lipid rafts that are cholesterol-rich and sensitive to M C D treatment.  60  3.2. Results 3.2.1. MCD disrupts lipid rafts and inhibits LFA-l-mediated adhesion of T cell lines To determine whether lipid rafts have a role in LFA-1 activation, the murine T cell leukemia line EL4 and the T cell hybridoma line T28 were treated with M C D to remove membrane cholesterol, and its effect on LFA-l-mediated cell adhesion to ICAM-1 (CD54) was tested. EL4 cells showed a low level of adhesion to ICAM-1, but upon stimulation with P M A they readily adhered to ICAM-1 immobilized on a plastic surface (figure 3.1a, left panel). This adhesion was mediated by LFA-1 as indicated by almost complete inhibition by an anti-LFA-1 mAb. M C D inhibited LFA-l-mediated T cell adhesion in a dose dependent manner. M C D at these concentrations did not cause cell death as determined by trypan blue staining. MCD-treated cells strongly adhered to ICAM-1 when treated with 2mM MnCl2, which binds to and directly activates L F A - 1 , indicating that the LFA-1 on MCD-treated cells was functional, but M C D treatment inhibited inside-out activation of L F A - 1 . Similar results were obtained with T28 cells (figure 3.1a, right panel). Flow cytometric analysis of MCD-treated cells showed that M C D had no effects on the expression levels of LFA-1 (figure 3.1b). The levels of the ganglioside GM1 and the GPI-anchored protein Thy-1, which together with cholesterol have been considered to be components of lipid rafts, were also unaffected by M C D treatment. Thus, lipid rafts are important for LFA-1 function. To determine whether LFA-1 on T cell lines associates with lipid rafts, EL4 cells were solubilized with different percentages of Triton X-100 on ice and fractionated by sucrose gradient centrifugation at 4°C. Under these conditions, lipid rafts are detergent-insoluble and recovered in low-density fractions (fractions 2 and 3 in figure 3.1c, left side). As expected, GM1 and Thy-1 are detected in the low-density fractions (see below). It should be noted that LFA-1 is  61  the only 0 2 integrin on these cells, and no other leukocyte integrins are detected by anti-CD 18 mAb on T cells. Western blot analysis of each fraction using anti-CD18 mAb detected no LFA-1 in the low-density fractions when 1% Triton X-100 was used. However, when the concentration of Triton X-100 was lowered to 0.05%, which is thought to allow better preservation of lipid rafts (Langlet et al., 2000), the majority of CD 18 was recovered in the low-density fraction (figure 3.1c). Similar results were also obtained with T28 cells (fractions 2 and 3 in figure 3.1c, right side). Triton X-100 at less than 0.05%, i.e. 0.01%, caused incomplete cell lysis since almost all of the C D 18 was found in the pellet. These results suggest that LFA-1 on these T cell lines associates with lipid rafts, but that the association is disrupted by high concentration (>0.05%) of Triton X-100. To confirm that M C D treatment disrupts lipid rafts of T cell lines, EL4 cells treated with M C D were analyzed by sucrose gradient centrifugation and Western analysis. As shown in figure 3.1d, all the molecules in the low-density fractions, including Thy-1, GM1 and L F A - 1 , were shifted to detergent-soluble high-density fractions by M C D treatment, whereas CD45 remained in the high-density fractions regardless of M C D treatment. Without M C D treatment 95% of LFA-1 was in low density fractions and after treatment with M C D the percentage of LFA-1 in low density fractions was decreased to 28% (as measured by densitometry). Similar results were obtained with T28 cells (figure 3.1d, right), from 54% without M C D treatment to 16% after treatment. Therefore, M C D treatment indeed disrupts lipid rafts of the murine T cell lines EL-4 and T28, and the association of LFA-1 with lipid rafts appeared to be important for its functions.  62  Figure 3.1.  T28  EL4 |  cn c Y  | unstimulated  mm  60  I  PMA  I PMA+anti-LFA-1  40  JD o  0  2.5  5  7.5  M C D (mM)  10  10 +  M  BSA  0  2.5  5  Fractions  Triton 2 3 4  5  (-)  6  8  Thy-1  CD18  —  *  T28  4 5 6 7 8 P  --  1  2  3  «  4  5  6  7  8  P  mwm  mm  —  1  —  +  —  •  —  CD45  +  63  2  M  BSA n  Fractions 3  4  5  6  7  8  P  :  3»  + —  GM1  —  + —  Thy1  3  +  '  0.01%  EL4 1 2  P  >mmm  mm  d  20 20  LFA-1 7  mm  G M 1 LFA-1 C D 4 5 C D 3  10  M C D (mM)  b  Ctrl  7.5  n  _  •  •  s  Figure 3.1. Disruption of lipid rafts and inhibition of LFA-1 activation by M C D treatment. (a) EL4 cells (left panel) or T28 cells (right panel) were incubated with (solid bars) or without (open bas) 50 ng/ml PMA in the presence of the indicated concentrations of MCD in serum free HBSS and incubated at 37°C for 30 rninutes, and their adhesion to immobilized soluble ICAM-1 was analyzed. Cells treated with/without PMA, 10 mM or 20 mM MCD and 2 mM MnCl were also tested (shown as 10+Mn and 20+Mn). PMAactivated cells blocked with anti-LFA-1 were tested as specificity control (gray bars). For control cell adhesion, BSA was immobilized in place of ICAM-1. The results are representative of five independent experiments, each done in triplicates, (b) FACS analysis of control (-) and 10 mM MCD treated (+) EL4 cells. Ganglioside GM1 was stained with FITC-conjugated CTxB. All other molecules were stained with appropriate mAb with secondary FITC-conjugated Abs. Ctrl shows unstained control, (c) EL4 (left) and T28 (right) cells were solubilized with the indicated concentrations of Triton X-100 and subjected to sucrose gradient centrifugation. Proteins in the sucrose gradientfractionswere separated by SDS-PAGE and CD 18 was detected by Western blotting. Fractions 2 and 3 are low-densityfractionsand contain lipid rafts whereasfractions5-8 are high-density fractions, and P indicates pellet, (d) EL4 cells were either treated (+) or not (-) with 10 mM MCD as in (a), solubilized with 0.05% Triton X-100, subjected to sucrose gradient centrifugation and analyzed by Western blotting for the indicated molecules. The numbers indicate sucrose gradientfractions.Fractions 2 and 3 are low-densityfractionscontaining lipid rafts. CD 18, Thy-1 and CD45 were detected by specific mAb and horseradish peroxidase-conjugated secondary anti-rat Ig Ab. GM1 was detected by horseradish peroxidase-conjugated CTxB. 2  64  3.2.2. MCD inhibits LFA-1 on primary T cells but does not disrupt Triton X-100-insoluble lipid rafts  To extend the above findings to freshly isolated primary T cells, the effect of M C D on LFA-1 of splenic T cells was tested. Similar to the above results with T cell lines, M C D inhibited LFA-1-mediated adhesion of PMA-activated splenic T cells in a dose dependent manner, and the inhibition was overcome by manganese treatment (figure 3.2a). Flow cytometric analysis confirmed that M C D treatment did not alter the expression levels of LFA-1 or other molecules tested (figure 3.2b). However, sucrose gradient centrifugation analysis showed that LFA-1 on splenic T cells differed from that on T cell lines, as no LFA-1 on splenic T cells was detected in the detergent-insoluble low-density fractions isolated with 1% or 0.05% Triton X-100 (figure 3.2c). Activation of LFA-1 on primary T cells with P M A - or Mn -treatment for 30 min or by 2+  CD3 crosslinking for 3 days or 1-week or Con A stimulation for 1 day failed to induce the association of LFA-1 with rafts (figure 3.2d). Triton X-100 at concentrations lower than 0.05% failed to solubilize splenic T cells. Furthermore, quite unexpectedly, sucrose gradient centrifugation analysis showed that the treatment of splenic T cells with M C D did not change the distribution of any of the molecules tested, including GM1 (83-99% in raft fractions as measured by densitometry) and Thy-1 (100%) (figure 3.2c). The Src family of protein tyrosine kinase 5 6 , which was found in rafts isolated with 0.05%, but not with 1% Triton X-100, remained in lck  P  rafts fractions with M C D treatment. CD45 was found to be outside of rafts regardless of Triton X-100 concentrations or M C D treatment (figure 3.2c).  65  Figure 3.2.  a ^ n  unstimulated PMA ' PMA+oc-LFAl  LLILLLLUU (+)  0  2.5  5 7.5 10 10+ BSA M C D (mM) M  GM1  Ctrl  n  GM1  0 . 0 5 % Triton X-100  +  —  —  CD45  d  1 2 3 4 5 6 7 8  ———  +  —  _  P  — «  LFA-1  —  <M ,  1 week  _  —  —  • » «•  — •  ——  +  mm  -  —  Thy-1  CD3 Thy-1  3 days  _ Lck  LFA-1 CD45  T r i t o n - X 0.05%  1 % Triton X-100  Fractions  OUlo  LUUUUU  •  +  -- -  ConA  mm •»  -  +  PMA  M  OTV  (flp •  Figure 3.2. Effects of M C D treatment on primary T cells, (a) Purified splenic T cells (>98% CD3 ) were treated with the indicated concentrations of M C D and their adhesion to immobilized ICAM-1 was analyzed as in Fig. 3.1a. Open bars represent no P M A stimulation, solid bars represent P M A (50 ng/ml) stimulated cells and gray bar represents P M A stimulation with anti LFA-1 blocking A b (TIB 213). Cells treated with 10 m M M C D and 2mM M n C l (10+Mn) were included. B S A instead of ICAM-1 was used as control. The results are representative of four independent experiments, each done in triplicates, (b) Purified splenic T cells treated (+) or not (-) with 10 m M M C D were stained for the indicated cell surface molecules and analyzed by FACS as in Fig. 3.1b. (c) Splenic T cells were treated with or without (-) 10 m M M C D , lysed with 1% or 0.05% Triton X-100, subjected to sucrose gradient centrifugation, and indicated molecules in each fraction were detected by Western blotting as in Fig. 3. ld. (d) Splenic T cells were grown in anti CD3e A b coated culture plate with 20U IL-2 for 3 days and 1 week, then lipid rafts were isolated as in figure 2c using 0.05% Triton X-1000 (upper panels). Lipid rafts were isolated as in 2c, after stimulation with 2mM M n C l (Mn) and P M A stimulation (PMA) for 30 minutes at 37°C and ConA (ConA) stimulation. (-) represents no treatment control. +  2  2  66  3.2.3. Specificity of M C D treatment The above results showed that LFA-1 on splenic T cells does not seem to associate with lipid rafts isolated with Triton X-100 and that M C D does not seem to disrupt lipid rafts of splenic T cells. Nevertheless, M C D treatment profoundly inhibited LFA-l-mediated T cell adhesion to ICAM-1. To determine whether the effects of M C D treatment was solely due to removal of cholesterol from the membrane or whether it is due to unrelated effects of M C D , we first confirmed that M C D indeed removed cholesterol from the membrane of T cells. Filipin III was used to stain cholesterol of splenic T cells. Filipin is a fluorescent polyene antibiotic from Saccharomyces filipinensis and it forms multimeric globular complex with cholesterol in the cell membrane (Hassall and Graham, 1995). Flow cytometric analysis showed that treatment of primary T cells with M C D reduced the staining with filipin to approximately 50% of control cells (figure 3.3a), indicating that M C D indeed patially removed cholesterol from the membrane of primary T cells. If the inhibition of LFA-1 by M C D treatment was solely due to cholesterol depletion, it should be reversed by restoring the cholesterol level of the cell membrane (Gomez-Mouton et al., 2001). To test this, water-soluble cholesterol was added to MCD-treated splenic T cells, and whether the inhibition of LFA-1 by M C D could be reversed by cholesterol reconstitution was examined. As seen in figure 3.3b, left, LFA-1 dependent adhesion of splenic T cells to ICAM-1, induced by P M A treatment, was strongly inhibited by 10 m M M C D . Addition of water-soluble cholesterol to MCD-treated splenic T cells effectively reversed the inhibitory effects of M C D . Similar results were also obtained with EL4 cells (figure 3.3 b, right). To confirm that M C D does not inhibit intracellular signaling induced by P M A , the effects of M C D on PMA-induced activation of the M A P kinase pathway were tested.  67  Figure 3.3.  FUJFTN  Fluorescence Intensity  b  I  I unstimulated  PMA  I—I  PMA+anti-LFA-1  ^  PMA + MCD + Cholesterol (60 ug/ml)  PMA MCD 40 kDa •  40 k D a ^  +  ^  +  PMA+MCD  + + pMAPK  MAPK  Figure 3.3. Specificity of M C D treatment, (a) Splenic T cells were treated with 10 m M M C D (filled histogram) or not (open histogram with solid line), and cholesterol in the plasma membrane was stained with 12.5 pg/ml filipin and analyzed by FACS. The open histogram with broken line shows autofluorescence of unstained splenic T cells, (b) Splenic T cells (left) and EL-4 cells (right) were treated with 10 m M M C D , and after washing away M C D , they were incubated with water-soluble cholesterol (60 pg/ml) at 37°C for 30 min for cholesterol reconstitution. The treated cells were analyzed for L F A 1-mediated adhesion to ICAM-1 as in Fig. 3.1a. Open bar represents unstimulated cells (unstimulated), solid bar represents P M A stimulation (PMA), gray bar represents P M A stimulation and anti LFA-1 A b blocking (PMA + anti-LFA-1), horizontally striped bar represents 10 m M M C D treatment after P M A stimulation (PMA + MCD) and diagonally striped bar represents P M A stimulation and M C D treatment followed by cholesterol reconstitution (PMA + M C D + Cholesterol 60 pg/ ml) (c) Splenic T cells were stimulated with (+) or without (-) P M A in the presence (+) or absence (-) of M C D and the phosphorylation of M A P kinase was analyzed by Western blotting using anti-phosphoErk-1/2 A b (top panel). The same blot was stripped and probed with anti- Erk 1/2 antibody to confirm equal loading of the samples (bottom panel). 68  Phosphorylation of Erk-1/2 was strongly induced by P M A treatment of splenic T cells and it was not affected by M C D treatment (figure 3.3b), indicating that the inhibitory effects of M C D on PMA-induced LFA-1 activation was not due to inhibition of PMA-induced signaling pathways. Taken together, these results indicate that the inhibition of LFA-1-mediated T cell adhesion by M C D is indeed due to depletion of cholesterol, not due to some other unrelated effects.  