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Regulation and function of hyaluronan binding by CD44 in the immune system Ruffell, Brian 2008

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REGULATION AND FUNCTION OF HYALURONAN BINDING BY CD44 IN THE IMMUNE SYSTEM by BRIAN RUFFELL B.Sc., University of British Columbia, Vancouver, B.C., Canada 2001  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Microbiology and Immunology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August 2008 © Brian Ruffell, 2008            ABSTRACT  The proteoglycan CD44 is a widely expressed cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan, and is involved in processes ranging from metastasis to wound healing. In the immune system, leukocyte activation induces hyaluronan binding through changes in CD44 post-translational modification, but these changes have not been well characterized. Here I identify chondroitin sulfate addition to CD44 as a negative regulator of hyaluronan binding. Chondroitin sulfate addition was analyzed by sulfate incorporation and Western blotting and determined to occur at serine 180 in human CD44 using site-directed mutagenesis. Mutation of serine 180 increased hyaluronan binding by both a CD44immunoglobulin fusion protein expressed in HEK293 cells, and full-length CD44 expressed in murine L fibroblast cells. In bone marrow-derived macrophages, hyaluronan binding induced by the inflammatory cytokines tumor necrosis factor-α and interferon-γ corresponded with reduced chondroitin sulfate addition to CD44. Retroviral infection of CD44-/- macrophages with mouse CD44 containing a mutation at serine 183, equivalent to serine 180 in human CD44, resulted in hyaluronan binding that was constitutively high and no longer enhanced by stimulation. These results demonstrate that hyaluronan binding by CD44 is regulated by chondroitin sulfate addition in macrophages. A functional consequence of altered chondroitin sulfate addition and increased hyaluronan binding was observed in Jurkat T cells, which became more susceptible to activationinduced cell death when transfected with mutant CD44. The extent of cell death was dependent upon both the hyaluronan binding ability of CD44 and the size of hyaluronan itself, with high molecular mass hyaluronan having a greater effect than intermediate or low molecular mass hyaluronan. The addition of hyaluronan to pre-activated Jurkat T cells induced rapid cell death independently of Fas and caspase activation, identifying a unique Fas-independent mechanism ii     for inducing cell death in activated cells. Results were comparable in splenic T cells, where high hyaluronan binding correlated with increased phosphatidylserine exposure, and hyaluronandependent cell death occurred in a population of restimulated cells in the absence of Fasdependent cell death. Together these results reveal a novel mechanism for regulating hyaluronan binding and demonstrate that altered chondroitin sulfate addition can affect CD44 function.  iii     TABLE OF CONTENTS Abstract……………………………………………………………………………………………ii Table of Contents…………………………………………...…………………………………….iv List of Tables………………………………………………………………………………….viii List of Figures…………………………………………………………………..………………ix List of Abbreviations…………………………………………………………………………..xii Acknowledgements…………………………………………………………………………...xiv Dedication………………………………………………………………………………………..xv Co-Authorship Statement……………………………………………….…………………….xvi  CHAPTER ONE: Introduction………...……………………………………….………………1 1.1 The immune system………………………………………………………………...………..2 1.1.1 The innate immune response…………………………………………………………...2 1.1.2 The adaptive immune response.………………………………………………………..4 1.2 Cell adhesion and migration………………………………………………………...………5 1.2.1 Cell adhesion in the immune system…………………..……………………………….5 1.2.2 Cell adhesion molecules in extravasation..……………………..………………………7 1.3 CD44 and hyaluronan………………………………………………………………….…10 1.3.1 Introduction to HA………………….………………………………………………..10 1.3.2 Introduction to CD44………………………………………………………………...13 1.3.3 CD44 function in the immune system……………………………………………….15 1.3.3a Leukocyte recruitment to the inflammatory site…………………...………….15 1.3.3b Resolution of inflammation……………………………………………………..21 1.3.3c T cell response….……………………………………………………………….22 1.3.4 Regulation of HA binding by CD44…………………………………………………..26 1.3.4a HA binding regulation in T cells……...………………………………………...26 1.3.4b HA binding regulation in macrophages…………….…………………………...28 1.4 Research objectives…………………………………………………………………………30 1.5 References…………………………………………………………………………………...31  iv     CHAPTER TWO: CS addition to CD44 negatively regulates HA binding………………...39 2.1 Introduction…………………………………………………………………………………40 2.2 Materials and methods……………………………………………………………………..42 2.2.1 Cell lines and transfections……………………………………………………………42 2.2.2 Antibodies and reagents…………………………………………………………….…42 2.2.3 Generation of point mutations………………………………………………………...43 2.2.4 Flow cytometry………………………………………………………………………..44 2.2.5 Immunoprecipitation, sulfate labeling and western blotting………………………….44 2.2.6 Detection of CS stub…………………………………………………………………..46 2.3 Results……………………………………………………………………………………….46 2.3.1 Serine residue 180 is the primary site of CS addition in CD44H……………………..46 2.3.2 CS inhibits HA binding by CD44-Fc…………………………………………………48 2.3.2 CS inhibits HA binding by cell surface CD44H………………………………………51 2.4 Discussion…………………………………………………………………………………...53 2.5 References…………………………………………………………………………………...55  CHAPTER THREE: CS addition to CD44 regulates HA binding in macrophages……….58 3.1 Introduction…………………………………………………………………………………59 3.2 Materials and methods……………………………………………………………………..60 3.2.1 Cell and cell lines……………………………………………………………………..60 3.2.2 Antibodies and reagents……………………………………………………………….61 3.2.3 Generation of point mutations………………………………………………………...62 3.2.4 Retroviral infection……………………………………………………………………62 3.2.5 Flow cytometry………………………………………………………………………..63 3.2.6 Analysis of CS on CD44……………………………………………………………...63 3.2.7 Semi-quantitative RT-PCR……………………………………………………………64 3.2.8 Statistics……………………………………………………………………………….64 3.3 Results……………………………………………………………………………………….65 3.3.1 Pro-inflammatory cytokines induce HA binding in BMDM………………………….65 3.3.2 TNFα stimulation reduces CS addition to CD44 in BMDM………………………….68 3.3.3 CS addition to CD44 regulates HA binding during TNFα stimulation……………….73 v     3.3.4 CS addition to CD44 is regulated by multiple cytokines……………………………..75 3.4 Discussion…………………………………………………………………………………...77 3.5 References…………………………………………………………………………………...80  CHAPTER FOUR: HA induces cell death in activated T cells through CD44……………..83 4.1 Introduction…………………………………………………………………………………84 4.2 Materials and methods……………………………………………………………………..85 4.2.1 Cell lines………………………………………………………………………………85 4.2.1 Antibodies and reagents……………………………………………………………….86 4.2.3 Generation of point mutations………………………………………………………...87 4.2.4 Flow cytometry………………………………………………………………………..87 4.2.5 Analysis of CS on CD44……………………………………………………………...88 4.2.6 Low molecular mass HA generation and analysis…………………………….……....88 4.2.7 Cell death analysis…………………………………………………………………….89 4.2.8 Induction of cell death…...……………………………………………………………90 4.2.9 Cell stimulation………………………………………………………………………..90 4.2.10 FasL RT-PCR………………………………………………………………………..91 4.2.11 Primary cells…………………………………………………………………………91 4.2.12 Statistics……………………………………………………………………………...92 4.3 Results……………………………………………………………………………………….92 4.3.1 CS addition to CD44 regulates HA binding in Jurkat T cells…………………………92 4.3.2 High HA binding T cells are more susceptible to AICD……………………………...95 4.3.4 HA binding by CD44 enhances AICD in T cells…..…………………...…….............97 4.3.5 The size of HA affects its ability to enhance cell death………………………………99 4.3.6 HA rapidly induces cell death in PMA stimulated Jurkat T cells………………….101 4.3.7 HA-induced cell death occurs independently of Fas-and caspase-mediated apoptosis…………………………………………………….…104 4.3.8 CD44 and HA can mediate AICD in ex-vivo T cells…………………………..……106 4.4 Discussion…………………………………………………………...……………………110 4.5 References………………………………………………………………………………….114  vi     CHAPTER FIVE: Summary and perspectives……………………………………………...118 5.1 Results and future directions……………………………………………………………..119 5.1.1 Mechanism of CS regulation………………………………………………………...119 5.1.2 Role of CS addition and HA binding in macrophages……………….……………123 5.1.3 Functional consequences of HA binding by CD44 in T lymphocytes………………127 5.2 Concluding remarks …………………………..………………………………………….131 5.3 References………………………………………………………………………………….133 APPENDIX…………………………………………………………………………………….138 Animal Care Certificates…………………………………………………………………139 Biohazard Approval Certificates…………………………………………………………144  vii     LIST OF TABLES  CHAPTER 1  TABLE 1.1 Role of HA binding by CD44 in the immune system ……………………………...19  TABLE 1.2 CD44 in autoimmune and inflammatory diseases………………………………….20  viii     LIST OF FIGURES  CHAPTER 1  FIGURE 1.1 Haematopoiesis………………………………………….………………..………...3  FIGURE 1.2 T lymphocyte differentiation………………………………………………………..6  FIGURE 1.3 Adhesion molecule families…………………………………………….…………..8  FIGURE 1.4 Leukocyte adhesion to activated endothelium………………………….…………..9  FIGURE 1.5 Structure of the different glycosaminoglycan disaccharides……………………..11  FIGURE 1.6 Structure of hyaluronan binding proteins………………………………………..12  FIGURE 1.7 Structure of CD44……………………………………………………..…………..14  FIGURE 1.8 Fas signaling pathway……………………………………………………………..24  FIGURE 1.9 Role of cell death in the T lymphocyte life cycle………………………………..25  FIGURE 1.10 Chondroitin sulfate synthesis and structure………………………………...…..29  ix     CHAPTER 2  FIGURE 2.1 CS addition to CD44 occurs primarily at serine 180……………………………...47  FIGURE 2.2 HA binding ability of wild-type and mutant CD44-Fc fusion proteins…………..50  FIGURE 2.3 CS addition and HA binding in mouse L cells transfected with human CD44H….…………………………………………………………………………..52  CHAPTER 3  FIGURE 3.1 Pro-inflammatory cytokines induce HA binding in BMDM……………………...66  FIGURE 3.2 TNFα does not induce expression of sulfated epitopes in BMDM…………….….67  FIGURE 3.3 CS addition to CD44 negatively correlates with HA binding in KG1a cells……………………………………………………………………………69 FIGURE 3.4 β-D-xyloside increases HA binding in BMDM…………………………………...70  FIGURE 3.5 TNFα stimulation reduces CS addition to CD44 in BMDM………………………72  FIGURE 3.6 CS addition to CD44 regulates HA binding during TNFα stimulation……………74  FIGURE 3.7 CS addition to CD44 is regulated by multiple cytokines………………………….76  x     CHAPTER 4  FIGURE 4.1 A S180A mutation in CD44 prevents CS addition and results in constitutive HA binding in Jurkat cells………………………………………………..94  FIGURE 4.2 Cell death is preferentially induced in TCR or PMA stimulated Jurkat cells expressing a high HA binding form of CD44……………………………………….96  FIGURE 4.3 CD44 mAbs can induce or prevent cell death of transfected Jurkat cells during PMA stimulation…………………………………………………………….98  FIGURE 4.4 Enhanced AICD in Jurkat transfectants is dependent upon HA binding by CD44……………………………………………………………………100  FIGURE 4.5 The size of HA affects its ability to induce cell death in PMA stimulated cells…………………………………………………………………………...102  FIGURE 4.6 HA can rapidly induce cell death in CD44 expressing cells after PMA activation……………………………………………………………………………103  FIGURE 4.7 HA-induced cell death does not occur via the Fas/FasL pathway……………..105  FIGURE 4.8 HA-induced cell death does not occur via the mitochondrial pathway………..…107  FIGURE 4.9 AICD in ex-vivo activated murine splenic T cells is enhanced by the presence of HA………………………………………………………………………….109  xi     LIST OF ABBREVIATIONS Ab AICD APC BSA BMDM β-D-xyloside β-Me CD44H CD44-Fc cDNA CS cm Da DMDM DNA EAE EAU ECL ECM EDTA FACS FasL FCS FITC Fl-HA GAG HA HBSS HEPES HRP hrs HS ICAM IFN Ig IL IRES LCCM LFA-1 LPS LYVE-1 mAb MAdCAM M-CSF  antibody activation-induced cell death antigen-presenting cell bovine serum albumin bone marrow derived macrophages p-nitrophenyl β-D-xylopyranoside β- mercaptoethanol CD44 hematopoietic/standard form immunoglobulin Fc-fusion protein complementary deoxyribonucleic acid chondroitin sulfate centimeter Daltons Dublbecco’s minimum essential medium deoxyribonucleic acid experimental autoimmune encephalomyelitis experimental autoimmune uveoretinitis enhanced chemiluminescence extracellular matrix ethylenediaminetetraacetic acid fluorescence activated cell sorting Fas ligand fetal calf serum fluorescein isothiocyanate fluorescein-conjugated hyaluronan glycosaminoglycan Hyaluronan Hank’s balanced salt solution 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid horse radish peroxidase hours heparan sulfate intercellular cell adhesion molecule interferon immunoglobulin interleukin internal ribosomal entry site L929 cell-conditioned media leukocyte function-associated antigen-1 lipopolysaccharide lymphatic vessel endothelial receptor-1 monoclonal antibody mucosal addressin cell adhesion molecule macrophage colony-stimulating factor xii      MFI min mg ml MMP mM mRNA MSCV µg µl µCi ng PAGE PAMPS PBM PBS PCR PE PI PMA PRR PSGL-1 PS RNA RPMI RT-PCR SD SDS SEM TBST TCR TGF TLR TNF TSG-6 VCAM-1 VLA-4 YFP  mean fluorescence intensity minute milligram milliliter matrix metalloproteinase millimolar messenger ribonucleic acid murine stem cell virus microgram microliter microcurie nanogram polyacrylamide gel electrophoresis pathogen-associated molecular patterns peripheral blood monocytes phosphate buffered saline PDGF platelet-derived growth factor polymerase chain reaction phycoerythrin propidium iodide phorbol 12-myristate 13-acetate pattern recognition receptors P-selectin glycoprotein ligand-1 phosphatidylserine ribonucleic acid Roswell Park Memorial Institute reverse transcriptase polymerase chain reaction standard deviation sodium dodecyl sulfate standard error of the mean Tris-buffered saline with tween T cell receptor transforming growth factor toll-like receptor tumor necrosis factor tumor necrosis factor alpha stimulated gene 6 vascular cell adhesion molecule-1 very late antigen-4 yellow fluorescent protein  xiii     ACKNOWLEDGEMENTS  I would like to sincerely thank Dr. Pauline Johnson for her support, assistance and guidance during my time in her laboratory. The intellectual contribution of my supervisory committee, Dr. Kelly McNagny, Dr. Hermann Ziltener, and Dr. Rachel Fernandez is also gratefully acknowledged. Technical assistance and reagents provided by Andy Johnson, Jennifer Cox and Bob Argiropoulos were invaluable. I wish to thank past and present members of Dr. Pauline Johnson’s lab for assistance and the Department of Microbiology and Immunology and UBC for support. Personal financial assistance from the Heart and Stroke Foundation of Canada, John Richard Turner Fellowship in Microbiology, Armauer-Hansen Memorial Scholarship, and the Robert Emmanuel and Mary Day Endowment was greatly appreciated.  xiv     DEDICATION Dedicated to my wife for her continuing encouragement  xv     CO-AUTHORSHIP STATEMENT Research design, data analysis and manuscript preparation were completed with the assistance of Dr. Pauline Johnson. Experimental research was conducted by the author with the following exceptions: Sie-Lung Tjew was responsible for figures 3.2B, 3.2C, and 3.5C; Darlene Birkenhead was responsible for figure 3.1B; some bone marrow isolations were performed by Jen Cross, Darlene Birkenhead, and Grace Poon; some T cell preparations were performed by Nina Maeshima.  xvi     CHAPTER ONE Introduction  A version of this chapter will be submitted for publication: Ruffell, B. and Johnson, P (2008) Hyaluronan and CD44 in the immune system      1.1 THE IMMUNE SYSTEM The importance of a functional immune system is readily observable in humans suffering from immune deficiency. These individuals are highly susceptible to infection by pathogenic organisms and experience greatly diminished life expectancies (1). However, even individuals with fully competent immune systems can succumb to infection, particularly in cases where pathogens are able to regulate the immune response (2-4). Interestingly, while pathogens are usually the causative agent that initiates an immune response, it is the immune system itself that is responsible for a majority of the symptoms associated with infection (5, 6). An overactive immune response results in uncontrolled inflammation and tissue damage, while recognition of self-antigens can lead to the development of autoimmune diseases (7). Appropriate regulation and targeting of the immune response is therefore as critical to the clearance of a pathogen as it is to resolving inflammation and recovery.  1.1.1 The innate immune response The immune system is divided into innate and adaptive immunity, with the evolutionary conserved innate immune system providing the initial response against pathogens (8). Innate immune cells derive from hematopoietic stem cells in the bone marrow and include neutrophils, macrophages, natural killer cells, basophils, mast cells, eosinophils, and dendritic cells (Fig. 1.1). Neutrophils and macrophages provide defence against bacterial infection through phagocytosis and the production of reactive oxygen species (9), natural killer cells are cytotoxic towards virally-infected or cancer cells (10), and mast cells, basophils, and eosinophils are important for resisting parasitic infection (11). Dendritic cells meanwhile do not act directly upon pathogens, but are instead professional antigen-presenting cells (APC) responsible for initiating and  2     FIGURE 1.1 Haematopoiesis. Mature cells of the innate immune system are highlighted with a ( ) and mature cells of the adaptive immune system are highlighted with a ( ). Modified from Wikipedia.org (http://en.wikipedia.org/wiki/Hematopoiesis)  !  regulating the adaptive immune response (12). With the exception of neutrophils and basophils, small numbers of innate immune cells exist as long-lived tissue-resident cells under homeostatic conditions (10-16). Upon tissue damage or pathogen detection, the release of inflammatory mediators induces molecular changes on the surface of nearby endothelial cells, resulting in a significant increase in leukocyte recruitment to the area (17, 18). This influx of innate immune cells is necessary to combat infection, and in the case of macrophage recruitment, is also important for subsequent wound healing (19).  1.1.2 The adaptive immune response Innate immune cells utilize pattern-recognition receptors (PRR) to identify important and highly conserved molecules synthesized by pathogens and other microbes termed pathogen-associated molecular patterns (PAMPS) (20). In contrast, cells of the adaptive immune system generate unique receptors capable of binding an array of possible peptides through a combination of gene rearrangement and random nucleotide insertion (21, 22). This allows recognition of novel pathogens and may help reduce immune evasion by rapidly evolving pathogens. Because few cells capable of recognizing a particular pathogen will exist at any one time, the height of the adaptive immune response occurs following clonal expansion and effector cell differentiation approximately 5-8 days after initial exposure to a pathogen. However, the formation of longlived memory cells, the basis of immunity, allows a more rapid response to be generated against pathogens to which a host has been previously exposed (23). T and B lymphocytes, so named due to their predominant localization to secondary lymphoid organs, are the two cell types belonging to the adaptive immune system. B cells are responsible for the production of antibodies (Abs), soluble proteins that can bind to a pathogen  4     and initiate the complement cascade or activate effector functions, such as phagocytosis, through receptors expressed on innate immune cells. Most vaccines are designed to induce a strong Ab response, which is effective at providing humoral-based immunity to most extracellular and some intracellular pathogens (24). Both humoral and cellular immunity is dependent upon T cells (Fig. 1.2). Helper T cells can provide the second signal required for B cell activation (25) and can activate macrophages to promote destruction of intracellular pathogens attempting to reside within these phagocytic cells (26). Cytotoxic T cells also act on cells infected with an intracellular pathogen by killing target cells displaying the appropriate peptide (27). Mounting the appropriate T cell response to a given pathogen is necessary for an effective immune response (4).  1.2 CELL ADHESION AND MIGRATION 1.2.1 Cell adhesion in the immune system Cell-cell and cell-extracellular matrix (ECM) interactions are critical for both the development and function of the immune system. For example, haematopoiesis in the bone marrow requires stable contact within the microenvironment (28), while the development of T lymphocytes involves transient adhesive interactions during thymus localization and migration within thymic compartments (29). Leukocyte activation, trafficking and effector function are also dependent upon adhesion and migration. Long-term contacts are formed between T cells and dendritic cells during lymphocyte activation, while cytotoxic T cells and natural killer cells are unable to kill target cells in the absence of adhesion molecule interactions (30, 31). Immune surveillance and trafficking to inflammatory sites meanwhile require both cell-cell and cell-ECM interactions. Circulating leukocytes must undergo a rapid series of transient interactions prior to adhering to the luminal side of the endothelium, transmigrating out of the blood, and migrating through the 5     Cytotoxic Kills cells expressing Ag (virus, cancer)  T lymphocyte  Th1 Activates macrophages (intracellular bacteria) Th2 Activates B cells (parasites, bacteria)  Dendritic cell  Th17 Produces IL-17 (bacteria, fungus) FIGURE 1.2 T lymphocyte differentiation. Following activation by dendritic cells, CD8+ T cells differentiate into cytotoxic T cells and CD4+ T cells differentiate into T helper cells (Th1, Th2, Th17).  $  tissues (17, 32). This process of extravasation, as with other adhesion-dependent events in the immune system, involves molecules from several different families (33).  1.2.2 Cell adhesion molecules in extravasation Most cell adhesion molecules belong to 4 main protein families: cadherins, immunoglobulin (Ig) superfamily proteins, integrins, and selectins (Fig. 1.3). Molecules from all of these families are involved in the process of extravasation. The initial interaction between a leukocyte and the endothelium is mediated by selectins and integrins binding to Ig superfamily or other proteins expressed on the opposing cell (33), while cadherin interactions must be disrupted to facilitate transmigration by creating space within the cellular junction between endothelial cells (34). Differential expression of selectins, integrins, and Ig superfamily proteins is important for tissue specific localization of leukocytes (35). One of the most well known examples of this is the binding of L-selectin on naïve lymphocytes to the carbohydrate epitopes expressed on the high endothelial venules of lymph nodes. Similarly, induced expression of P-and E-selectin by activated endothelial cells directs leukocytes to inflamed tissues by binding to P-selectin glycoprotein ligand-1 (PSGL-1) and other ligands expressed on the leukocyte (36-38). Selective expression of integrins can also mediate T cell localization, with lymphocyte expression of α4β7 directing cells to gut-associated tissues expressing mucosal addressin cell adhesion molecule (MAdCAM). Selectin-ligand interactions are primarily responsible for the “tethering” and “rolling” interactions that initiate the leukocyte adhesion cascade (Fig. 1.4), although integrins containing the α4 subunit can also mediate a rolling interaction. Subsequent firm adhesion and transmigration are highly dependent upon the interaction of integrins with members of the Ig  7     Ca2+  E-, P-, N-, Cadherins  P- E- LSelectins  MAdCAM  NCAM  ICAMs  Integrins  Immunoglobulins  FIGURE 1.3 Adhesion molecule families. MAdCAM, mucosal addressin cell adhesion molecule; ICAM intercellular cell adhesion molecule; NCAM, neural cell adhesion molecule. Modified from: Petruzzelli L, Takami M, and HD Humes (1999) Structure and function of cell adhesion molecules. Am J Med 106(4): 467-476.  &  CD44? Integrins Selectins  Chemokines  Blood Flow  Rolling Adhesion  Arrest  Diapedesis  FIGURE 1.4 Leukocyte adhesion to activated endothelium. The molecules implicated at each stage are indicated above. Circles ( ) represent inflammatory agents such as chemokines. Modified from: Johnson P, Maiti A, Brown KL and R Li (2000) A role for the cell adhesion molecule CD44 and sulfation in leukocyte-endothelial cell adhesion during an inflammatory response? Biochem Pharmacol 59 (5): 455-465  '  superfamily (39, 40). This includes the binding of leukocyte function-associated antigen-1 (LFA1) to intercellular cell adhesion molecule (ICAM) and very late antigen-4 (VLA-4) to vascular cell adhesion molecule-1 (VCAM-1). Normally inactive integrins become activated during rolling due to binding of chemokines on the surface of the endothelium to G protein-linked receptors (41). Chemokines are selectively expressed in a tissue and condition specific manner, and binding of these polypeptides to their receptors, which are differentially expressed on leukocyte subsets, provides an additional level of control over cell localization (42). Integrins are also important for tissue migration, as in addition to mediating cell-cell contact, they are largely responsible for connecting the cell to the ECM. Another molecule involved in both extravasation and migration is CD44, a member of the hyaladerin family of extracellular and transmembrane proteins that bind to the ECM component hyaluronan (HA).  1.3 CD44 AND HYALURONAN 1.3.1 Introduction to HA The ECM glycosaminoglycan (GAG) HA is an unbranched polymer composed of a repeating β(1-4)-D-glucuronic acid-β-(1-3)-N-acetyl-D-glucosamine disaccharide (Fig. 1.5). Unlike other GAGs that are made as proteoglycans, HA is not linked to a protein core and is instead synthesized by three hyaluronan synthases on the inner surface of the plasma membrane. Transportation of HA to the cell surface during synthesis allows up to 25,000 disaccharides to be linked together, creating a polysaccharide with a molecular mass that normally ranges between 106-107 Daltons (43-45). HA is a critical component of the ECM, demonstrated by the embryonic lethality of hyaluronan synthase-2 knockout mice (46), and is bound by a number of ECM and cell surface proteins. These HA-binding proteins are termed hyaladerins (Fig. 1.6) and share a homologous 10     GAG  Hexuronic or Iduronic acid  Galactose  Hexosamine  Disaccharide composition COO  Heparan Sulfate / Heparin  D-glucuronic acid (GlcA)  -  D-glucosamine (GlcNAc)  O  CH2OH O  OH  OH  O  OH  NHCOCH3  GlcA β(1-4) GlcNAc α(1-4) CH2OH  L-iduronic acid (IdoA)  -  D-glucosamine (GlcNAc)  O  COO OH  O  OH  O  OH  NHCOCH3  IdoA β(1-4) GlcNAc α(1-4) CH2OH  Keratan Sulfate  CH2OH HO  HO  -  Galactose (Gal)  D-glucosamine (GlcNAc)  O  OH  O  OH  OH  NHCOCH3  Gal β(1-4) GlcNAc β(1-4) COO-  Chondroitin Sulfate  D-glucuronic acid (GlcA)  CH2OH HO  -  D-galactosamine (GalNAc)  O  O  O  OH OH  NHCOCH3  GlcA β(1-3) GalNAc β(1-4) CH2OH  Dermatan Sulfate  L-iduronic acid (IdoA)  -  D-galactosamine (GalNAc)  O  HO  COOOH  O  O OH  NHCOCH3  IdoA β(1-3) GalNAc β(1-4) COO  Hyaluronan / Hyaluronic acid  D-glucuronic acid (GlcA)  -  D-glucosamine (GlcNAc)  O  CH2OH O  OH  O  HO OH  NHCOCH3  GlcA β(1-3) GlcNAc β(1-4)  FIGURE 1.5 Structure of the different glycosaminoglycan (GAG) disaccharides. Potential sulfation positions are marked with a star ( ). Modified from: Prydz K and KT Dalen (2000) Synthesis and sorting of proteoglycans. J Cell Sci 113 pt 2: 193-205.    