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Investigating the expression of N-acetylglucosamine 6-0 sulfotransferases in myeloid cells and its implications… Tjew, Sie Lung 2006

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INVESTIGATING T H E EXPRESSION OF A / - A C E T Y L G L U C O S A M I N E 6-0 SULFOTRANSFERASES IN M Y E L O I D C E L L S A N D ITS IMPLICATIONS O N CD44 SULFATION by SIE L U N G TJEW B. Sc., York University, 2001 A THESIS SUBMITTED IN P A R T I A L F U L F I L L M E N T OF THE REQUIREMENTS FOR THE D E G R E E OF M A S T E R OF SCIENCE in T H E F A C U L T Y OF G R A D U A T E STUDIES (Microbiology and Immunology)  T H E UNIVERSITY OF BRITISH C O L U M B I A August 2006 © Sie Lung Tjew, 2006  ABSTRACT The sulfation of proteoglycans and glycoproteins has been implicated in many biological processes. Glycoproteins on high-walled endothelial venules present sialyl 6-sulfo Lewis x determinants that can mediate lymphocyte homing to lymph nodes by serving as ligands for L-selectin on circulating lymphocytes. CD44 represents a family of glycoproteins that is expressed on most cell types. Interactions between CD44 and hyaluronic acid (HA) have been implicated in many facets of the inflammatory response, including leukocyte recruitment. CD44-HA binding is tightly regulated by several mechanisms including changes in CD44 glycosylation. Recently, increased sulfation of CD44 was correlated with increased H A binding induced by the pro-inflammatory cytokine, tumour necrosis factor (TNF)a, in the myeloid cell line, SR91, and in human peripheral blood monocytes (PBM). Additional experiments determined 6-sulfo iV-acetyllactosamine (LacNAc)/Lewis x carbohydrate epitopes, as defined by the monoclonal antibody A G 107, were induced on Nand O-linked glycans of CD44 by T N F a in SR91 cells. This led to the hypotheses that the induction of the A G 107 epitope on CD44 was associated with increased expression of a specific JV-acetylglucosamine 6-0 sulfotransferase (GlcNAc6ST) and that the A G 107 epitope was involved in the inflammatory process by mediating CD44-ligand interactions. Semi-quantitative reverse-transcriptase polymerase chain reaction revealed that T N F a upregulated the expression of GlcNAc6ST-l in SR91 cells and GlcNAc6ST-l and -4 in human P B M . In addition, GlcNAc6ST-l was implicated in the synthesis of the A G 107 epitope on CD44. Transcripts for GlcNAc6STs were not increased in mouse macrophages and human M l macrophages exposed to pro-inflammatory mediators. By contrast,  ii  GlcNAc6ST-l and GlcNAc6ST-4 transcripts were upregulated in human M2 macrophages incubated with T N F a . However, the A G 107 epitope was not detected in these cells. Overall, this work suggests that GlcNAc6ST-l and GlcNAc6ST-4 are involved the inflammatory process by generating 6-sulfo LacNAc/Lewis x epitopes on CD44 of human P B M . However, the results presented herein do not support a role for the A G 107 epitope in TNFa-induced H A binding or L-selectin mediated rolling. In addition, GlcNAc6ST-l and GlcNAc6ST-4 may also play a role in functions associated with human M2 macrophages, such as Th2-mediated immunity and wound healing, but not by generating the A G 107 epitope.  iii  TABLE OF CONTENTS  Abstract  ii  Table of contents  iv  List of tables  viii  List of figures  :  ix  List of abbreviations  xii  List of publications  xvi  Acknowledgements  xvii  C H A P T E R 1: Introduction  1  1.1  Overview of inflammation  2  1.2  Role of macrophages in inflammation.  2  1.2.1  Development of macrophages  3  1.2.2  Initiation of inflammation  3  1.2.3  Macrophage activation/polarization  3  1.2.4  Resolution of inflammation  6  1.3  1.4  Recruitment of leukocytes....  