3.2.4. Filipin inhibits LFA-1 activation Filipin, through its ability to form complexes with cholesterol, has been used to sequester cholesterol in the plasma membrane (Simons and Toomre, 2000). We examined whether sequestering cholesterol in the plasma membrane by filipin treatment has similar effects on L F A 1 as those of cholesterol depletion by M C D treatment. Cell adhesion assays showed that P M A induced LFA-1 activation on EL-4 cells and splenic T cells was inhibited by filipin in a dose dependent manner (figure 3.4a). Inhibition of LFA-1 on EL-4 cells required four-fold higher concentration of filipin (0.4 mg/ml versus 0.1 mg/ml) than that for primary T cells, presumably due to higher cholesterol content of EL-4 cells than primary T cells (see below). In the range of 0.2 mg/ml to 0.4 mg/ml of filipin, the inhibition of LFA-1 was dose dependent (figure 3.4a). Sucrose gradient centrifugation analysis of Triton X-100-solubilized EL4 cells showed that cholesterol sequestration by filipin disrupted lipid rafts, and LFA-1 and Thy-1 in the low-density detergent-insoluble fractions were shifted to the high-density fractions (figure 3.4b, left panel). With primary T cells, similar to M C D treatment, filipin treatment did not significantly change the distribution of any of the molecules tested (figure 3.4b, right panel). These results indicate that the effects of cholesterol sequestering by filipin are similar to those of cholesterol depletion by M C D .  69  Figure 3.4.  EL4  PMA  _  mAb  _  P (mg/ml)  0  f i l i  i n  + _ 0  b  +  +  Primary T cells  +  +  +  +  + 0  0.025 0.05 0.1 0.3 0.4  0  EL4  Thyl  LFA-1  + •  —  + -  +  Lck  m  0  0.025 0.05 0.1  -  Thyl  + -  ~  + GM1  CD45  6 7 8 P  -  +  MI •  mm-.  0  1 2 3 4 5  -  CD45  + + + + + - - -  Primary T cells  1 2 3 4 5 6 7 8 P LFA-1  +  —  m — —  +  mw--  —  m +  Figure 3.4. Effects of filipin on LFA-1. (a) EL-4 cells and primary T cells were incubated with (+) or without (-) P M A in the presence of the indicated amount of filipin, and their adhesion to immobilized ICAM-1 in the presence (+) or absence (-) of blocking anti-LFA-1 antibody was assessed, (b) EL-4 cells and splenic T cells were treated with (+) or without (-) 0.4 mg/ml or 0.1 mg/ml filipin, respectively, and were lysed with 0.05% Triton X-100, subjected to sucrose gradient centrifugation and the indicated molecules in the sucrose gradient were detected by Western blotting. The numbers indicate fractions starting from the low-density fraction. 70  In both cases, lipid rafts are disrupted only in T cell lines but not primary T cells and yet LFA-1 activation is inhibited in both cell types.  3.2.5. Lipid rafts of T cell lines and primary T cells are different The levels of ganglioside GM1 and cholesterol of the plasma membrane of primary T cells and T cell lines were directly compared. GM1 was stained with FITC-CTxB whereas cholesterol was stained with filipin. Flow cytometry analysis of the stained cells showed that splenic T cells are very rich in GM1 and have approximately 10 to 100 fold higher level of GM1 than T28 or EL4 cells (figure 3.5). On the other hand, the level of cholesterol in the plasma membrane of splenic T cells is nearly 10 fold lower than those of the T cell lines.  3.2.6. LFA-1 in primary T cells are found in Brij 35-insoluble and MCD-sensitive lipid rafts The above results suggest that lipid rafts of primary T cells may significantly differ from those of T cell lines, and it is possible that LFA-1 on primary T cells may localize in lipid rafts that are soluble in cold 0.05% Triton X-100 whereas lipid rafts of T cell lines containing LFA-1 are rich in cholesterol and insoluble in Triton X-100. Therefore, we tested other non-ionic detergents. Results with CHAPS, Brij 99, 58 and 56 were the same as that with Triton X-100 (figure 3.6c). In contrast, significant amounts (48-65% as measured by densitometer) of LFA-1 were detected in the low-density fractions of sucrose gradient centrifugation when 1% Brij 35 was used to solubilize primary T cells (figure 3.6a, first row), as confirmed in six independent experiments. The low-density fractions also contained G M 1 , Thy-1 and Lck but not CD45. Furthermore, upon treatment with 10 m M M C D to extract cholesterol,  71  Figure 3 . 5 . Cholesterol  G M 1  Figure 3.5. Comparison of levels of GM1 and cholesterol in the plasma membrane of splenic T cells and T cell lines. Splenic T cells, EL-4 and T28 cell lines were stained with FITC-CTxB that binds to GM1 or with filipin (12.5 pg/ml) that binds to cholesterol and analyzed by FACS. The machine setting for the fluorescence detection was the same for all the cell types to allow direct comparison of the expression levels. Open histograms showfluorescenceof unstained cells and filled histograms show those of stained cells. The results are representative of three independent experiments. 72  Figure 3. 6. LFA-1 1 2 3 4 5 6 7 8 P ST r tr LFA-1 •  r  1 2 3 4 5  mm • • mm  1% Brij 99  •  1% Brij58 Thyl  —  + —  Lck  GM1  CD45  1  1% C H A P S  • -  l % B r i j 56  +  +  —  »  •  1 1 2 3 4 5 6 7 8 P  Figure 3.6. Detection of LFA-1 in Brij 35-insoluble, MCD- andfilipin-sensitivelipid rafts of primary T cells, (a) Primary T cells, untreated (-) or treated (+) with 10 m M M C D , were solubilized with 1% Brij 35 and subjected to sucrose gradient centrifugation. Fractions were blotted with the corresponding antibodies, (b) Primary T cells were either untreated (-) or treated (+) with 0.1 mg/ml filipin, lysed with 1% Brij 35, subjected to sucrose gradient centrifugation and fractions were probed with anti-CD 18 (LFA-1). The results are representative of three independent experiments, (c) Primary splenic T cells were lyzed with the indicated non-ionic detergent then lipid rafts were isolated as in a, then detected in western blot for the indicated molecules.  73  almost 50 % of LFA-1 in the low-density fractions were shifted to the high-density fractions (figure 3.6a, second row). This result was confirmed in three independent experiments. Similarly, M C D treatment reduced GM1 in the low-density fractions by approximately 10% with corresponding increase in the high-density fractions (figure 3.6a, seventh and eighth rows). However, the distribution of Thy-1 and Lck was not significantly disrupted by M C D treatment (figure 3.6a, third to sixth rows). CD45 was found outside rafts regardless of M C D treatment (figure 3.6a, ninth and tenth rows). Filipin treatment shifted LFA-1 not only to soluble fractions but also to the pellet (figure 3.6b), possibly due to formation of multimeric complex of filipincholesterol-LFA-1. These results suggest that some LFA-1 on primary T cells indeed localizes in lipid rafts, and cholesterol depletion with M C D or sequestration by filipin disrupts those containing L F A - 1 . However, lipid rafts containing LFA-1 seem to be different from those containing Thy-1 or Lck. The former are disrupted by M C D treatment whereas the latter are resistant. Experiments using Brij 35 at a concentration of 0.05% resulted in incomplete lysis of cells, making the results hard to interpret.  3.2.7. LFA-1 and cholesterol on primary T cells co-cap To further compare lipid rafts of T cell lines and primary T cells, we examined the codistribution of L F A - 1 , cholesterol and GM1 by confocal microscopy. LFA-1 on primary T cells and T28 cells was crosslinked to induce capping of LFA-1 and the cells were triple stained for LFA-1 (red), cholesterol (blue) and ganglioside GM1 (green). When capping of LFA-1 on primary T cells was induced (figure 3.7a, upper row and 3.7b, row 2), cholesterol co-capped with LFA-1 whereas GM1 was more evenly distributed on the cell surface, suggesting that LFA-1 localizes in cholesterol-rich but GMl-poor rafts. However, cholesterol-rich lipid rafts containing  74  LFA-1 were not totally devoid of GM1 as some GM1 co-localized with L F A - 1 , resulting in yellow-white caps on the merged images. The results shown are representatives of three independent experiments, in which 92± 7% (n=26) of capped cells demonstrated similar features. When the capping of the GPI-anchored protein Thy-1 on primary T cells and T28 cells was induced, both GM1 and most, but not all, cholesterol co-capped with Thy-1 (figure 3.7b and c, row 4), suggesting that Thy-1 localizes in lipid rafts that are different from those containing LFA-1 (see discussion). The results were representative of 3 independent experiments in which all the cells have similar features (n=9). CD45 was used as a control protein that does not localize in lipid rafts. As expected, capping of CD45 did not induce changes in the distribution of cholesterol or GM1 (figure 3.7b, row 6) in any (n=9) of the cells examined. With T28 cells, capping of LFA-1 resulted in co-capping of both GM1 and cholesterol (figure 3.7c row 2), indicating that lipid rafts of T28 cells containing LFA-1 are rich in both. The results are representatives of three independent experiments, in which 94 ± 9% (n=17) of the cells showed similar features. Thy-1 similarly co-capped with both GM1 and cholesterol (figure 3.7c, row 4), whereas no such co-capping was seen with CD45 (row 6). Confocal microscopic analysis of EL-4 cells was difficult due to very low level of GM1 on the surface.  75  Figure 3. .7 a  LFA-1  cholesterol  GM1  merge  Figure 3.7. Confocal microscopic analysis of co-capping of LFA-1 and cholesterol. (a) Splenic T cells (upper row) and T28 cells (lower row) were stained with anti-LFA-1 and Alexa Fluor 568-conjugated goat anti-rat secondary A b (red) and incubated at 37C for 30 min to induce capping of LFA-1. The cells were then stained at 4 °C for cholesterol with filipin (blue) and for GM1 with FITC-CTxB (green), fixed with formaldehyde and analyzed by confocal microscopy. Images of mid level sections of the cells are shown. Merge images are shown in the right most columns. Results are representatives of multiple cells in 3 independent experiments, (b) Primary T cells were either untreated (row 1, 3 and 5) or subjected to Ab crosslinking to induce capping of LFA-1 (row 2), Thy-1 (row 4) or CD45 (row 6). The cells were then stained in three colors for cholesterol (blue), GM1 (green) and either LFA-1, Thy-1 or CD45 (red) and analyzed by confocal microscopy as in (a), (c) T28 cell line was analyzed as in (b). 76  3.3. Discussion We have shown here that cholesterol is important for the activation of the leukocyte integrin L F A - 1 . Cholesterol depletion or sequestration by M C D or filipin treatment, respectively, results in profound inhibition of LFA-l-mediated adhesion of T cell lines and primary T cells to I C A M 1. The inhibition of LFA-1 by M C D and filipin does not seem to be due to their non-specific effects. Pizzo et al. reported that M C D treatment of the human T cell line Jurkat inhibits C a  2+  release from intracellular stores (Pizzo et al., 2002). However, the inhibitory effects of M C D treatment in our study was reversed by cholesterol reconstitution, and M C D treatment did not inhibit PMA-induced M A P kinase phosphorylation. Therefore, inhibition of LFA-1 by M C D is most probably due to depletion of cholesterol and not due to unrelated, non-specific effects. It is generally believed that cholesterol depletion/sequestration disrupts lipid rafts in general (Simons and Toomre, 2000). However, our results showed that lipid rafts of T cells are heterogeneous and not all of them are disrupted by cholesterol depletion. Those isolated from primary T cells with Triton X-100 are not disrupted by M C D or filipin treatment. In contrast, lipid rafts isolated from T cell lines by the same procedure are sensitive to M C D or filipin. Furthermore, LFA-1 is detected in lipid rafts isolated with 0.05% Triton X-100 for T cell lines or with 1% Brij 35 for primary T cells. Importantly, cholesterol depletion with M C D disrupted lipid rafts that contain LFA-1 in both cases. Thus, it seems likely that the inhibition of L F A - l mediated T cell adhesion to ICAM-1 by M C D or filipin treatment is due to disruption of lipid rafts containing L F A - 1 . However, it should be noted that a substantial portion of LFA-1 on primary T cells is found in non-raft fractions, and M C D treatment reduces the amount of LFA-1 in lipid rafts and cholesterol in the plasma membrane by only about 50%. Nevertheless, the same M C D treatment almost completely inhibited LFA-l-mediated T cell adhesion. It is possible that  77  the density of LFA-1 or cholesterol in lipid rafts may have to exceed a critical threshold for the activation of LFA-1 and that treatment with M C D may lower the levels of LFA-1 and/or cholesterol in lipid rafts below the threshold. The precise role of lipid rafts in the regulation of LFA-1 remains to be determined. It is possible that lipid rafts may fuse to form larger rafts upon cell activation and induce clustering of L F A - 1 , which enhances avidity of L F A - 1 . Cholesterol may mediate lipid raft association of L F A - 1 , and dissociating LFA-1 from rafts may impair its ability to cluster and hence disturb its avidity. Our results have demonstrated significant differences between primary T cells and T cell lines. Primary T cells are rich in GM1 but have only small amounts of cholesterol whereas T cell lines have high levels of cholesterol and relatively low amount of G M 1 . Tuosto et al. have reported that GM1 in resting human peripheral blood T cells is stored in the intracellular compartment and mobilized to the cell surface upon cell activation (Tuosto et al., 2001). Therefore, we tested the level of GM1 in murine splenic and peripheral blood T cells by cell surface staining of intact cells as well as intracellular staining of permeabilized and fixed cells with FITC-CTxB. Confocal microscopy of the unfixed cells showed that both splenic and peripheral blood T cells expressed high level of GM1 on the cell surface, and the expression level did not change upon activation with ConA. When the cells were fixed with paraformaldehyde and permeabilized with Triton X as described by Tuosto et al., all the cell surface staining was lost, leaving only weak intracellular staining (figure 3.8). Therefore, we were unable to properly examine intracellular G M 1 . Lipid rafts of primary T cells seem to be rather heterogeneous with respect to their lipid and protein compositions. Most of them seem to mainly consist of GM1 and perhaps only small amount of cholesterol. They are insoluble in Triton X-100 or Brij 35 and are resistant to cholesterol depletion due to low cholesterol content,  78  F i g u r e 3.8.  