Aggrecan Versican Neurocan Brevican RHAMM Link Protein TSG-6 CD44 ~100 amino acids  LYVE-1  Link  C-type lectin  CUB  Transmembrane  Cytoplasmic  CCP  Unknown  EGF  GAG attachment  Ig  FIGURE 1.6 Structure of hyaluronan binding proteins. Only members of the Link module superfamily are shown, with the exception of RHAMM (receptor for hyaluronan-mediated motility). Modified from: Day AJ (2001) Understanding hyaluronan-protein interactions. Glycoforum website (http://www.glycoforum.gr.jp/science/hyaluronan/HA16/HA16E.html).    globular region at the amino-terminal end of the protein called the Link homology domain (47). The majority of these proteins are found within the ECM, including aggregan, versican, and tumor necrosis factor alpha stimulated gene 6 (TSG-6). Only 2 transmembrane hyaladerins have been described, and one of these, lymphatic vessel endothelial receptor-1 (LYVE-1), is predominately expressed by lymphatic endothelium (48). The other more widely expressed molecule, CD44, has been implicated in a range of processes that are largely, but not exclusively, dependent on its function as a HA-binding protein (49).  1.3.2 Introduction to CD44 CD44 is a type I transmembrane protein expressed by most cell types, including all mature immune cells such as T and B lymphocytes, monocytes, macrophages, dendritic cells, natural killer cells, neutrophils, and eosinophils. These cells predominately express the standard (CD44s) or hematopoietic (CD44H) form of CD44, as opposed to one of the many isoforms that can result from alternative splicing within the membrane proximal region (Fig. 1.7). However, even in the absence of alternative isoform expression, there is a high degree of heterogeneity in the structure of CD44 due to extensive post-translational modifications. While the protein backbone of CD44 has an expected size of 36 kDa, CD44 is usually found in a 80-90 kDa form due to N-and O-linked glycosylation, as well as the addition of glycosaminoglycans, sialic acid, and sulfate groups (49-51). These modifications result in the formation of a number of epitopes, including well characterized blood group antigens (52, 53) and ligands for the selectin family of adhesion molecules (54-56). These modifications also provide a means to tightly regulate HA binding by CD44. The importance of this can be observed in immune cells, which although express CD44 constitutively, do not bind soluble HA unless activated or stimulated by  13     FIGURE 1.7 Structure of CD44. Regions and domains of CD44 are shown on the left with the amino acid numbers on the scale bar. A graphical interpretation of the CD44 structure is shown on the right. Cysteine disulfide bonds are represented in the CD44 globular domain by a (S--S). Potential serine phosphorylation sites are indicated in the cytoplasmic domain by a ( ). Modified from: Lesley, J and R Hyman (1998) CD44 structure and function. Front Biosci 3: d616-630.  "  inflammatory conditions (57-59). The precise function of the CD44-HA interaction in the immune system is not clear. While CD44 was originally classified as a homing receptor due to the ability of Abs against CD44 to inhibit cell recruitment under homeostatic conditions (60, 61), this conclusion has been questioned by experiments with CD44 knockout mice (62). However, other studies have demonstrated that CD44 is important for recruitment of immune cells to sites of inflammation (63) and have also indicated a possible role for CD44 in activation (64, 65) and immune cell function (66-69).  1.3.3 CD44 function in the immune system 1.3.3a Leukocyte recruitment to the inflammatory site CD44 was first described as a homing receptor due to the ability of CD44 monoclonal Abs (mAbs) to inhibit T cell binding to high endothelial venules from the lymph node (60). Following this, CD44 mAbs were also shown to inhibit recruitment of progenitor cells to the thymus (61). However, no obvious defects in the development of the immune system were observed in CD44 knockout mice (62), and direct competition experiments between wild type and CD44 knockout cells have indicated that CD44 is not important for T cell recruitment to the lymph node (70) and has only a small effect on the seeding of progenitor cells in the thymus (71). While a function for CD44 under homeostatic conditions is debatable, CD44 does appear to play a key role in the recruitment of T cells to sites of inflammation in specific organs. This discovery began with the observation that the interaction of CD44 with HA can mediate the initial rolling interaction between lymphocytes and endothelial cells (72, 73). The rolling interaction is required for cells to then firmly adhere and subsequently extravasate into the tissue, and blocking the CD44-HA interaction reduces the number of T cells recruited to an inflamed 15     peritoneum (63). HA can be expressed on the surface of endothelial cells, with an increase in the amount of HA occurring when the cells are subjected to inflammatory conditions (74). As T cells display CD44-dependent HA binding upon activation (57, 75), it has been proposed that extravasation of activated T cells to sites of inflammation is initiated by lymphocyte CD44 interacting with HA expressed on inflamed endothelium (76). Endothelial cells also express CD44 capable of binding HA, particularly following treatment with inflammatory cytokines, and HA bound to CD44 can support cell rolling in vitro (77). This has led to the suggestion of a “sandwich” model, whereby CD44 on endothelial cells allows HA to be presented to CD44 on activated T cells in the blood. The importance of CD44 for T cell extravasation has been demonstrated in several models of autoimmune diseases including collagen-induced arthritis (78), allergic dermatitis (79), experimental autoimmune uveoretinitis (EAU) (80) and experimental autoimmune encephalomyelitis (EAE) (81, 82). In the arthritis model, CD44 knockout mice were less susceptible to the induction of the disease, while affected animals displayed reduced severity in the absence of CD44. Using adoptive transfer to analyze T cell trafficking patterns, it was found that CD44-/- T cells had delayed entry into the joints of arthritic mice, suggesting that CD44dependent T cell extravasation is important in the progression of arthritis (70, 78). In models of EAE, administration of Abs against CD44 reduced T cell trafficking to the spinal cord (81) and brain (82) and prevented the development of EAE. Unfortunately, disease prevention required continuous administration of the anti-CD44 Ab as the incidence of EAE returned to normal once treatment was stopped. Interestingly, CD44-dependent extravasation to the inflamed peritoneum requires firm adhesion mediated by the α4β1 integrin VLA-4 (83, 84), and an Ab against α4 integrin can reduce leukocyte recruitment to the brain and prevent the  16     occurrence of EAE (82). This could indicate that, in addition to the peritoneum, a cooperative interaction between CD44 and VLA-4 is involved in leukocyte recruitment to the brain. CD44 expression may also be important for the recruitment of neutrophils (85) and macrophages (86) to sites of inflammation. Unlike activated T cells however, neither of these cells bind to HA when in circulation, suggesting that the sandwich model may not apply. Neutrophils for example, do not roll on HA in vitro and do not display a defect in rolling in vivo. Despite this, neutrophil accumulation in the tissues is reduced in CD44 knockout mice in a chemokine model of recruitment (85) and within liver sinusoids of endotoxemic mice (87). An analysis of CD44 function using chimeric mice demonstrated that while endothelial CD44 was crucial for neutrophil adhesion in the chemokine model, the absence of CD44 on neutrophils did not greatly affect their adhesion to the endothelium. CD44 expression was required however for normal neutrophil emigration out of the vasculature. This may be explained by the reduced ability of CD44 negative neutrophils to migrate towards a chemotactic signal in vitro (88), although neutrophils did appear to migrate normally in the tissues once they have managed to extravasate (85). Surprisingly, neutrophils do not bind detectable levels of soluble HA following activation with a number of inflammatory mediators, even though blocking the CD44-HA interaction reduces neutrophil adhesion in vivo (87). This discrepancy may be explained by two studies that have shown that CD44 binding to HA is increased when HA is associated with other proteins (89, 90). This suggests that the ability of cells to bind soluble HA in vitro may not accurately reflect the ability of cells to bind HA in vivo. Given the discrepancies between potential CD44 functions resulting from Ab cross-linking and demonstrated functions for the CD44-HA interaction, the use of a more physiological form of HA for in vitro studies may expand the number of functions attributable to HA binding by CD44.  17     The function of CD44 on the endothelium during neutrophil recruitment is unclear. One possibility is that proteoglycan forms of CD44 are acting as receptors for chemokines (91, 92). Chemokine activation of circulating leukocytes is necessary for firm adhesion to occur and reduced chemokine accumulation could explain the reduction in neutrophil adhesion. However, as hyaluronidase treatment prevents neutrophil adhesion in vivo, this indicates that the ability of CD44 to support an HA rich extracellular matrix is of crucial importance (85). This is also likely due to chemokine accumulation, as HA forms the scaffold for numerous proteoglycans in the extracellular matrix. Interestingly, the presence of HA at a wound site is also important for recruitment of macrophages (93). While the importance of CD44 was not investigated in this case, reduced macrophage recruitment into inflamed lungs following Mycobacterium tuberculosis infection (94) and lipopolysaccharide (LPS) inhalation (95) has been observed in CD44-/- mice. However, it is not clear from these models that macrophage recruitment is impaired, as reduced overall inflammation would also result in reduced leukocyte recruitment. CD44 null macrophages have been shown to have reduced ability to migrate to atherosclerotic lesions in a mouse model of atherosclerosis (86) and monocytes, the macrophage precursor cell found in the blood, can bind to HA following stimulation with inflammatory mediators (59). However, monocytes are not likely to experience long term exposure to inflammatory cytokines in the blood, so the importance of HA binding for macrophage recruitment may be similar to that observed in neutrophils, in which CD44-HA interaction plays a role in adhesion and migration within the tissues, but not on the endothelium.  18     TABLE 1.1 Role of HA binding by CD44 in the immune system  Function  Leukocyte  Mechanism  Inflammation  References  in CD44 KO Recruitment to  T lymphocyte  Rolling on endothelium  Reduced  (63, 73)  site of  Neutrophil  Emigration/sequestration  Reduced  (85, 87)  Macrophage  Unknown  Reduced  (86)  Activation  T lymphocyte  Unknown  Unknown  (66, 96)  AICD  T lymphocyte  Unknown  Unknown  (65, 97)  HA uptake  Macrophage  CD44-mediated endocytosis  Increased  (98, 99)  inflammation  19     TABLE 1.2 CD44 in autoimmune and inflammatory diseases Severity in  Disease Model  Leukocyte CD44 Function  Methodology  Arthritis  T cell recruitment  CD44 KO  Reduced  (78)  EAE  T cell recruitment  CD44 Abs  Reduced?  (81, 82)  EAU  T cell recruitment  CD44 Abs  Reduced?  (80)  Dermatitis  T cell recruitment  CD44 KO  Reduced  (79)  Atherosclerosis  Macrophage recruitment  CD44 KO  Reduced  (86)  Hepatitis  Neutrophil sequestration  CD44 KO  Reduced  (87)  Colitis  Reduce cell death  CD44v7 KO  Reduced  (100)  Hepatitis  Increase cell death  CD44v7 KO  Increased  (101)  Hepatitis  Increase cell death  CD44 KO  Increased  (102)  Lung Inflammation  Fragmented HA uptake  CD44 KO  Increased  (98)  Endotoxic Shock  Reduce TLR signaling  CD44 KO  Increased  (103)  20     References  CD44 KO  1.3.3b Resolution of inflammation The role of CD44 in the recruitment of immune cells suggests a largely pro-inflammatory role for CD44. However, in a lung injury model using intratracheal administration of bleomycin, in which mice normally recover, it was observed that 75% of CD44 knockout mice died within 2 weeks (98). The increase in death was attributed to a failure to resolve the inflammation, specifically due to reduced processing of transforming growth factor-β1 (TGF-β1), impaired removal of apoptotic neutrophils, and accumulation of both high molecular mass and fragmented hyaluronan. CD44 is involved in the activation of TGF-β1 through its association with matrix metalloproteinase-9 (MMP-9) (104). Soluble MMP-9 is localized to the cell surface by its interaction with CD44, allowing it to cleave and activate TGF-β1. Whether this process occurs primarily on immune cells or the surrounding tissue is unclear, but TGF-β1 is crucial for downregulating the immune response in many experimental systems (105), including lung injury (106). CD44 may also be involved phagocytosis. Incubation of macrophages with an anti-CD44 Ab increases the ability of cells to phagocytose apoptotic neutrophils (107), while artificially coating cells with anti-CD44 antibodies allows them to be phagocytosed by macrophages (108). However, the only direct evidence that CD44 may be a phagocytic receptor for apoptotic cells involves CD44 Ab blocking in endothelial cells (109). Thus, whether CD44 is an apoptotic receptor, and the possible identity of the CD44 ligand on apoptotic cells, remains unknown. Interestingly, it has been shown that CD44 expression is important for the ability of macrophages to bind and internalize Mycobacterium tuberculosis (94). Fragmented HA accumulates at sites of injury and can activate cells, including macrophages, through toll-like receptor (TLR)-2 and -4 (110). The majority of HA is normally  21     turned over in the tissues by removal via the lymphatic system. However, local turnover of HA can account for over 25% of HA degradation (45). The importance of CD44 for this local turnover has been demonstrated in the skin, where the inhibition of CD44 expression by keratinocytes results in the accumulation of HA (111). HA uptake in the skin and other tissues, although not in the lymph nodes, occurs via CD44 (98, 112). Macrophage CD44 in particular has been implicated in the local turnover of HA in the lungs (99). Consistent with this, HA accumulation in the lungs of CD44 knockout mice treated with bleomycin was reduced in chimeric mice reconstituted with wild type bone marrow (98). These studies suggest an important role for CD44 in the removal of fragmented HA from inflammatory sites. In addition, while CD44 is not the receptor by which fragmented HA can activate macrophage and dendritic cells, it does appear that CD44 can act as a negative regulator of TLR signaling (103, 113). Therefore, through the removal of pro-inflammatory fragmented HA and the inhibition of TLR signaling, CD44 may inhibit the inflammatory response and assist in the resolution of inflammation.  1.3.3c T cell response During T cell development, CD44 expression can be used to distinguish between different double negative subsets, although the relevance of these changes in CD44 expression is unclear. CD44 is constitutively expressed in mature T cells; however, expression is increased upon activation and high CD44 levels are commonly used as a marker for activated and memory T cells in mice. Along with high CD44 expression, a percentage of activated T cells are able to bind HA. HA binding cells are observed in both the CD4+ and CD8+ T cell populations, and it does not appear that HA binding occurs in a specific subset of T cells (57). Memory T cells also  22     do not bind HA unless reactivated, thereby demonstrating that while CD44 expression is important, it is not the only determining factor in whether cells are able to bind HA. CD44 has the potential to provide co-stimulatory activity during T cell activation, as cross-linking CD44 together with the T cell receptor (TCR) increases proliferation and the expression of activation markers, as well as augmenting interleukin-2 (IL-2) production (64, 65). HA binding is induced upon T cell activation and there is some evidence that HA can enhance CD3-mediated activation of human peripheral T cells (66). Other data suggest an indirect effect with HA binding to dendritic cells (114) and affecting conjugate formation (96), or by HA oligosaccharides increasing dendritic cell maturation via TLR-4 (115). Given that T cells do not bind HA until they have been activated, it remains to be shown if and how the interaction of HA with CD44 on T cells can increase activation. Of interest however, is that HA binding is a marker of functionally active T regulatory cells (68), with high molecular weight HA enhancing the suppressive ability of these cells (69). As there is some evidence that HA (66) and CD44 cross-linking (67) can enhance the effector function of cytotoxic T cells, it is possible that HA binding could be a marker for highly activated T cells. There is also some evidence that CD44 may be involved in activation-induced cell death (AICD). AICD occurs following secondary stimulation of activated T cells that have experienced prolonged exposure to IL-2 and primarily involves the interaction of the death receptor Fas with Fas Ligand (Fig. 1.8). The process of AICD, together with activated cellautonomous death (ACAD), is responsible for removal of activated T cells and maintenance of immune homeostasis (Fig. 1.9) (116). Two studies have found that AICD in T cell lines incubated on immobilized anti-CD3 mAb is reduced by CD44 cross-linking (97, 117). In addition, the absence of CD44 has been found to partially reduce the lymphoproliferative  23     FIGURE 1.8 Fas (CD95) signaling pathway. The binding of Fas Ligand (FasL) to Fas results in caspase 8 activation. This leads to direct activation of effector caspases and/or indirect activation of effector caspases through mitochondrial membrane depolarization and the release of cytochrome C. Subsequent changes within the cell include externalization of phosphotidylserine (PS), chromatin condensation, and DNA fragmentation, followed by the formation of apoptotic bodies and loss of plasma membrane integrity.  "  Bone Marrow  T lymphocyte development  Thymus Positive selection  Apoptosis  Cell survival  Negative selection  Central Tolerance  Periphery / Lymphoid organs  T lymphocyte activation  Antigen encounter  Apoptosis  Activation  Anergy / Apoptosis  Peripheral Tolerance  Proliferation / Differentiation  Tissues / Lymphoid organs Effector Function  Persistent antigen  Antigen clearance  Activation-induced cell death (AICD)  Activated-cell autonomous death (ACAD)  Contraction Phase  FIGURE 1.9 Role of cell death in the T lymphocyte life cycle. T cell precursors enter the thymus and survival signals are provided by recognition of MHC-peptide complexes through the T cell receptor (TCR). Following this positive selection step, T cells expressing TCRs that bind with high affinity to self-peptide are induced to undergo apoptosis (negative selection). Cells that survive enter the periphery and circulate between the blood and lymphoid organs. Upon encountering the appropriate MHC-peptide complex, cells will either become activated or anergic, depending upon the level of co-stimulation being provided by the antigen-presenting cell. After proliferation and differentiation, T cells enter the tissues or different lymphoid compartments and perform their effector function. Following antigen clearance, a reduction in pro-survival cytokines and TCR stimulation results in ACAD. During instances of persistent antigen, prolonged exposure to IL-2 causes cells to become susceptible to AICD, and TCR stimulation results in Fas- and Fas Ligand-dependent apoptosis.  #  symptoms found in mice that do not express Fas (118). However, in direct contrast to this, another study found increased lymphoproliferative and autoimmune symptoms in mice lacking expression of both CD44 and Fas (119), and CD44 cross-linking in another T cell line increases AICD (65). Therefore, the role of CD44 in AICD, and whether this is mediated by the interaction of CD44 with HA, remains to be determined.  1.3.4 Regulation of HA binding by CD44 Despite constitutive expression of CD44, mature immune cells are unable to bind soluble HA under homeostatic conditions. Instead, HA binding by T cells (57), B cells (120), macrophages (59) and eosinophils (121) is upregulated when these cells are activated or exposed to proinflammatory mediators. The unique ability of CD44 to act as both a pro- and anti-inflammatory molecule, depending on the context of the pathological condition being examined, indicates the importance of understanding the CD44-HA interaction.  1.3.4a HA binding regulation in T cells CD44 expression is increased in primary T cells upon activation (57) and HA binding is proportional to CD44 expression in transfected T cell lines (122), provided that a threshold level of CD44 expression is exceeded. However, CD44 expression is not the only factor that can affect the avidity of the CD44-HA interaction, as transfection of a CD44 cytoplasmic domain deletion mutant into mouse AKR T lymphoma cells results in a relatively low level of binding to soluble HA (123). This is not due to an inability of CD44 lacking the cytoplasmic domain to bind HA, as there is no observable defect in HA binding by CD44-immunoglobulin fusion proteins (124). Instead, as some CD44 antibodies are able to induce HA binding (123) it has been suggested that  26     CD44 clustering may be a mechanism by which HA binding is regulated (125). Clustering is observed in the mouse T lymphoma BW5147 cell line following incubation with HA (126) and the induction of HA binding in transfected Jurkat cells by PMA is dependent upon the presence of the CD44 cytoplasmic domain (127). Furthermore, in both these cell lines HA binding is disrupted by treatment with cytochalasin D, an inhibitor of actin polymerization. This suggests that the interaction of the CD44 cytoplasmic domain with the cytoskeleton is important for receptor localization and that CD44 clustering may be a mechanism used to regulate HA binding. HA binding can be induced in primary T cells by Ab treatment (128), although it remains to be seen whether CD44 clustering occurs naturally in T cells following activation and is important for HA binding in these cells. HA binding by CD44 can also be influenced by the various forms of post-translational modifications that occur on CD44. These modifications can have both positive and negative effects, depending on the type of glycosylation involved (129). The 5 N-glycosylation sites in CD44 are found within the amino-terminal HA-binding domain, and it is generally found that reduced N-glycosylation correlates with increased binding to HA and that treatment with an inhibitor of N-glycosylation, tunicamycin, increases HA binding (51). Part of this effect may be due to the terminal sialic acid residues found on N-linked glycans on CD44, which when removed by enzymatic digestion, also results in increased HA binding (130). However, there is limited data concerning the effect of glycosylation on the CD44-HA interaction specifically in T cells (122) and no obvious glycosylation changes on CD44 were observed following superantigen activation of lymph node T cells (75). It therefore remains to be shown that CD44 glycosylation changes occur following T cell activation and that these are responsible for the increase in HA binding observed.  27     1.3.4b HA binding regulation in macrophages Regulation of HA binding by CD44 has been more thoroughly examined in monocytes and macrophages. Human peripheral blood monocytes (PBM) are induced to bind HA upon stimulation with the inflammatory cytokine tumor necrosis factor-α (TNFα), in addition to several other cytokines (59, 131). This increase in HA binding is associated with increased CD44 expression as well as reduced CD44 glycosylation (132). Sialic acid residues also negatively affect HA binding in PBM (133). Intriguingly, this appears to be a regulated process as sialidase activity was induced by LPS, while HA binding was reduced when a sialidase inhibitor was used. As sialidase activity was not detected at the plasma membrane or in the culture supernatant, it suggests that the removal of sialic acid occurs during intracellular transport. Whether this digestion occurs during the transport of newly synthesized CD44 to the plasma membrane, or as suggested, during possible recycling of CD44, remains to be determined. HA binding in human monocytes also correlates with increased levels of CD44 sulfation following TNFα stimulation (134). In both a human monocytic cell line and PBM (131) an inhibitor of sulfation prevents TNFα inducible HA binding. How CD44 sulfation could increase HA binding is unclear and further work is required to determine if sulfation can alter either CD44 conformation or localization. HA binding can also be negatively regulated in TNFα stimulated PBM by incubation with IL-4 or IL-13 (59), and this is prevented when GAG addition is inhibited (132). The addition of the heavily sulfated GAG chondroitin sulfate (CS) occurs within the mucin-like membrane proximal domain of CD44 during protein synthesis (Fig. 1.10) (135). However, the specific site of covalent CS modification is unknown, as is the effect that this modification has on the HA binding ability of CD44.  