6  1.3.1  Rolling....  8  1.3.2  Firm adhesion  10  1.3.3  Diapedesis  11  CD44  •  11  1.4.1  Structure  H  1.4.2  Ligands  1.4.3  Function in inflammation  14  1.4.4  Regulation of CD44-mediated hyaluronic acid binding  16  "•.  13  1.5  Glycosylation  -17  1.6  Sulfation  19  1.7  jV-acetylglucosamine 6-0 sulfotransferases (GlcNAc6STs)  22  1.8  Thesis objectives  27  iv  C H A P T E R 2: Materials and methods  30  2.1  Cell lines  ....31  2.2  Primary cells  32  2.2.1  Peripheral blood monocytes  32  2.2.2  Peripheral blood monocyte-derived macrophages  32  2.2.3  Bone marrow-derived macrophages  33  2.2.4  Thioglycollate-elicited peritoneal macrophages  34  2.2.5  Splenic macrophages  35  2.2.6  Brain and kidney  35  2.3  Reagents  35  2.4  Antibodies  36  2.5  Flow cytometry and fluorescence-associated cell sorting  36  2.6  Neuraminidase treatment  39  2.7  Immunoprecipitation, PNGase F digestion and western blot analysis  39  2.8  [ S]Sulfate labeling  40  2.9  Semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR)  41  2.10  Isolation of genomic D N A  43  2.11  Production of L-selectin/IgM tissue-cultured supernatant  43  2.12  Parallel plate flow chamber  44  2.13  Enzyme-linked immunosorbant assay (ELISA)  45  35  C H A P T E R 3: Results  46  3.1  GlcNAc6ST-l is upregulated in the SR91 myeloid cell line by TNFct  47  3.2  T N F a upregulates expression of the AG107 epitope on CD44 in human peripheral blood monocytes  3.3  GlcNAc6ST-l and GlcNAc6ST-4 are upregulated in human monocytes by T N F a  3.4  49  :  51  GlcN Ac6ST-1 is implicated in the generation of the A G 107 epitope on CD44  51  3.4.1  Expression of GlcNAc6ST-l leads to increased sulfation of CD44  51  3.4.2  Expression of GlcNAc6ST-l leads to the expression of the A G 107 epitope, predominantly on the A/-linked gly cans of CD44  54  3.5  Expression of the A G 107 epitope did not correlate TNFa-induced H A binding  3.6  Expression of the A G 107 epitope did not lead to enhanced rolling on L-selectin  3.7  58  Expression of GlcNAc6STs, 6-sulfo LacNAc-containing epitopes and CD44 sulfation in human macrophages 3.7.1  61  Morphology and cytokine profile of human GM-CSF- and M-CSF-cultured macrophages  3.7.2  62  GlcNAc6ST-l and GlcNAc6ST-4 are upregulated by inflammatory mediators in human M-CSF- but not GM-CSF-cultured macrophages  3.7.3  3.7.4  66  Human M-CSF-cultured macrophages did not express the AG107 epitope in response to IFNy plus LPS or to T N F a  3.9  66  Human M-CSF-cultured macrophages bind more H A and expressed higher levels of CD 14 than GM-CSF-cultured macrophages  68  Expression of other 6-sulfo TV-acetyllactosamine containing epitopes  68  3.9.1  Expression of other 6-sulfo JV-acetyllactosamine containing epitopes in SR91 cells  3.9.2  71  Expression of other 6-sulfo JV-acetyllactosamine containing epitopes inTHP-1 cells  3.9.3  73  Expression of other 6-sulfo JV-acetyllactosamine containing epitopes inU937cells  3.9.4  '.  75  Effect of GlcNAc6ST-l on the expression of other 6-sulfo Af-acetyllactosamine containing epitopes in ECV304 cells  3.10  64  No increase in CD44 sulfation in human M-CSF-cultured macrophages in response to IFNy plus LPS or to T N F a  3.8  56  77  Expression of GlcNAc6ST-1 and GlcNAc6ST-4 were not induced in mouse macrophages under inflammatory conditions  77  3.10.1 Expression of GlcNAc6ST-l and GlcNAc6ST-4 were not induced while C6ST-1 was downregulated by T N F a in bone marrow-derived macrophages  77  3.10.2 No expression of 2F3, AG107 and DD-2 epitopes in bone marrowderived macrophages stimulated with or without T N F a  vi  80  3.10.3 Expression of GlcNAc6ST-l and GlcNAc6ST-4 were not induced by T N F a in