Surface staining  rermeaDUizea+stamea  fixed+CTxB/FITC  0.1% Tx-100+fixed+CTxB/FITC  F i g u r e 3.8. Intracellular staining caused extraction o f membrane GM1. T cells  were isolated from spleen (upper row) and from intravascular or peripheral T cell (lower row). Then they were either directly stained (left panel) or permeabilized with 0.1% Triton X-100 (Tx-100) (right panel) then stained with CtxB/FITC (green) and DAPI (red).  79  and they contain Thy-1 and Lck but not L F A - 1 . However, some lipid rafts of primary T cells seem to contain higher amount of cholesterol and only a small amount of G M 1 . These cholesterol-rich lipid rafts contain LFA-1 but not Thy-1 or Lck, and they are sensitive to cholesterol depletion. They are also insoluble in Brij 35 but not other detergents tested in our study, including Triton X-100, C H A P S , Brij 99, Brij 98, Brij 58 and Brij 56. On the other hand, lipid rafts of T cell lines seem to have higher amount of cholesterol and substantially less G M 1 and they contain L F A - 1 , Thy-1 and Lck. They are mostly insoluble in Triton X-100 and sensitive to M C D or filipin. Sphingolipids have long saturated acyl chains and thought to be critical for the formation of liquid ordered phase of lipid rafts (Schroeder et al., 1994; Simons and Ikonen, 1997; Langlet et al., 2000). Ganglioside-rich membrane microdomains are not disrupted by M C D treatment (Hansen et al., 2001). The existence of a glycosphingolipid core rich in ganglioside GM1 and GPI-anchored proteins that is resistant to Triton X-100 treatment has also been suggested (Uangumaran and Hoessli, 1998; Waheed et al., 2001). Our finding that the membranes of primary T cells are rich in GM1 and resistant to cholesterol depletion is consistent with this idea. In contrast to primary T cells, T cell lines have high levels of cholesterol and their lipid rafts are susceptible to disruption by M C D or filipin treatment. Results from confocal microscopy also support the idea of the existence of heterogeneous lipid rafts in primary T cells. LFA-1 co-caps with cholesterol but not as much with G M 1 , suggesting that LFA-1 is associated with cholesterol-rich, but low in G M 1 , lipid rafts. GPI-anchored Thyl co-caps with both GM1 and cholesterol, suggesting that Thy-1 localizes in lipid rafts containing both. Based on our current results, we propose a model for lipid raft heterogeneity in primary T cells (figure 3.9), where different rafts are not distinctly separated, but rather they exist as a continuous spectrum, ranging from those consisting of mostly GM1 to those mostly cholesterol.  80  Figure 3. 9.  Figure 3.9. A model for lipid rafts heterogeneity in primary T cells. The solid line represents the GM1 content of lipid rafts and the dashed line represents cholesterol content. Thy-1 and Lck localize in GMl-rich lipid rafts (darker gray bar) while LFA-1 partially localizes in cholesterolrich rafts (lighter gray bar). Solid bars represent lipid rafts insolubility in Triton X-100 (Tx-100) (1% and 0.05%) and Brij 35 (1%). The M C D sensitive bar shows the portion of lipid rafts that is insoluble in 1% Brij 35 and sensitive to M C D treatment. The range of lipid rafts that remains detergent-insoluble in the cold depends on the detergent. A much wider range of lipid rafts seem to be insoluble in Brij35 than in Triton X-100 or other detergents. This model explains why cholesterol, but not most G M 1 , co-caps with LFA-1 whereas some, but not all, cholesterol and GM1 co-caps with Thy-1. The model predicts that the sensitivity of lipid rafts to M C D treatment depends on the cholesterol content of the rafts. In primary T cells, which have relatively low level of cholesterol, only those with the highest level of cholesterol are MCD-sensitive, and LFA-1 seems to be associated with these lipid rafts.  81  The role of lipid rafts in LFA-1 regulation has been controversial. Krauss and Altevogt reported that L F A - 1 on mouse thymocytes is found in lipid rafts isolated with 1% Triton X-100 (Krauss and Altevogt, 1999). However, we failed to detect a significant amount of LFA-1 in lipid rafts isolated from murine thymocytes with 1% or 0.05% Triton X-100 (figure 3.10). It is possible that only a very small proportion of total LFA-1 on thymocytes is in lipid rafts. Leitinger and Hogg reported that LFA-1 transfected into the human T cell line Jurkat does not localize in lipid rafts unless it is activated by M n  2 +  or its I-domain is deleted (Leitinger and  Hogg, 2002). As shown in our current study, T cell lines and primary T cells significantly differ in lipid rafts, and findings with T cell lines do not necessarily apply to primary T cells. Shamri et al. reported that M C D did not inhibit LFA-l-mediated adhesion of chemokine-stimulated human peripheral blood T cells to ICAM-1 (Shamri et ah, 2002) whereas M C D strongly inhibited L F A 1 activation with P M A in our study. This may be due to differences in the inside-out signaling pathways involved in P M A - and chemokine-induced activation of L F A - 1 . The redistribution of LFA-1 on the surface of T cells is thought to be important for the formation of the IS between T cells and antigen presenting cells (Dustin and Chan, 2000). LFA-1 on T cells have been shown to initially form a cluster in the centre of T cell-APC contact point and subsequently move to the periphery forming a ring around the central core of the TCR cluster (Monks et al., 1998; Grakoui et al., 1999). Since many intracellular signaling molecules involved in TCR-mediated signaling are thought to associate with lipid rafts, lipid rafts may play an active role in the formation of the IS. Our results suggest that in primary T cells, LFA-1 and Lck may associate with different subsets of lipid rafts. This may explain why in the IS formation LFA-1 is found in the p-SMAC, while TCR and Lck are in the c - S M A C (Monks et al, 1998; Grakoui et al., 1999). Recently it has been demonstrated that GM1 accumulates in the center-  82  S M A C and it is resistant to M C D treatment whereas peripheral-SMAC, which contains L F A - 1 , seems to be sensitive to M C D treatment (Burack et al., 2002). These results further supported the idea of lipid rafts heterogeneity and its involvement in the IS. Taken together with our data, we predict that cholesterol accumulates in peripheral-SMAC. The relationship between the distributions of L F A - 1 , cholesterol, lipid rafts and the IS requires further investigation. The results presented in this report have important implications for studies on lipid rafts of T cells. We have demonstrated that lipid rafts of primary T cells are quite different from those of T cell lines. Therefore, many studies on lipid rafts using T cell lines will have to be re-examined with primary T cells.  83  Figure 3.10. Thymocyte  1 2 3 4 5 6 7 8 P LFA-1 1% TxlOO  GM1 CD45  LFA-1 0.05% TxlOO  GM1 CD45  Figure 3.10. Most of LFA-1 on thymocytes is detected in high density fractions when extracted with 1% and 0.05% Triton X-100. Thymocytes were isolated from thymus and lyzed with 1% (upper panel) and 0.05% (lower panel) Triton X-100 in ice. Then cell lysates were subjected to sucrose gradient centrifugations. Fractions were collected and probed for the indicated molecules.  84  CHAPTER 4 LIPID RAFTS MEDIATE ASSOCIATION OF LFA-1 AND CD3 AND FORMATION OF THE IMMUNOLOGICAL SYNAPSE OF CTL  The material presented in this chapter is essentially as reported in: Marwali M R , MacLeod M , Muzia D, Takei F. (2004). Lipid rafts mediate LFA-1 and CD3 association and formation of the immunological synapse of C T L . J. Immunol.; In press.  85  4.1. Introduction Lipid rafts are plasma membrane microdomains that are rich in sphingolipids with saturated acyl chains and cholesterol in the outer leaflet (Simons and Ehehalt, 2002). Lipid rafts are thought to be important for cell signaling and membrane trafficking since certain surface receptors and signaling molecules localize in lipid rafts (Dykstra et al., 2003). Recent reports suggest that lipid rafts in T cells do not simply exist as one distinct type of membrane microdomain but rather they may be heterogeneous (Gomez-Mouton et al., 2001; Schade and Levine, 2002; Marwali et al., 2003). On primary resting T cells, lipid rafts can be divided into ganglioside G M l - r i c h and cholesterol-rich domains (Marwali et al., 2003). LFA-1 on resting T cells associates with cholesterol-rich rafts whereas Lck and Thy-1 associate with GMl-rich rafts. The significance of the heterogeneity of lipid rafts in T cell functions is still unclear. The IS is a supra molecular structure that is formed in the contact area between T cells and A P C (Montoya et al., 2002). In the mature IS, LFA-1 forms an outer ring called the pS M A C while the TCR and signaling molecules are in the c-SMAC (Bromley et ah, 2001; Sims and Dustin, 2002; Huppa and Davis, 2003). The mature IS lasts for several hours and is thought to be important for sustained TCR signaling (Freiberg et al., 2002). At the same time, TCR signaling seems to be important for the maintenance of the IS (Wulfing et al., 2002; Lee et al., 2002a; Lee et al, 2002b; Huppa et al, 2003). The IS formed upon CTL-target cell interaction has also been reported. Similar to the IS formed in resting T cell-APC interaction, the C T L IS also has LFA-1 in the periphery with a secretory domain in the center (Stinchcombe et al, 2001; Bossi et al, 2002). However, unlike activation of resting T cells, which requires prolonged activation, CTLs become activated and release cytotoxic granules within minutes following  86  recognition of appropriate targets. Therefore, the activation machinery in CTLs may be preassembled prior to target cell recognition. Lipid rafts accumulate in the IS (Viola et al., 1999a; Burack et al., 2002; Dupre et al., 2002). This has been demonstrated by staining of GM1 with fluorescent CTxB (Viola et al., 1999a; Burack et al., 2002). However, the mechanisms by which lipid rafts accumulate in the IS are unknown. Moreover, lipid rafts are heterogeneous, and some cholesterol-rich rafts of T cells contain little G M 1 . Conversely, cholesterol depletion with M C D , which is often used to disrupt lipid rafts, does not disrupt GMl-rich lipid rafts. Thus, the heterogeneity of lipid rafts complicates our understanding of their role in the formation of the IS. We have examined the role of lipid rafts in formation of the IS of C T L . Our results show that the formation of the IS of C T L is different from that of resting T cells. CD3 on C T L , but not resting T cells, localizes in lipid rafts and associates with L F A - 1 , which is already in its high avidity state and which readily mediates the binding of C T L to target cells. Through the association with L F A - 1 , the TCR/CD3 complex is recruited to the contact site to form the C T L IS, independent of antigen/MHC recognition. Within the IS, L F A - 1 , CD3 and GM1 remains in the periphery whereas cholesterol is more widely distributed.  87  4.2. Results 4.2.1. Lipid rafts are important for cytotoxicity of CTL Lymphocytes from female HY-specific TCR transgenic Rag2* mice were stimulated A  with male dendritic cells to generate C T L specific for the H Y peptide presented by D . The C T L b  thus generated were almost pure (figure 4.1a) and effectively killed the murine lymphoma line R M A (H-2 ) that had been pulsed with the H Y peptide, but not with control randomized peptide b  or with no peptide (figure 4.1b). To examine the role of lipid rafts in cytotoxicity of CTL, we disrupted lipid rafts by extracting cholesterol with M C D (Simons and Toomre, 2000). The maximum concentration of M C D that did not cause cell death of C T L , as determined by trypan blue staining, was 20 mM. At this concentration, M C D strongly inhibited CTL-mediated cytotoxicity (figure 4.1b) and this inhibition was reversed by cholesterol reconstitution, indicating the specificity of M C D treatment. The treatment of C T L with 20 m M M C D removed 50% of membrane cholesterol but did not affect the level of G M 1 , as determined by flow cytometric analysis (figure 4.1e). Therefore, lipid rafts seem to be important for CTL-mediated cytotoxicity. Next we tested whether this inhibition was due to inhibition of L F A - 1 . We have previously shown that LFA-l-mediated adhesion of resting T cells is strongly inhibited by M C D (Marwali et al., 2003). LFA-1 on C T L was significantly different from that on resting T cells. The former seemed to be already in active state and readily mediated adhesion of C T L to I C A M 1 (figure 4.1c), whereas LFA-1 on resting T cells had to be activated to mediate adhesion (figure 4.1d). Furthermore, the adhesion of C T L to ICAM-1 was relatively resistant to M C D . Even with 20 m M M C D , which killed resting T cells, the adhesion of C T L was only partially inhibited,  88  Figure 4.1.  — • - HY peptide —A- HY peptide + MCD - A - HY peptide +MCD+ Choi —D— control peptide — X — no peptide 2.5:1  CD3E  1.25:1  0.6 : 1  E : T ratio  +MCD (mM)  +MCD (mM)  Figure 4.1. Cholesterol depletion inhibits CTX. cytotoxicity. CTL were generated from H-Y TCR Tg Rag 2~'~ mice as described in Material and Methods, (a) CTL were analyzed for purity by flow cytometry. Open histogram shows staining with isotype control Ab and solid histogram shows staining with FITC-conjugated anti-CD3s Ab. (b) Cytotoxicity of CTL was analyzed by Cr-release assay using RMA cells as target. RMA cells were pulsed with HY peptide (solid squares), control peptide (open squares) or no peptide (crosses). Some CTL were treated with 20 mM MCD at 37°C for 30 min (solid triangles) to deplete cholesterol or cholesterol-depleted and reconstituted with 60 pg/ml watersoluble cholesterol (chol) at 37°C for additional 30 min (open triangles), and tested against HY-peptide pulsed RMA cells, (c) CTLs were fluorescence labeled with CalceinAM, treated with the indicated concentration of MCD for 30 min, and then incubated with (solid bars) or without (open bars) 50 ng/ml PMA for 30 min at 37°C in serum-free HBSS. The adhesion of the treated cells to ICAM-1 was determined as described in Materials and Methods. Anti-LFA-1 Ab (Ab; 10 pg/ml) was added to inhibit cell adhesion to confirm LFA-1 dependent the cell adhesion, and BSA coated wells were used as control, (d) Adhesion of primary splenic T cells to ICAM-1 was tested as above. The results are representative of three independent experiments. 51  89  20 m M M C D e  Cholesterol  GM1  Figure 4.1. (e) CTLs with 20 m M M C D (interrupted line histogram) and without M C D treatment (uninterrupted line histogram) were stained with filipin III for cholesterol (left panel) and FITC-CTxB for GM1 (right panel) and analyzed by F A C S . For filipin staining, cells were treated with 12.5 pg/ml of fresh filipin in HBSS at 4°C in the dark for 2 hr and analyzed by a FACStar Plus (Becton Dickinson) equipped with a 360-nm Coherent Enterprise Argon laser. Emissions were collected via a 640-nm dichroic long pass filter with 424/44-nm band pass filter.  whereas LFA-l-mediated adhesion of resting T cells was almost completely inhibited with 10 m M M C D . Therefore, it is unlikely that the inhibition of the cytotoxicity of C T L with M C D was due to inhibition of adhesion of C T L to target cells (Doucey et al, 2001). It is more likely that TCR-mediated signaling events may be inhibited by cholesterol depletion with M C D .  4.2.2. L F A - 1 and CD3 on C T L localize in MCD-sensitive lipid rafts To examine the distribution of LFA-1 and CD3 in lipid rafts, C T L and resting T cells were lyzed with cold non-ionic detergent Brij 35 (1%) followed by sucrose gradient ultracentrifugation, and individual fractions were analyzed by western blotting. The low-density  90  fractions contained detergent insoluble lipid rafts, as indicated by the presence of ganglioside G M 1 , a commonly used marker for lipid rafts. About 60% of L F A - 1 on C T L and 50% on resting T cells were found in the detergent-insoluble low-density lipid raft fractions (figure 4.2). Cholesterol depletion with M C D shifted about 50% of LFA-1 in the raft fractions to detergentsoluble non-raft fractions, indicating that a half of LFA-1 in lipid rafts is MCD-sensitive. Thus, LFA-1 on resting T cells and C T L is similarly distributed in lipid rafts. In contrast, the distribution of CD3 on C T L is significantly different from that on resting T cells. About 30% of CD3 on C T L is found in the low-density raft fractions whereas no CD3 of resting T cells is found in the raft fractions. This is consistent with earlier studies reporting that TCR becomes associated with lipid rafts upon activation by Ab crosslinking (Xavier et al., 1998). M C D treatment almost completely shifted CD3 in the raft fractions to non-raft fractions, indicating that cholesterol is critical for the integrity of the lipid rafts containing CD3. The distribution of GM1 was not affected by cholesterol depletion. CD45, which is not found in lipid rafts, was used as negative control. When Triton X-100 was used in place of Brij35, G M 1 , but not CD3 or L F A - 1 , was found in detergent-insoluble low density fractions, suggesting that the lipid rafts containing CD3 and LFA-1 are soluble in 1% Triton X-100. Thus, as we previously reported with resting T cells (Marwali et al., 2003), the lipid rafts of C T L are likely heterogeneous. Some lipid rafts of C T L contain high amount of GM1 but low in cholesterol and are resistant to cholesterol depletion, whereas those with high amounts of cholesterol but are low GM1 are readily disrupted by M C D treatment. LFA-1 is found in both MCD-sensitive and MCD-resistant lipid rafts whereas CD3 of C T L , but not resting T cells, is in MCD-sensitive lipid rafts that are thought to be cholesterol-rich.  91  Figure 4.2.  Figure 4. 2. LFA-1 and CD3 are found in MCD-sensitive detergent insoluble fractions. C T L (left panel) and resting primary splenic T cells (right panel) were incubated with 20 m M (for CTL) or 10 m M (for splenic T cells) M C D (+) or no M C D (-) as in Fig. 4.1, solubilized with ice cold 1% Brij 35 and subjected to sucrose gradient centrifugation for 18 hr at 4°C. Eight fractions were collected from top to bottom. Detergent resistant light density fractions were recovered in fraction 2-3 , and material at the bottom was referred to as pellet (p). Indicated molecules in each fraction were detected by western blot using anti-CD18 Ab, anti-CD3C Ab, biotinylated CtxB and anti-CD45 A b with anti-rat IgG secondary Abs or streptavidin conjugated with horseradish peroxidase and the E C L chemiluminescence. The results are representative of three independent experiments.  92  4.2.3. LFA-1 and CD3 on CTLs, but not resting T cells, co-cap The above results suggest that some LFA-1 and  CD3E  on C T L may co-localize in lipid  rafts. To confirm this, we induced capping of LFA-1 on C T L by Ab crosslinking and examined the distribution of CD3e. Both LFA-1 and CD3e were evenly distributed on the surface of untreated CTLs (figure 4.3, row 1). Crosslinking of LFA-1 (red) induced the redistribution of virtually all of the LFA-1 to one side of the cell, forming a distinct "cap." This induced redistribution of CD3e (green) to the same site, and the resultant co-distribution of LFA-1 and CD3e formed a yellow cap in the merged image (row 2). This co-capping was seen in 92% ± 5% (n=50) of the cells examined. Conversely, when capping of CD3e was induced by Ab crosslinking, some, but not all, LFA-1 co-capped with CD3e in the majority (82% ± 7 %; n= 50) of the cells examined (row 3). It should be noted that LFA-1 is much more abundant than CD3e on CTLs, and that some LFA-1 did not seem to associate with lipid rafts (see above). Treatment of CTLs with M C D did not affect the capping of LFA-1 induced by anti-LFA-1 mAb crosslinking, but it almost completely (91% ± 4, n=50) inhibited co-capping of C D 3 E with L F A 1 (figure 4.3, row 4). Therefore, lipid rafts seem to be important for the association between LFA-1 and CD3 on CTLs. LFA-1 and CD3 on resting T cells showed very different distributions from those on C T L . Capping of LFA-1 on resting T cells did not cause co-capping of CD3e (figure 4.3, row 6). Similarly, capping of CD3e did not result in co-capping of LFA-1 on resting T cells (row 7). These findings support the notion that LFA-1 and CD3 co-localize in the same membrane microdomains on C T L whereas they are in different microdomains on resting T cells.  93  LFA-1  C D 3e  merge /• """""" "^k  2  co  CD3e 3 LFA-1  ->  4  i  LFA-1  merge  CD3e  merge  f *  LFA-1  '  CD3s  merge  5  CD3s 7  "*  \  6  f  merge  LFA-1 ' /  % J|  Figure 4.3. LFA-1 and CD3 co-cap on C T L but not on resting T cells. C T L (rows 14) and splenic T cells (rows 5-7) were untreated (rows 1 and 5) or incubated with either anti- LFA-1 A b (rows 2 and 6) or anti-CD3s A b (rows 3 and 7) on ice for 30 min. After washing, appropriate secondary Abs were added for cross linking and incubated at 37°C for 30 min. The cells were then fixed with 4 % paraformaldehyde and stained with antiCD3 or anti-LFA-1 Abs where indicated. CTLs were also treated with 20 m M M C D for 30 minutes at 37°C, prior to LFA-1 capping by crosslinking, then fixed and stained for CD3s, (row 4). The stained cells were examined by confocal microscope, and images at the middle sections of the cells were obtained. Each set of images is representative of 50 cells examined in 3 independent experiments. 94  4.2.4. L F A - 1 and CD3 co-distribute with cholesterol and G M 1 on C T L Ganglioside GM1 and cholesterol are two major components of lipid rafts. To examine the association of LFA-1 and CD3 with lipid rafts, we tested co-capping of LFA-1 and lipid rafts. Fluorescent CtxB was used to detect G M 1 , whereas cholesterol was stained with filipin Til, a fluorescent compound derived from Saccharomyces filipinensis that is known to specifically bind to membrane cholesterol (Hassall and Graham, 1995). Before crosslinking of L F A - 1 , GM1 and cholesterol were evenly distributed on the surface of C T L (figure 4.4, row 1). Crosslinking of LFA-1 induced co-capping of most, but not all, GM1 and cholesterol, forming a white cap in the merged image (row 2) with most (92% ± 4%; n=50) of the cells examined. Crosslinking of CD3 also induced its capping, but no detectable co-capping of GM1 and cholesterol was detected (88 ± 6%; n=40) (row 3). It should be noted that the amount of CD3 on C T L is much lower than GM1 or cholesterol, and the area of capped CD3 was not devoid of cholesterol. Capping of GM1 induced co-capping of LFA-1 (row 5) on most C T L (86% ± 5%; n=50). In contrast, CD3 did not co-cap with GM1 (row 7) on most C T L ( 93 ± 3%; n=50) and cholesterol did not co-cap with GM1 (row 9) on most C T L (95% ± 2%; n=50). This is consistent with the notion that lipid rafts of C T L are heterogeneous. It should be noted that the area of capped GM1 is not devoid of cholesterol suggesting that GMl-rich rafts contain a low amount of cholesterol.  95  Figure 4.4. LFA-1  I  1  CD3e  <  G M 1 cholesterol  j 1  *  merge  m  oM  GM1  CD3  J 0 merge  G M 1 cholesterol merge \  1 1 .  J  GM1  cholestero1 merge  3 Figure 4.4. LFA-1 co-cap with both GM1 and cholesterol but CD3 and cholesterol do not co-cap with GM1. C T L were either untreated (rows 1, 4, 6, 8), treated to crosslink LFA-1 (row 2) and CD3e (row3) as in Fig. 4.3 or treated with FITC-CTxB and anti-CTx antibodies to crosslink GM1 (rows 5, 7, 9) for 30 min. They were then fixed and stained for indicated molecules, filipin III (125 pg/ml) was used to stain cholesterol. The stained cells were analyzed by confocal microscopy as in Fig. 4.3. Each set of images is representative of 50 cells examined in 2 or 3 independent experiments. Over 90% of the cells showed similar stainings.  96  4.2.5. LFA-1, CD3 and lipid rafts form the immunological synapse of CTL The above results suggest that LFA-1 and CD3 co-localize in a subset of lipid rafts on CTL. To investigate the physiological significance of this finding, we examined the distribution of these molecules in the IS formed on C T L upon interaction with target cells. We generated TCR-transgenic C T L specific for the H Y peptide presented by D . Murine fibroblast line L cells b  (H-2 ), which lack expression of the LFA-1 ligand ICAM-1, were transfected with ICAM-1 k  alone, D alone or both D and ICAM-1 (figure 4.5a) and used as target cells. Prolonged b  b  incubation (over 30 min) of the C T L with the targets resulted in lysis of L cells expressing D  b  and ICAM-1, pulsed with the H Y peptide, but not a control peptide, whereas those expressing ICAM-1 alone or D alone were not killed. The C T L were centrifuged onto the transfected L cell b  monolayers, incubated for 5, 15 and 20 min, fixed and stained for L F A - 1 , CD3, GM1 and cholesterol. Differential interference contrast (DIC) microscopy showed that the C T L formed tight cell contact with L cells transfected with D and ICAM-1 (figure 4.5b, left panel) or I C A M b  1 alone (right panel), whereas the C T L did not bind to untransfected L cells or those transfected only with D , and they were not analyzed in this study. The distribution of various molecules on b  the C T L at the cell contact sites was further analyzed by deconvolution fluorescence microscopy and 3D reconstruction of the fluorescence images (see Materials and Methods). 18 to 20 images viewed en face from the C T L side were analyzed in blind by two individuals unfamiliar with the study for each time point and CTL-target combination. For all the molecules tested, no difference in their distribution was seen between the ISs formed when C T L bound to L cells expressing ICAM-1 and those with both ICAM-1 and D The distribution of LFA-1 (red) and GM1 (green) b  in the IS was very similar to each other in most of the IS analyzed and often formed yellow ringlike or partial ring distribution in the merged two-color images (figure 4.6).  97  Figure 4.5.  