28     A trans-Golgi network  Sulfotransferases  CS Polymerizing Enzymes  trans-Golgi  GlcA transferase  medial-Golgi  Gal transferases I and II  HS Polymerizing Enzymes  cis-Golgi cis-Golgi network  Y  ER-Golgi intermediate complex (ERGIC)  Y  Xylotransferase  Endoplasmic Reticulum (ER)  B  Linker Tetrasaccharide COO-  O  HO O  OH OH  COO-  CH2OH O  HO O  OH  NHCOCH3  OH  COO-  CH2OH O  HO O  OH  NHCOCH3  COO-  CH2OH  OH  O  HO O  OH OH  NHCOCH3  COO-  CH2OH O  HO O  OH  NHCOCH3  Chondroitinase ABC  O  HO O  OH OH  O  HO O  OH  NHCOCH3  OH  CH2OH  CH2OH  NH O  CH2 CH C=O  OH  Gal HO O  O  OH  OH  Gal  COO-  CH2OH  HO O  OH  OH  GlcA COO-  CH2OH  Xylose  CH2OH  OH  β-D-xyloside  NH O  O  OH  CH2 CH C=O  OH  OH  NH O  O  OH  CH2 CH  OH  FIGURE 1.10 Chondroitin sulfate (CS) synthesis and structure. A, Enzymes responsible for synthesis of the CS linker tetrasaccharide. CS polymerizing enzymes are found in the trans-Golgi and trans-Golgi network. Modified from: Prydz K and KT Dalen (2000) Synthesis and sorting of proteoglycans. J Cell Sci 113 pt 2: 193-205. B, Structure of CS and the linker tetrasaccharide. The effect of treatment with Chondroitinase ABC or the competitive inhibitor β-D-xyloside on the number of carbohydrates is shown.  '  C=O  1.4 RESEARCH OBJECTIVES HA binding by CD44 clearly plays an important role in the recruitment of leukocytes to an inflammatory site. As symptoms in autoimmune disease models are reduced in CD44 knockout mice, this makes CD44 an attractive therapeutic target. However, given that CD44 is also important in resolving inflammation, any treatment is likely to have unintended consequences in variable and multi-factored human diseases. 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Bimolecular Complex between Rolling and Firm Adhesion Receptors Required for Cell Arrest; CD44 Association with VLA-4 in T Cell Extravasation. Immunity 20:455-465. Siegelman, M. H., D. Stanescu, and P. Estess. 2000. The CD44-initiated pathway of Tcell extravasation uses VLA-4 but not LFA-1 for firm adhesion. J. Clin. Invest. 105:683691. Khan, A. I., S. M. Kerfoot, B. Heit, L. Liu, G. Andonegui, B. Ruffell, P. Johnson, and P. Kubes. 2004. Role of CD44 and hyaluronan in neutrophil recruitment. J Immunol 173:7594-7601. Cuff, C. A., D. Kothapalli, I. Azonobi, S. Chun, Y. M. Zhang, R. Belkin, C. Yeh, A. Secreto, R. K. Assoian, D. J. Rader, and E. Pure. 2001. The adhesion receptor CD44 promotes atherosclerosis by mediating inflammatory cell recruitment and vascular cell activation. J Clin Invest 108:1031-1040. McDonald, B., E. F. McAvoy, F. Lam, V. Gill, C. de la Motte, R. C. Savani, and P. Kubes. 2008. 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Abrogation of experimental colitis correlates with increased apoptosis in mice deficient for CD44 variant exon 7 (CD44v7). Journal of Experimental Medicine 191:2053-2063. McKallip, R. J., M. Fisher, U. Gunthert, A. K. Szakal, P. S. Nagarkatti, and M. Nagarkatti. 2005. Role of CD44 and its v7 isoform in staphylococcal enterotoxin Binduced toxic shock: CD44 deficiency on hepatic mononuclear cells leads to reduced activation-induced apoptosis that results in increased liver damage. Infect Immun 73:5061. Chen, D. W., R. J. McKallip, A. Zeytun, Y. K. Do, C. Lombard, J. L. Robertson, T. W. Mak, P. S. Nagarkatti, and M. Nagarkatti. 2001. CD44-deficient mice exhibit enhanced hepatitis after concanavalin a injection: Evidence for involvement of CD44 in activationinduced cell death. Journal of Immunology 166:5889-5897. Liang, J., D. Jiang, J. Griffith, S. Yu, J. Fan, X. Zhao, R. Bucala, and P. W. Noble. 2007. 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The Hyaluronan Receptor (CD44) Participates in the Uptake and Degradation of Hyaluronan. J Cell Biol 116:1055-1062. Kawana, H., H. Karaki, M. Higashi, M. Miyazaki, F. Hilberg, M. Kitagawa, and K. Harigaya. 2008. CD44 Suppresses TLR-Mediated Inflammation. J Immunol 180:42354245. Mummert, M. E., D. Mummert, D. Edelbaum, F. Hui, H. Matsue, and A. Takashima. 2002. Synthesis and surface expression of hyaluronan by dendritic cells and its potential role in antigen presentation. J Immunol 169:4322-4331. Termeer, C., F. Benedix, J. Sleeman, C. Fieber, U. Voith, T. Ahrens, K. Miyake, M. Freudenberg, C. Galanos, and J. C. Simon. 2002. Oligosaccharides of Hyaluronan activate dendritic cells via toll-like receptor 4. J Exp Med 195:99-111. Krammer, P. H., R. Arnold, and I. N. Lavrik. 2007. Life and death in peripheral T cells. Nat Rev Immunol 7:532-542. Larkin, J., G. J. Renukaradhya, V. Sriram, W. Du, J. Gervay-Hague, and R. R. Brutkiewicz. 2006. CD44 differentially activates mouse NK T cells and conventional T cells. J Immunol 177:268-279. Weber, G. F. 2004. The absence of CD44 ameliorates Fas(lpr/lpr) disease. Autoimmunity 37:1-8. Do, Y., A. Q. Rafi-Janajreh, R. J. McKallip, P. S. Nagarkatti, and M. Nagarkatti. 2003. Combined deficiency in CD44 and Fas leads to exacerbation of lymphoproliferative and autoimmune disease. Int Immunol 15:1327-1340. Kryworuchko, M., F. Diaz-Mitoma, and A. Kumar. 1999. Interferon-gamma inhibits CD44-hyaluronan interactions in normal human B lymphocytes. Experimental Cell Research 250:241-252. Watanabe, Y., M. Hashizume, S. Kataoka, E. Hamaguchi, N. Morimoto, S. Tsuru, S. Katoh, K. Miyake, K. Matsushima, M. Tominaga, T. Kurashige, S. Fujimoto, P. W. Kincade, and A. Tominaga. 2001. Differentiation stages of eosinophils characterized by hyaluronic acid binding via CD44 and responsiveness to stimuli. DNA & Cell Biology 20:189-202.  37     122.  123.  124. 125.  126.  127.  128. 129.  130. 131.  132.  133.  134. 135.  English, N. M., J. F. Lesley, and R. Hyman. 1998. Site-specific de-N-glycosylation of CD44 can activate hyaluronan binding, and CD44 activation states show distinct threshold densities for hyaluronan binding. Cancer Res. 58:3736-3742. Lesley, J., Q. He, K. Miyake, A. Hamann, R. Hyman, and P. W. Kincade. 1992. Requirements for hyaluronic acid binding by CD44: a role for the cytoplasmic domain and activation by antibody. J. Exp. Med. 175:257-266. Aruffo, A., I. Stamenkovic, M. Melnick, C. B. Underhill, and B. Seed. 1990. CD44 is the principle cell surface receptor for hyaluronate. Cell 61:1303-1313. Lesley, J., P. W. Kincade, and R. Hyman. 1993. Antibody-induced activation of the hyaluronan receptor function of CD44 requires multivalent binding by antibody. Eur. J. Immunol. 23:1902-1909. Bourguignon, L. Y. W., V. B. Lokeshwar, X. Chen, and W. G. L. Kerrick. 1993. Hyaluronic acid-induced lymphocyte signal transduction and HA receptor (GP85/CD44)cytoskeleton interaction. J. Immunol. 151:6634-6644. Liu, D., D. Zhang, H. Mori, and M.-S. Sy. 1996. Binding of CD44 to hyaluronic acid can be induced by multiple signals and requires the CD44 cytoplasmic domain. Cell. Immunol. 174:73-83. Lesley, J., and R. Hyman. 1992. CD44 can be activated to function as an hyaluronic acid receptor in normal murine T cells. Eur. J. Immunol. 22:2719-2723. Skelton, T. P., C. X. Zeng, A. Nocks, and I. Stamenkovic. 1998. Glycosylation provides both stimulatory and inhibitory effects on cell surface and soluble CD44 binding to hyaluronan. J. Cell Biol. 140:431-446. Katoh, S., Z. Zheng, K. Oritani, T. Shimozato, and P. W. Kincade. 1995. Glycosylation of CD44 negatively regulates its recognition of hyaluronan. J. Exp. Med. 182:419-429. Brown, K. L., A. Maiti, and P. Johnson. 2001. Role of sulfation in CD44-mediated hyaluronan binding induced by inflammatory mediators in human CD14+ peripheral blood monocytes. J. Immunol. 167:5367-5374. Levesque, M. C., and B. F. Haynes. 1999. TNF alpha and IL-4 regulation of hyaluronan binding to monocyte CD44 involves posttranslational modification of CD44. Cell. Immunol. 193:209-218. Katoh, S., T. Miyagi, H. Taniguchi, Y. Matsubara, J. Kadota, A. Tominaga, P. W. Kincade, S. Matsukura, and S. Kohno. 1999. Cutting edge: An inducible sialidase regulates the hyaluronic acid binding ability of CD44-bearing human monocytes. J. Immunol. 162:5058-5061. Maiti, A., G. Maki, and P. Johnson. 1998. TNF-a induction of CD44-mediated leukocyte adhesion by sulfation. Science 282:941-943. Greenfield, B., W. C. Wang, H. Marquardt, M. Piepkorn, E. A. Wolff, A. Aruffo, and K. L. Bennett. 1999. Characterization of the heparan sulfate and chondroitin sulfate assembly sites in CD44. J. Biol. Chem. 274:2511-2517.  38     CHAPTER TWO Chondroitin sulfate addition to CD44 negatively regulates hyaluronan binding  A version of this chapter has been published: Ruffell, B. and Johnson, P. (2005) Chondroitin sulfate addition to CD44H negatively regulates hyaluronan binding. Biochem. Biophys. Res. Commun. 334(2): 306-312 39     2.1 INTRODUCTION CD44 binding to the extracellular matrix and cell surface glycosaminoglycan (GAG), hyaluronan (HA), is tightly regulated. The interaction between CD44 and HA has been implicated in a variety of processes, including inflammation, cancer metastasis, wound healing and angiogenesis (reviewed in (1-4)). CD44 can exist as several isoforms due to the alternative splicing of 10 variably expressed exons, but all forms contain the HA binding region, including the standard form of CD44, CD44H, which contains none of the alternatively spliced exons. While CD44 is expressed on most cell types, the majority of cells do not bind HA constitutively. In peripheral blood monocytes, inflammatory cytokines upregulate CD44 expression and induce CD44 mediated HA binding (5, 6). Likewise, antigen induced T cell activation transiently induces HA binding (7). The ability of CD44 to bind HA is influenced by the expression level of CD44, as well as other factors such as CD44 clustering and linkage to the cytoskeleton (8), sialylation (9), sulfation (10), and N- and O-linked glycosylation on CD44 (11, 12). CD44 also binds to the chondroitin sulfate (CS) chains of the CS proteoglycans serglycin (13), versican (14) and aggrecan (15). Antibodies (Abs) that block the binding of HA to CD44 also block the binding of these proteoglycans, suggesting that they bind to the same site on CD44 (13-15). Although free CS chains have a low affinity for CD44, they can also compete with HA for binding to CD44 (16, 17). In addition to binding glycosaminoglycans, CD44 can also be covalently modified by them (18). Both CS and heparan sulfate (HS) addition occurs via O-linkages to serine residues that are most commonly followed by a glycine. However, not all Ser-Gly sequences are modified by glycosaminoglycans and there is specificity between CS and HS addition. The standard form of human CD44 (CD44H) contains four Ser-Gly sequences, two in exon 5 and one each in exon  40     15 and 16 (19). Using CD44-Fc fusion proteins, it has been shown in Cos-7 cells that CS addition occurs only in the sequence encoded by exon 5 (18). In contrast, HS addition did not occur at any of these sites in CD44H, but occurred in the alternatively spliced form of CD44 containing variable exon 3 (18). In this case, acidic residues surrounding the Ser-Gly sequence were found to be important for HS addition (18). Studies investigating the effect of CS addition on the CD44-HA interaction have produced conflicting results. Murine L cell fibroblasts express a CS modified form of CD44 and do not bind HA, but can be induced to bind by the anti-CD44 Ab, IRAWB 14 (20). L cells deficient in both CS and HS synthesis, but not just HS synthesis, lacked this IRAWB 14 inducible HA binding (20). However, another study found that L cells and pre-B cells were unable to bind HA, but became inducible HA binders after treatment with p-nitrophenyl β-D-xylopyranoside (β-D-xyloside), a competitive inhibitor of CS and HS addition, or after digestion with chondroitinase ABC (21). In human cells, chondroitinase ABC treatment of a colon carcinoma cell line had little effect on adhesion to HA, despite expression of CS modified CD44 (22), whereas treatment of human peripheral blood monocytes with β-D-xyloside increased the level of tumor necrosis factor-α (TNFα) induced HA binding (23). These studies suggest that CS can affect HA binding. However, it is not clear whether the effect is due to GAG removal from the entire cell surface or CD44, as mutations in GAG synthesis and inhibition of GAG addition affect the whole cell. Here it is demonstrated that CS modification of CD44 occurs primarily at serine residue 180 in human CD44H. Mutation of this Ser-Gly sequence in CD44 results in loss of CS addition and an increase in HA binding in both a soluble CD44-immunoglobulin fusion protein and full length CD44.  41     2.2 MATERIALS AND METHODS 2.2.1 Cell lines and transfections The human embryonic kidney 293 cell line (HEK293) and murine L fibroblasts were from the American Type Culture Collection (ATCC, Rockville, MD) and were cultured in Dulbecco’s minimum essential medium (DMEM, Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum (FCS, Hyclone, Logan, UT), 1 mM sodium pyruvate (Life Technologies, Burlington, Ontario), and 2 mM L-glutamine (Sigma-Aldrich, St. Louis, MO). HEK293 and L cells were transfected using the GenePORTER2 transfection reagent (Gene Therapy Systems, San Diego, CA) according to the manufacturer’s instructions. Transfected HEK293 cells were selected using 450 µg/ml active G418 (Invitrogen) followed by cloning and maintenance in 300 µg/ml. L cells were selected and maintained in 300 µg/ml hygromycin B (Calbiochem, La Jolla, CA) and sorted once by flow cytometry to select for transfectants expressing high levels of human CD44.  2.2.2 Antibodies and reagents Purified rat anti-human/mouse CD44 monoclonal Ab (mAb), IM7.8.1 (ATCC# TIB-235) was conjugated to Alexa 488 (Molecular Probes, Eugene, OR) or coupled to CNBr-activated Sepharose 4B (Amersham Biosciences, Piscataway, NJ) according to the manufacturer’s instructions. The mouse anti-human CD44 mAb, 3G12 was from G. Dougherty (24) and the rat anti-human CD44 mAb, Hermes-1, was from the Development Studies Hybridoma Bank (University of Iowa, Iowa City, IA). 3B3 and 2B6 mAbs that recognize terminal 6- or 4- sulfated CS disaccharides generated after chondroitinase ABC treatment (25) were from Seikagaku America (East Falmouth, MA). Fluorescein-conjugated HA (Fl-HA) was made as described (26) using rooster comb HA from Sigma-Aldrich. Fluorescein isothiocyanate (FITC)-conjugated goat  42     anti-rat Ab and horse radish peroxidase (HRP)-conjugated goat anti-mouse Ab were purchased from Jackson ImmunoResearch (West Grove, PA). Prestained molecular mass standards were from New England Biolabs (Beverly, MA).  2.2.3 Generation of point mutations Human CD44H cDNA (27) was expressed as a full-length protein in the plasmid pCEP4 or as an immunoglobulin (Ig) Fc-fusion protein (CD44-Fc) in the plasmid pIRESneo. The CD44-Fc fusion protein consists of the extracellular domain of human CD44 fused to the constant region of human IgG1 (16). S180A, G181A, and G181V mutations in CD44 were created by oligonucleotide site directed mutagenesis. Eight primers were used: primer 1 containing a HpaI site (5’-ATAACTATTGTTAACCGTGATGGCACCCGC-3’); primer 2 containing a PpuMI site (5’-GGTATGGGACCCCCCACTGGG-3’); complementary primers 3 (5’GATGACGTGAGCGCCGGCTCCTCCAGT-3’) and 4 (5’ACTGGAGGAGCCGGCGCTCACGTCATC-3’) containing the S180A mutation; complementary primers 5 (5’- GATGACGTGAGCAGCGCTTCCTCCAGT-3’) and 6 (5’ACTGGAGGAAGCGCTGCTCACGTCATC-3’) containing the G181A mutation; and complementary primers 7 (5’-GATGACGTGAGCAGCGTTTCCTCCAGT-3’) and 8 (5’ACTGGAGGAAACGCTGCTCACGTCATC-3’) containing the G181V mutation. The mutated sequences are underlined in each case. Fragments were generated by PCR using primers 1 with 4, 6 or 8, and 2 with 3, 5 or 7, then each pair was mixed and PCR performed again. Each product was inserted into the CD44 sequence using HpaI and PpuMI.  43     2.2.4 Flow cytometry L cells were incubated with and without Hermes-1 mAb tissue culture supernatant for 30 min on ice. Cells were washed once and incubated for 30 min with FITC-conjugated goat anti-rat Ab or with Fl-HA in PBS/2%FCS/5 mM EDTA (FACS buffer). Cells were washed again and resuspended in FACS buffer containing 5 µg/ml propidium iodide. Cells were analyzed on a FACScan using CellQuest software (BD Biosciences, Mississauga, Ontario). For experiments involving CD44-Fc fusion proteins, protein A polystyrene particles (Spherotech, Libertyville, IL) were washed with PBS/1 mg/ml bovine serum albumin (BSA) and incubated for 1 hr at 4ºC with dilutions of tissue culture supernatant from HEK293 cells producing CD44-Fc protein. Two clones of transfected HEK293 cells (1x105) were grown in 2 ml with or without 2 mM β-Dxyloside for 24 hours, followed by replacement of the media and 2 days incubation prior to harvesting the supernatant. For experiments involving chondroitinase ABC, CD44-Fc coated protein A beads were first incubated with 300 mU of hyaluronidase in PBS/1 mg/ml BSA for 1 hr at 37°C, then washed with chondroitinase ABC buffer (40 mM Tris, 40 mM NaOAc, 0.01% BSA, pH 8.0), and incubated with or without 20 mU of chondroitinase ABC for 2 hrs at 37°C. Beads were then blocked with DMEM/10% horse serum for 30 min, washed and split into replicates. Replicates were incubated with either 2.5 µg/ml of Alexa 488 conjugated IM7 or FlHA for 1 hr, washed, then resuspended in PBS/1 mg/ml BSA. Fl-HA binding and CD44 levels were determined by flow cytometry.  2.2.5 Immunoprecipitation, sulfate labeling and Western blotting HEK293 cells transfected with the CD44-Fc plasmid were pre-incubated with β-D-xyloside as described above and Na2[35SO4] at 100 µCi/ml was added to the media after 24 hrs. After 2 days,  44     1 ml of supernatant was harvested and incubated with 30 µl of immobilized recombinant protein A Sepharose beads (Repligen, Cambridge, MA) rotating end-over-end for 2 hrs at 4°C. For L cells, 100 µCi/ml of Na2[35SO4] was added to 3x105 cells after a 2 hr incubation with β-Dxyloside. After 2 days cells were lysed in 1% Triton X-100, 10 mM Tris, 140 mM KCl, pH 7.5, 200 μM phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 μg/ml pepstatin. Lysates were centrifuged at 12,000 g for 10 minutes, and incubated with 25 µg of Hermes-1 rotating end-over-end for 1 hr at 4°C to immunoprecipitate human but not mouse CD44, then incubated with 20 µl protein G Sepharose beads (Amersham Biosciences) for 1 hr. Beads were washed twice and incubated with 30 µl of buffer or either 20 mU chondroitinase ABC in 40 mM Tris-HCl pH 8.0, 40 mM NaOAc, 0.01% BSA, or 1 mU of Flavobacterium heparinum heparitinase (Seikagaku America) in 50 mM NaOAc, 5 mM Ca(OAc)2, pH 7.0 for 2 hours at 37°C. Following digestion, CD44- or CD44-Fc coated beads were washed twice and boiled for 10 min in 3x reducing sample buffer. Samples were loaded onto a 7.5% SDS-polyacrylamide gel and transferred to a PVDF membrane (Millipore, Billerica, MA). Membranes were exposed to Kodak BioMax MR film at -80°C for 3 to 7 days. For experiments involving CD44-Fc, 1/5 the sample volume was loaded onto a separate gel for use in a CD44 western blot. To determine relative CD44 levels, membranes were blotted with 3G12 supernatant diluted 1:100 in 150 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.1% Tween 20 (TBST) containing 5% skim milk. Blots were washed with TBST, incubated with HRP-conjugated goat anti-mouse Ab at 1:5000 and washed again. Proteins were detected using enhanced chemiluminescence (ECL, Amersham Biosciences).  45     2.2.6 Detection of chondroitin sulfate stub CD44-Fc or CD44 was immunoprecipitated, digested with chondroitinase ABC, and transferred to a PVDF membrane as described above. Chondroitinase ABC cleaves CS chains but leaves a hexasaccharide ‘stub’ on the protein. The terminal 6- or 4- sulfated CS disaccharide can be recognized by 3B3 or 2B6 mAbs respectively (25). 1/10 of the sample was loaded and blotted for CD44 while 9/10 was blotted for CS stubs using either 1:200 dilution of mAb 3B3 in TBST containing 5% BSA, or 1:1000 dilution of mAb 2B6. Membranes were then washed and incubated with HRP-conjugated goat anti-mouse Ab at 1:5000, washed again, and detected using enhanced chemiluminescence.  2.3 RESULTS 2.3.1 Serine residue 180 is the primary site of CS addition in CD44H Previous data had localized CS addition on CD44 to the sequence encoded by exon 5 (18). This exon encodes two Ser-Gly sequences (residues 180-181 and 190-191 in human CD44). Since Ser-Gly 180-181 is conserved between several species, serine 180 was mutated to an alanine (S180A). HEK293 cells were stably transfected with CD44-Fc or CD44-Fc mutated at S180A then incubated with or without 2 mM β-D-xyloside in addition to sodium [35S]sulfate. CD44-Fc was isolated from supernatant using protein A Sepharose beads and separated by SDS-PAGE (see Materials and methods for details). As seen in Fig. 2.1A, the majority of CD44-Fc existed as a ~90 kDa form, but a highly sulfated, higher molecular mass form was also present. This high molecular mass form was recognized by the anti-CD44 mAb, 3G12, indicating that it was CD44 (Fig. 2.1B). Heavily sulfated, heterogeneous, high molecular mass forms are indicative of large chain GAG addition and a characteristic of several proteoglycans. Treatment with β-D-xyloside substantially reduced the presence of this high molecular mass isoform and reduced total 46     1V  G  18  A  1A  18  80  G  [35S] sulfate  S1  175 83 62  175 2B6  175 83 62  B  D  xyloside wt mut wt mut  w t  A  83  CD44  175 3B3 83  xyloside wt mut wt mut  175 CD44  175  83  CD44  83 62  C  ABC Hep -  + -  wt  -  +  -  175  + -  mut -  + [35S] sulfate  83 62 175  CD44  83 62  FIGURE 2.1 CS addition to CD44 occurs primarily at serine 180. A, [35S] sulfate labeled wild type (wt) or S180A mutant (mut) CD44-Fc proteins were purified from the supernatant of transfected HEK293 cells grown in the presence or absence of 2 mM β-D-xyloside and resolved by SDS-PAGE. The top panel shows [35S] sulfate incorporation by CD44-Fc, while the bottom panel shows CD44-Fc levels, determined by immunoblotting with the anti-CD44 mAb, 3G12. B, Same as A, except that the CD44 blot was overexposed and [35S] sulfate incorporation is not shown. C, Same as A, except that immunoprecipitated CD44-Fc was digested with chondroitinase ABC (ABC) or heparitinase (Hep) prior to SDS-PAGE. D, Detection of chondroitin sulfate on wild-type (wt) and S180A, G181A and G181V CD44-Fc fusion proteins using 2B6 and 3B3 antibodies that recognize 4- and 6- sulfated CS stubs respectively, after chondroitinase ABC treatment. The bottom panel shows CD44-Fc levels, as determined in A. Each experiment was repeated at least three times and a representative experiment is shown. Molecular mass markers are indicated at the left in kiloDaltons.  "%  [35S]sulfate incorporation by over 80%, indicating that CD44-Fc was modified by GAGs. In contrast, S180A CD44-Fc did not have this higher molecular mass form and incorporated approximately 80% less [35S]sulfate than wild type CD44-Fc. Furthermore, [35S]sulfate incorporation by S180A CD44-Fc was minimally affected by β-D-xyloside, demonstrating that the majority of GAG addition to CD44-Fc occurs at serine 180. To determine if the GAG was HS or CS, immunoprecipitated CD44-Fc was digested with either chondroitinase ABC or heparitinase. Chondroitinase ABC, but not heparitinase, eliminated the high molecular weight forms of CD44-Fc and reduced [35S]sulfate incorporation by over 80% (Fig. 2.1C), indicating that CD44-Fc from HEK293 cells is modified by CS and not HS. Digestion with chondroitinase ABC had no effect on [35S]sulfate incorporation by mutant CD44Fc, indicating that serine 180 is the primary site of CS addition in CD44H. Since serine residues can also be modified by O-linked carbohydrates, and since Ser-Gly is thought to be important for CS addition, we mutated the glycine residue at position 181 to an alanine or a valine and expressed these mutants as CD44-Fc fusion proteins as well. As an alternative method for detecting CS modification, CD44-Fc was digested with chondroitinase ABC, which generates a 4- or 6-sulfated CS ‘stub’ that can be recognized by mAbs, 2B6 and 3B3 respectively (25). Both the S180A mutation and the two glycine 181 mutations resulted in loss of Ab reactivity to both 2B6 and 3B3 (Fig. 2.1D), supporting the data that Ser-Gly 180-181 is the site of CS addition and that both residues are important for the addition of CS.  2.3.2 CS inhibits HA binding by CD44-Fc CD44-Fc fusion proteins often reflect the HA binding capability of the cells that produce them (11) and provide an opportunity to study the effects of post-translational modifications of CD44  48     on its ability to bind HA in the absence of other cellular components. To determine whether CS addition specifically to CD44 alters its ability to bind HA, CD44-Fc and mutant CD44-Fc produced in the presence or absence of β-D-xyloside were bound to protein A-coated polystyrene beads for use in flow cytometry (see Materials and methods for details). The amount of CD44 on the beads was monitored using an Alexa 488 conjugated anti-CD44 mAb and the HA binding ability determined by Fl-HA binding (Fig. 2.2A). CS modified CD44 bound low levels of Fl-HA whereas β-D-xyloside treatment and the S180A mutation substantially increased HA binding over a range of CD44-Fc levels, and did so to the same degree. This suggests that both the S180A mutation and β-D-xyloside increased HA binding specifically by preventing CS addition. To provide additional evidence for this, CD44-Fc was digested with chondroitinase ABC and HA binding assessed. As chondroitinase ABC has some hyaluronidase activity, the fusion protein was first pre-treated with hyaluronidase. Chondroitinase ABC treatment increased HA binding by CD44-Fc (Fig. 2.2B), but had no effect on mutant CD44-Fc. Notably however, chondroitinase ABC did not increase HA binding as well as β-D-xyloside or the mutation. This is possibly due to the presence of the hexasaccharide CS stub that remains attached to the protein core following chondroitinase ABC digestion. Finally, the G181V and G181A mutations had the same effect on HA binding as the S180A mutation (Fig. 2.2C). The data show that all the mutations resulted in the loss of CS addition and increased HA binding to a level that is comparable to the HA binding observed with β-D-xyloside treated wild-type CD44. These data are consistent with the interpretation that it is the loss of CS addition, and not the mutations themselves, which is responsible for the observed increase in HA binding.  49     A 500 Fl-HA (MFI)  400  wt mut w t + xyloside mu t + xyloside  300 200 100 0 0  B 350 300  200 300 400 CD44 (MFI)  500  wt mut wt + ABC mut + ABC  Fl-HA (MFI)  250  100  200 150 100 50 0 0  C 300 250  wt S180A G181A G181V  Fl-HA (MFI)  200  50 100 150 200 250 300 350 CD44 (MFI)  150 100 50 0 0  50  100 150 200 250 300 CD44 (MFI)  FIGURE 2.2 HA binding ability of wild-type and mutant CD44-Fc fusion proteins. CD44-Fc fusion proteins were coupled to protein A beads and CD44 levels and HA binding was assessed by flow cytometry using directly conjugated CD44 antibody and fluorescently labeled HA (Fl-HA). Graphs show the amount of Fl-HA bound with increasing levels of CD44. In each case, the amount of fluorescence was expressed as mean fluorescence intensity units (MFI). A, Fl-HA binding of wild-type (wt) and S180A (mut) CD44-Fc fusion proteins derived from HEK293 cells grown with or without β-D-xyloside. B, Fl-HA binding of wild-type (wt) and S180A (mut) CD44-Fc fusion proteins incubated with or without with chondroitinase ABC (ABC) prior to addition of Fl-HA or CD44 antibody. C, Fl-HA binding of wild-type (wt) and S180A, G181A and G181V CD44-Fc fusion proteins. Experiments were performed at least three times and a representative figure is shown.  #  2.3.3 CS inhibits HA binding by cell surface CD44H To determine whether CS addition to CD44 is also able to inhibit HA binding in a cellular context, murine L cells were transfected with full-length human CD44H and S180A mutant CD44H and sorted one time for high expression. L cells express mouse CD44 modified by CS (20) and do not bind HA (20, 21). Immunoprecipitated [35S]sulfate labeled human CD44 is similar to the previously characterized mouse CD44 with a ~90 kDa form and heterogeneous, higher molecular mass forms ranging from 90 to over 175 kDa (Fig. 2.3A). The treatment of cells with β-D-xyloside reduces the higher molecular mass forms as well as the amount of [35S]sulfate incorporation, whereas treatment of the immunoprecipitate with chondroitinase ABC abolishes the high molecular mass forms, converting them to the 90 kDa form of CD44 containing the hexasaccharide CS stub. This is observed by a small increase in CD44 protein and [35S]sulfate levels at 90 kDa (Fig. 2.3A). This indicates that human CD44 is modified by CS in mouse L cells. Expression of the S180A mutant revealed a reduction in the sulfated, higher molecular mass form indicating that serine 180 was being used as a site of CS addition in CD44H in L cells. Chondroitinase ABC revealed a small amount of sensitive material in the mutant suggesting that additional sites may be used in L cells. However, Western blotting with the 2B6 Ab was unable to detect CS stubs on S180A mutant CD44 after chondroitinase ABC treatment (Fig 2.3B), confirming that the mutation prevents the majority of CS addition. Consistent with our findings for CD44-Fc, mutant CD44H transfected cells had a higher level of Fl-HA binding compared to cells transfected with CD44H, as determined by flow cytometry (Fig. 2.3C). This difference was not due to differences in expression levels of transfected CD44, as the levels were comparable (Fig. 2.3C). This indicates that CS addition negatively influences HA binding by cell surface CD44H.  51     A  xyloside ABC Hep vec wt mut wt mut wt mut wt mut 175  [35S] sulfate  83 62 175  CD44  83 62  175 83  vec  wt  C  mut 2B6  mut  hCD44 -  Fl-HA -  wt  Cell number  B  175 83  CD44  vec 101102103 101 102103 Fluorescence intensity  FIGURE 2.3 CS addition and HA binding in mouse L cells transfected with human CD44H. A, Human CD44H immunoprecipitated from [35S]sulfate labeled L cells grown in the presence or absence of 2 mM β-D-xyloside and treated with or without chondroitinase ABC (ABC) or heparitinase (Hep) and resolved by SDS-PAGE. The top panel shows [35S]sulfate incorporation, while the bottom panel shows CD44 levels as determined by immunoblotting with the anti-CD44 mAb 3G12. B, Human CD44H was immunoprecipitated, digested with chondroitinase ABC, and then immunoblotted with 2B6, the mAb that detects 4-sulfated CS stubs. The bottom panel shows CD44 loading. C, Flow cytometry showing CD44 levels and HA binding ability of L cells transfected with S180A CD44H (mut), CD44H (wt), or vector alone (vec). The left panel shows expression levels of human CD44, detected using an anti-human CD44 antibody, while the right panel shows Fl-HA binding. The graph shows fluorescence intensity on a log scale versus cell number. This is a representative figure of an experiment that was reproduced at least three times.  #  2.4 DISCUSSION Here we provide data supporting a negative role for CS addition at serine 180 of CD44H in regulating HA binding. This negative effect of CS is observed on both cell surface CD44 and secreted CD44-Fc fusion protein indicating a direct effect of CS on the ability of CD44 to bind HA. While the precise mechanism remains to be determined, data indicating that CS can compete for HA binding, albeit at a low affinity, suggests that the CS chain from serine 180 may bind to the HA binding site of CD44, or nearby, and block HA binding. To determine if this is the case, crystallization of CS modified CD44 would be required. Previous data using GAG-deficient cells or cells where GAG addition was prevented suggested both positive and negative roles for CS in the IRAWB 14 Ab inducible binding of CD44, although in these cases all proteoglycans were affected (20, 21). In another study, all 4 Ser-Gly motifs were individually mutated to Gly-Gly in a CD44 transfected human melanoma line, which resulted in a similar decrease in HA binding in each case (28). However, the effect of these mutations on CS addition was not assessed and mutation of serine 180 to glycine generated another Ser-Gly motif at 179-180. Here, we localize CS addition in CD44H to serine 180 and show that this site is modified by CS in CD44-Fc secreted from HEK293 cells and on CD44H expressed in murine L cells. We then go on to show that mutation of serine 180 or glycine 181 prevents CS addition on CD44 and results in increased HA binding. Since CS addition at serine 180 negatively affects HA binding, factors that influence CS addition may be predicted to regulate HA binding by CD44. In addition, CD44 modified by the hexasaccharide CS stub had intermediate HA binding between the CS modified and β-D-xyloside treated CD44, suggesting that chain length or extent of sulfation may also affect the extent of HA binding. Interestingly, the anti-inflammatory cytokine transforming growth factor-β (TGF-β)  53     has been shown to increase the length of CS chains on CD44 in human lung fibroblasts (29) and mouse melanoma cells (30). Likewise, platelet-derived growth factor (PDGF) can increase the frequency of CS addition to CD44 in human dermal fibroblasts (31). However, in these cases the effect on HA binding was not assessed. In one study, oncostatin M treatment of lung-epithelial derived tumor cells increased CS addition on CD44 and HA binding (32). In contrast to the present study, this suggested a positive correlation between CS addition and HA binding in these cells, although it does not exclude the possibility that other changes are also affecting HA binding. Here we show that CS addition to serine 180 on CD44H has a direct negative effect on its ability to bind HA.  54     2.5 REFERENCES 1. 2.  3. 4. 5.  6.  7.  8.  9.  10. 11.  12.  13.  14.  15.  16.  Lesley, J., R. Hyman, N. English, J. B. Catterall, and G. A. Turner. 1997. CD44 in inflammation and metastasis. Glycoconj. J. 14:611-622. Johnson, P., A. Maiti, K. L. Brown, and R. Li. 2000. A role for the cell adhesion molecule CD44 and sulfation in leukocyte-endothelial cell adhesion during an inflammatory response? Biochem. Pharm. 59:455-465. Pure, E., and C. A. Cuff. 2001. A crucial role for CD44 in inflammation. Trends Mol. Med. 7:213-221. Ponta, H., L. Sherman, and P. A. Herrlich. 2003. CD44: from adhesion molecules to signalling regulators. Nat. Rev. Mol. Cell. 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Bell, D. G. Jackson, F. B. Cornelis, U. Gerth, and J. I. Bell. 1992. Genomic structure of DNA encoding the lymphocyte homing receptor CD44 reveals at least 12 alternatively spliced exons. Proc Natl Acad Sci (U.S.A.) 89:12160-12164. Esford, L. E., A. Maiti, S. A. Badar, F. Tufaro, and P. Johnson. 1998. Analysis of CD44 interactions with hyaluronan in murine L cell fibroblasts deficient in glycosaminoglycan synthesis - A role for chondroitin sulfate. J. Cell Sci. 111:1021-1029. Lesley, J., N. English, A. Perschl, J. Gregoroff, and R. Hyman. 1995. Variant cell lines selected for alterations in the function of the hyaluronan receptor CD44 show differences in glycosylation. J. Exp. Med. 182:431-437. Takahashi, K., I. Stamenkovic, M. Cutler, A. Dasgupta, and K. K. Tanabe. 1996. Keratan sulfate modification of CD44 modulates adhesion to hyaluronate. J. Biol. Chem. 271:9490-9496. Levesque, M. C., and B. F. Haynes. 1999. 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Glycosylation of CD44 is implicated in CD44-mediated cell adhesion to hyaluronan. J. Cell Biol. 132:1199-1208. Romaris, M., A. Bassols, and G. David. 1995. Effect of transforming growth factor-beta1 and basic fibroblast growth factor on the expression of cell surface proteoglycans in human lung fibroblasts - Enhanced glycanation and fibronectin-binding of CD44 proteoglycan, and down-regulation of glypican. Biochemical Journal 310:73-81. Faassen, A. E., D. L. Mooradian, R. T. Tranquillo, R. B. Dickinson, P. C. Letourneau, T. R. Oegema, and J. B. McCarthy. 1993. Cell surface CD44-related chondroitin sulfate proteoglycan is required for transforming growth factor-beta-stimulated mouse melanoma cell motility and invasive behavior on type-I collagen. J Cell Sci 105:501-511. Clark, R. A., F. Lin, D. Greiling, J. An, and J. R. Couchman. 2004. Fibroblast invasive migration into fibronectin/fibrin gels requires a previously uncharacterized dermatan sulfate-CD44 proteoglycan. J Invest Dermatol 122:266-277.  56     32.  Cichy, J., and E. Pure. 2000. Oncostatin M and transforming growth factor-beta 1 induce post-translational modification and hyaluronan binding to CD44 in lung-derived epithelial tumor cells. J. Biol. Chem. 275:18061-18069.     57     CHAPTER THREE Chondroitin sulfate addition to CD44 regulates hyaluronan binding in macrophages  A version of this chapter will be submitted for publication: Ruffell, B., Brown, K.L., Tjew, S.L., and Johnson, P. (2008) Chondroitin sulfate addition to CD44 regulates hyaluronan binding in macrophages. 58     3.1 INTRODUCTION As professional antigen-presenting cells and secretors of inflammatory mediators, macrophages are a key component of both the innate and adaptive immune response. Macrophages are also crucial for the tissue repair process to proceed normally. Macrophages produce cytokines and chemokines to promote tissue growth, and play a subsequent role in angiogenesis and tissue remodeling (1). They are also responsible for the phagocytosis of debris resulting from cell death and extracellular matrix (ECM) degradation (2, 3). One of the degraded components of the ECM that must be removed is the glycosaminoglycan hyaluronan (HA). HA accumulates at sites of injury (4) and is important in recruiting macrophages to skin wounds (5). However, fragments of HA can activate cells, including macrophages, through toll-like receptor (TLR)-2 and -4 and failure to clear fragmented HA can lead to a sustained inflammatory response (6). HA internalization by macrophages and other cell types occurs via the cell surface receptor CD44 (6, 7), and it has been suggested that HA uptake by macrophages is important for the local turnover of HA (8). CD44 is also involved in leukocyte recruitment (9) and can affect the severity of arthritis (10) and atherosclerosis (11) in mouse disease models. However, while CD44 is the primary cell surface receptor for HA, the majority of CD44 expressing immune cells do not bind HA. Inflammatory cytokines such as tumor necrosis factor-α (TNFα) induce HA binding in monocytes (12, 13) and endothelial cells (14), while T cells bind to HA following activation with antigen (15). Binding of CD44 to HA is highly regulated by expression levels of CD44 (16) and CD44 clustering and linkage to the cytoskeleton (17), in addition to post-translational modifications on CD44 including sialylation (18), sulfation (19), and N- and O-linked glycosylation (16, 20). We have also shown that HA binding by CD44 is negatively affected by  59     the addition of the glycosaminoglycan chondroitin sulfate (CS) to CD44 (21). CS is covalently linked to a specific serine residue (S180A) in the mucin-like region of CD44 and preventing this addition, either through use of a mutation or inhibitors, increases the binding affinity of CD44 for HA. In human monocytes, HA binding correlates with increased sulfation (13) and reduced CS addition (22), while TNFα induces the formation of sulfated carbohydrate epitopes (23). We were therefore interested in what molecules were responsible for the induction of HA binding in mouse macrophages. Here we find that an inhibitor of glycosaminoglycan addition increases HA binding by CD44 in bone marrow derived macrophages (BMDM). We also observe that stimulation with TNFα, or lipopolysaccharide (LPS) and interferon-γ (IFNγ), increases HA binding and reduces CS addition to CD44. Finally, we demonstrate using a CD44 point mutation that loss of CS addition results in increased binding of CD44 to HA, and that TNFα stimulation does not affect HA binding in the absence of CS modification. Together, these results indicate that CS addition to CD44 reduces HA binding in BMDM and that inflammatory cytokine stimulation increases HA binding by reducing the level of CS modification of CD44.  3.2 MATERIALS AND METHODS 3.2.1 Cell and cell lines The human myeloid cell line KG1a from the American Type Culture Collection (ATCC #CRL246.1, Rockville, MD) was cultured in RPMI-1640 supplemented with 10% FCS, 1 mM sodium pyruvate (Invitrogen, Burlington, ON), 2 mM L-glutamine (Sigma-Aldrich, Oakville, ON), and 50 U/ml Penicillin/Streptomycin (Invitrogen). High and low HA binding KG1a cells were previously established by cell sorting (17). Murine BMDM were generated by isolating bone marrow from the tibia and femurs of C57BL/6 and CD44 knockout mice, lysing red blood 60     cells with 0.84% ammonium chloride, and then plating 1-2x107 cells in a 10 cm2 Petri dish with DMEM (Invitrogen) supplemented with 20% FCS, 1 mM sodium pyruvate, 2 mM L-glutamine, 50 U/ml Penicillin/Streptomycin and 5% L cell conditioned media (LCCM). After 4 days cells were re-plated at a 1:4 dilution and stimulated with 20 ng/ml mouse recombinant TNFα (R&D Systems, Minneapolis, MN) and/or grown in the presence of 2 mM p-nitrophenyl β-Dxylopyranoside (β-D-xyloside, Sigma-Aldrich) for 3 days. In some experiments, cells were stimulated for 2 days with 10 ng/ml mouse recombinant interleukin-4 (IL-4) or 10 ng/ml mouse recombinant IFNγ (eBioscience, San Diego, CA) and 100 ng/ml of ultrapure LPS (SigmaAldrich). Animal experimentation was conducted in accordance with protocols approved by the University Animal Care Committee and Canadian Council of Animal Care guidelines.  3.2.2 Antibodies and reagents Purified rat anti-human/mouse CD44 mAb, IM7.8.1 (ATCC #TIB-235) was conjugated to Alexa 488 and Alexa 647 (Molecular Probes, Eugene, OR), or coupled to CNBr-activated Sepharose 4B (Amersham Biosciences, Piscataway, NJ) according to the manufacturer’s instructions. The rat anti-mouse CD44 mAb KM201 (ATCC #TIB-240) was a gift from P. Kincade. The antichondroitin sulfate mAbs 2B6 and 3B3 (24) were from Seikagaku America (East Falmouth, MA). FITC-conjugated goat anti-mouse Ab was from Caltag (Burlingame, CA) and HRPconjugated goat anti-mouse and HRP-conjugated goat anti-rabbit Ab were from Jackson ImmunoResearch (West Grove, PA). Fluorescein-conjugated HA (Fl-HA) was made as described (25) using rooster comb HA from Sigma-Aldrich. Prestained molecular mass standards were purchased from New England Biolabs (Beverly, MA). LCCM is tissue culture supernatant from mouse L929 cells and is a source of murine M-CSF.  61     3.2.3 Generation of point mutations Mouse CD44H was expressed as a full-length protein in pBS (Stratagene, La Jolla, CA) lacking the BamHI cleavage site. R43A and S183A mutations in CD44 were created by oligonucleotide site-directed mutagenesis. Six primers were used: primer 1 containing the XhoI site in the pBS vector (5’-CCCCCCCTCGAGGTCGAC-3’); primer 2 containing the BamHI site in CD44 (5’TCCAGCTAATTCGGATCCATGAGTCACAGT-3’), complementary primers 3 (5’CCGGGAGATACTGTAGGCGCCATTTTT-3’) and 4 (5’AAAAATGGCGCCTACAGTATCTCCCGG-3’) containing the R43A mutation; complementary primers 5 (5’-ATGGTGGAGCCGGCGCTGACATCGTC-3’) and 6 (5’GACGATGTCAGCGCCGGCTCCACCAT-3’) containing the S183A mutation. The mutated sequences are underlined in each case. Fragments were generated by PCR using primer 1 with primers 3 or 5, and using primer 2 with primers 4 or 6. Each fragment pair was then mixed and PCR was performed again. The final product was inserted into the CD44 sequence using XhoI and BamHI.  3.2.4 Retroviral infection Full length mouse CD44 was inserted into MIY, a murine stem cell virus (MSCV) based vector with the internal ribosomal entry site (IRES) sequence and the gene for yellow fluorescent protein (YFP). Amphotropic Pheonix packaging cells (26) were transfected with the vectors using Lipofectamine 2000 (Invitrogen) and the resulting supernatants were used to infect the ecotropic packaging cell line GP+E86 (27). Virus-producing cells were subsequently sorted for high expression of YFP. MIY and the packaging cell lines were a gift from R.K. Humphries. For infection of bone marrow, GP+E86 cells were treated with 25 µg/ml of Mitomycin C  62     (Calbiochem, La Jolla, CA) for 30 min, washed twice, and then incubated overnight in fresh media. The next day, freshly isolated bone marrow cells were added to the GP+E86 monolayer in the presence 5 µg/ml protamine sulfate (Sigma-Aldrich). After 2 days, non-adherent bone marrow cells were removed, resuspended in fresh media, and plated on Petri dishes. On day 4 cells were re-plated as described above.  3.2.5 Flow cytometry Cells (~2x105) were incubated with 2.4G2 (ATCC #HB-197) tissue culture supernatant for 20 min on ice to block Fc receptors. After removal of supernatant, cells were incubated for 20 min with approximately 5 µg/ml IM7-Alexa 488 or Fl-HA in PBS containing 2% FCS and 5 mM EDTA. Alternatively, cells were incubated with both IM7-Alexa 647 and Fl-HA. After washing, cells were resuspended in buffer containing 5 µg/ml propidium iodide (PI; Sigma-Aldrich) and a minimum of 5000 live events were collected on a FACScan or FACSVantage and analyzed using CellQuest (Becton Dickinson) or FlowJo (Tree Star, Ashland, OR) software. Detection of sulfated epitopes on the cell surface was performed as described (23). AG107 recognizes 6-sulfo N-acetyllactosamine/Lewis X, 2F3 recognizes sialyl Lewis X/sialyl 6-sulfo Lewis X, and DD-2 recognizes 6-sulfo N-acetyllactosamine.  3.2.6 Analysis of chondroitin sulfate on CD44 Immunoprecipitation, sulfate labeling and Western blotting of CD44 was done as described (21). Briefly, cells were cultured for 2 days in Na2[35SO4] following a 1 day incubation with TNFα and/or β-D-xyloside. Cells (~2x106) were lysed, incubated with IM7-coupled beads, and then immunoprecipitated CD44 was resolved on a 7.5% SDS-polyacrylamide gel and transferred to a  63     polyvinylidene difluoride membrane (Millipore, Mississauga, ON). Membranes were exposed to Kodak BioMax MR film (Interscience, Markham, ON) at -80°C for 7 to 10 days. To determine relative CD44 levels, membranes were blotted with a polyclonal Ab against the cytoplasmic domain of mouse CD44 (JIWBB) and HRP-goat anti-rabbit Ab. To detect chondroitin sulfate, immunoprecipitated CD44 from ~2.5x107 cells was digested with chondroitinase ABC (Seikagaku America) and blotted with the mAb 2B6 or 3B3. Following incubation with HRPgoat anti-mouse Ab, membranes were developed with ECL (Amersham Biosciences) according to the manufacture’s instructions.  3.2.7 Semi-quantitative RT-PCR Sulfotransferase expression levels were measured as described (23). Primers for mouse GlcNAc6ST-1 (5’-GAGGTGTTCTTCCTCTATGAGCC-3’ and 5’CCACGAAAGGCTTGGAGGAGG-3’) formed an 847 bp fragment, primers for mouse GlcNAc6ST-4 (5’-ACCCAGGAAAAGCAACACATCTATG-3’ and 5’GGTTAAGAAGAAATCAGCGCGTGG-3’) formed a 735 bp fragment, and primers for mouse C6ST-1 (5’-GGACCTTGTACACAGCCTAAAGATTCG-3’ and 5’CTCGGACAGCCACTTCTTCCA-3’) formed a 928 bp fragment.  3.2.8 Statistics Data are shown as the mean +/- SD. Significance was determined by the Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001.  64     3.3 RESULTS 3.3.1 Pro-inflammatory cytokines induce HA binding in BMDM We have previously shown that TNFα induces CD44-dependent HA binding in human peripheral blood monocytes (PBM) as well as a human monocytic cell line (13, 19). This induction of HA binding correlated with an increase in CD44 sulfation and could be reduced by an inhibitor of sulfation. We were therefore interested in determining whether the same mechanism was responsible for the regulation of HA binding in mouse macrophages. To this end, BMDM were generated from the bone marrow of WT and CD44 knockout mice by incubation in 5% L929 cell-conditioned medium (LCCM) as a source of M-CSF. Subsequent stimulation of cells with TNFα for 3 days induced high levels of HA binding in BMDM derived from WT but not CD44 knockout mice, indicating that HA binding was CD44-dependent (Fig. 3.1A). HA binding was also induced following treatment with other inflammatory mediators such as LPS and IFNγ, although not to the same extent as with TNFα (Fig. 3.1B). A comparison of HA binding over a range of CD44 expression showed a clear difference between unstimulated and TNFα stimulated cells (Fig. 3.1C), suggesting that increased expression of CD44 is only partially responsible for the increase in HA binding and that additional changes must be occurring upon TNFα stimulation. To determine if CD44 sulfation was increased upon TNFα stimulation, [35S]sulfate was added to BMDM during the final 2 days of incubation prior to immunoprecipitation of CD44 and analysis of sulfate incorporation by autoradiography. Surprisingly, sulfate incorporation by CD44 was actually reduced by TNFα stimulation (Fig. 3.2A). We also did not detect the sulfated epitopes in either unstimulated or TNFα stimulated BMDM (Fig. 3.2B) that we had previously found to be upregulated upon TNFα stimulation of human PBM. In addition, GlcNAc6ST-1 and -4, the sulfotransferases responsible for the  65     A  CD44 80  80 % of max  100  % of max  100  60 40 20 0 100  Fl-HA  60 40 20  101  0 104 100 101 Fluorescence intensity  102  103  KO  B  WT  102  103  104  WT + TNFα Fl-HA  IFN-γ Cell number  LPS LPS + IFN-γ TNFα 101 102 103 101 102 103 Fluorescence intensity  C  3000  Unstim TNFα  Fl-HA (MFI)  2500 2000 1500 1000 500 0  400  1200 2000 CD44 (MFI)  2800  FIGURE 3.1 Pro-inflammatory cytokines induce HA binding in BMDM. A, Day 7 unstimulated or TNFα stimulated BMDM derived from wild type or CD44 knockout mice were analyzed by flow cytometry for CD44 expression or HA binding. The two left panels show expression levels of CD44, detected using Alexa 488 or Alexa 647 conjugated IM7, while the two right panels show binding to fluorescently labeled HA (Fl-HA). B, HA binding by BMDM was measured following incubation with TNFα, LPS, IFN-γ, or both LPS and IFN-γ. The left panel shows unstained cells, while the right panel shows cells stained with Fl-HA. C, CD44 expression and Fl-HA binding was measured as in A, and the fold increase in mean fluorescence intensity (MFI) following TNFα stimulation is shown. D, Numerous gates were drawn based on CD44 expression levels and the MFI for Fl-HA binding was determined for each of these gates. The two values were then plotted against each other.  $$  BMDM -  TNFα  175  [35S]  +  Br ain  C  +  GlcNAc6ST-1  sulfate  β-actin BMDM  83  -  TNFα  +  y  -  ne  TNFα  Kid  A  GlcNAc6ST-4 β-actin  CD44  83  B  Unstimulated Neuraminidase DD-2 Cell number  AG107 2F3 Ab control 101  102  101 103 Fluorescence intensity  102  103  TNFα Neuraminidase DD-2 Cell number  AG107 2F3 Ab control 101  102  103 101 Fluorescence intensity  102  103  FIGURE 3.2 TNFα does not induce expression of sulfated epitopes in BMDM. A, [35S]sulfate was added to BMDM during stimulation and following immunoprecipitation of CD44 and separation by SDSPAGE, sulfate incorporation was detected by autoradiography and CD44 loading was determined by Western blotting with JIWBB. B, Unstimulated or TNFα stimulated BMDM were stained with the 2F3, AG107 or DD-2 mAbs and analyzed by flow cytometry. Some samples were treated with neuraminidase to remove terminal sialic acid residues. The Ab control represents staining with FITC-labeled secondary only. C, RNA was isolated from BMDM and subjected to semiquantitative RT-PCR using primers for GlcNAc6ST-1, GlcNAc6ST-4, or β-actin. RNA isolated from mouse brain or kidney was used as a positive control for GlcNAc6ST-1 or GlcNAc6ST-4, respectively.  $%  formation of these epitopes, were not expressed in BMDM (Fig. 3.2C). Interestingly, we also failed to detect sialyl Lewis X epitopes on BMDM, which contrasts with the high levels observed in the human myeloid-leukemic cell line SR91 (22). These results suggest that the posttranslational modifications used to regulate HA binding differ between mouse BMDM and human PBM.  3.3.2 TNFα stimulation reduces CS addition to CD44 in BMDM In addition to the increase in overall CD44 sulfation that was observed in the human monocytic cell line, SR91, there was also a relative decrease in sulfation due to CS following TNFα stimulation (22). In KG1a cells that had been previously sorted (17) to establish high and low HA binding populations (Fig. 3.3A), we observed reduced CS addition to CD44 as detected by Western blotting with the anti-CS mAb 2B6 (Fig. 3.3B). The 2B6 mAb recognizes the terminal 4-sulfated CS disaccharide remaining after digestion with chondroitinase ABC and so measures the frequency that CD44 is modified by CS as opposed to the length of CS chains. Densitometric analysis of the 2B6 Western blots revealed that CS modification of CD44 was approximately 3fold higher in low HA binding cells compared to the unsorted parental cells (Fig. 3.3C), showing an inverse correlation between HA binding and CS addition to CD44. As we have previously established that CS addition to CD44 decreases its affinity for HA, we hypothesized that cytokine regulation of HA binding in macrophages could occur via regulation of CS addition. To determine if HA binding by BMDM was affected by CS addition, BMDM were incubated in the presence of 2 mM β-D-xyloside, a competitive inhibitor of glycosaminoglycan addition, and then analyzed for their ability to bind HA (Fig. 3.4). Few unstimulated cells bound to HA (5±2%) while treatment with β-D-xyloside substantially  68     A  CD44  100  % of max  80  60 40 20  60  Low High  40 20  101  Low  102  0 103 104 100 101 Fluorescence intensity  C  High  83  2B6  83  CD44  D  4 3 2 1 0  102  ** Low High  103  100 80 60 40 20 0  HA binding (% +ve)  0 100  Relative 2B6 levels  % of max  80  B  Fl-HA  100  104  Low High  FIGURE 3.3 CS addition to CD44 negatively correlates with HA binding in KG1a cells. A, KG1a cells sorted for either low or high HA binding were analyzed for CD44 expression and binding to Fl-HA by flow cytometry. B, CS addition to CD44 was detected by Western blotting with the 2B6 mAb after immunoprecipitation of CD44 and digestion with chondroitinase ABC. CD44 loading was determined by blotting with the anti-human mAb 3G12. C, Densitometry was performed on the Western blots from B, and after controlling for differences in CD44 loading, the amount of 2B6 detected was set to 1 for the parental cell line. Data is the mean +/- SD of 4 experiments and statistical significance (**F < 0.01) is shown compared to low cells. D, As in A, except the percent of cells that stained positive with Fl-HA is shown. Data is the mean +/- SD of 3 experiments.  $'  -  Xyloside  100  % of max  80 60  Unstim  40 20 0 100  101  102  103 104 100 101 Fluorescence intensity  102  103  104  100  % of max  80 60  TNFα  40 20 0 100  101  102  Unstained  101 103 104 100 Fluorescence intensity  Fl-HA  102  103  104  KM201 + Fl-HA  FIGURE 3.4 β-D-xyloside increases HA binding in BMDM. BMDM were incubated in the presence or absence of TNFα and/or 2 mM β-D-xyloside for 3 days. Cells were then harvested and analyzed for Fl-HA binding by flow cytometry. Some samples were incubated with the CD44 blocking mAb KM201 prior to staining with Fl-HA. One of three representative experiments is shown.  %  increased both the percentage of HA binding cells (47±8%) and the MFI of cells incubated with Fl-HA. Binding to HA by β-D-xyloside treated cells remained CD44 specific as it could be blocked with the anti-CD44 mAb KM201. In TNFα stimulated cells, β-D-xyloside had minimal effect on the percent of HA binding cells (93±2% versus 99±0.3%), but did cause approximately a 1.5-fold increase in the MFI of cells incubated with Fl-HA. These results demonstrate that inhibiting glycosaminoglycan addition can induce HA binding in unstimulated cells and cause a small increase in HA binding in TNFα stimulated BMDM, suggesting that CS addition to CD44 is negatively affecting its ability to bind HA in these cells. To directly test whether CS addition to CD44 was reduced by TNFα stimulation, CD44 was immunoprecipitated from unstimulated and TNFα stimulated cells and analyzed for CS addition by Western blotting with the anti-CS mAb 3B3 (Fig. 3.5A). The 3B3 mAb is similar to the 2B6 mAb, except that it recognizes the 6sulfated rather than the 4-sulfated terminal disaccharide. A 3B3 signal could be detected with CD44 from both 4-day and 7-day old unstimulated cells, while no signal was detected when CD44 was immunoprecipitated from TNFα stimulated BMDM. In addition to the loss of 3B3 staining, we also found reduced expression of the enzyme responsible for 6-sulfation of CS, chondroitin 6-sulfotransferase-1, following TNFα stimulation (Fig. 3.5B). As an alternative method to demonstrate reduced CS addition to CD44 upon TNFα stimulation, [35S]sulfate was added to BMDM during the final 2 days of incubation in the presence or absence of β-D-xyloside. As β-D-xyloside prevents glycosaminoglycan addition, this allowed the relative contribution that CS makes to overall CD44 sulfation to be determined (Fig. 3.5C). Analysis of sulfate incorporation by autoradiography revealed that CS was responsible for about half of CD44 sulfation in both unstimulated (44±9%) and TNFα stimulated (49±12%) cells.  71     Day 4  TNFα  -  -  + 3B3  83  CD44  83  B  C  Day 7  100  Relative sulfate incorporation (%)  A  80  NT Xylo  60 40 20 0  Unstim TNFα  BMDM TNFα  -  +  C6ST-1 β-actin  FIGURE 3.5 TNFα stimulation reduces CS addition to CD44 in BMDM. A, CS addition to CD44 was detected in unstimulated and TNFα stimulated BMDM by Western blotting with the 3B3 mAb after immunoprecipitation of CD44 and digestion with chondroitinase ABC. CD44 loading was determined by blotting with the anti-mouse CD44 Ab JIWBB. B, RNA was isolated from BMDM and subjected to semiquantitative RT-PCR using primers for C6ST-1 and β-actin. C, [35S]sulfate was added to BMDM during stimulation in the presence or absence of β-D-xyloside (xylo). Following immunoprecipitation of CD44 and separation by SDS-PAGE, sulfate incorporation was detected by autoradiography and CD44 loading was determined by Western blotting with JIWBB. After controlling for differences in CD44 loading, sulfate incorporation by unstimulated and non-treated (NT) cells was set to 100%. Data is shown as the mean +/- SD of three experiments.  %  However, as TNFα stimulation reduced overall CD44 sulfation by 38±7%, this worked out to approximately a 1.5-fold reduction in the total amount CS-dependent sulfation. Therefore, results from both sulfate labeling and Western blotting with an anti-CS mAb indicate a reduction in CS addition and/or CS-dependent sulfation on CD44 upon TNFα stimulation of BMDM.  3.3.3 CS addition to CD44 regulates HA binding during TNFα stimulation While β-D-xyloside is an extremely useful tool for studying glycosaminoglycan addition, it is not specific to CD44 and it increases formation of CS in the cell, potentially leading to other glycosylation changes. We therefore used a retroviral infection system to infect CD44 null BMDM with CD44 or CD44 containing a serine to alanine point mutation at residue 183. A mutation (R43A) that prevents HA binding was also used as a control. S183 in mouse is equivalent to S180 in human CD44, which we have previously established to be the site of CS addition (21). This was confirmed in BMDM expressing S183A mutant CD44 by the absence of staining with the 3B3 mAb (Fig. 3.6A). Cells expressing wild type CD44 behaved as expected, with a greater percentage of TNFα stimulated cells binding to Fl-HA (Fig. 3.6B) and the amount of HA binding being proportional to the level of CD44 expression (Fig. 3.6C). In contrast, approximately half of BMDM expressing S183A-CD44 bound Fl-HA constitutively, with only a minimal increase in the percent of HA binding cells occurring upon TNFα stimulation. After controlling for differences in CD44 expression, unstimulated BMDM expressing S183A-CD44 displayed higher HA binding than even TNFα stimulated CD44 infected cells. In addition, TNFα stimulation of S183A-CD44 infected cells did not appear to have a significant effect on HA binding. In support of these observations, comparable results were obtained when CD44 infected  73     A  B  CD44 S183A  Unstim 80  83  Fl-HA binding (% +ve)  3B3  83  CD44  70 60 50 40 30 20 10 0  C  TNFα  CD44  S183A  R43A  7000 6000  Fl-HA (MFI)  5000 4000 3000 2000 1000  2600  2200  1800  1400  1000  600  200  0  CD44 (MFI)  CD44 CD44 + Xylo CD44 + TNFα CD44 + TNFα + Xylo  S183A S183A + Xylo S183A + TNFα S183A + TNFα + Xylo  FIGURE 3.6 CS addition to CD44 regulates HA binding during TNFα stimulation. During the generation of BMDM, bone marrow from CD44 knockout mice was infected with a retrovirus vector expressing a YFP marker and wild type, S183A mutant CD44, or R43A mutant CD44. A, CD44 was immunoprecipitated from CD44 and S183A-CD44 infected BMDM and digested with chondroitinase ABC. CS addition was then detected by Western blotting with the 3B3 mAb. CD44 loading was determined by blotting with the anti-mouse CD44 Ab JIWBB. B, BMDM were stimulated with TNFα and then analyzed by flow cytometry after staining with Alexa 647 conjugated IM7 and Fl-HA. The percent of HA binding cells was determined from the CD44 positive population. Data is shown as the mean +/- SD of 5 experiments. C, BMDM were stimulated in the presence or absence of β-D-xyloside (xylo) for 3 days and then analyzed by flow cytometry as above. Numerous gates were drawn based on CD44 expression levels and the MFI for Fl-HA binding was determined for each of these gates. To control for leakage of the YFP signal, the MFI of cells infected with CD44 containing the R43A mutation was subtracted from the original values. The final MFI value for Fl-HA was then plotted against the MFI used for each CD44 gate.  %"  cells were incubated with β-D-xyloside. These data suggest that CS addition is the primary mechanism that regulates CD44 binding to HA in BMDM upon TNFα stimulation.  3.3.4 CS addition to CD44 is regulated by multiple cytokines We next wished to determine if CS modification of CD44 was responsible for regulation of HA binding in response to cytokines other than TNFα. As LPS induces HA binding and IL-4 inhibits HA binding by human monocytes (12), BMDM were stimulated with TNFα, IL-4, or LPS and IFNγ for 48 hrs in the presence or absence of β-D-xyloside and analyzed for their HA binding ability (Fig. 3.7A). As before, non-binding unstimulated cells were able to bind HA after incubation in β-D-xyloside; while β-D-xyloside had minimal effect on the already high HA binding TNFα stimulated cells. A high percent of HA binding cells were also observed following stimulation with LPS and IFNγ, with β-D-xyloside having no discernable effect on the level of HA binding. In contrast, only a small percentage of cells bound to HA following IL-4 treatment, while there was a substantial increase in HA binding in cells incubated with β-D-xyloside. These results led us to hypothesize that LPS/IFNγ stimulation would reduce CS addition and that IL-4 treatment would increase CS addition. Detection of CS modified CD44 by Western blotting with the 3B3 mAb supported this hypothesis, with a detectable band observed from unstimulated, but not TNFα or LPS/IFNγ stimulated BMDM. IL-4 treatment meanwhile, increased the intensity of 3B3 mAb staining by approximately 5-fold (Fig. 3.7B). These results suggest that HA binding is increased following TNFα or LPS/IFNγ stimulation by a reduction in CS modification, while HA binding in IL-4 treated cells is minimized by high levels of CS addition to CD44.  75     A  Unstimulated  TNFα  100  % of max  80 60 40 20 0 100  101  102  103 104 100 101 Fluorescence intensity  IL-4  102  103  104  LPS + IFNγ  100  % of max  80 60 40 20 0 100  101  102  103 104 100 101 Fluorescence intensity  Unstained  10 9 8 7 6 5 4 3 2 1 0  103  104  Xyloside  *  Relative 3B3 staining  B  Untreated  102  * **  **  - TNFα IL-4 LPS IFNγ  FIGURE 3.7 CS addition to CD44 is regulated by multiple cytokines. A, BMDM were stimulated with TNFα, IL-4, or LPS and IFN-γ for 48 hrs in the presence or absence of β-D-xyloside. Cells were then stained with Fl-HA and analyzed by flow cytometry. B, BMDM were stimulated as in A, followed by immunoprecipitation of CD44 and digestion with chondrointase ABC. CS addition was detected by Western blotting with the 3B3 mAb and CD44 loading was determined by blotting with the anti-mouse CD44 Ab JIWBB. Western blots were analyzed by densitometry and, after taking into account variations in CD44 loading, the intensity of 3B3 staining for CD44 from unstimulated BMDM was set to 1. Data is shown as the mean +/- SD of 3 experiments with significance indicated as: *F < 0.05, **F < 0.01, ***F < 0.001.  %$  3.4 DISCUSSION Here we show that CS addition to CD44 is reduced by TNFα stimulation of BMDM, and that this reduction contributes to the increased HA binding by CD44 in TNFα activated cells. Several inflammatory mediators increased CD44 expression and induced HA binding, but this increase could not be explained solely by an increase in CD44 expression. In human PBM, TNFα induced HA binding correlates with increased carbohydrate sulfation and expression of sulfated determinants on N-linked carbohydrates (13, 17). In mouse BMDM, we observed a decrease in overall CD44 sulfation upon TNFα stimulation. Further analysis indicated less sulfation of Nand O-linked carbohydrates (data not shown) as well as an absence of sulfated carbohydrate epitopes detectable with specific Abs. Glycosaminoglycan-dependent sulfation was also reduced; while HA binding could be increased by β-D-xyloside, a competitive inhibitor of glycosaminoglycan addition. When BMDM expressed a CD44 mutant that could not be modified by CS, they displayed constitutively high binding to HA that was not further affected by TNFα stimulation β-D-xyloside treatment. This contrasts with cells expressing wild type CD44, which did not bind HA in any significant amount prior to stimulation, but showed an increase in HA binding upon TNFα stimulation. Taken together, these data show that HA binding can be negatively regulated by CS modification of CD44 and that downregulation of CS addition to CD44 is a physiological event induced by TNFα stimulation. In human monocytes HA binding can be regulated by induced expression of sialidase and removal of sialic acid residues from CD44 (18). This did not appear important during activation of BMDM as TNFα did not affect HA binding in the absence of CS modification. We have found that treatment with neuraminidase, an enzyme that removes sialic acid resides, increases HA binding in BMDM (Ruffell, B unpublished observations). However, a similar increase was  77     observed in both unstimulated and TNFα stimulated cells, supporting the conclusion that it is not a major regulatory mechanism in BMDM. It is interesting to note that β-D-xyloside increases HA binding in human monocytes as well as prevents interleukin-4 treatment from downregulating HA binding (28). This suggests that CS modification of CD44 may be a mechanism for HA binding regulation in these cells and is consistent with our finding that IL-4 treatment of BMDM increases the amount of CS modified CD44. While IL-4 treatment actually increased HA binding in BMDM, this increase was small when compared to the increase in CD44 expression. Furthermore, the ability of CS addition to inhibit HA binding in IL-4 treated BMDM was demonstrated by the large increase in HA binding that occurred following incubation of the cells in β-D-xyloside. As TNFα and LPS/IFNγ treatment reduced CS addition to CD44, while IL-4 treatment increased CS addition to CD44, it demonstrates that CS modification is responsible for both cytokine-mediated upregulation and downregulation of HA binding in these cells. There are several other examples of CS synthesis being responsive to cytokine treatment. Transforming growth factor-β (TGF-β) modifies the expression of various CS proteoglycans and increases the length of CS chains on biglycan (29), decorin (29, 30) and versican (31). CS chain length is also increased on CD44 in human lung fibroblasts (32) and mouse melanoma cells (33), inferring that treatment with this anti-inflammatory cytokine may reduce HA binding. Meanwhile, platelet-derived growth factor (PDGF) increases CS addition to CD44 in human dermal fibroblasts (34) and can increase the ratio of 6-to 4-sulfation (29, 31), although this has not been shown for CD44. Here we find that TNFα decreased the amount of glycosaminoglycandependent sulfation on CD44 and reduced expression of chondroitin 6-sulfotransferase-1, leading to reduced staining with a mAb against 6-sulfation of CS.  78     While modification of CD44 by CS reduces binding to HA, CS addition is necessary for CD44-mediated binding to other ECM components, including fibronectin (35) and collagen XIV (36), and may also be involved in binding to collagen IV (37). The induced changes in CS seen following cytokine treatment are important in CD44 mediated motility on these substrates. PDGF induces invasive migration of human dermal fibroblasts into a fibronectin/fibrin gel that can be blocked by both β-D-xyloside and anti-CD44 mAbs (34), while β-D-xyloside also inhibits invasion of rabbit wound microvascular endothelial cells into a fibrin matrix (38). Similarly, the invasion and migration of mouse melanoma on type I collagen is increased following TGF-β treatment and blocked by β-D-xyloside (33). Human melanoma cell migration on type IV collagen also involves CS modified CD44 and possibly a cooperative role with integrins (37). Thus the presence of CS can promote cell migration on the ECM, whereas we have shown a reduction in CS leads to increased adhesion to the ECM component HA.  79     3.5 REFERENCES 1.  2. 3.  4. 5.  6. 7. 8. 9. 10.  11.  12.  13.  14.  15.  16.  Crowther, M., N. J. Brown, E. T. Bishop, and C. E. Lewis. 2001. Microenvironmental influence on macrophage regulation of angiogenesis in wounds and malignant tumors. J Leukoc Biol 70:478-490. Leibovich, S. J., and R. Ross. 1975. The role of the macrophage in wound repair. A study with hydrocortisone and antimacrophage serum. Am J Pathol 78:71-100. Duffield, J. S., S. J. Forbes, C. M. Constandinou, S. Clay, M. Partolina, S. Vuthoori, S. Wu, R. Lang, and J. P. Iredale. 2005. Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair. J Clin Invest 115:56-65. Jiang, D., J. Liang, and P. W. Noble. 2007. Hyaluronan in tissue injury and repair. Annu Rev Cell Dev Biol 23:435-461. Jameson, J. M., G. Cauvi, L. L. Sharp, D. A. Witherden, and W. L. Havran. 2005. Gammadelta T cell-induced hyaluronan production by epithelial cells regulates inflammation. J Exp Med 201:1269-1279. Teder, P., R. W. Vandivier, D. Jiang, J. Liang, L. Cohn, E. Pure, P. M. Henson, and P. W. Noble. 2002. Resolution of lung inflammation by CD44. Science 296:155-158. Culty, M., H. A. Nguyen, and C. B. Underhill. 1992. The Hyaluronan Receptor (CD44) Participates in the Uptake and Degradation of Hyaluronan. J Cell Biol 116:1055-1062. Underhill, C. B., H. A. Nguyen, M. Shizari, and M. Culty. 1993. CD44 Positive Macrophages Take Up Hyaluronan During Lung Development. Dev Biol 155:324-336. DeGrendele, H. C., P. Estess, and M. H. Siegelman. 1997. Requirement for CD44 in activated T cell extravasation into an inflammatory site. Science 278:672-675. Stoop, R., H. Kotani, J. D. McNeish, I. G. Otterness, and K. Mikecz. 2001. Increased resistance to collagen-induced arthritis in CD44-deficient DBA/1 mice. Arthritis Rheum 44:2922-2931. Cuff, C. A., D. Kothapalli, I. Azonobi, S. Chun, Y. M. Zhang, R. Belkin, C. Yeh, A. Secreto, R. K. Assoian, D. J. Rader, and E. Pure. 2001. The adhesion receptor CD44 promotes atherosclerosis by mediating inflammatory cell recruitment and vascular cell activation. J Clin Invest 108:1031-1040. Levesque, M. C., and B. F. Haynes. 1997. Cytokine induction of the ability of human monocyte CD44 to bind hyaluronan is mediated primarily by TNF-alpha and is inhibited by IL-4 and IL-13. J. Immunol. 159:6184-6194. Brown, K. L., A. Maiti, and P. Johnson. 2001. Role of sulfation in CD44-mediated hyaluronan binding induced by inflammatory mediators in human CD14+ peripheral blood monocytes. J. Immunol. 167:5367-5374. Mohamadzadeh, M., H. Degrendele, H. Arizpe, P. Estess, and M. Siegelman. 1998. Proinflammatory stimuli regulate endothelial hyaluronan expression and CD44/HAdependent primary adhesion. J. Clin. Invest. 101:97-108. Lesley, J., N. Howes, A. Perschl, and R. Hyman. 1994. Hyaluronan binding function of CD44 is transiently activated on T cells during an in vivo immune response. J. Exp. Med. 180:383-387. English, N. M., J. F. Lesley, and R. Hyman. 1998. Site-specific de-N-glycosylation of CD44 can activate hyaluronan binding, and CD44 activation states show distinct threshold densities for hyaluronan binding. Cancer Res. 58:3736-3742.  80     17.  18.  19. 20.  21. 22.  23.  24.  25. 26. 27. 28.  29.  30.  31.  Brown, K. L., D. Birkenhead, J. C. Lai, L. Li, R. Li, and P. Johnson. 2005. Regulation of hyaluronan binding by F-actin and colocalization of CD44 and phosphorylated ezrin/radixin/moesin (ERM) proteins in myeloid cells. Exp. Cell Res. 303:400-414. Katoh, S., T. Miyagi, H. Taniguchi, Y. Matsubara, J. Kadota, A. Tominaga, P. W. Kincade, S. Matsukura, and S. Kohno. 1999. Cutting edge: An inducible sialidase regulates the hyaluronic acid binding ability of CD44-bearing human monocytes. J. Immunol. 162:5058-5061. Maiti, A., G. Maki, and P. Johnson. 1998. TNF- induction of CD44-mediated leukocyte adhesion by sulfation. Science 282:941-943. Skelton, T. P., C. X. Zeng, A. Nocks, and I. Stamenkovic. 1998. Glycosylation provides both stimulatory and inhibitory effects on cell surface and soluble CD44 binding to hyaluronan. J. Cell Biol. 140:431-446. Ruffell, B., and P. Johnson. 2005. Chondroitin sulfate addition to CD44H negatively regulates hyaluronan binding. Biochem Biophys Res Commun 334:306-312. Delcommenne, M., R. Kannagi, and P. Johnson. 2002. TNF-alpha increases the carbohydrate sulfation of CD44: induction of 6-sulfo N-acetyl lactosamine on N- and Olinked glycans. Glycobiology 12:613-622. Tjew, S. L., K. L. Brown, R. Kannagi, and P. Johnson. 2005. Expression of Nacetylglucosamine 6-O-sulfotransferases (GlcNAc6STs)-1 and -4 in human monocytes: GlcNAc6ST-1 is implicated in the generation of the 6-sulfo N-acetyllactosamine/Lewis x epitope on CD44 and is induced by TNF-alpha. Glycobiology 15:7C-13C. Couchman, J. R., B. Caterson, J. E. Christner, and J. R. Baker. 1984. Mapping by monoclonal antibody detection of glycosaminoglycans in connective tissues. Nature 307:650-652. de Belder, A. N., and K. O. Wik. 1975. Preparation and properties of fluorescein-labelled hyaluronate. Carbohydr. Res. 44:251-257. Kinsella, T. M., and G. P. Nolan. 1996. Episomal vectors rapidly and stably produce high-titer recombinant retrovirus. Hum Gene Ther 7:1405-1413. Markowitz, D., S. Goff, and A. Bank. 1988. A safe packaging line for gene transfer: separating viral genes on two different plasmids. J Virol 62:1120-1124. Levesque, M. C., and B. F. Haynes. 1999. TNF alpha and IL-4 regulation of hyaluronan binding to monocyte CD44 involves posttranslational modification of CD44. Cell. Immunol. 193:209-218. Schonherr, E., H. T. Jarvelainen, M. G. Kinsella, L. J. Sandell, and T. N. Wight. 1993. Platelet-derived growth factor and transforming growth factor-beta 1 differentially affect the synthesis of biglycan and decorin by monkey arterial smooth muscle cells. Arterioscler Thromb 13:1026-1036. Kahari, V. M., H. Larjava, and J. Uitto. 1991. Differential regulation of extracellular matrix proteoglycan (PG) gene expression. Transforming growth factor-beta 1 upregulates biglycan (PGI), and versican (large fibroblast PG) but down-regulates decorin (PGII) mRNA levels in human fibroblasts in culture. J Biol Chem 266:10608-10615. Schonherr, E., H. T. Jarvelainen, L. J. Sandell, and T. N. Wight. 1991. Effects of plateletderived growth factor and transforming growth factor-beta 1 on the synthesis of a large versican-like chondroitin sulfate proteoglycan by arterial smooth muscle cells. J Biol Chem 266:17640-17647.  81     32.  33.  34.  35. 36.  37.  38.  Romaris, M., A. Bassols, and G. David. 1995. Effect of transforming growth factor-beta 1 and basic fibroblast growth factor on the expression of cell surface proteoglycans in human lung fibroblasts. Enhanced glycanation and fibronectin-binding of CD44 proteoglycan, and down-regulation of glypican. Biochem J 310 ( Pt 1):73-81. Faassen, A. E., D. L. Mooradian, R. T. Tranquillo, R. B. Dickinson, P. C. Letourneau, T. R. Oegema, and J. B. McCarthy. 1993. Cell surface CD44-related chondroitin sulfate proteoglycan is required for transforming growth factor-beta-stimulated mouse melanoma cell motility and invasive behavior on type I collagen. J Cell Sci 105 ( Pt 2):501-511. Clark, R. A., F. Lin, D. Greiling, J. An, and J. R. Couchman. 2004. Fibroblast invasive migration into fibronectin/fibrin gels requires a previously uncharacterized dermatan sulfate-CD44 proteoglycan. J Invest Dermatol 122:266-277. Jalkanen, S., and M. Jalkanen. 1992. Lymphocyte CD44 binds the COOH-terminal heparin-binding domain of fibronectin. J Cell Biol 116:817-825. Ehnis, T., W. Dieterich, M. Bauer, B. Lampe, and D. Schuppan. 1996. A chondroitin/dermatan sulfate form of CD44 is a receptor for collagen XIV (undulin). Exp Cell Res 229:388-397. Knutson, J. R., J. Iida, G. B. Fields, and J. B. McCarthy. 1996. CD44/chondroitin sulfate proteoglycan and alpha 2 beta 1 integrin mediate human melanoma cell migration on type IV collagen and invasion of basement membranes. Mol Biol Cell 7:383-396. Henke, C. A., U. Roongta, D. J. Mickelson, J. R. Knutson, and J. B. McCarthy. 1996. CD44-related chondroitin sulfate proteoglycan, a cell surface receptor implicated with tumor cell invasion, mediates endothelial cell migration on fibrinogen and invasion into a fibrin matrix. J Clin Invest 97:2541-2552.     82     CHAPTER FOUR Hyaluronan induces cell death in activated T cells through CD44  A version of this chapter has been submitted for publication: Ruffell, B. and Johnson, P. (2008) Hyaluronan induces cell death in activated T cells through CD44  83     4.1 INTRODUCTION CD44 and its ligand hyaluronan (HA), an extracellular matrix glycosaminoglycan, have been implicated in a number of processes including wound healing, inflammation, angiogenesis and metastasis (1). In immune-mediated diseases, CD44 can either augment or inhibit inflammation and autoimmunity. For example, CD44-/- mice have reduced disease severity in models of atherosclerosis (2) and arthritis (3), whereas disease severity increases in models of lung inflammation (4) and inflammatory bone loss (5). In T cells, the evidence suggests a largely proinflammatory role for CD44. Increased expression of CD44 is a marker for activated and memory T cells in mice (6), and a population of activated T cells is able to bind HA (7). CD44 has the potential to provide co-stimulatory activity during T cell activation, as cross-linking CD44 together with the T cell receptor (TCR) increases proliferation and expression of activation markers, as well as augmenting interleukin-2 (IL-2) production (8, 9). HA binding is induced upon T cell activation and there is some evidence to suggest that HA can enhance CD3mediated activation of human peripheral T cells (10). Other data suggest an indirect effect with HA binding to dendritic cells (11) and affecting conjugate formation (12), or by HA oligosaccharides increasing dendritic cell maturation via toll-like receptor-4 (TLR-4) (13). Better established is a role for CD44 and HA in the extravasation of T cells to sites of inflammation. CD44 allows a mouse T cell line to roll on HA coated surfaces (14) and on tumor necrosis factor-α stimulated endothelial cells in vitro (15), with similar observations being made in vivo with Th1 and Th2 polarized cells (16). Rolling on endothelial cells is necessary for subsequent firm adhesion and extravasation of T cells, and CD44 has been shown to facilitate this process by enhancing VLA-4 mediated adhesion (17). Furthermore, T cell recruitment to inflammatory  84     sites in staphylococcal enterotoxin B-induced peritonitis (18) and collagen-induced arthritis (3) is CD44-dependent. There is also some evidence that CD44 may be involved in activation-induced cell death (AICD). AICD occurs following secondary stimulation of activated T cells that have experienced prolonged exposure to IL-2 and primarily involves the interaction of the death receptor Fas with Fas Ligand (FasL). The process of AICD, together with activated cellautonomous death (ACAD), is responsible for removal of activated T cells and maintenance of immune homeostasis (19). Two studies have found that AICD in T cell lines incubated on immobilized anti-CD3 mAb is reduced by CD44 cross-linking (20, 21). In addition, the absence of CD44 has been found to partially reduce the lymphoproliferative symptoms found in mice that do not express Fas (22). However, in direct contrast to this, another study found increased lymphoproliferative and autoimmune symptoms in mice lacking expression of both CD44 and Fas (23), and CD44 cross-linking in another T cell line increases AICD (9). These conflicting studies make it hard to interpret the role for CD44 in AICD. Furthermore, the effect of HA, the primary physiological ligand for CD44, has not been examined. In this study, we use two CD44 mutants that either increase or prevent HA binding to show that the interaction between CD44 and HA causes cell death in activated human Jurkat T cells. This HA-dependent cell death occurred independently of both Fas and caspases, and was observed in primary T cells in the absence of Fas-dependent apoptosis.  