thioglycollate-elicited peritoneal macrophages  80  3.10.4 Expression of GlcNAc6ST-l and GlcNAc6ST-4 transcripts were not induced in splenic macrophages from mice infected with Listeria monocytogenes  82  C H A P T E R 4: Discussion 4.1  85  GlcNAc6ST-l and GlcNAc6ST-4 expression were inducible in human monocytes  86  4.2  GlcNAc6ST-l was implicated in the synthesis of the AG107 epitope on CD44  87  4.3  Expression of the AG107 epitope did not correlate with H A binding  89  4.4  CD44 and AG107 did not enhance rolling on L-selectin  91  4.5  Expression of GlcNAc6ST-1 and GlcNAc6ST-4 in human GM-CSF- and M-CSF-cultured macrophages  4.6  .93  CD44 sulfation and the AG107 epitope in human M-CSF-cultured macrophages.  96  4.7  H A binding in human GM-CSF- and M-CSF-cultured macrophages  97  4.8  Expression of other 6-sulfo LacNAc-containing epitopes  99  4.9  Expression of GlcNAc6ST-l and GlcNAc6ST-4 in mouse macrophages  101  C H A P T E R 5: Conclusion  103  5.1  Conclusion  104  C H A P T E R 6: References  107  vii  LIST OF T A B L E S  Table 1.1  Comparison of activated macrophages  Table 1.2  Structure of the major carbohydrate determinant recognized by selectins and their physiological ligands  Table 1.3  9  Summary of cloned human 7V-acetylglucosamine 6-0 sulfotransferases (GlcNAc6STs)  Table 2.1  5  23  Carbohydrate determinants recognized by monoclonal antibodies (mAbs) used in this study  37  viii  LIST OF FIGURES  Figure 1.1  Overview of leukocyte recruitment  7  Figure 1.2  Schematic diagram of CD44  12  Figure 1.3  Biosynthesis of iV-linked glyans  18  Figure 1.4  Biosynthesis of 0-linked glycans  20  Figure 1.5  Classification of glycosaminoglycans and their microheterogeneities  21  Figure 1.6  Sequence alignment of the five cloned human JV-acetylglucosamine 6-0 sulfotransferase  Figure 3.1  25  Effect of T N F a on the expression of JV-acetylglucosamine 6-0 sulfotransferase (GlcNAc6ST) transcripts in SR91 cells  Figure 3.2  48  Effect of T N F a on the expression of the AG107 epitope (6-sulfo JV-acetyllactosamine [LacNAc]/Lewis x on human peripheral blood Monocytes (PBM) and on CD44  Figure 3.3  50  Effect of T N F a on the expression of JV-acetylglucosamine 6-0 sulfotransfere (GlcNAc6ST) transcripts  Figure 3.4  52  Effect of exogenous expression of 7V-acetylglucosamine 6-0 sulfotransferase-1 (GlcNAc6ST-l) on [ S]sulfate incorporation 35  into CD44 from ECV304 cells Figure 3.5  ..53  The expression of the A G 107 epitope (6-sulfo Af-acetyllactosamine [LacNAc]/Lewis x) on CD44 and on N-acetylglucosamine 6-0 sulfotransferase-1 (GlcNAc6ST-l) and a 1,3 fucosyltransferase VII (Fuc-T VII) transfected ECV304 cells  Figure 3.6  ..55  Induction of binding to fluoresceinated hyaluronic acid (FL-HA) in SR91 cells sorted for expression of the AG107 epitope (6-sulfo N-acetyllactosamine [LacNAc]/Lewis x)  Figure 3.7  57  Expression of the AG107 epitope (6-sulfo JV-acetyllactosamine [LacNAc]/Lewis x) in TNFa-stimulated SR91 cells sorted for low and high binding to fluoresceinated hyaluronic acid (FL-HA)  Figure 3.8  Effect of the A G 107 epitope (6-sulfo TV-acetyllactosamine [LacNAc]/ Lewis x) and CD44 on rolling on immobilized L-selectin/  ix  59  immunoglobulin M (L-sel/IgM) in SR91 cells Figure 3.9  60  Isolation of human peripheral blood monocytes (PBM) and morphology and cytokine profile of GM-CSF ( M l ) - and M-CSF (M2)-cultured macrophages  Figure 3.10  63  Effect of IFNy plus LPS and T N F a on the expression of JV-acetylglucosamine 6-0 sulfotransferase (GlcNAc6ST)-l and GlcNAc6ST-4 transcripts in human GM-CSF ( M l ) - and M-CSF (M2)-cultured macrophages  Figure 3.11  65  Effect of IFNy plus LPS and T N F a on CD44 sulfation in human M-CSF-cultured (M2) macrophages.  