a  L-IC anti-ICAM-1  L-IC+D anu-ICAM-1 „ T-i  '  1  T  b  anti-D  b  ;  Figure 4.5. CTL tightly bind to L cells transfected with ICAM-1 or ICAM-1 and MHC class I D . (a) L cells were transfected with ICAM-1 alone (L-IC) or with both M H C class I D and ICAM-1 (L-IC+D ). The expression of D and ICAM-1 on the transfected cells was analyzed by flow cytometry. Solid histograms show I C A M 1 staining (left and center) or D staining (right) and open histograms show isotype control staining. These cells were used as target for C T L in the IS experiments, (b) L cells transfected with D and ICAM-1 (D +IC-l) or ICAM-1 alone (IC-1) were pulsed with H Y peptide and incubated with HY-specific C T L for 5 min. They were then fixed and stained with filipin III for cholesterol (blue), FITC-CtxB for GM1 (green) and anti-LFA-1 (red). Top pictures show DIC images and bottom pictures show fluorescence staining. L cells express very low amounts of GM1 and are not stained with CTxB but they express high amount of cholesterol and are readily visualized by staining with filipin III. White scale bars represent 10 pm. b  b  b  b  b  b  98  b  On the other hand, cholesterol (blue) distribution was often (but not always) quite different from those of LFA-1 and G M 1 . Figure 4.6 shows representative images of C T L IS where the target was transfected either with ICAM-1 alone (figure 4.6a) or with D and ICAM-1 (figure 4.6.b). A b  similar distribution was observed when the target was transfected with either condition. In some of the ISs, cholesterol (blue) was found in the center whereas LFA-1 (red) and GM1 (green) formed a ring in the peripheral (figure 4.6, row 1). This distribution was observed with about 30% (n=40) of the C T L incubated for 5 min with the targets and was seen more often (about 60%, n=36) with those incubated for 15 or 20 min. Staining with CD3e (green), GM1 (red) and cholesterol (blue) also showed the same patterns of distribution (figure 4.6, row 2). CD3 and GM1 co-localized, often forming yellow rings (lower row), while cholesterol (blue) showed mostly different patterns. Co-staining with CD3 and LFA-1 further confirmed the co-distribution of CD3 and LFA-1 in the IS (figure 4.6, row 3). The distribution of these molecules on the C T L surface at non-cell contact sites was also examined as controls and they were found to be randomly distributed (figure 4.6, row 4 ). We also used the murine T cell line R M A (Ff-2 ) b  pulsed with the H Y peptide as a target and examined the distribution of L F A - 1 , CD3e and GM1 on C T L , and the results were identical to those in figure 4.6 (figure 4.7). LFA-1 and CD3e were distributed in the p - S M A C and upon IS analysis they were co-distributed.  99  Figure 4.6. LFA-1, CD3 and lipid rafts form CTL immunological synapse. L cells transfected with ICAM-1 (a) or D and ICAM-1 (b) were pulsed with H Y peptide and incubated with CTL. After incubation for 5, 15 or 20 min at 37°C cells, the cells were fixed and stained for GM1, L F A - 1 , CD3 and cholesterol. The stained cells were analyzed by deconvolution microscope. The stacks were 3-D reconstructed, and the C T L - L cell interphase was cropped and rotated to obtain a view from the C T L side. In row 1, the synapse was stained with filipin HI for cholesterol (Cho) (blue) CtxB-biotin and streptavidin-FITC for GM1 (green) and anti-LFA-1 and Alexa Fluor 568-conjugated secondary A b (red). In row 2, staining with filipin IH (blue), CTxB-biotin and streptavidin-Alexa Fluor 568 (red) and anti-CD3s-FITC (green) is shown. In row 3, staining with anti-LFA-1 and Alexa Fluor 568 -conjugated secondary A b (red) and antiCD3s-FITC (green) is shown. In row 4, a section of the C T L membrane that was not making contact with the L cell was stained as in row 1. The merged images of various color combinations (r=red, g=green, b=blue) are also shown. Each image is representative of 18-20 synapses examined for each condition of target cell (D +IGAM-1 or ICAM-1 alone), LFA-1 or CD3e staining or both and different incubation time course (5, 15 and 20 min). No difference was seen between the synapse formed when C T L were incubated with L-cells expressing ICAM-1 and those expressing ICAM-1 and D . Similar images were obtained with cells incubated for 5, 15 or 20 min, although the distribution patterns shown in this figure were seen less frequently with 5 min incubation. b  b  b  101  Figure 4.7.  CD3e  JCTL  merge  Target  Figure 4.7. CTLs were conjugated with Calcein labeled R M A target cells (green intracellular staining) and incubated at 37°C for 5 and 20 minutes before fixation with 4% formaldehyde on a poly-L-lysine coated glass coverslip. The conjugates werethen stained with anti LFA-1 A b and Alexa Fluor 568 conjugated secondary Ab (red membrane staining) and with FITC-conjugated anti CD3e A b (green membrane staining) or FITC-conjugated CTxB (results not shown). Then z-stacks were collected by BioRad Multiphoton confocal microscopy and IS were 3D reconstructed and analyzed from the C T L side. A l l results with various staining and conditions showed distribution of LFA-1 and CD3 and GM1 in the outer ring of IS. A representative image is shown above.  102  4.3. Discussion We have demonstrated that LFA-1 and CD3 on C T L co-localize in lipid rafts. When C T L bind to target cells, both CD3 and LFA-1 are distributed to the periphery of the cell contact site. This pattern is in contrast to that in the IS formed on C D 4 T cells interacting with A P C +  (Bromley et al, 2001; Sims and Dustin, 2002; Huppa and Davis, 2003). In the latter IS, the TCR and LFA-1 show differential distribution. Initially, LFA-1 distributes in the center of the IS surrounded by the TCR whereas the mature IS has the TCR in the center and LFA-1 in the periphery. Furthermore, the formation of the C T L IS is antigen-independent whereas the recognition of specific peptide-lVLHC complex by the TCR is essential for the formation of the IS on C D 4 T cells, although antigen-independent IS on CD4 T cells interacting with D C has been +  reported (Revy et al, 2001). These differences between the IS of C T L and that on resting CD4 T cells are likely due to differences in the distribution and activation status of LFA-1 and CD3 on these cells. LFA-1 on C T L is functionally active and readily mediates cell adhesion to ICAM-1 whereas LFA-1 on resting T cells is in inactive form and has to be activated to mediate cell adhesion. Moreover, some CD3 on C T L is found in cold detergent insoluble light density fractions whereas no CD3 on resting T cells can be detected in the same fractions. Crosslinking of LFA-1 on C T L induces co-capping of L F A - 1 , CD3, GM1 and cholesterol whereas no cocapping of LFA-1 and CD3 or GM1 can be induced with resting T cells. GM1 and cholesterol are thought to be major components of lipid rafts, and the co-capping LFA-1 and CD3 is inhibited by cholesterol depletion with M C D . Therefore, these results suggest that LFA-1 and CD3 on C T L co-localize in lipid rafts whereas CD3 on resting T cells is not in lipid rafts and is not associated with L F A - 1 . It appears that binding of functionally active LFA-1 on C T L to ICAM-1 on target cells initiates accumulation of LFA-1 in the cell contact site and brings the  103  TCR/CD3 complex that associates with LFA-1 through lipid rafts to the cell contact site forming the C T L IS. The inhibition of cytotoxicity by M C D treatment may be due to inhibition of TCRmediated calcium signaling as reported by Doucey et al. (Doucey et al., 2001). However, M C D treatment also disrupts the association between LFA-1 and CD3 and may inhibit localization of CD3 in the IS. It is also possible that M C D inhibits LFA-l-mediated signaling. The association of LFA-1 and CD3 on C T L implies that the recruitment of the TCR/CD3 complex to the IS is highly efficient and antigen-independent. The TCR recruited to the IS would scan the M H C peptide complex on target cells and transduce activation signals upon interaction with cognate peptide-MHC. This would be followed by the movement of lytic granules that dock within the secretory domain of the IS mediated by adaptor protein-3 (AP-3) (Kuhn and Poenie, 2002; Clark et al., 2003). The association of CD3 and LFA-1 with lipid rafts on C T L also suggests that activation complexes consisting of the TCR, LFA-1 and signaling molecules such as the Src family of protein tyrosine kinases, which are also found in lipid rafts, might be pre-assembled, which would result in rapid activation of C T L upon encountering target cells expressing cognate peptide-MHC complex. Indeed, activation of C T L as indicated by exocytosis of cytotoxic granules, is rapid and takes only few minutes following binding to target cells. In contrast, activation of resting T cells requires prolonged interaction with antigen presenting cells (Freiberg et al, 2002; Schrum and Turka, 2002; Lee et al, 2002a; Huppa et al, 2003; Huppa and Davis, 2003). The formation of the IS on resting T cells is also rather complex. It requires functional activation of L F A - 1 , recognition of cognate peptide/MHC complex and recruitment of lipid rafts and signaling molecules (Huppa and Davis, 2003).  104  Our results are in general agreement with recent reports of the C T L IS. Using supported planar lipid bilayers containing ICAM-1, Somersalo et al. reported that human C T L clones form an antigen-independent ring junction of LFA-1 (Somersalo et al., 2004). Although the distribution of the TCR and GM1 in the C T L IS in their report is different from that in our study, it maybe due to differences in the targets (cells versus planar membranes). Stinchcombe at al. reported that C T L form mature IS with adhesion molecules forming a ring surrounding an inner signaling domain (Stinchcombe et al., 2001). However, the distribution of the CD3/TCR complex was not directly examined in their study. Faroudi et al. reported that multiple molecules, including CD2, phosphotyrosine, tubulin and perforin, accumulate at the CTL-target interphase in an antigen-dependent manner, but the distribution of L F A - 1 and CD3 in the IS was not studied (Faroudi et al., 2003a). Purbhoo et al. recently examined the distribution of MHC/peptide and ICAM-1 on target cells as they interact with C T L and found that a ring-like distribution of ICAM-1 in the IS is dependent on the accumulation of a high level of MHC/peptide whereas C T L killing requires only three MHC/peptide complexes, suggesting that formation of stable IS is not required for C T L killing (Purbhoo et al., 2004). It has been suggested that CD3 and LFA-1 have to be physically separated in the IS to allow binding to their respective ligands, because LFA-1 and ICAM-1 are considerably larger than the TCR and M H C . Thus, co-distribution of LFA-1 and CD3 may cause physical constraints to their ligand binding. However, LFA-1 on C T L seems to be already activated and its conformation may be significantly different from that of inactive L F A - 1 on resting T cells. Crystal structure studies have shown that the ligand-bound integrin oc P3 has a bent structure v  (Xiong et al., 2001; Xiong et al., 2002). This bending substantially reduces its height on the surface of the plasma membrane. ICAM-1 also has a 90° bend in its structure at its 3 and 4 Igrd  105  th  like domains. It is estimated that the binding of bent LFA-1 to ICAM-1 will generate about 15 nm of separation between the membranes of two interacting cells. This may allow the TCR to interact with M H C , which is thought to require a distance of 15 nm or less between them (Sims and Dustin, 2002). Thus, co-distribution of LFA-1 and CD3 in the C T L IS may actually facilitate the binding of the TCR/CD3 complex to MHC-peptide. It remains to be determined whether activated LFA-1 on the cell surface exists in a bent form or an extended form, which is demonstrated by electron micrographs of purified soluble LFA-1 (Takagi and Springer, 2002). Our results suggest that some LFA-1 and CD3 on C T L localize in membrane microdomains rich in cholesterol and ganglioside G M 1 . In the plasma membrane, cholesterol and GM1 are thought to cluster and form liquid-ordered microdomains, termed lipid rafts. Since these putative microdomains are too small for optical microscopy, the existence of lipid rafts in intact cells is difficult to demonstrate, and whether they actually form unique microdomains on intact cells has been questioned (Munro, 2003). In our study, we used a combination of density separation of cold non-ionic detergent insoluble membrane, cholesterol depletion and co-capping experiments to demonstrate that LFA-1 somehow associates with CD3, cholesterol and GM1 on the surface of CTL. Interestingly, crosslinking of GM1 induces co-capping of some L F A - 1 , but not cholesterol, suggesting that GM1 and cholesterol may not necessarily co-exist in the same microdomains. This is consistent with our previous results that suggested a heterogeneity of lipid rafts of primary T cells. They seem to consist of a spectrum of microdomains ranging from those containing high amount of GM1 but only small amount of cholesterol to those with the opposite lipid composition (Marwali et al., 2003). LFA-1 on C T L seems to be distributed among a wide range of lipid rafts as it co-caps with both GM1 and cholesterol. Cholesterol depletion with M C D treatment also results in about 50% reduction in the amount of LFA-1 in detergent insoluble low  106  density raft fractions, suggesting that some LFA-1 on C T L is in MCD-sensitive cholesterol-rich lipid rafts and some in MCD-resistant, presumably cholesterol-poor but GMl-rich, rafts. On the other hand, CD3 co-caps with L F A - 1 , but it does not co-cap with G M 1 . CD3 in the detergentinsoluble low-density raft fractions becomes almost completely detergent-soluble by cholesterol depletion, whereas GM1 is unaffected. Therefore, CD3 on C T L seems to associate with lipid rafts containing high amounts of cholesterol but relatively small amount of G M 1 . In the C T L IS, GM1 mainly co-localizes in the periphery with LFA-1 and CD3 whereas cholesterol is more widely distributed, including the center of the IS. This distribution of GM1 in the C T L IS is different from that in the IS of resting CD4 T cells. In the latter, GM1 is enriched in the center of the IS (Burack et al., 2002), again reflecting significant differences between these two types of the ISs. It is currently unknown why the distribution of cholesterol is different from that of CD3, LFA-1 and GM1 in the C T L IS, but it seems likely that cholesterol-rich lipid rafts contain unidentified molecules that participate in the formation of the IS.  107  C H A P T E R  A N T I G E N - I N D E P E N D E N T  R E O R G A N I Z A T I O N  M T O C  A C T I N C Y T O S K E L E T O N  A N D  A N T I G E N - D E P E N D E N T  T R A N S L O C A T I O N A N D  T H E  5  C E L L  C T L I M M U N O L O G I C A L  108  S I G N A L I N G  S Y N A P S E  IN  5.1. Introduction Killing of target cells by C T L is a multi-step process. The first step is the binding of C T L to target cell, which is mediated by the adhesion molecules L F A - l - I