4.2 MATERIALS AND METHODS 4.2.1 Cell lines The human Jurkat T lymphoma cell line, clone E6.1, was from the American Type Culture Collection (ATCC; Manassas, VA) and was cultured in RPMI-1640 supplemented with 10% 85     FCS, 1 mM sodium pyruvate (Invitrogen, Burlington, ON), 2 mM L-glutamine (Sigma-Aldrich, Oakville, ON), and 50 U/ml Penicillin/Streptomycin (Invitrogen). To obtain CD44-positive cells, Jurkat cells were electroporated with 20 µg of CD44H-pCEP4 plasmid DNA using the Gene Pulser apparatus (Bio-Rad, Mississauga, ON) at 250 V and 950 µF and selected in 300 µg/ml hygromycin B (Calbiochem, San Diego, CA). Cells were then sorted for high CD44 expression on a FACS Vantage SE Turbo (Becton Dickinson, Mississauga, ON). All experiments were performed with cells grown in the absence of selection for 3-10 days.  4.2.2 Antibodies and reagents. Purified rat anti-human/mouse CD44 mAb IM7.8.1 (ATCC #TIB-235) was conjugated to Alexa 488 (Molecular Probes, Eugene, OR) or coupled to CNBr-activated Sepharose 4B (Amersham Biosciences, Piscataway, NJ) according to the manufacturer’s instructions. The mouse antihuman CD44 mAb 3G12 was from G. Dougherty (24) and the mouse anti-human CD44 mAb Hermes-3 was from the ATCC (#HB-9480). Hermes-1, a rat anti-human CD44 mAb capable of blocking binding to HA, was from the Development Studies Hybridoma Bank (University of Iowa, Iowa City, IA). The mouse anti-human TCR mAb C305 (#CRL-2424) and anti-CD3 mAb OKT3 (#CRL-8001) were from the ATCC. The anti-chondroitin sulfate (CS) mAb 2B6 was from Seikagaku America (East Falmouth, MA). The mouse anti-human Fas mAb DX2 was purchased from Southern Biotech (Birmingham, AL), while 7C11, an IgM mAb capable of inducing Fas-mediated apoptosis, was from Immunotech (Marseille, France). The mouse antihuman blocking mAb against Fas (ZB4) was from Stressgen (Victoria, BC), while the mouse anti-human (NOK-1) and hamster anti-mouse (MFL3) blocking mAbs against FasL were from eBioscience (San Diego, CA). Unlabeled goat anti-rat Ab and goat anti-mouse Ab were a gift  86     from I. Trowbridge. FITC-conjugated goat anti-mouse Ab was from Caltag (Burlingame, CA) and HRP-conjugated goat anti-mouse Ab was from Jackson ImmunoResearch (West Grove, PA). Fluorescein-conjugated HA (Fl-HA) was made as described (25) using rooster comb HA from Sigma-Aldrich. Prestained molecular mass standards were purchased from New England Biolabs (Beverly, MA) and the pan-caspase inhibitor z-VAD-fmk was from Calbiochem.  4.2.3 Generation of point mutations Human CD44H cDNA (26) in pCEP4 (Invitrogen) and S180A and G181A mutations in human CD44 were described previously (27). The R41A mutation was created by oligonucleotide site directed mutagenesis using four primers: primer 1 containing a NcoI site (forward, 5’CGCTCCGGACACCATGGACAAG-3’); primer 2 containing a HpaI site (reverse, 5’CGGGTGCCATCACGGTTAACAATAGT-3’); and complementary primers 3 (forward, 5’AAAAATGGTGCCTACAGCATCTCTCGG-3’) and 4 (reverse, 5’CCGAGAGATGCTGTAGGCACCATTTTT-3’) containing the R41A mutation. The mutated sequences are underlined. Two fragments were generated by PCR using primers 1 with 4 and 2 with 3. The fragments were then mixed and PCR performed again. The final product was inserted into the CD44 sequence using NcoI and HpaI.  4.2.4 Flow cytometry Cells (2x105) were incubated with 1 µg/ml of DX2 in PBS containing 2% FCS and 5 mM EDTA for 30 min on ice, washed once, and incubated with 10 µg/ml of FITC-goat anti-mouse Ab. Alternatively, cells were incubated with approximately 5 µg/ml IM7-Alexa 488 or Fl-HA. After washing, cells were resuspended in buffer containing 5 µg/ml propidium iodide (PI; Sigma-  87     Aldrich). A minimum of 5000 live events was collected on a FACScan and analyzed using CellQuest (Becton Dickinson) or FlowJo (Tree Star, Ashland, OR) software.  4.2.5 Analysis of chondroitin sulfate on CD44 Immunoprecipitation, sulfate labeling and Western blotting of CD44 was done as described (27). Briefly, cells were cultured for 2 days in Na2[35SO4] following a 1 day incubation in the presence or absence of 2 mM p-nitrophenyl β-D-xylopyranoside (β-D-xyloside, Sigma-Aldrich). Cells were lysed and incubated with IM7-coupled beads. Some samples were digested with Proteus vulgaris chondroitinase ABC or Flavobacterium heparinum heparitinase (Seikagaku America). Immunoprecipitated CD44 was resolved on a 7.5% SDS polyacrylamide gel and transferred to a polyvinylidene fluoride membrane (Millipore, Mississauga, ON). Membranes were exposed to Kodak BioMax MR film (Interscience, Markham, ON) at -80°C for 3 to 10 days. To determine relative CD44 levels, membranes were blotted with 3G12. To detect CS, immunoprecipitated CD44 from 1x107 cells was digested with chondroitinase ABC to expose the epitope for the antiCS mAb 2B6. Following sequential incubations with the 2B6 mAb and HRP-goat anti-mouse Ab, membranes were developed with ECL (Amersham Biosciences) according to the manufacture’s instructions.  4.2.6 Low molecular mass HA generation and analysis HA was dissolved in 150 mM NaCl, 100 mM NaOAc, 1 mM EDTA, pH 5.0 at a concentration of 5 mg/ml and bovine testes hyaluronidase (Calbiochem) was added at 200 U/ml. The mixture was incubated at 37ºC for 10 sec or 16 hrs prior to inactivation of the enzyme by boiling for 10 min, followed by adjustment of the pH to 7.0. HA was analyzed by PAGE and silver staining as  88     described (28). From the observed pattern, HA digested for 10 sec was termed intermediate molecular mass HA, while HA digested for 16 hrs was low molecular mass HA. To compare relative binding of CD44 to different sizes of HA a competitive binding assay was setup in which cells stimulated for 8 hrs with 10 ng/ml PMA (Sigma-Aldrich) were incubated with 0.5 µg/ml Fl-HA mixed with 0.5 µg/ml to 5 mg/ml of unlabeled HA, intermediate HA, or low molecular mass HA on ice for 30 min. Cells were analyzed by flow cytometry as described above.  4.2.7 Cell death analysis Cell viability was assessed by flow cytometry following PI and Annexin V-FITC (Southern Biotech) staining done according to the manufacturer’s instructions. Annexin V-PE from Becton Dickinson was also used in one experiment. The percentage of viable cells was determined by the percent of cells negative for both Annexin V and PI. In one experiment, cells were stained with PI alone and the number of live events collected in 30 sec was counted. Data was normalized by setting the number of live events in the untreated sample to 100%. Caspase activation was determined by Western blotting to detect cleaved fragments with an anti-caspase 3 Ab or an anti-caspase 8 mAb from Cell Signaling (Danvers, MA). Mitochondrial membrane polarization was measured using JC-1 (Molecular Probes) according to the manufacturer’s instructions. Cell polarization was measured by staining with DiOC6(3) (Calbiochem) at a final concentration of 40 nM followed by the addition of PI and analysis by flow cytometry.  89     4.2.8 Induction of cell death Cells were suspended at 5x105 cells/ml and treated with 100 nM staurosporine (Sigma-Aldrich) or 10-100 ng/ml of 7C11, an anti-Fas mAb, for various times. CD44 was cross-linked either by incubating cells with 10 µg/ml of Hermes-1 or Hermes-3 for 20 min on ice, washing once, and culturing cells for 16 hrs with 10 µg/ml goat anti-rat or goat anti-mouse Ab, or by incubating cells for 0 to 8 hrs with both 5 µg/ml Hermes-1 and 25 µg/ml of goat anti-rat Ab. Serum starvation was conducted by incubating 1x105 cells/ml in RPMI1640 without FCS for 0 to 3 days.  4.2.9 Cell stimulation Cells were suspended at 5x105 cells/ml in RPMI-10% FCS and stimulated with 10 ng/ml PMA for various times in the presence or absence of the mAbs Hermes-1 or Hermes-3 at 10 µg/ml. Alternatively, cells were added to 96-well plates coated with 50 µl of 5 µg/ml C305 or OKT3. To control the amount of HA in the culture medium cells were resuspended in AIMV (Invitrogen), which is a defined serum free media that does not contain HA. Various concentrations of HA, intermediate HA or low molecular mass HA along with 10 µg/ml Hermes1 or 1 U/ml Streptomyces hyaluronidase (Calbiochem) were then added to the cells for 30 min prior to PMA stimulation for 16 hrs. Alternatively, cells were stimulated in AIMV for 12 hrs with PMA, and then incubated with or without blocking Abs or inhibitors for 20 min prior to the addition of various amounts of HA for up to 8 hrs.  90     4.2.10 FasL RT-PCR RNA from ~4x106 cells was isolated using RNeasy Mini kit (Qiagen, Chatsworth, CA) and 10 µg was reverse transcribed with Superscript II (Invitrogen) according to the manufacturer’s instructions. PCR using ~2.5 µg of cDNA was performed with Taq DNA Polymerase in 50 µl with 1 mM Mg2+ as follows: 94ºC for 2 min; 25-40 cycles at 94ºC for 30 sec, 58ºC for 30 sec and 72ºC for 50 sec; 72ºC for 10 min. β-actin primers (forward, 5’GACTACCTCATGAAGATCCT-3’; reverse, 5’-ATCCACATCTGCTGGAAGGT-3’) formed a 512 bp fragment and amplification was done for 25 cycles. FasL primers (forward, 5’CACTACCGCTGCCACCCC-3’; reverse, 5’-CCAGAGAGAGCTCAGATACGTTG-3’) formed a 606 bp fragment and amplification was done for 40 cycles. PCR product (20 µl) was electrophoresed in 1.5% agarose gel containing SYBR Safe (Invitrogen) and visualized under ultraviolet light.  4.2.11 Primary cells Splenic T cells were purified from 6-12 week old C57BL/6 (The Jackson Laboratory, Bar Harbor, ME) and CD44-/- mice (29) by negative selection. Briefly, spleens were ground up using glass slides and red blood cells were lysed using 0.83% ammonium chloride. Cells were then suspended in PBS containing 2 mM EDTA and 0.5% BSA and incubated with biotin-conjugated mAbs against B220, CD11b and Ter119, washed, then incubated with anti-biotin magnetic microbeads (Miltenyi Biotec, Auburn, CA). After washing, cells were run through a MACS LS separation column (Miltenyi Biotec) to remove non-T cells. Preparations were over 90% positive for CD3 expression immediately after purification and over 98% positive by day 6, as determined by flow cytometry. Fas negative T cells from MRL/lpr mice were purified as above,  91     except that an anti-CD19 mAb was used in place of the B220 mAb. Following purification, cells at 106/ml were stimulated with 2.5 ng/ml PMA and 500 ng/ml ionomycin (Sigma-Aldrich) in complete RPMI with 10 mM HEPES, and 55 µM β-mercaptoethanol. After 2 days, 20 U/ml of IL-2 (R&D Systems, Minneapolis, MN) was added to the media and the cells were maintained in IL-2 at 1-2x106 cells/ml. On day 5, cells were resuspended at 1x106 cells/ml in AIMV with or without 1 µg/ml of the Fas blocking mAb MFL3 and added to wells coated with 0.1-5 µg/ml of the anti-CD3 mAb 2C11 and/or 5 µg/ml of the anti-CD44 mAb IM7 for 24 hrs. HA at 500 ng/ml was added to some samples at various times during stimulation. Cell viability and HA binding was assessed as described above. Animal experimentation was conducted in accordance with protocols approved by the University Animal Care Committee and Canadian Council of Animal Care guidelines.  4.2.12 Statistics Data are shown as the mean +/- SD of three experiments, unless otherwise indicated. Significance was determined by the Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001.  4.3 RESULTS 4.3.1 Chondroitin sulfate addition to CD44 regulates HA binding in Jurkat T cells. To evaluate the effect of HA binding in T cells, CD44 negative Jurkat T lymphoma cells were transfected with human CD44 or CD44 containing S180A or R41A point mutations. R41, located within the HA binding site, is critical for HA binding and mutation to an alanine abolishes HA binding (30, 31). The S180A mutation acts as a gain-of-function mutant as it prevents chondroitin sulfate (CS) addition and increases the affinity of CD44 for HA when made  92     as an Ig-fusion protein or when expressed in mouse fibroblast L cells (27). However, it was not known whether CS modification negatively regulates HA binding in T cells. To first determine if S180A-CD44 could be used as a constitutive HA binding mutant in T cells, HA binding was assessed in the transfected cells. Levels of CD44 expression were approximately equivalent, yet S180A-CD44 expressing cells bound significantly higher levels of fluorescent-HA (Fl-HA) compared to wild type CD44, as determined by flow cytometry (Fig. 4.1A). Stimulation of CD44 transfected Jurkat cells with 10 ng/ml of PMA for 8 hrs induced HA binding in CD44 expressing cells and further enhanced binding in S180A-CD44 expressing cells, while R41A-CD44 transfected cells did not bind HA under any condition tested. This suggested that CS addition was down regulating HA binding by CD44 in Jurkat T cells. To confirm CS modification of residue S180, CD44 was immunoprecipitated from cells grown in the presence of sodium [35S]sulfate (Figs. 4.1B and 4.1C). While CD44 did not display the heavily sulfated, heterogeneous, high molecular mass forms normally indicative of large chain CS addition; sulfate incorporation by CD44 was reduced by growth in the presence of β-Dxyloside, an inhibitor of glycosaminoglycan addition, and by treatment with chondroitinase ABC. This suggested that CD44 in Jurkat T cells is modified by short chains of CS. Wild type CD44 was sulfated to a much greater extent than S180A-CD44, which was also unaffected by βD-xyloside  or chondroitinase ABC, indicating that S180 is the major site of CS addition. This  was further supported by Western blotting CD44 with the anti-CS mAb 2B6 (Fig. 4.1D), which bound to wild type and R41A-CD44, but not to CD44 containing either the S180A or G181A mutations that prevent CS addition (27). Together, these data indicate that CS addition occurs on CD44 in Jurkat T cells and negatively regulates HA binding. Notably, even low molecular mass forms of CS on CD44 are able to exert this effect.  93     A  -  CD44  Fl-HA  -  PMA  PMA  R41A Cell number  S180A CD44 Vector  B  101 102 103 101 102 103 Fluorescence intensity  C  175  110 100 90 80 70 60 50 40 30 20 10 0  S180A  S180A CD44  CD44  xyloside  Relative Sulfate Incorporation (%)  101 102 103  [35S] sulfate  83 62 175  CD44  83  101 102 103  * **  * **  ABC - + Hep - - + CD44  - + - - + S180A  R41A RA;SA  Vector  D  CD44 S180A G181A  62  83  2B6  83  CD44  FIGURE 4.1 S180A mutation in CD44 prevents CS addition and results in constitutive HA binding in Jurkat cells. A, CD44 expression and HA binding were analyzed by flow cytometry in transfected Jurkat cells either unstimulated or stimulated for 8 hrs with PMA. The two left panels show expression levels of CD44, detected using Alexa 488 conjugated IM7, while the two right panels show binding to FlHA. B, CD44 immunoprecipitated from sodium [35S]sulfate labeled cells grown in the presence or absence of Β-D-xyloside and resolved by SDS-PAGE. C, Relative quantitation of sulfate incorporation by CD44 before and after digestion with chondroitinase ABC (ABC) or heparitinase (Hep) as measured by densitometry. Sulfate incorporation by untreated wild type CD44 was set to 100% after adjustment for the level of CD44. Data is shown as the mean +/- SD of three experiments with significance determined CS on immunoprecipitated CD44 by Western blotting with the anti-CS mAb 2B6. CD44 loading levels were determined by blotting with the mAb 3G12.  '"  4.3.2 High HA binding T cells are more susceptible to AICD The generation of Jurkat cells expressing CD44 with different binding abilities for HA (high, low, and not detectable) allowed us to investigate the consequences of HA binding. Stimulation with immobilized TCR mAb induced cell death in both Jurkat T cells and CD44 expressing Jurkat T cells, however, this AICD was noticeably more pronounced in both S180A-CD44 and G181A-CD44 expressing cells (Fig. 4.2A). Subsequent analysis of cell death by staining with Annexin V-FITC and propidium iodide (PI) revealed a percentage of Annexin V single positive cells, indicating that phosphatidylserine (PS) exposure, a hallmark of apoptotic cells, was occurring either prior to, or in the absence of, cell death (Fig. 4.2B). Activation of Jurkat cells with immobilized anti-TCR or anti-CD3 mAb results in a small amount of Fas-dependent AICD due to the induction of FasL expression (32). While similar results are obtained upon stimulation with PMA and ionomycin (33), PMA stimulation alone is able to protect Jurkat cells from Fasdependent apoptosis (34). In accordance with this, minimal cell death was observed in vector control and wild type CD44 transfectants following PMA stimulation (Fig. 4.2C). However, Annexin V binding and cell death was still observed in cells expressing the high HA binding CD44 mutant (Fig. 4.2D). Interestingly, enhanced survival, not death was observed in cells expressing R41A-CD44, suggesting that it is the ability to bind HA, not the loss of CS addition that is a factor in PMA induced T cell death (Fig. 4.2C). The data showed a good correlation between the HA binding ability of CD44 and the degree of cell death observed during the activation of Jurkat cells with anti-TCR mAb or PMA. Furthermore, as PMA activation protects against Fas-mediated apoptosis in Jurkat cells it suggests that this cell death may occur independently of Fas.  95     A 120  C 100 90 80 70 60  Viable cells (%)  Relative cell number (%)  110 100 90 80 70 60 50 40 30 20 10 0  0  4  8 12 16 Time (h)  20  V ector CD44  0  S180A G181A  D  100  4  90  80  80  60 50 40 30 20 10  16  100  90 70  8 12 Time (h)  R41A R41A;S180A  Percent of cells (%)  Percent of cells (%)  B  50 40 30 20 10 0  70 60 50 40 30 20 10  0 Time (h) 0 8 16  0 8 16  0 Time (h) 0 8 16  0 8 16  Vector  S180A  Vector  S180A  Viable  Annexin V  PI / Annexin V  FIGURE 4.2 Cell death is preferentially induced in TCR or PMA stimulated Jurkat cells expressing a high HA binding form of CD44. A, Time course showing percentage live cells after stimulation with the anti-TCR mAb C305. Data is shown as the mean +/- SD of three experiments and was normalized by setting the number of live events in the untreated samples to 100%. B, Representative experiment showing the percent of cells that were unstained (viable), single positive for Annexin V, or double positive for both Annexin V and PI following anti-TCR stimulation. C, Time course of cell viability during PMA stimulation as determined by lack of Annexin V-FITC and PI staining. Data is shown as the mean +/- SD of three experiments. D, Same as B, except cells were stimulated with PMA.  '$  4.3.4 HA binding by CD44 enhances AICD in T cells To determine that the observed effects of S180A-CD44 or R41A-CD44 expression were not due to altered signaling, multivalent binding by HA was mimicked by cross-linking CD44 with Hermes-1, a mAb that binds to the HA binding site of CD44. This induced a similar level of Annexin V binding in all unstimulated CD44 expressing Jurkat T cells (Fig. 4.3A). Similar results were observed in PMA activated Jurkat T cells cultured in serum free conditions, where CD44 cross-linking induced PS exposure and cell death (Fig. 4.3B). This indicates that all mutant forms of CD44 have a similar capacity to induce PS exposure and cell death, and that PMA stimulation increases the percent of dead cells after CD44 cross-linking. Interestingly, CD44 cross-linking with Hermes-3, a CD44 mAb that does not bind to the HA binding site, did not induce PS exposure (Fig. 4.3A). To further determine that it was HA binding by CD44 that was important for mediating AICD, soluble Hermes-1 and Hermes-3 mAbs were added to cells during PMA stimulation. Hermes-1 completely prevented cell death induced by PMA stimulation in S180A-CD44 cells, whereas Hermes-3 had no effect (Fig. 4.3C). These results strongly suggest that the binding of serum HA by wild type and S180A-CD44 enhances cell death during PMA stimulation. Unexpectedly, the small amount of death observed in the vector control cells as a result of PMA stimulation was not observed in the CD44-R41A transfectants (Fig. 4.3C). Similarly, Hermes-1 completely blocked cell death in CD44 transfected cells, despite wild type CD44 expression alone having a minimal effect on cell death during PMA stimulation. This implies that the inability of CD44 to bind HA protects against cell death whereas high HA binding forms of CD44 promote cell death. As Jurkat T cells do not produce HA (35), the likely source of HA in these experiments was the FCS present in the media. To examine this, 500 ng/ml of HA purified from rooster comb  97     A  Viable  Annexin V  PI / Annexin V  100  Percent of cells (%)  90 80 70 60 50 40 30 20 10 0  Hermes-1 - + Hermes-3 - - + Secondary + + +  - + - - + + + +  - + - - + + + +  - + - - + + + +  Vector  CD44  S180A  R41A  B  100  Percent of cells (%)  90 80 70  Viable Annexin V PI / Annexin V  60 50 40 30 20 10 0  CD44  S180A  CD44 -  PMA  100 90 80 Viable cells (%)  C  S180A  - + - + - + - +  Hermes-1 - + - + Secondary - + - +  ** ** **  70  ** * **  **  60 50 40  * **  30  * **  20 10 0  PMA - + + + Hermes-1 - - + Hermes-3 - - - + Vector  - + + + - - + - - - + CD44  - + + + - - + - - - + S180A  - + + + - + + + - - + - - + - - - + - - - + R41A R41A;S180A  FIGURE 4.3 CD44 mAbs can induce or prevent cell death of transfected Jurkat cells during PMA stimulation. A, Representative experiment showing the percent of cells that were viable, single positive for Annexin V, or double positive for both Annexin V and PI following incubation with Hermes-1 or Hermes-3 for 20 min and cross-linking with secondary Ab for 16 hrs. B, Same as A, except unstimulated or PMA stimulated cells were grown in AIMV serum free media prior to co-incubation of Hermes-1 and secondary Ab for 8 hrs. C, Graph showing percent viable cells following PMA stimulation for 16 hrs in the presence or absence of the HA blocking anti-CD44 mAb Hermes-1 or the non-blocking anti-CD44 mAb Hermes-3. Data is shown as the mean +/- SD of three experiments with significance determined by the  '&  was added to CD44-S180A transfectants cultured in media without FCS. In the absence of FCS, only a small amount of cell death occurred following PMA stimulation for 16 hrs, whereas the addition of HA greatly increased cell death (Fig. 4.4A). This was not due to contaminants in the purified HA, as cell death was prevented by digestion of the HA by hyaluronidase prior to PMA stimulation. Titration of purified HA into serum free AIMV media revealed that concentrations of HA as low as 10 ng/ml augmented AICD significantly in both CD44 and S180A-CD44 transfectants, with the maximum amount of cell death occurring with HA concentrations below 500 ng/ml (Fig. 4.4B). CD44 and HA dependency was again demonstrated by the ability of Hermes-1 mAb to completely block death from occurring, even in the presence of 5000 ng/ml of HA. Therefore, the data show that HA binding by both wild type and mutant CD44 results in a substantial increase in AICD upon PMA stimulation of Jurkat transfectants. Analysis of CD44 expression and HA binding in CD44 and S180A-CD44 expressing cells revealed a time dependent increase in both upon PMA stimulation (Fig. 4.4C). HA was significantly induced in wild type CD44 transfectants and increased in S180A-CD44 transfectants such that HA binding was always greater in S180A-CD44 expressing cells (Fig. 4.4C middle panel). Furthermore, S180A-CD44 expressing cells showed consistently higher HA binding when equivalent CD44 levels were compared (Fig. 4.4C lower panel) suggesting that cells expressing this mutated form of CD44 have a higher overall avidity for HA than those expressing wild type CD44.  4.3.5 The size of HA affects its ability to enhance cell death HA in the extracellular matrix normally consists of high molecular mass chains (>106 Da), but in inflamed tissues HA is degraded and lower molecular mass HA fragments can be detected.  99     * **  * **  C  * ** * **  **  *  500 0  - ++ - - + HA  CD44 S180A CD44 + Hermes-1 S180A + Hermes-1  2  4 6 8 10 12 Time (h)  0  2  4 6 8 10 12 Time (h)  1000 800 600 400 200 0  80 70  1200 1000  60 50 40 30 20 10 0 100  0  1200  Fl-HA (MFI)  Viable Cells (%)  2000  1000  PMA - + + - + + - - + - - + Serum Media  100 90  * **  1500  50 40 30 20 10 0  B  CD44 S180A  2500  CD44 (MFI)  Viable cells (%)  100 90 80 70 60  Fl-HA (MFI)  A  800 600 400 200  101 102 HA (ng/mL)  103  104  0  0  500 1000 1500 2000 2500 CD44 (MFI)  FIGURE 4.4 Enhanced AICD in Jurkat transfectants is dependent upon HA binding by CD44. A, Graph showing cell viability in S180A-CD44 cells following PMA stimulation for 16 hrs in RPMI supplemented with 10% FCS, or in RPMI ml HA. Hyaluronidase ( ) treatment was done for 30 min prior to PMA stimulation. Data is shown as the mean +/- SD of three experiments shown following PMA stimulation for 16 hrs in AIMV serum free media with various concentrations of HA. The HA-blocking anti-CD44 mAb Hermes-1 was added to some samples. C, The mean fluorescence intensity (MFI) for S180A-CD44 expression (top panel) and Fl-HA binding (middle panel) at various time points during PMA stimulation is shown. The bottom panel shows the relationship between Fl-HA binding and the level of CD44 expression for CD44 and S180A-CD44 at each time point. Data in B and C is shown as the mean +/- SD of three experiments.    