C A M - 1 and CD2-LFA-3. These interactions allow the TCR to interact with MHC-peptide more efficiently since the affinity of this interaction is generally low. Recognition of agonist antigen peptide presented on M H C class I on the target cell by the TCR triggers activation signals that lead to polarized release of cytotoxic granules towards the target cell (Lieberman, 2003). The cytoskeleton is thought to play important roles in these processes since inhibitors of the actin cytoskeleton and microtubules inhibit the killing of target cells by CTL. The results in the previous chapter showed that the IS is formed on C T L as they bind to target cells in an LFA-1-dependent but antigen-independent manner. Stinchcombe et al. demonstrated that the actin cytoskeleton is distributed in the periphery of C T L IS together with talin and L F A - 1 , whereas secretory granules and signaling molecules, such as Lck and PKC9, occupy the c-SMAC (Stinchcombe et al., 2001). Somersalo et al. used planar supported lipid bilayers inserted with fluorescently labeled ligands (ICAM-1, M H C class I and MICA) and a human C T L clone to examine IS formation. Antigen-independent IS of L F A - l / I C A M - 1 forming outer ring was observed (Somersalo et al., 2004). A human C T L clone also seem to have a dual activation threshold, where mature IS is not necessary for C T L killing but required for I F N Y production (Faroudi et al., 2003a). After engagement the C T L quickly polarizes its actin, M T O C , Golgi complex and lytic granules towards the target cell. The dynamics of tubulin reorganization in CTL-target cell conjugate has been studied by modulated polarization microscopy (MPM) (Kuhn and Poenie, 2002). Using a sliding mechanism, the M T O C is drawn vectorially to the contact site, followed by lateral oscillation. Microtubules loop through and anchor to the  109  p-SMAC where LFA-1 is localized, so far there is no clear evidence of a direct tubulin-LFA-1 interaction. In C T L , unlike N K cells, actin accumulation and M T O C reorientation towards the target invariably occurs (Wulfing et al., 2003). However, the mechanisms underlying these processes are unclear. Whether actin and microtubule are differentially regulated and which surface receptors initiate the processes are not well understood. CTL-target cell conjugate formation induces rapid polarization of the M T O C towards the contact site (Geiger et al., 1982; Kupfer and Dennert, 1984; Kupfer et al., 1985). Yet which surface receptors dictate this process is still unclear. The results in the previous chapter showed that LFA-1 initiates the formation of the IS on C T L in an antigen-independent manner. However, it is insufficient for the triggering of cytotoxicity, which is initiated by the TCR. In this chapter, the role of L F A - 1 and the TCR in the activation of C T L leading to killing of target cells is further investigated, with particular focus on the reorganization of the cytoskeleton.  110  5.2. Results 5.2.1. Actin cytoskeleton accumulates in the IS in an antigen-independent manner We examined the distribution of the actin cytoskeleton in our CTL-target cell system to determine whether this was dictated in an antigen-dependent or -independent manner. As described in the previous chapter, the conjugates were incubated for 5 and 20 minutes and were fixed, permeabilized and stained with rhodamine-conjugated phalloidin for F-actin (red) and anti-LFA-1 plus FITC-conjugated secondary antibody (green). Twenty CTL-target cell conjugates were examined for each condition, i.e. target cells transfected with D + ICAM-1 and b  I C A M alone and for 5 and 20 minutes incubation. Figure 5.1a shows representative images of a mid-section of CTL-target cell conjugate. When C T L encountered a target cell with D and b  ICAM-1 pulsed with H Y peptide (upper panel), F-actin was enriched in the IS area, which showed complete co-distribution with LFA-1 staining on the merged images, ruling out the possibility of contribution of F-actin from the target cell (right side of each images). When compared to target cells transfected with ICAM-1 alone and also pulsed with H Y peptide (lower panel), a similar accumulation of F-actin from C T L side was observed (target cell on the right). This finding is consistent in all of the conditions tested, i.e. D +IC-l 5 minutes, 20 minutes and b  IC-1 alone 5 minutes and 20 minutes (80-90% of conjugates). The distribution of F-actin (red) and LFA-1 (green) in the IS was further analyzed by 3-D reconstruction of the confocal images. The results showed that F-actin co-distributed with LFA-1 to form an outer-ring, although they did not completely overlap on the merged images, regardless of whether the TCR recognized (upper panel) or did not recognize (lower panel) the target cells (figure 5.1b). These data suggest that F-actin distribution in the IS is antigen-independent.  Ill  Figure 5.1  D +IC-l b  CTL TC  IC-1  CTL  Actin  LFA-1  merge H  D +IC-l b  IC-1  Figure 5.1. Actin cytoskeleton accumulates in IS in antigen-independent manner. CTLs were allowed to bind to target cell (TC) and C T L - T C conjugates were fixed with 4% formaldehyde and then permeabilized with 0.5% saponin then stained with rhodamine-phalloidin for F-actin staining (red) and anti-LFA-1 primary Ab and FITCconjugated secondary A b (green). Z-sections of C T L - T C conjugates were collected by confocal microscopy and then 3-D reconstructions were done by Volocity software program, (a) Mid-section images of target cell transfected with D and ICAM-1 (D +IC1) (upper panel) and transfected with ICAM-1 alone (IC-1) (lower panel), (b) 3-D reconstruction of IS area viewed en face from C T L side. b  112  b  5.2.2. LFA-1 capping does not induce co-capping of F-actin cytoskeleton and they are independent of lipid rafts in CTL To determine whether the accumulation of F-actin in the IS is mediated by its association with L F A - 1 , we did co-capping experiment. LFA-1 capping was induced by antibody crosslinking and incubation at 37°C for 30 minutes as described in chapter 4, C T L were then fixed and permeabilized and stained for F-actin with rhodamine-conjugated phalloidin. Cocapping was assessed by confocal microscopy. Figure 5.2a demonstrates representative images from 100 cells analyzed for each condition. Capping of L F A - 1 (green) did not induce co-capping of F-actin (upper panel). This was observed in 94 ± 5 % of capped cells. The lower panel shows a control condition where LFA-1 capping was not induced. It is worth noting that LFA-1 capping induced clustering of F-actin at different sites rather than inducing co-capping with L F A - 1 . Factin did not show clustering in control cells without LFA-1 capping. To test whether lipid rafts play a role in the clustering of F-actin, lipid rafts were disrupted by M C D treatment. CTLs were treated with 20 m M M C D , and then resuspended in BSA-containing media, and LFA-1 capping was induced in a similar manner as in figure 5a. M C D treatment did not have any effects on capping of L F A - 1 or clustering of F-actin. (figure 5.2b, upper panel). This was observed in 90 ± 6 % of capped cells.  5.2.3. Accumulation of phospho-tyrosine in the IS is antigen-dependent L cells transfected either with ICAM-1 and D or with ICAM-1 alone were pulsed with b  the H Y peptide before incubation with C T L generated from H Y T C R transgenic Rag 2' ' mouse 1  spleen. CTL-target cell conjugates were incubated for 5 and 20 minutes then fixed and  113  Figure 5.2 LFA-1  Actin  a  9  -~  I  -  i• -•  _____  .  •  i  -  . . ...  ."  sswIB?  '  . .. ...  " "• -" ; . .- . " ;  - 1  '.' . ... u .  P  ___\ ' .  1  -• §| - =•".  ^^^^^^^^^^^^^  LFA-1  Actin  merge  4  r o  o  0?\  o °  Figure 5.2. LFA-1 capping does not induce co-capping of F-actin cytoskeleton and they are independent of lipid rafts in CTL. (a) LFA-1 capping was induced by anti-LFA-1 A b crosslinking and FITC-secondary A b (green) and incubation at 37°C for 30 minutes. Then cells were fixed and permeabilized with 0.5% saponin and stained for F-actin with rhodamine-phalloidin (red). Representative images of mid-section confocal image of LFA-1 capped (upper panel) and uncapped control (lower panel) were shown, (b) CTLs were treated with 20 m M M C D then LFA-1 (green) capping was induced as in (a) then fixed and permeabilized with 0.5% saponin and stained with rhodamine-phalloidin (red). Upper panel: LFA-1 capped; lower panel: uncapped control  114  permeabilized with 4% formaldehyde and 0.5% saponin before staining with filipin HI for membrane cholesterol (blue) and FITC conjugated CtxB (green) and the anti-phospho-tyrosine mAb, 4G10 (red). Conjugates were examined by Deltavision deconvolution microscopy. Zstacks were collected, 3-D reconstruction of CTL-target cell contacts was generated and images were cropped and viewed from C T L side for phospho-tyrosine (pY) distribution on CTL. Figure 5.3a demonstrates representative images of CTL-target cell conjugates from each condition. The binding of C T L to L cells transfected with both D and ICAM-1 induced p Y accumulation in the b  IS area (upper panel). With L cells expressing ICAM-1 but not D , p Y in C T L was evenly b  distributed in the cytoplasm with much lower intensity (lower panel). This finding was the same at the 5 minute and 20 minute incubation periods. Therefore, the engagement of LFA-1 with ICAM-1 does not induce significant accumulation of pY, which instead requires the recognition of peptide/MHC by TCR. When target cells were transfected with D alone without ICAM-1, b  CTLs did not bind to target cell and were washed away during the sample preparation. We then examined whether p Y co-distributes with a specific subset of lipid rafts in the IS. We found that p Y distribution was equally distributed in both cholesterol-rich and GMl-rich regions of the IS after 5 and 20 minute incubation (n=40 of each condition) (Figure 5.3b). The upper panel illustrates a representative image from the target cell transfected with D and I C A M b  1. GM1 formed an outer ring whereas cholesterol was distributed more widely, and p Y was evenly distributed without specific patterns. As a control, C T L IS images taken from target cell transfected with ICAM-1 alone are also shown (lower panel). The phospho-tyrosine signal in this condition was very weak as seen in figure 5.3.a lower panel, since the T C R did not recognize M H C class I-HY peptide. Phospho-tyrosine signals (red) of figure 5.3.b lower panel has been enhanced by increasing the sensitivity setting of the deconvolution microscopy.  115  Figure 5.3. merge  1  a.  TC  L-  IC+D  b  CTL  V  (  7  J  ?  b. cholesterol  GM-1  IO  L-  IC+D  b/g  b  g  r/b  r/g/b  1 ^  Lr  IC-1  r /  •w  1  •  Figure 5.3. (a) Tyrosine-phosphorylated proteins accumulate in antigen-specific C T L I S . C T L were incubated with H Y peptide-pulsed L cells transfected with ICAM-1 (L-IC) or ICAM-1 and D (L-IC+Db) for 5 min, fixed, permeabilized and stained for the indicated molecules. Cholesterol (blue)and GM1 (green) were stained as in chapter 4, figure 4.6 whereas phospho-tyrosine was stained with the anti-phosphotyrosine mAb 4G10 and Alexa Fluor 568-conjugated secondary antibody. The stained cells were analyzed by deconvolution microscope. Images at the mid section of the C T L are shown. The images are representative of more than 40, all showing similar patterns of staining for each condition, ( b ) Distribution of phospho-tyrosine in the IS. Images from 3-D reconstruction of IS from (a) viewed frontally from C T L side. Upper panel is representative images from distribution of cholesterol (blue), GM1 (green) and phospho-tyrosine (red) when the target cell was transfected with D +IC-l. Lower panel is representative image of C T L IS when target was transfected with IC-1 alone. Combinations of any of two colors and three colors merges were also shown. r=red, g=green, b=blue b  b  116  5.2.4. Lck is enriched in the IS and co-localizes with GMl-rich rafts in an antigenindependent manner It has been well established that Lck is involved in the initial T cell activation. Lck has also been reported to accumulate in the c-SMAC of C T L IS (Stinchcombe et al., 2001). Therefore, we examined whether Lck co-distributes with lipid rafts in the C T L IS. First, Lck association with lipid rafts was evaluated by the sucrose gradient centrifugation method. Two non-ionic detergents, Tx-100 and Brij 35, were compared. Lipid rafts containing Lck in both resting T cells and CTLs were isolated with 1% Brij 35, but not 1% Tx-100 (Figure 5.4a). In both cells, the lipid rafts containing Lck were similarly resistant to cholesterol extraction by 20 m M M C D treatment (right side). The percentage of Lck shifted to the soluble fractions were 9% and 11% for resting T cells and CTLs, respectively, as measured by densitometry. Thus, the majority (-90%) of Lck constitutively associates with GMl-rich rafts regardless of the activation state of the cells. Next we examined the distribution of Lck in CTLs after engagement with target cells. CTLs incubated with target cells for 5 minutes or 20 minutes were fixed, permeabilized and stained with rabbit anti mouse Lck polyclonal antibodies with FITC-conjugated secondary antibody (green) and biotin-conjugated CtxB with streptavidin-conjugated Alexa-Fluor 568 (red) (figure 5.4b). The upper panel shows representative images of Lck distribution in the CTLs incubated with L cells transfected with both ICAM-1 and D pulsed with H Y peptide. The target b  cells are on the left side and the area where C T L formed contact with target cell can be seen as a flattened area. Analysis of 20 conjugates each, from 5 minute and 20 minute incubation, showed similar results as represented in the upper panel. Lck was distributed all over the cytoplasm of C T L , but it was more enriched in the p-SMAC. A similar distribution was also observed  117  Figure 5.4 resting T cells 1% Brij 35  1% Tx-100 1  a  1  2  3  4  5  6  i-lr  LCK  7  8  P  1  2  3  • -  4  5  6  7  8  P  —  w  C T L •. i . LCK  +  Figure 5.4. Lck is found in lipid rafts isolated by 1% Brij 35 and resistant to MCD treatment in both resting T cells and CTL. (a)Sucrose gradient centrifugation of resting T cells (upper panel) and CTLs (lower panel) lyzed with 1% Triton X-100 (left) and 1% Brij 35 (right). Lipid rafts localized to low density fractions, fraction 2 and 3. (-) represents without M C D treatment and (+) represents treatment with 10 m M and 20 m M M C D for resting T cells and CTLs respectively.  