These smaller forms of HA have been shown to be pro-inflammatory when added to dendritic cells or macrophages (36). It was therefore of interest to determine if the size of HA could affect its ability to enhance AICD in T cells. To generate intermediate or low molecular mass HA, bovine testicular hyaluronidase was used to digest HA (Fig. 4.5A). Both intermediate and low molecular mass HA displayed an ability to compete for Fl-HA binding to CD44 (data not shown) and S180A-CD44 (Fig. 4.5B). However, while unlabeled HA effectively competed with Fl-HA when used in 10-fold excess, a 1000-fold excess was required for intermediate, and a 10,000fold excess for low molecular mass HA. During PMA stimulation 500 ng/ml of intermediate sized HA caused significant death in S180A-CD44 cells. However, cell death was only a fraction of the cell death observed with high molecular mass HA, even when the concentration was increased as high as 50 µg/ml (Fig. 4.5C). Low molecular mass HA did not affect viability at any concentration tested. This experiment demonstrates that the size of HA is important in determining its ability to enhance AICD.  4.3.6 HA rapidly induces cell death in PMA stimulated Jurkat T cells As CD44 cross-linking has been reported to provide co-stimulatory activity during T cell activation (8, 37) it was possible that the HA-mediated augmentation of cell death observed during the activation of Jurkat cells was due to HA providing a co-stimulatory signal through CD44 and enhancing T cell activation. To determine if the HA signal had to be given simultaneously with the T cell receptor or PMA signal, CD44-S180A cells were stimulated with PMA for 12 hrs, washed twice, and then incubated with HA for 4 hrs (Fig. 4.6A). Incubation of cells with HA for 4 hrs subsequent to PMA stimulation had the same effect on cell viability as incubation with both HA and PMA for 16 hrs. Cell death following the addition of HA occurred  101     HA BPB High Int Low  B Relative Fl-HA binding (%)  A  High In t Low  High + Hermes-1 In t + Hermes-1 Low + Hermes-1  0  100 101 102 103 104 Ratio of HA to Fl-HA (x:1)  100 90 80 70 60  Viable cells (%)  C  120 110 100 90 80 70 60 50 40 30 20 10 0  50 40 30 20 10 0  0  .005 .05 .5 5 HA (μg/mL)  50  FIGURE 4.5 The size of HA affects its ability to induce cell death in PMA stimulated cells. A, Visualization of high, intermediate (int) or low molecular mass HA by silver staining samples run on a 15% acrylamide gel. Bromophenol blue (BPB) was used as a marker dye. B, Competition assay between FlHA and increasing amounts of unlabeled high, intermediate and low molecular mass HA in S180A-CD44 cells. C, Cell viability is shown in S180A-CD44 cells following PMA stimulation for 16 hrs in AIMV serum free media with various concentrations of high, intermediate, or low molecular mass HA. The HA-blocking anti-CD44 mAb Hermes-1 was added to some samples. Data in B and C is shown as the mean +/- SD of three experiments.    A  100  * **  90  60 50 40 30 20 10 0  100 50 500 + Hermes-1  100 90 80  70  HA PMA HA PMA  500 10  Viable cells (%)  Viable cells (%)  80  B  * * **  -  + + -  + +  + + + +  + -  0-12h + + 12-16h -  70 60 50 40 30 20 10 0  0  .5  1  2 3 4 Time (h)  6  8  FIGURE 4.6 HA can rapidly induce cell death in CD44 expressing cells after PMA activation. A, Graph showing cell viability in S180A-CD44 cells suspended in AIMV media and stimulated with PMA in the presence or absence of 500 ng/ml HA. Cells were initially stimulated for 12 hrs, then washed twice and incubated for an additional 4 hrs in the presence of PMA and/or HA. Data is shown as the mean +/Time course of cell viability following the addition of HA to S180A-CD44 cells pre-stimulated for 12 hrs with PMA. Hermes-1 mAb was added 20 min prior to the addition of HA for one sample set. Data is shown as the mean +/- SD of three experiments.  !  rapidly within 30 min, with the degree and time of cell death dependent on the amount of HA being added (Fig. 4.6B). These data demonstrate that HA binding induces cell death, and that this is not due to co-stimulation, but instead occurs independently in activated cells capable of binding high levels of HA.  4.3.7 HA-induced cell death occurs independently of Fas- and caspase-mediated apoptosis Fas susceptible Jurkat T cells become resistant to Fas mediated apoptosis upon PMA stimulation (34), despite an upregulation of FasL (38). It was therefore possible that HA binding by CD44 was causing cell death in Jurkat cells by reversing the effects of PMA on Fas signaling or otherwise inducing Fas signaling. In unstimulated cells, neither CD44 expression (Fig. 4.7A) nor the presence of exogenous HA (data not shown) affected sensitivity to apoptosis induced with an anti-Fas mAb. The expression of Fas (Fig. 4.7B) and FasL mRNA (Fig. 4.7C) were also unaffected by the presence of HA although, as expected, FasL mRNA was increased by PMA stimulation. As these results did not rule out an effect of HA on the Fas signaling pathway, HA was also added to PMA activated cells pre-treated with blocking mAbs against Fas (ZB4) or FasL (NOK-1). Neither mAb had an effect on HA-induced cell death (Fig. 4.7D) indicating that HA was not acting via the Fas/FasL interaction. This was further confirmed by anti-CD3 activation of S180A-CD44 expressing Jurkat cells with various combinations of HA or blocking mAbs (Fig. 4.7E). While blocking Fas largely prevented cell death in the absence of HA, it had minimal effect on HA-induced cell death. Notably, blocking Fas consistently reduced cell death in activated cells by approximately 10%, whereas blocking the CD44-HA interaction reduced cell death by an additional 30%. This indicates that HA- and Fas-dependent cell death are additive and suggests that the two signaling pathways are distinct. In support of this, the addition  104     A  C  PMA + HA  E  90  8  **  ** **  70 Viable cells (%)  Viable cells (%)  **  80  D  60 50 40 30 20 10 0  B  100  ZB4 Hermes-1 HA OKT3  CD44  % of Max  80  G  60 40  90  103  S180A  F  Time (h) 62 48 33 25 17  60 40 20 101  Control  102 Fas  NT  7  103  0  1 2 4 8 16 7C11  Viable cells (%)  102 Fas  80 % of Max  + +  * **  + +  + + + * **  + + +  + + + +  * **  70 101  100  0  + +  80  20 0  - - - - +  60 50 40 30 20 10 0  z-VAD-fmk 0 0 25 50 0 0 25 50 Caspase 8  HA  7C11  PMA  FIGURE 4.7 HA induced cell death does not occur via the Fas/FasL pathway. A, Cell viability was assessed in unstimulated cells incubated with the anti-Fas mAb 7C11 for various times. Data is shown as the mean +/- SD of three experiments. B, Fas expression as determined by flow cytometry in cells cultured in 10% FCS and stimulated with PMA for 8 hrs. C, FasL mRNA expression in AIMV cultured S180A-CD44 cells as determined by semi-quantitative PCR following PMA stimulation in the presence or absence of HA. B-actin was used as a loading control. D, Cell viability of S180A-CD44 transfectants following the addition of HA for 2 hrs to cells pre-stimulated with PMA. Cells were pre-incubated for 20 min with the Fas blocking mAb ZB4 or the FasL blocking mAb NOK-1 prior to the addition of HA. E, Cell viability of S180A-CD44 cells activated for 16 hrs with immobilized anti-CD3 mAb OKT3 in the presence of various combinations of HA, ZB4, or Hermes-1. F, Western blot analysis of caspase 8 activation following the addition of HA to PMA stimulated S180A-CD44 cells. Incubation of unstimulated cells with the anti-Fas mAb 7C11 for 2 hrs was used as a positive control. G, Same as D, except cells were prepan-caspase inhibitor z-VAD-fmk. As a control, unstimulated cells were incubated with z-VAD-fmk prior to the addition of the 7C11 mAb. For D, E and G data is shown ***p < 0.001).  #  of HA to PMA stimulated cells did not result in the activation of caspase 8 (Fig. 4.7F), and even high concentrations of the pan-caspase inhibitor zFAD-fmk failed to inhibit HA-induced cell death (Fig. 4.7G). While AICD occurs primarily through the extrinsic Fas-dependent apoptotic pathway, the intrinsic mitochondrial-dependent pathway can also play a role (39). This is particularly true in Jurkat T cells, where even effective Fas signaling requires the mitochondrial pathway (40). As CD44 did not appear to be enhancing AICD via the extrinsic pathway, we investigated whether CD44 expression enhanced cell death via the intrinsic pathway. Death induced by either staurosporine or serum withdrawal was equal between the different Jurkat transfectants (Fig. 4.8A), with the addition of exogenous HA having no effect on apoptosis (data not shown). More importantly, membrane depolarization during HA-induced cell death was not detected prior to loss of membrane integrity, as measured with the mitochondrial specific JC-1 probe (Fig. 4.8B) or the more general DiOC6(3) probe (Fig. 4.8C). Caspase 3 activation, which can result from cytochrome C release from the mitochondria, was also not observed following the addition of HA (Fig. 4.8D). These results indicate that HA-induced cell death in activated Jurkat cells does not activate this intrinsic, mitochondrial-dependent, apoptotic pathway.  4.3.8 CD44 and HA can mediate AICD in ex vivo T cells Although AICD occurs upon TCR-mediated stimulation of Jurkat T cells, it requires secondary TCR stimulation preceded by culture in IL-2 in ex vivo T cells (41). This has led to the development of an in vitro system for studying AICD where T cells are activated and then incubated for several days in IL-2 prior to restimulation. Using this procedure we determined whether HA was able to induce cell death in activated splenic T cells purified from wild type  106     V ector CD44 S180A 0  B  1 2 Time (h)  3  0  1 2 Time (days)  3  35 30  50 40 30 20 10 0  V ector CD44 S180A  C  10 ng/ml HA 100 ng/ml HA Hermes-1 + 2o 100 ng/ml 7C11  100 90 80 70 60  Mitochondrial Polarization (%)  100 90 80 70 60 50 40 30 20 10 0  % Depolarized  90 80 70 60 50 40 30 20 10 0  Viable cells (%)  Viable cells (%)  A 100  25 20 15 10 5  0  2  4 6 Time (h)  D  0 7C11 PMA HA Hermes-1 20  8  -  + -  + -  + + -  + + + -  + + +  Time (h) 48 33 25 17  0  1 2  4 8 16 7C11  7  Caspase 3  FIGURE 4.8 HA induced cell death does not occur via the mitochondrial pathway. A, Cell viability of unstimulated Jurkat transfectants incubated with staurosporine for 1-3 hrs (left panel) or incubated in RPMI without FCS for 1-3 days (right panel). B, Time course of mitochondrial depolarization in S180ACD44 cells. PMA stimulated cells were incubated with either HA or Hermes-1 mAb and goat anti-rat Ab (2o) and then stained with the mitochondrial membrane specific dye JC-1. Incubation of unstimulated cells with the 7C11 mAb was used as a positive control. C, Same as B, except cells were analyzed after 2 hrs with the membrane dye DiOC6(3) and the percent of live depolarized (PI negative, DiOC6(3) low) cells is shown. D, Western blot analysis of caspase 3 activation following the addition of HA to PMA stimulated S180A-CD44 cells. Incubation of unstimulated cells with the anti-Fas mAb 7C11 for 2 hrs was used as a positive control. Data from A, B and C is shown as the mean +/- SD of three experiments.  %  C57BL/6 or CD44-/- mice. Equal amounts of death were observed after restimulation with immobilized anti-CD3 between wild type and CD44 null cells, and the addition of HA had no effect (Fig. 4.9A). However, cell death was greatly enhanced when wild type T cells were activated in the presence of immobilized anti-CD44 mAb IM7. Increased death was observed even with low concentrations of immobilized anti-CD3, yet there was no effect when the cells were not stimulated. This shows that CD44 can significantly augment AICD. However, this was not observed with HA, suggesting that the level of HA binding by restimulated T cells may be insufficient to induce significant cell death. While the levels of CD44 were increased upon restimulation, the activated T cells showed a range of HA binding (Fig. 4.9B). To determine if HA augmented AICD in the high HA binding cells, T cells were stimulated with sub-optimal levels of anti-CD3, then incubated with Fl-HA for 30 min at 37ºC and finally labeled with Annexin V-PE. Analysis of live cells revealed that Fl-HA binding cells were also positive for Annexin V (Fig. 4.9C). Fl-HA binding cells were not Annexin V positive after primary stimulation of T cells (data not shown). Since we had previously found in the Jurkat T cells that HA-induced cell death occurred independently of Fas-dependent cell death, and given that the high HA binding population represented only a fraction of the activated T cells, we set out to determine whether HA-induced cell death could be observed more readily in the absence of Fasdependent cell death. Fas-mediated AICD was blocked with an anti-FasL mAb added prior to stimulation and a comparison of cells stimulated in the presence versus the absence of HA revealed approximately a 10% reduction in the percent of viable cells (Fig. 4.9D). This decrease was not observed when HA was added to CD44-/- T cells. Death occurred within 30 min of adding HA to the T cells, recapitulating the timeframe observed in Jurkat T cells. Furthermore, CD44 and HA-dependent cell death was observed when AICD was induced in splenic T cells  108     B  30 20 10 0  * *  90  90 80 70 60 50 40  80 70  MFI  %  6  *  5  60  4  50 40  3  30  2  20  1  10 0 0  1  2 3 2C11 (μg/ml)  4  5  Non Low High  0  Annexin V  Fl-HA Binding  D  100  7  Annexin V 101 102 103  100  Counts  C  KO KO + HA KO + IM7  Annexin V Positive (%)  Relative percent of live cells (%)  100  WT WT + HA WT + IM7  Fluorescence Intensity (Arbitrary Units)  A  Annexin V  E  40  12  20 0  10  101  102 103 CD44  100 % of Max  80 60 40 20 0  WT ** KO  14  101  **  **  8 6 4 2 0  0.5  2 Time (h)  24  Relative decrease in viability (%)  16  60  Relative decrease in viability (%)  % of Max  80 60  * **  50 40 30  * **  20  * **  10 0  HA IM7  -  HA IM7 2C11  102 103 Fl-HA  FIGURE 4.9 AICD in ex vivo activated murine splenic T cells is enhanced by the presence of HA. A, Representative experiment showing the percentage of live day 6 splenic T cells from wild type (WT) and CD44 knockout (KO) mice following re-activation with immobilized anti-CD3 mAb 145-2C11 for 24 hrs (see materials and methods for details). Some samples were incubated with 500 ng/ml HA or with both immobilized anti-CD3 and anti-CD44 (IM7). Data is shown as the mean +/- SD of three experiments. B, Analysis of CD44 expression and Fl-HA binding of day 6 activated splenic T cells either unstimulated (thin line) or restimulated (thick line) for 24 hrs. The cells alone negative control (shaded) is also shown. C, Analysis of PS exposure and Fl-HA binding in day 6 wild type splenic T cells. After restimulation for 24 ml of immobilized anti-CD3 mAb, cells were incubated with Fl-HA hrs stained with Annexin V-PE on ice, and then analyzed by flow cytometry. Live cells were divided into non-, low-, and high-HA binding populations and analyzed for levels of Annexin V-PE staining. The percent of cells positive for Annexin V within each population is indicated. MFI was normalized between experiments by setting the intensity of the non-HA binding population to 1 and data is shown as the ml of the Fas blocking mAb MFL3 for 24 hrs on immobilized anti-CD3 mAb. HA at 500 ng/ml was added to the cells for 0.5 hrs, 2 hrs, or 24 hrs. To normalize between experiments, the percent loss of cell viability between cells stimulated in the absence versus the presence of HA is shown. Data is the mean +/- SEM of 4 experiments with pools of 2 mice per experiment. Significance (**p<0.01) is shown compared to CD44 knockout (KO) cells. E, Graph showing the relative decrease in cell viability in Fas negative T cells from MRL/lpr mice that were cultured and stimulated as in A. The data shown is the mean +/- SEM of 3 experiments with a total of 6 mice. Significance (***p < 0.001) is shown compared to the preceding sample.  '  from MRL/lpr mice, which lack functional Fas (Fig. 4.9E). This demonstrates that HA-induced cell death occurs independently of Fas in a subset of activated T cells.  4.4 DISCUSSION Here we have shown that HA induces cell death in activated T cells via CD44. HA-induced cell death was dependent on the ability of CD44 to bind HA, as a loss-of-function mutation in CD44 prevented cell death and a gain-of-function mutation increased cell death. Inhibition of HAinduced cell death by soluble CD44 mAbs and the induction of cell death by CD44 cross-linking suggests that the ability of HA to cross-link CD44 is an important factor in its function. This is also supported by the fact that intermediate to low molecular mass HA had a decreased ability to induce cell death. CD44 cross-linking has previously been shown to both increase (9) and decrease (20, 21) AICD in T cell lines. These conflicting results may be due to the state of the cell examined, as we have shown that the outcome of CD44 ligation can depend on the activation state of the cell. CD44 cross-linking caused PS exposure in both unstimulated and PMA stimulated Jurkat T cells, yet significant cell death only occurred in activated cells. This demonstrates that activation is required to make the cells susceptible to CD44-dependent cell death. Similarly, S180A-CD44 expressing Jurkat T cells bound HA constitutively, yet HA only induced cell death in PMA activated cells, again indicating that cells must first be activated to become susceptible to HA-induced cell death. This corresponds well to our observations with activated splenic T cells, in which neither incubation on immobilized anti-CD44 mAb nor the presence of HA induced cell death unless the cells were re-activated via the TCR. Furthermore, there was a correlation between the extent of HA binding and the percent of cells showing PS  110     exposure following secondary, but not primary, stimulation. This indicates that HA-induced cell death only occurs in T cells primed to undergo AICD. This requirement of secondary activation for T cells to be susceptible to HA-dependent AICD is also observed for Fas-dependent AICD (19). Despite this, we found that Fas and HA acted independently to induce AICD. This was demonstrated most clearly in transfected Jurkat cells where both anti-Fas and anti-CD44 mAbs were required to completely block cell death. HA induced more AICD than Fas in activated CD44+ Jurkat T cells, whereas the opposite appeared to be true in reactivated splenic T cells. This may relate to the extent of HA binding in activated T cells, as CD44-dependent AICD induced with immobilized CD44 mAbs was dramatic in both cell types. HA-induced cell death occurred in about 10% of activated T cells, but this only became evident after blockage of Fas-mediated AICD. The independence of these two pathways was further demonstrated using MRL/lpr T cells that lack functional Fas, but were still susceptible to CD44 and HA-dependent AICD. This independence is also observed in vivo where the loss of CD44 increased the severity of lymphoproliferative and autoimmune disease in Fas deficient (lpr/lpr) mice (23). HA-induced death in Jurkat cells occurred in the absence of caspase 3 and 8 activation and was not blocked by a pan-caspase inhibitor. This suggests that CD44 induces a form of nonapoptotic programmed cell death, as caspase activation is required for cells to be considered apoptotic (42). Caspase-independent, non-apoptotic death has been previously observed in a model of ACAD and was attributed to reactive oxygen species (ROS) formation (39). However, treatment with a ROS inhibitor did not reduce CD44-induced death in Jurkat cells (data not shown). Caspase-independent death has also been described in an erythroleukemia cell line following treatment with an anti-CD44 mAb (43). In this case, death was linked to release of  111     apoptosis-inducing factor and activation of calpain. However, we did not see mitochondrial depolarization, which is necessary for the release of apoptosis-inducing factor (44), and death occurred over a much longer time period than we observed in our experiments. Having eliminated the most likely mechanisms for CD44 induced cell death, determining the precise mechanism leading to PS exposure and cell death will require further study. The difference between the percentage of cell death observed in T cells upon CD44 cross-linking versus the addition of HA suggests that HA induced death may be limited to cells that exhibit high levels of HA binding. In vitro, using purified HA, this appears to be approximately 10% of activated T cells. However, this percentage may be greater in vivo as HA binding proteins present in the extracellular matrix have been shown to enhance HA binding to CD44 (45, 46). Indeed, an in vivo role for CD44 in AICD has been suggested as CD44-/- mice show increased severity of concanavalin A induced hepatitis (47) and the delayed-type hypersensitivity response (48), which was attributed to reduced AICD. CD44 mAbs that bind to the HA binding site of CD44 were efficient at inducing cell death in activated T cells, whereas Hermes-3, a mAb that binds to CD44 at another site, did not. This infers that a specific interaction with the HA binding site of CD44 is required to induce cell death. HA binding may facilitate a conformational change in CD44, or its repeating structure may facilitate clustering, which then transmits a signal to the cell. The response to HA may relate to the avidity of the interaction with CD44, as there was a correlation between the level of HA binding and cell death. A reduced ability of low molecular mass HA to engage multiple CD44 molecules or facilitate clustering may help explain its inability to enhance AICD in the transfected Jurkat cells. It is tempting to speculate that low molecular mass or fragmented HA in inflamed tissue could reduce CD44-dependent AICD, thereby maintaining T cell activation and  112     promoting the inflammatory response, whereas newly synthesized high molecular mass HA, produced to facilitate tissue repair, could increase AICD and thus promote contraction of the immune response and restoration of immune homeostasis. PS exposure is sufficient to target cells for recognition, phagocytosis and subsequent degradation by macrophages (49). In tissues, high HA binding by activated T cells may trigger PS exposure and flag these cells for removal by macrophages. It is possible that high HA binding is a characteristic of highly active T cells, as increased CD44 expression is a marker for activated T cells and HA binding identifies highly active T regulatory cells (50). Levels of CD44 expression and HA binding by T cells could therefore be important for the initiation of inflammation due to their role in extravasation, and important for the resolution of inflammation due to their ability to mediate AICD in T cells.  113     4.5 REFERENCES 1. 2.  3.  4. 5.  6.  7.  8. 9. 10.  11.  12.  13.  14.  15.  Ponta, H., L. Sherman, and P. A. Herrlich. 2003. CD44: from adhesion molecules to signalling regulators. Nat. Rev. Mol. Cell. Biol. 4:33-45. Cuff, C. A., D. Kothapalli, I. Azonobi, S. Chun, Y. M. Zhang, R. Belkin, C. Yeh, A. Secreto, R. K. Assoian, D. J. Rader, and E. Pure. 2001. The adhesion receptor CD44 promotes atherosclerosis by mediating inflammatory cell recruitment and vascular cell activation. J. Clin. Invest. 108:1031-1040. Stoop, R., H. 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Use of CD44 by CD4+ Th1 and Th2 lymphocytes to roll and adhere. Blood 107:4798-4806. Nandi, A., P. Estess, and M. Siegelman. 2004. Bimolecular Complex between Rolling and Firm Adhesion Receptors Required for Cell Arrest; CD44 Association with VLA-4 in T Cell Extravasation. Immunity 20:455-465. DeGrendele, H. C., P. Estess, and M. H. Siegelman. 1997. Requirement for CD44 in activated T cell extravasation into an inflammatory site. Science 278:672-675. Krammer, P. H., R. Arnold, and I. N. Lavrik. 2007. Life and death in peripheral T cells. Nat Rev Immunol 7:532-542. Ayroldi, E., L. Cannarile, G. Migliorati, A. Bartoli, I. Nicoletti, and C. Riccardi. 1995. Cd44 (pgp-1) inhibits cd3 and dexamethasone-induced apoptosis. Blood 86:2672-2678. Larkin, J., G. J. Renukaradhya, V. Sriram, W. Du, J. Gervay-Hague, and R. R. Brutkiewicz. 2006. CD44 differentially activates mouse NK T cells and conventional T cells. J. Immunol. 177:268-279. Weber, G. F. 2004. 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Biophys. Res. Commun. 334:306-312. Ikegami-Kawai, M., and T. Takahashi. 2002. Microanalysis of hyaluronan oligosaccharides by polyacrylamide gel electrophoresis and its application to assay of hyaluronidase activity. Anal. Biochem. 311:157-165. Schmits, R., J. Filmus, N. Gerwin, G. Senaldi, F. Kiefer, T. Kundig, A. Wakeham, A. Shahinian, C. Catzavelos, J. Rak, C. Furlonger, A. Zakarian, J. J. L. Simard, P. S. Ohashi, C. J. Paige, J. C. Gutierrezramos, and T. W. Mak. 1997. CD44 regulates hematopoietic progenitor distribution, granuloma formation and tumorigenicity. Blood 90:2217-2233. Peach, R. J., D. Hollenbaugh, I. Stamenkovic, and A. Aruffo. 1993. Identification of hyaluronic acid binding sites in the extracellular domain of CD44. J. Cell Biol. 122:257264. Bajorath, J., B. Greenfield, S. B. Munro, A. J. Day, and A. Aruffo. 1998. Identification of CD44 residues important for hyaluronan binding and delineation of the binding site. J. Biol. Chem. 273:338-343. Dhein, J., H. Walczak, C. Baumler, K. M. Debatin, and P. H. Krammer. 1995. Autocrine T-cell suicide mediated by APO-1/(Fas/CD95). Nature 373:438-441.  115     33.  34.  35.  36. 37.  38.  39.  40.  41. 42. 43.  44. 45.  46.  47.  48.  Chwae, Y. J., M. J. Chang, S. M. Park, H. Yoon, H. J. Park, S. J. Kim, and J. Kim. 2002. Molecular mechanism of the activation-induced cell death inhibition mediated by a p70 inhibitory killer cell Ig-like receptor in Jurkat T cells. J. Immunol. 169:3726-3735. Holmstrom, T. H., S. C. Chow, I. Elo, E. T. Coffey, S. Orrenius, L. Sistonen, and J. E. Eriksson. 1998. Suppression of Fas/APO-1-mediated apoptosis by mitogen-activated kinase signaling. J. Immunol. 160:2626-2636. Makatsori, E., N. K. Karamanos, N. Papadogiannakis, A. Hjerpe, E. D. Anastassiou, and T. Tsegenidis. 2001. Synthesis and distribution of glycosaminoglycans in human leukemic B- and T-cells and monocytes studied using specific enzymic treatments and high-performance liquid chromatography. Biomed. Chromatogr. 15:413-417. Stern, R., A. A. Asari, and K. N. Sugahara. 2006. Hyaluronan fragments: an informationrich system. Eur. J. Cell Biol. 85:699-715. Li, R. H., N. Wong, M. D. Jabali, and P. Johnson. 2001. CD44-initiated cell spreading induces Pyk2 phosphorylation, is mediated by Src family kinases, and is negatively regulated by CD45. J. Biol. Chem. 276:28767-28773. Latinis, K. M., L. L. Carr, E. J. Peterson, L. A. Norian, S. L. Eliason, and G. A. Koretzky. 1997. Regulation of CD95 (Fas) ligand expression by TCR-mediated signaling events. J. Immunol. 158:4602-4611. Hildeman, D. A., T. Mitchell, T. K. Teague, P. Henson, B. J. Day, J. Kappler, and P. C. Marrack. 1999. Reactive oxygen species regulate activation-induced T cell apoptosis. Immunity 10:735-744. Scaffidi, C., S. Fulda, A. Srinivasan, C. Friesen, F. Li, K. J. Tomaselli, K. M. Debatin, P. H. Krammer, and M. E. Peter. 1998. Two CD95 (APO-1/Fas) signaling pathways. EMBO J. 17:1675-1687. Lenardo, M. J. 1991. Interleukin-2 programs mouse alpha beta T lymphocytes for apoptosis. Nature 353:858-861. Jaattela, M., and J. Tschopp. 2003. Caspase-independent cell death in T lymphocytes. Nat. Immunol. 4:416-423. Artus, C., E. Maquarre, R. S. Moubarak, C. Delettre, C. Jasmin, S. A. Susin, and J. Robert-Lezenes. 2006. CD44 ligation induces caspase-independent cell death via a novel calpain/AIF pathway in human erythroleukemia cells. Oncogene 25:5741-5751. Ly, J. D., D. R. Grubb, and A. Lawen. 2003. The mitochondrial membrane potential (deltapsi(m)) in apoptosis; an update. Apoptosis 8:115-128. Lesley, J., I. Gal, D. J. Mahoney, M. R. Cordell, M. S. Rugg, R. Hyman, A. J. Day, and K. Mikecz. 2004. TSG-6 modulates the interaction between hyaluronan and cell surface CD44. J. Biol. Chem. 279:25745-25754. Zhuo, L., A. Kanamori, R. Kannagi, N. Itano, J. Wu, M. Hamaguchi, N. Ishiguro, and K. Kimata. 