118  Figure 5.4 Lck  GM1  merge  L(D +IC) b  Figure 5.4. Lck is enriched in the IS and co-localizes with GMl-rich rafts in antigen-independent manner, (b) C T L - T C conjugates were fixed and permeabilized and stained with rabbit anti mouse Lck polyclonal serum and stained with FITCconjugated anti rabbit (green) and stained with biotin-CTxB and streptavidin-Alexa Fluor 568 (red). Then z sections were collected with confocal microscopy. The upper panel illustrates conjugates where target cells were transfected with D and ICAM-1 and lower panel is representative images of conjugates where target cells were transfected with ICAM-1 alone (c) C T L IS images were 3-D reconstructed and ISs were viewed frontally from C T L side. The left side shows two representative ISs (upper and lower panels) from target cells transfected with D and ICAM-1. The right side is two representatives of ISs from target cells transfected with ICAM-1 alone b  b  119  in conditions where target cell was only transfected with ICAM-1 alone and then pulsed with H Y peptide (lower panel, mid section of conjugate is shown). Analysis of the distribution of Lck in the IS showed that with both L cells transfected with D +ICAM-1 (left side) and with ICAM-1 alone (right side), the majority of Lck molecules b  co-localized with GM1 (figure 5.4c). This is in agreement with the sucrose gradient experiment result, which showed around 90% of the Lck associated with M C D resistant rafts representing GMl-rich rafts. Lck and GM1 were distributed in the outer ring in most synapses in an antigenindependent manner. However, it is unknown whether this result represents the distribution of activated Lck, because the antibody did not specifically recognize the activated form of Lck.  5.2.5. M T O C translocation to the IS is dictated by T C R recognition of target Translocation of the M T O C in C T L towards target cells is thought to be a critical step in the exocytosis of cytotoxic granules (Kuhn and Poenie, 2002). Therefore, we examined whether it is dictated by the TCR or L F A - 1 . Conjugates of C T L and target cell were incubated for 5 and 20 minutes, fixed, permeabilized and stained with anti-0 tubulin mAb plus Alexa-Fluor 568conjugated secondary antibody (red) and stained with anti LFA-1 mAb plus FITC-conjugated secondary Ab (blue). We examined 40 conjugates for each condition and found that when the targets were recognized by the TCR, the M T O C translocated to the IS area (left side, figure 5.5). In contrast, without D expression on L cells, the M T O C was found away from the IS (right side, b  figure 5.5). This was observed in almost all of the conjugates (95-100% of n=40). Therefore, M T O C translocation seems to be dictated by TCR engagement with cognate MHC-peptide rather than by LFA-1 binding to ICAM-1.  120  Figure 5.5.  Figure 5.5. MTOC translocation to the IS is dictated by TCR recognition of target. Target cells (L-IC and L-IC+ Db) were incubated with C T L and both were stained for ptubulin with anti-mouse tubulin P and Alexa Fluor 568-conjugated secondary antibody (red) and anti-LFA-1 A b (blue). The stained cells were analyzed by confocal microscope. Images at the mid section of the C T L are shown. The images are representative of more than 40, all showing similar patterns of staining for each condition. White arrows point to MTOC  121  5.3. Discussion Here we have shown the differential regulation of actin cytoskeleton reorganization and M T O C translocation upon C T L engagement with target cells. Protein tyrosine-phosphorylation and M T O C translocation towards the IS takes place upon TCR engagement, whereas cell polarization by accumulation of F-actin in the synapse area is independent of TCR signals and mediated by LFA-1 ligation. It is unlikely that F-actin accumulates at the synapse by a passive mechanism, due to its association with L F A - 1 . Other studies have suggested that LFA-1 directly interacts with talin and possibly the actin cytoskeleton (Pardi et al., 1992; K i m et al., 2003). K i m et al. transfected LFA-1 into the human erythroleukemia line K562 to demonstrate the direct interaction between LFA-1 and talin (Kim et al., 2003). Pardi et al. used human peripheral blood T lymphocytes and showed that LFA-1 was in the pellet after detergent solubilization, consistent with cytoskeletal association. LFA-1 and actin also seemed to co-localize in a study that used fluorescence microscopy (Pardi et al., 1992). However, the co-capping studies in this chapter showed that LFA-1 on mouse C T L does not directly associate with F-actin. Instead, capping of LFA-1 induces clustering of F-actin in separate sites, suggesting that L F A - 1 clustering induces polymerization of actin. Furthermore, upon binding of C T L to L cells expressing ICAM-1, Factin accumulates in the IS in an antigen-independent manner. Therefore, it seems likely that the binding of LFA-1 to ICAM-1 triggers outside-in signals that lead to actin polymerization at the cell contact site. Detailed examination of the distribution of LFA-1 and F-actin in the synapse revealed that although both LFA-1 and F-actin formed the outer ring of the IS, their distributions only partially overlapped. Outside-in signals from LFA-1 to F-actin is possibly mediated by signaling molecules such as V a v l , W A S P and Racl and also by lipid rafts (Sasahara et al., 2002; Dupre et al, 2002; Riteau et al, 2003; Sanchez-Martin et al, 2004). We ruled out the  122  involvement of lipid rafts in this event, since M C D disruption of rafts did not have any effect on F-actin clustering after LFA-1 capping (figure 5.2b compared to 5.2a). This is consistent with studies by Dupre et al. and Sasahara et al. which showed that lipid rafts are crucial for TCR stimulation but not for LFA-1 signaling (Sasahara et al., 2002; Dupre et al., 2002). Using N K cells, Riteau et al. demonstrated that LFA-1 binding to ICAM-1 transfected into insect cells induces actin reorganization through V a v l activation. However, participation of other surface receptors such as CD2 and LFA-3 cannot be excluded (Shaw et al., 1986; Selvaraj et al., 1987). M T O C translocation to the C T L IS is an essential step for killing function. Secretory granules are thought to slide along the microtubules towards the plus end (away from MTOC) to be secreted at the IS (Stinchcombe et al., 2001; Bossi et al., 2002; Kuhn and Poenie, 2002; Trambas and Griffiths, 2003). Although it has been known for a long time that the M T O C is redirected towards the target cells, it is not completely clear whether this is an antigenindependent or antigen-dependent process (Geiger et al., 1982; Kupfer and Dennett, 1984; Kupfer et al., 1985), because the M T O C may reorient toward the IS without necessarily causing cytotoxicity. The killing of target cells requires additional processes of fusion of the granules with the plasma membrane, as evident in several human diseases and murine models (Trambas and Griffiths, 2003). The delivery of secretory granules to the contact site requires the involvement of particular gene products, which are defective in several human diseases or mouse models (Trambas and Griffiths, 2003). Lyst is defective in Chediak-Higashi syndrome and in beige mice (Nagle et al., 1996; Perou et al., 1996). Rab27a is non-functional in Griscelli syndrome and ashen mice (Wilson et al., 2000; Menasche et al., 2000), whereas Rab geranylgeranyl transferase malfunctions in Hermansky-Pudlak syndrome and gunmetal mice (Detter et al., 2000). Individuals with Hermansky-Pudlak type 2 lack the cytosolic adaptor  123  protein 3 (AP-3), which is essential for lysosomal sorting (Clark et al., 2003). In patients with familial hemophagocytic lymphohystiocytosis type 3 (FHL3) there is a defect in cytotoxicity function but normal perforin. A member of vesicle priming proteins, HMunc 13-4, were found to be involved in the defect in exocytosis of cytolytic granules (Feldmann et al., 2003). Recent work by Kuhn et al. described the dynamics of microtubules and M T O C (Kuhn and Poenie, 2002). Faroudi et al. observed M T O C translocation in a C T L clone. However, it was not clear whether this was an antigen-dependent event (Faroudi et al., 2003a). We have shown in this chapter that M T O C translocation is indeed an antigen-dependent consequence of TCR recognition. This may provide C T L with more control of its killing capability, avoiding nonspecific cytotoxicity. We also described in this study that phospho-tyrosine signaling accumulation in the IS was dependent on TCR recognition (figure 5.3a), supporting the idea that L F A - 1 engagement with ICAM-1 alone is not sufficient to induce strong tyrosine phosphorylation signals. Most integrin P chains have conserved N P X Y motifs in the cytoplasmic domain and tyrosinephosphorylation of the N P X Y motif is thought to be important for the regulation of integrin functions. However, the P chain of LFA-1 lacks the N P X Y motif and has N P L F residues instead. It is currently unclear how ligation of LFA-1 induces outside-in signaling. It is possible that phosphorylation at serine 745 induces the release of JAB1 from the LFA-1 cytoplasmic domain, which results in c-Jun activation (Perez et al., 2003). Analysis of phospho-tyrosine distribution in the IS suggested that phospho-tyrosine signaling might occur in both in G M 1 - and cholesterol-rich rafts. Studies on naive T cell also concluded that tyrosine phophorylated active Lck is distributed in the c-SMAC, which is G M l rich, and the p-SMAC, which is M C D sensitive (Burack et al, 2002; Lee et al, 2003). In our  124  study of Lck distribution, no difference between Lck distributions after target recognition was observed. This maybe due to the antibody used, since the antibody was against Lck but not the phophorylated Lck (pY394). However, the enrichment of Lck in the IS agrees with result from a study of C T L IS in vivo (McGavern et al., 2002). Stinchcombe et al reported that Lck is distributed in the c-SMAC of C T L IS, which is different than our findings in p-SMAC). This maybe due to the different system used, since Stinchcombe et.al. used C T L clone. Sedwick et al. reported that TCR regulates M T O C and tubulin reorganization while LFA-1 dictates F-actin accumulation by using beads and A P C simultaneously in naive resting T cell (Sedwick et al., 1999). Our results with activated C T L are consistent with these studies. The role of this differential cytoskeletal control by LFA-1 and T C R is more clearly demonstrated with C T L in this chapter.  125  CHAPTER 6 SUMMARY AND DISCUSSION  126  Summary and discussion The regulation of LFA-1 and other integrins is unique. It does not involve up-regulation of surface expression level but is mediated by increase in affinity caused by conformational changes and in avidity caused by increase in clustering, which likely involves inside-out and outside-in signalling. In the last several years, significant progress has been made in integrin structure-function studies that revealed the nature of affinity changes in integrins. X-ray crystallography of ccvP3 and the I-domain of LFA-1 provided compelling evidence that conformational changes take place when integrins interact with ligand (Xiong et al., 2001; Xiong et al., 2002; Shimaoka et al., 2003b). On the other hand, the mechanisms by which the avidity of integrins is regulated were unclear at the beginning of this thesis. MAbs that recognize clustered LFA-1 were reported earlier (Binnerts and van Kooyk, 1999). More recently, X-ray crystallography revealed that ICAM-1 formed a W-shaped tetramer mediated by D l - D l and D4D4 Ig-like domain interactions (Yang et al., 2004). This further suggested the importance of clustering in LFA-1 regulation. However, little was known about the actual mechanisms involved in the clustering of L F A - 1 . Lipid raft microdomains were reported to have an important role in T cell activation by facilitating clustering of receptors required for cell signalling (Xavier et al, 1998). It has also been reported that LFA-1 binding to ICAM-1 is increased when lipid raft clustering is induced by crosslinking GPI-anchored CD24 with antibody or by CTx (Krauss and Altevogt, 1999).Thus, it was tempting to speculate that lipid rafts may play a role in clustering of LFA-1 increasing its avidity. The main objective of this thesis work was to elucidate the role lipid rafts in the regulation of LFA-1 functions. In chapter 3, whether LFA-1 localizes in lipid rafts and whether lipid rafts regulate LFA-l-mediated cell adhesion to ICAM-1 was studied using T cell lines and primary T cells. The results showed that disruption of lipid rafts by  127  membrane cholesterol extraction by M C D inhibits LFA-1-mediated cell adhesion, suggesting an important role of lipid rafts in LFA-1 function. However, when lipid rafts are isolated by insolubility in Triton X-100, the most commonly used detergent for the isolation of lipid rafts, LFA-1 is not found in lipid raft fractions. Among several non-ionic detergents tested, only Brij35 maintains LFA-1 in lipid raft fractions. Co-capping of LFA-1 and membrane cholesterol further supported the notion that some LFA-1 on T cells localizes in lipid rafts. The results with various non-ionic detergents as well as the co-capping experiments also suggested that lipid rafts of primary T cells are heterogeneous. LFA-1 seems to associate with cholesterol-rich lipid rafts while GPI-anchored Thy-1 associates with GMl-rich rafts. In human peripheral blood T cell lymphoblast, Lck associates with cholesterol extraction-sensitive lipid rafts, whereas lipid rafts containing L A T are resistant (Schade and Levine, 2002). Using 1% Brij 98 at 37 °C to isolate raft from Jurkat T cells, Munoz et al. demonstrated the existence of at least three types of Brij 98-resistant raft subsets: CD38 rafts, which are enriched in CD38, CD3£, and Lck; TCR/CD3 rafts, which are enriched in CD3£, CD3e, and Lck; and L A T rafts, which are primarily enriched in Lck. CD38 is a N A D ( P ) glycohydrolase and plays a role in lymphocyte activation. CD38 may +  also act as an ectocyclase that converts N A D to the Ca -releasing second messenger cyclic +  2+  ADP-ribose (Munoz et al., 2003). Upon stimulation of Ramos B cell, B cell antigen receptor (BCR) associates with lipid rafts together with CD20 and subsequently dissociates from CD20 in distinct rafts before endocytosis, which can be inhibited by M C D (Petrie and Deans, 2002). Other studies also suggest the heterogeneity of lipid rafts. Although both ganglioside GM1 and GM3 partition into detergent-resistant membrane, they were differentially polarized in migrating Jurkat T cell. GM1 distributes to the uropod whereas GM3 moves to the leading edge. The polarized morphology is inhibited by M C D treatment that can be reversed by cholesterol  128  reconstitution (Gomez-Mouton et al., 2001). Glycosphingolipids GM1 and GD3 segregate to distinct lipid rafts in neuronal cells (Vyas et al., 2001). Tetraspanins are thought to form membrane microdomain distinct from lipid rafts (Hemler, 2003). M H C class II-peptides were demonstrated to be differentially distributed in distinct membrane microdomains of A P C . The few M H C molecules found in lipid rafts bind peptides representative of the whole repertoire, whereas those in tetraspan in microdomains are enriched for specific peptide-MHC class II complexes, which colocalize with the M H C class II editor H L A - D M (DM) and the costimulatory molecule CD86 (Kropshofer et al., 2002). Thus, the results in chapter 3 agree with the rapidly accumulating evidence for lipid raft heterogeneity. Furthermore, they have clarified the relationship between lipid rafts and L F A - 1 . Lipid rafts are important for L F A - 1 function possibly by facilitating clustering to increase avidity for its ligand. However, the exact nature of the lipid raft microdomain is still unclear. The prevailing theory is that microdomains are so small in size, as determined by photonic force microscopy to be 26±13 nm (Pralle et al., 2000), that they cannot be observed by light microscopy (Simons and Ehehalt, 2002). A study using a combination of homo-FRET and fluorescence anisotropy of GPI-anchored proteins revealed that they are present as monomers and a smaller fraction (20%-40%) as nanoscale (<5 nm) cholesterol-sensitive clusters (Sharma et al., 2004). These findings may be consistent with the proposed idea of a "lipid shell". Surface receptors are thought to preferentially associate with small sphingolipid-cholesterol "condensed complexes", comprised of 15 to 30 molecules, which interact through hydrogen bonds. Upon interaction with surface proteins these condensed complexes form a bigger sized cluster (~ 7 nm) possibly containing around 80 lipid molecules (Anderson and Jacobson, 2002). In this thesis work lipid rafts were evaluated by using not only  129  non-ionic detergent solubility but also confocal microscopy. Therefore, results with detergentinsolubility were corroborated with microscopy studies. Although the inhibitory effects of M C D treatment on LFA-1 functions suggest that lipid rafts are important for L F A - 1 functions, how lipid rafts regulate L F A - 1 functions is unknown. As discussed above, it is possible that lipid rafts facilitate the clustering of L F A - 1 , thus increasing the avidity. It is also possible that membrane cholesterol and glycosphingolipids alter the local thickness and curvature of the membrane (Pande, 2000), and this may contribute to the function of L F A - 1 . Interestingly, bi-directional signaling of LFA-1 was demonstrated to involve the separation of oc and 02 chains (Kim et al., 2003). It is possible that lipid composition of the L  plasma membrane plays an important role here. Consistent with the data in chapter 3, other reports also revealed the importance of membrane cholesterol and glycosphingolipid in integrin functions (Green et al., 1999; Gopalakrishna et al., 2000; del Pozo et al., 2004; McDonald et al., 2004). Further biophysical and microscopy study or other methods are required to elucidate the nature of lipid rafts microdomain as well as the mechanism(s) by which surface proteins and membrane lipids interact. This area of research warrants closer attention since many receptor functions are shown to be dependent on membrane cholesterol and other membrane lipids. In addition, more and more studies have reported that lipid rafts are required for the pathogenicity of a variety of microorganisms (Simons and Ehehalt, 2002; Manes et al., 2003). Lipid rafts have been demonstrated to be important for T cell activation (Xavier et al., 1998). They have also been shown to accumulate in the contact area where T cell is activated by anti-CD3- and anti-CD28-coated beads (Viola et al., 1999a). Thus, I started the study on the the distribution of lipid rafts and their heterogeneity in the IS on C T L . The results of this study were presented in chapter 4 of this thesis. M y results showed that lipid rafts mediate the association  130  between LFA-1 and CD3 on C T L , but not on resting T cells. The raft-mediated association between LFA-1 and CD3 seems responsible for the formation of the IS on C T L , which is antigen-independent but LFA-1-dependent. It is thought that the binding of ICAM-1 on target cells to pre-activated LFA-1 on C T L initiates accumulation of L F A - 1 on cell contact sites and recruits the TCR/CD3 complex through lipid raft association. Thus, both L F A - 1 and the TCR/CD3 complex accumulate in the IS independent of antigen recognition by the TCR. This process likely facilitates rapid and efficient scanning of the target cell surface by the TCR for agonist peptide/MHC. Although LFA-1 and CD3 were co-distributed with GM1 rafts in the periphery of the IS, cholesterol distribution was different. What mediates the differential distribution of cholesterol and GM1 is still unclear. It is antigen-independent and, therefore, not mediated by the TCR. It is possible that other unknown molecule(s) co-distribute(s) with membrane cholesterol. Although the distribution of lipid rafts may also be regulated by the actin cytoskeleton, the results in chapter 5 showed that F-actin does not co-distribute with cholesterol in the IS. It is possible that unidentified GPI-anchored molecule, which is known to localize in cholesterol-rich lipid rafts, may regulate the distribution of cholesterol in the IS. Further investigation is necessary to test this possibility in the future. The description of the C T L synapse in chapter 4 is in agreement with other studies. Using lipid bilayers containing M H C class Il-peptide and I C A M - 1 , Burack et al. revealed that the p-SMAC, where LFA-1 is distributed, is sensitive to M C D extraction. In contrast, the cS M A C is resistant to M C D extraction and is enriched in GM1 (Burack et ah, 2002). A more recent published study showed that an antigen-independent ring is formed by L F A - l - I C A M - 1 interaction on the C T L synapse (Somersalo et al., 2004). It is also consistent with another recent  131  study which showed that a mature synapse was not necessary for the killing functions of C T L (Purbhoo et al., 2004). Future research may focus on the relationship of lipid raft heterogeneity to the secretory granules. Stinchcombe at al. showed that lytic granules are localized in the center of C T L synapse (Stinchcombe et al., 2001). It is also likely that the separation of membrane cholesterol from GM1 rafts is the result of lytic granule apposition to the IS. It is unlikely to be caused by fusion of the lytic granules since the separation is antigen-independent. However, it would be interesting to investigate the distribution of molecules known to be important in the delivery of lytic granules to the synapse, such as Lyst, Rab 27a and Rab geranyl geranyl transferase (Trambas and Griffiths, 2003). It is tempting to speculate that these molecules might associate with cholesterol-rich rafts in the synapse. Beige, ashen and gunmetal mice are the mouse models with defective genes for these molecules, respectively. Further investigation also can use these murine models to evaluate the distribution of lipid rafts heterogeneity in the C T L immunological synapse. LFA-1 in C T L is pre-activated, suggesting that it might have received an inside-out signal from TCR and/or IL-2 receptor during the generation of C T L . Interestingly, LFA-1 on C T L is different than that on primary T cell. Lipid raft disruption by M C D treatment of C T L does not inhibit L F A - 1 , which is already activated. In contrast, M C D treatment of resting T cells inhibits PMA-induced LFA-1 activation. It is possible that the activation pathways of P M A induced inside-out signalling may be sensitive to M C D whereas once activated, LFA-1 may become insensitive to M C D . It is also possible that PMA-induced and TCR- and IL-2R-induced activation pathways are different in the sensitivity to M C D treatment. P M A has been proposed to activate LFA-1 by inducing clustering rather than affinity changes (van Kooyk and Figdor, 2000). The single particle tracking method also has shown that P M A induces an increase in the  132  lateral mobility of LFA-1 possibly through the release of cytoskeletal constraints (Kucik et al., 1996). Taken together, these data suggest an important role for lipid rafts in avidity regulation. In chapter 5, the differential roles of antigen-independent and antigen-dependent cellular events in C T L were examined. The results suggested that F-actin clustering and Lck enrichment in the synapse are antigen-independent. In contrast, phospho-tyrosine accumulation and M T O C translocation to the synapse is TCR-mediated. As mentioned above, the distribution of cholesterol and GM1 does not seem to be dictated by actin cytoskeleton. LFA-1 has always been thought to associate with the actin cytoskeleton through its association with talin. However, the results with co-capping experiments suggest that LFA-1 does not directly associate with F-actin in C T L or resting T cells. The most likely explanation is that the accumulation of F-actin in the synapse is mediated by outside-in signalling through LFA-1 binding to I C A M - 1 . Further study is needed to confirm this notion and to verify that actin clustering is not due to mere LFA-1 clustering. Future research may focus on the signalling events involved in the outside-in signaling. V a v l and Pyk 2 have been demonstrated to be essential in L F A - 1 outside-in signal (Rodriguez-Fernandez et al., 1999; Rodriguez-Fernandez et ah, 2002; Riteau et al, 2003). Using transfected insect cells that express ligands of human N K cell receptors, Riteau et al. showed that engagement of LFA-1 on N K cells by ICAM-1 leads to tyrosine phosphorylation of V a v l and that this is not sensitive to cholesterol depletion or to inhibition of actin polymerization. V a v l phosphorylation is blocked by an inhibitor of Src-family kinases, and correlated with activation of its downstream effector, P A K . V a v l is also recruited to lipid rafts when the co-receptor 2B4 was engaged with ligand CD48, which enhanced V a v l phosphorylation (Riteau et al, 2003). Therefore V a v l seems to be a possible candidate molecule linking LFA-1 to actin polymerization. V a v l has also been demonstrated to mediate TCR activation of LFA-1  133  clustering (Krawczyk et al., 2002). These suggest the bi-directional nature of signals through Vavl. Talin has been shown to directly interact with a variety of integrins. X-ray crystallography has revealed the nature of talin binding to P3 integrin cytoplasmic domain (Garcia-Alvarez et al., 2003). N M R study of aubpS cytoplasmic domain also revealed that talin binding causes disturbance of heterodimeric interaction, which subsequently releases the "handshake" clasp between them, allowing inside-out signal to activate extracellular domain (Vinogradova et al., 2002). Using siRNA to knock down talin and mAb detection of high affinity integrin, Tadokoro et al. demonstrated that talin binding causes aL\\\$3 affinity changes (Tadokoro et al., 2003). The LFA-1 cytoplasmic domain also interacts with talin in a transfected cell line (Kim et al., 2003). Thus, talin very likely interacts with LFA-1 in a different way in C T L than in resting T cells. The exact molecular interactions of L F A - 1 , talin, V a v l and actin cytoskeleton or other molecules such as cytohesinl and RapL in the regulation of outside-in signaling remain to be investigated. The results presented in this thesis clarify a number of issues regarding the role of lipid rafts in the regulation of L F A - 1 . However, there are some limitations in this study. Image analysis was done in a qualitative manner rather than quantitavely. LFA-1 mediated cell adhesion assay could determine avidity but not able to asses affinity of L F A - 1 . Sucrose gradient centrifugation method to isolate lipid rafts as mentioned in the introduction was dependent on the non-ionic detergent used. The future studies will have to deal with these limitations to elucidate how lipid rafts regulate LFA-1 at the molecular level.  134  REFERENCES Ahmed, S.N., Brown, D.A., and London, E. (1997). 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