2006. SHAP potentiates the CD44-mediated leukocyte adhesion to the hyaluronan substratum. J. Biol. Chem. 281:20303-20314. Chen, D. W., R. J. McKallip, A. Zeytun, Y. K. Do, C. Lombard, J. L. Robertson, T. W. Mak, P. S. Nagarkatti, and M. Nagarkatti. 2001. CD44-deficient mice exhibit enhanced hepatitis after concanavalin a injection: Evidence for involvement of CD44 in activationinduced cell death. J. Immunol. 166:5889-5897. McKallip, R. J., Y. Do, M. T. Fisher, J. L. Robertson, P. S. Nagarkatti, and M. Nagarkatti. 2002. Role of CD44 in activation-induced cell death: CD44-deficient mice  116     49.  50.  exhibit enhanced T cell response to conventional and superantigens. Int. Immunol. 14:1015-1026. Schlegel, R. A., M. K. Callahan, and P. Williamson. 2000. The central role of phosphatidylserine in the phagocytosis of apoptotic thymocytes. Ann. N. Y. Acad. Sci. 926:217-225. Firan, M., S. Dhillon, P. Estess, and M. H. Siegelman. 2006. Suppressor activity and potency among regulatory T cells is discriminated by functionally active CD44. Blood 107:619-627.  117     CHAPTER FIVE Summary and Perspectives  118     5.1 RESULTS AND FUTURE DIRECTIONS 5.1.1 Mechanism of chondroitin sulfate regulation Here it has been shown that the covalent modification of CD44 by the glycosaminoglycan chondroitin sulfate (CS) reduces the ability of CD44 to bind to its primary ligand, the extracellular matrix (ECM) glycosaminoglycan hyaluronan (HA). Prior to this, there were conflicting reports regarding the effect of CS on the CD44-HA interaction (1‐4). These studies also failed to differentiate between CS modification of CD44 versus other proteoglycans on the cell surface. In this work, the creation of a S180A point mutation in human CD44 that specifically prevents CS addition demonstrated that CS modification of CD44 reduces its ability to bind HA. While it can not be ruled out that the mutation alters conformation independently of CS modification, supporting data involving an inhibitor of glycosaminoglycan addition and chondroitinase ABC supports the conclusion that HA binding by CD44 is inhibited by CS addition. The negative effect of CS on HA binding was observed in both CD44-immunoglobulin fusion proteins (CD44-Fc) and in transfected cells. This indicates that CS reduces the affinity of CD44 for HA, as the positioning of the fusion proteins during the in vitro assay used to examine HA binding would not be altered by the presence of CS. However, it remains possible that CS could affect clustering of full length CD44 on the cell surface. The CS addition site is located within the stem region of CD44, and CS chains parallel with the membrane could reduce CD44 aggregation. Preliminary experiments with confocal microscopy have not detected gross differences in localization between wild type and S180A mutant CD44 (Maeshima, N unpublished observations), but higher resolution experiments are required to determine if CD44 aggregation is affected by CS addition.  119     Other than the addition of sulfate groups, CS and HA have a high degree of structural similarity. Consequently, CD44 displays low affinity binding for CS (5) and can also bind serglycin and other CS proteoglycans via their CS chains (6, 7). Binding of CS proteoglycans to CD44 can be blocked by the same Abs that block binding to HA, indicating that CD44 binds to CS within, or close to, the HA binding groove. These data strongly favour a model in which CS chains on CD44 reduce affinity for HA by competing for the HA binding groove. It is unclear how CS could effectively compete for binding as the affinity of CD44 for CS is several magnitudes lower than it is for HA (5). However, all CS is synthesized attached to a protein core, and there is evidence that CD44 binds better to linked CS chains than to purified CS (7). Thus, through either an intramolecular or intermolecular interaction, CS linked to CD44 could be better able to compete with HA for binding. Another possibility is that the close proximity of CS chains to the HA binding groove in an intramolecular interaction would dramatically increase the probability of binding CS. Whether such an interaction is possible is currently unknown, as the crystal structure has not been determined for the entire CD44 extracellular domain (8). Alternatively, the presence of CS could cause a conformational change in CD44 that reduces its ability to bind to HA. As a first step to examine this possibility, a panel of mAbs directed against both the globular and stem region of CD44 could be examined for their ability to bind CD44. If a conformation change occurs in the presence of CS, then the affinity of some of these mAbs may be affected. In a preliminary experiment, no difference in mAb binding between CD44 and S180A-CD44 expressed in Jurkat T cells was observed for the anti-CD44 mAbs IM7, Hermes-1, Hermes-3, 3G12, 3C12 or A3D8 (data not shown); however, numerous additional mAbs would need to be examined to rule out a conformation change. To clearly demonstrate the molecular  120     mechanism by which CS addition to CD44 reduces binding to HA, one may need to create the complete crystal structure of CD44, including its post-translational modifications. In both HEK293 and L fibroblasts, CD44 was modified by long chains of CS that more than doubled the molecular weight of an already heavily glycosylated CD44. In contrast, although CD44 from both transfected Jurkat T cells and murine bone marrow-derived macrophages (BMDM) was modified by CS, little to no high molecular mass CD44 was observed. Yet in all cells tested to date, the expression of the S180A mutation (S183A in mouse CD44) resulted in a dramatic increase in HA binding compared to wild type CD44. These data suggest that both short and long CS chains can reduce HA binding by CD44. This was further demonstrated by digesting CD44-immunoglobulin fusion proteins with chondroitinase ABC, which leaves a CS “stub” consisting of six sugar residues. Complete digestion with chondroitinase ABC only partially increased HA binding compared to unmodified CD44. Therefore, both short and long CS chains reduce HA binding by CD44, with long chains of CS having a greater negative effect. The ability of short CS chains to affect HA binding favours the hypothesis that an intramolecular (or cis) interaction is responsible for CS modification reducing the affinity of CD44 for HA. The greater ability of undigested CS chains to reduce HA binding could therefore be due to a greater ability of slightly longer CS chains (>6 sugars) to bind to the HA binding region or cause a conformation change in CD44. However, the possibility that very long chains of CS could futher reduce HA binding through intermolecular (or trans) interactions cannot be excluded. The evidence presented here does not demonstrate that the very long CS chains responsible for generating a small amount of CD44 with a molecular mass of over 200 kiloDaltons have a greater effect on HA binding than shorter CS chains (>6 sugars). If a trans effect of CS could be demonstrated, however, very long chains of CS would most likely be  121     responsible for mediating this effect. The simplest way to look for a trans effect would be to generate CD44-Fc fusion proteins containing the R41A mutation or R41A;S180A double mutation. A binding assay could then be performed with beads coated with a 1:1 ratio of S180ACD44-Fc with R41A-CD44-Fc or R41A;S180A-CD44-Fc. If the presence of R41A-CD44-Fc caused a greater reduction in HA binding than the presence of R41A;S180A-CD44-Fc, it would suggest that CS can act in trans to reduce binding to HA. The addition of R41A-CD44-Fc digested with chondroitinase ABC could also indicate whether the size of CS chains is important for mediating this effect. Although not addressed here, it is likely that CS addition to S180 would have the same affect on HA binding by most CD44 isoforms as it does to CD44H. This is because the insertion of variable exons occurs within the CD44 stem region after S180, and should therefore not affect the relative position of CS compared to the globular domain of CD44. A possible exception to this would be the expression of variable exon 10 (v10), as a motif of basic residues within this exon has been shown to bind to CS on CD44H (9, 10). This occurs when CD44H and CD44v10 are expressed on different cells, but it is possible that v10 could also bind to CS on neighbouring CD44 molecules. This could potentially reduce the negative effect of CS modification on HA binding by CD44 and could be tested with CD44-Fc fusion proteins by generating v10 expression fusion proteins with the mutations that prevent HA binding and CS addition, as discussed above. The pronounced increase in HA binding in BMDM expressing mouse S183A-CD44 indicates that CS addition to CD44 is one of the primary post-translational modifications responsible for preventing HA binding in unstimulated macrophages. Similarly, the reduction in CS modified CD44 following stimulation with tumor necrosis factor-α (TNFα) or interferon-γ  122     (IFNγ) and lipopolysaccharide (LPS) indicates that downregulating CS addition is one of the primary mechanisms responsible for increasing HA binding in stimulated BMDM. CS addition could be regulated at the transcriptional level, which would result in reduced CS addition to all proteoglycans, including CD44. In support of this, TNFα stimulation reduced mRNA expression of the enzyme responsible for 6-sulfation of CS. Most or all of the enzymes responsible for CS synthesis have been cloned (11), and it will be useful to determine if expression of these enzymes is reduced by TNFα stimulation. Conversely, as CS addition to CD44 was increased by treatment of BMDM with interleukin-4 (IL-4), expression of these same enzymes might be expected to increase in IL-4 treated cells. However, as both [35S] sulfate labeling and Western blotting with the anti-CS mAbs are dependent upon the sulfation of CS, and the expression of chondroitin 6-sulfotransferase-1 (C6ST-1) was reduced by TNFα stimulation, it is difficult to conclude whether CS sulfation, CS chain length, or the frequency of CS addition is reduced. To differentiate between these possibilities, it will be necessary to perform chromatography on [35S]sulfate and [3H]glucosamine-labeled CD44, as has been described by others (12). CS addition to CD44 could also be specifically regulated at the post-translational level by altering the trafficking of CD44 though the endoplasmic reticulum and Golgi apparatus. Golgi localization is thought to allow specific CS structures to be formed on different core proteins (11), and by either reducing the frequency of CS modification, or reducing the length of CS chains, HA binding by CD44 could be increased.  5.1.2 Role of chondroitin sulfate addition and hyaluronan binding in macrophages In the bleomycin lung injury model CD44-/- mice, but not wild type mice, succumb due to a failure to resolve inflammation (13). This was at least partially attributed to a failure to remove  123     fragmented HA from the lungs; something that could be largely remedied by reconstitution of the hematopoietic system of CD44-/- mice with bone marrow from wild type mice (13). As macrophage CD44 has been implicated in the local turnover of HA in the lungs (14), this suggests that CD44 on alveolar macrophages is responsible for HA uptake, including the clearance of low molecular weight and fragmented HA during inflammation. Interestingly, the bleomycin model of non-infectious lung injury involves induced expression of TNFα (15), which is thought to be important for the observed pathology (16). TNFα increases HA binding by macrophages in vitro, and the increased level of TNFα in the lungs of bleomycin treated mice would likely induce HA binding by alveolar macrophages. It is thus interesting to note that coadministration of TNFα and bleomycin results in less lung injury than treatment with bleomycin alone (17). LPS/IFN-γ activated macrophages that produce TNFα are classified as M1, or classically activated macrophages, and are associated with the Th1 immune response, the production of inflammatory cytokines, and the release of reactive oxygen and nitrogen intermediates. IL-4 activated macrophages meanwhile represent a subset of M2, or alternatively activated macrophages, and are associated with the Th2 immune response, as well as tissue repair (18). Macrophages are crucial for the tissue repair process to proceed normally as they are responsible for the phagocytosis of debris resulting from cell death and ECM degradation (19, 20), as well as the production of cytokines and chemokines to promote tissue growth (21). M2 macrophages are more strongly associated with this repair process as they can support tissue growth in vitro and display greater endocytic ability than M1 macrophages (18, 22). It is therefore interesting that both TNFα and IL-4 activated BMDM bind HA, and are able to take up HA in a CD44 dependent fashion (Poon, G unpublished observations). Whether TNFα or IL-4 activated BMDM  124     are better able to uptake HA is not yet known. However, if HA binding is critical for HA uptake, then higher HA binding by TNFα stimulated cells should increase HA uptake. Furthermore, as CS addition to CD44 is reduced by TNFα stimulation of BMDM, and the absence of CS modification increases the affinity of CD44 for HA, then these cells should be better able to bind to, and take up, low molecular weight and fragmented HA. It is therefore possible that CS regulation could play a critical role in the resolution of inflammation. A useful approach to investigate the role of CD44 and CS regulation on HA uptake and the resolution of inflammation would be to generate BMDM expressing either wild type CD44 or S183A-CD44, and then examine their ability to take up fluorescent-HA (Fl-HA) by confocal microscopy. This could be done by using the retroviral expression system already set up, but replacing the yellow fluorescent protein marker with a cyan fluorescent protein. As CS addition is largely reduced by TNFα stimulation, it is expected that HA uptake will be similar between stimulated CD44 and S183A-CD44 expressing cells. In contrast, CS addition to CD44 is increased by IL-4 treatment and only low levels of HA binding are observed in IL-4 activated cells, despite a large increase in CD44 expression. Expression of S183A-CD44 should greatly increase HA binding in IL-4 treated cells as HA binding was significantly enhanced by incubating cells with an inhibitor of glycosaminoglycan addition. Therefore, if CS addition to CD44 is an important regulator of CD44-dependent HA uptake, expression of S183A-CD44 should enhance HA uptake by IL-4 treated BMDM. If this is the case, it is expected that TNFα stimulated BMDM will display a greater ability to uptake HA than IL-4 treated BMDM. Alternatively, if HA uptake is higher in IL-4 activated cells, it would suggest that factors other than CS addition to CD44 are important for regulating the ability of cells to uptake HA. Comparing HA uptake between unstimulated and activated BMDM expressing S183A-CD44  125     should provide additional insight into the relative importance of HA binding versus other factors such as endocytic capacity. Macrophage recruitment to an inflammatory sites helps contain infections, in addition to assisting wound healing, and is dependent upon both HA (23) and CD44 (24-26). CD44 allows to T cells to roll on the endothelium (27), with rolling being the initial step in a process of leukocyte recruitment that subsequently involves adhesion and extravasation into the tissues. However, while monocytes can bind to HA following stimulation with TNFα (28), they are not likely to experience sufficient exposure to inflammatory cytokines prior to extravasation. Instead, CD44 may be important for macrophage recruitment by playing a role in adhesion and migration within the tissues. CS addition is necessary for CD44 mediated binding to the ECM components fibronectin (29) and collagen (30, 31), as well as for migration on these molecules by endothelial cells, fibroblasts, and melanoma cells (31-34). This suggests that macrophage migration through the ECM may be mediated by CS modified CD44 prior to TNFα stimulation. Low level HA binding by unstimulated macrophages could also be important for migration, as has been observed for KG1a cells (Brown, K.L. PhD Thesis). Following activation with TNFα, reduced CS addition to CD44 would decrease the ability of macrophages to migrate on fibronectin and collagen. Furthermore, HA binding induced at the site of inflammation could assist in retaining elicited macrophages. In this scenario, migration would be impeded by a strong CD44-HA interaction and would require CD44 cleavage by metalloproteinases (35). This model predicts that IL-4 activated M2 macrophages, which express high levels of CS modified CD44 and bind low levels of HA, would be highly motile compared to their TNFα activated counterparts. Unfortunately, BMDM migrate poorly through a three-dimensional matrix in response to a serum gradient. Therefore, to examine the role of HA binding and CS modification  126     in macrophage migration, chimeric mice will need to be generated expressing CD44 containing the S183A and/or R43A mutations. These mice can be created by reconstituting the hematopoietic system of wild type irradiated mice with retrovirus infected CD44 null bone marrow. This will allow in vivo models of migration and recruitment to be examined in the context of gain and loss of function experiments for HA binding and CS addition. Corresponding in vitro experiments on specific substrata can be done using thioglycollate-elicited macrophages from the peritoneum.  5.1.3 Functional consequences of hyaluronan binding by CD44 in T lymphocytes The generation of chimeric mice expressing constitutively active (S183A) or non-binding (R43A) forms of CD44 will allow a detailed examination of the role that HA binding by CD44 plays in the T cell life cycle. While CD44 has been implicated in T cell development (36-38), activation (39-42), recruitment (43-47), and activation-induced cell death (AICD) (40, 48-51), results have often been conflicting and minimal attention has been given to HA as the primary physiological ligand for CD44. Firmly established is that CD44 can mediate T cell rolling on endothelial cells expressing HA (52-55) and that this is important for T cell recruitment to the peritoneum (43, 56, 57). Antibodies against CD44 can also reduce T cell trafficking to the central nervous system (45-47), and CD44 null T lymphocytes are delayed in their entrance into arthritic joints (58). For the selectin and integrin molecular families, the selective expression of these receptors, and their corresponding ligands, mediates tissue specific localization (59). The importance of CD44 for recruitment to the peritoneum and central nervous system could indicate that CD44 is also important for recruitment to select locations, but this remains to be determined. For several experimental systems, it has also not been clearly demonstrated whether HA binding  127     or CD44 expression alone that is important. This is a critical distinction as CD44 can act as a ligand for selectins, depending upon its glycosylation state (60, 61), and this has been shown to be important for stem cell recruitment (62, 63). Expression of high HA binding S180A-CD44 allows Jurkat T cells to adhere to an HA coated plate under physiological flow conditions (data not shown). Therefore, the use of CD44 chimeric mice should allow HA binding dependency to be examined for T cell trafficking. Utilizing multiple in vivo recruitment models should also indicate whether the CD44-HA interaction is important for tissue specific localization or recruitment of specific T cell subsets. CD44 cross-linking during T cell activation in vitro can increase expression of activation markers (39, 40, 64), however, T cell activation is not reduced in CD44 null lymphocytes (37), and exogenous HA is not co-stimulatory (Maeshima, N unpublished observations). There is evidence that CD44 expression is important for T cell activation by dendritic cells (DCs), possibly by playing a role in the formation of DC-T cell conjugates (42, 65). While naïve T cells do not bind HA, binding is induced during in vitro stimulation within 24 hours; the amount of time it takes for T cells to begin proliferating in vivo (66). It is therefore possible that HA expression by DCs could facilitate the later stages of T cell activation (41). Testing DCdependent T cell activation with cells from the chimeric mice should clearly demonstrate whether HA binding by CD44 is important for conjugate formation and overall T cell activation. Furthermore, measuring anti-CD3 driven activation of S183A-CD44 expressing T cells will determine if HA binding by CD44 can provide a co-stimulatory signal. If co-stimulation is observed, the next step will be to determine why HA is unable to enhance activation of wild type T cells in vitro. One possibility is that exogenous HA added to cells during in vitro experiments is relatively free of HA binding proteins, as tumor necrosis factor-stimulated gene 6 product  128     (TSG-6) and serum-derived HA-associated protein (SHAP) have both been shown to increase the ability of CD44 to bind HA (67, 68). In transfected Jurkat T cells, expression of the CD44 mutant lacking CS modification greatly enhanced the ability of HA to induce AICD. Thus, a requirement for HA-associated proteins might be bypassed when HA binding is enhanced by the absence of CS modification. However, protein modification of HA could be necessary for CSmodified CD44 to induce an intracellular signal sufficient to affect T cell activation. Protein modification of HA could also be important for HA-dependent AICD. Despite high levels of HA binding by both activated wild type CD44 and S180A-CD44 expressing Jurkat T cells, S180A-CD44 expressing cells were more sensitive to AICD in the presence of HA. HAdependent AICD was also affected by the size of HA, with high molecular weight HA, but not low molecular weight HA causing cell death. While the absence of CS modification increases the affinity of CD44 for HA, this would also directly increase its avidity. Therefore, as the size of HA would not affect CD44 binding affinity, the most parsimonious explanation is that CD44 binding avidity is the major factor that determines the strength of the intracellular signal induced by HA binding. This hypothesis predicts that the presence of either TSG-6 or SHAP will increase HA-dependent AICD, as both molecules are thought to increase the CD44 avidity (67, 68). It could also explain the significant effects of CD44 in the response of mice to Concanavalin A (50) and on the delayed-type hypersensitivity response (51), both of which were attributed to reduced AICD in CD44-/- mice. Elucidating the role that CS modification has in regulating CD44 and HA-dependent AICD will require a detailed examination of when and how CS addition is regulated during T cell activation. In Jurkat cells, CS addition was not affected by PMA stimulation (data not shown). If secondary stimulation of T cells does not reduce, or even increases, CS addition to CD44, it would suggest that CS modification assists in protecting cells  129     from HA-dependent AICD. By comparison, if CS addition was reduced, it would suggest that AICD is enhanced by reduced CS modification. Analyzing CS addition in naïve, activated, and memory T cells should provide additional insight into the importance of CS regulation and HA binding in T cell function. Formulated hypothesizes can then be tested by analyzing the T cell response in chimeric mice expressing CS mutant CD44. The expression of CD44 isoforms may also affect AICD independent of HA binding. In Jurkat T cells, expression of v2-10 reduced Fas-mediated apoptosis approximately 5-fold (69). This was largely attributed to exons v6 or v9, although the effect of other individually expressed exons was not examined. Interestingly, CD44v7 deficient mice are not susceptible to trinitrobenzene sulfonic acid-induced colitis and intestinal mononuclear cells displayed increased levels of apoptosis (70). Whether expression of CD44v7 reduces Fas-dependent apoptosis remains to be determined. However, the loss of the variable exons in reconstituted CD44-/- mice, and the potential positive effect this has on Fas-dependent apoptosis, must be taken into account when analyzing AICD in the chimeric mice. Apoptotic cell death is defined by the activation of caspases and is often considered synonymous with programmed cell death (PCD). However, caspase-independent PCD has also been described and classified into either apoptosis-like or necrotic-like categories based upon the degree of chromatin condensation (71, 72). In Jurkat T cells, HA-induced cell death occurred in the absence of caspase activation and could not be blocked by a caspase inhibitor. By definition, receptor-mediated cell death is not necrosis; therefore, HA-induced cell death must represent a form of non-apoptotic PCD. Further work is required to determine if HA induces apoptosis-like or necrosis-like PCD, as phosphatidylserine (PS) externalization prior to cell death can be observed in any form of PCD. Preliminary experiments favor necrosis-like PCD as significant  130     DNA fragmentation and chromatin condensation were not observed (data not shown). Release of apoptosis-inducing factor (AIF) or cytochrome C into the cytoplasm, events important for the induction of apoptosis or apoptosis-like PCD, were also not detected (data not shown). The rapid PS externalization and loss of membrane integrity that occurred following the addition of HA to activated T cells favors a non-classical pathway being responsible for CD44-mediated cell death. However, speculation is difficult given the current dearth of knowledge regarding caspaseindependent cell death. Rapid PS externalization could suggest early activation of a phospholipid scramblase (73, 74). Scramblase activation results from calcium influx and is believed to be responsible for PS exposure during apoptosis. While preliminary experiments did not detect calcium influx in response to HA, and cell death was not inhibited by the intracellular calcium chelator (data not shown), additional work is required to examine this possibility.  5.2 CONCLUDING REMARKS This work has identified one of the principal mechanisms used to regulate the association of CD44 with its primary ligand. Post-translational modification of CD44 with CS reduced HA binding in several human and mouse cell lines, as well as murine macrophages. The extent of CS modification was divergently regulated in response to Th1 or Th2 cytokines, highlighting prospective functions for HA binding in the immune response. Subsequent examination of the CD44-HA interaction in T cells resulted in the discovery of a novel Fas-independent pathway for mediating AICD. 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