Figure 3.12  :  67  Expression of 2F3, AG107 and DD-2 epitopes in human M-CSFcultured (M2) macrophages incubate with or without IFNy plus LPS  Figure 3.13  69  Effect of T N F a on binding to fluoresceinated hyaluronic acid (FL-HA) in human GM-CSF ( M l ) - and M-CSF (M2)-cultured macrophages...  Figure 3.14  70  Expression of other 6-sulfo A^-acetyllactosamine (LacNAc)-containing epitopes in SR91 cells  Figure 3.15  72  Effect of T N F a on binding to fluoresceinated hyaluronic acid (FL-HA) and expression of CD44 and 6-sulfo ./V-acetyllactosamine (LacNAc)containing epitopes in THP-1 cells incubated with or without P M A  Figure 3.16  74  Expression of 6-sulfo JV-acetyllactosamine (LacNAc)-containing epitopes in U937 cells incubated with or without P M A  Figure 3.17  76  Effect of expression of JV-acetylglucosamine 6-0 sulfotransferase-1 (GlcNAc6ST-1) on the expression of other 6-sulfo ./V-acetyllactosamine (LacNAc)-containing epitopes in ECV304 cells  Figure 3.18  Effect of T N F a on binding to fluoresceinated hyaluronic acid (FL-HA)  78 ,  and the expression of JV-acetylglucosamine 6-0 sulfotransferase ' (GlcNAc6ST)-l, GlcNAc6ST-4 and chondroitin sulfate sulfotransferase-1 (C6ST-1) transcripts in mouse bone marrow-derived macrophages (BMDM) Figure 3.19  79  Expression of 6-sulfo JV-acetyllactosamine (LacNAc)-containing epitopes 2F3, AG107, and DD-2 in mouse bone marrow-derived  x  macrophages (BMDM) stimulated with or without T N F a Figure 3.20  81  Effect of T N F a on the expression of N-acetylglucosamine 6-0 sulfotransferase (GlcNAc6ST)-l and GlcNAc6ST-4 transcripts in mouse thioglycollate-elicited peritoneal macrophages  Figure 3.21  83  Expression of Af-acetylglucosamine 6-0 sulfotransferase (GlcNAc6ST)-1 and GlcNAc6ST-4 in splenic macrophages from mice infected with Listeria monocytogenes  84  xi  LIST OF ABBREVIATIONS  aa  amino acid  Ab  antibody  APS  adenosine phophosulfate  ATCC  American Type Culture Collection  ATP  adenosine triphosphase  BMDM  bone marrow-derived macrophages  BSA  bovine serum albumin  C6ST-1  Chondroitin 6-0 sulfotransferase-1  CS  chondroitin sulfate  CSB  carbohydrate substrate binding site  DMEM  Dulbecco's modified eagle medium  ECM  extracellular matrix  EDTA  ethylenediaminetetraacetic acid  ER  endoplasmic reticulum  ESL-1  E-selectin ligand-1  FACS  fluorescein-associated cell sorter  FCS  fetal calf serum  FGF-2  fibroblast growth factor-2  FITC  fluorescein isothiocyanate  FL-HA  fluoresceinated hyaluronic acid  Fuc  fucose  Fuc-T VII  a 1,3 fucosytransferase VII  GAG  glycosaminoglycan  Gal  galactose  GalNAc  ^-acetylgalactosamine  Glc  glucose  GlcA  glucuronic acid  GlcNAc  iV-acetylglucosamine  GlcNAc6ST  N-acetylglucosamine 6-0 sulfotransferase  xii  <  GlyCAM-1  glycosylated cell adhesion molecule-1  GM-CSF  granulocyte-macrophage colony stimulating factor  HA  hyaluronic acid  HBSS  Hank's balanced salt solution  HCELL  hematopoietic cell E-/L-slectin ligand  HEPES  4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid  HEV  high-walled endothelial venules  HMW HA  high molecular weight hyaluronic acid  HRP  horse radish peroxidase  HS  heparan sulfate  ICAM-1  intercellular cell adhesion molecule-1  IdoA  idouronic acid  IFNy  interferon y  Ig  immunoglobulin  IL  interleukin  JAM  junctional adhesion molecule  kDa  kilodalton  KS  keratan sulfate  KSGal6ST  Keratan sulfate 6-0 sulfotransferase  LacNAc  JV-acetyllactosamine  LCCM  L-cell cultured medium  LFA-1  leukocyte function associated antigen-1  LMW HA  low molecular weight hyaluronic acid  LPS  lipopolysaccharide  L-sel/IgM  L-selectin/Immunoglobulin M  mAb  monoclonal antibody  MAdCAM-1  mucosal vascular addressin cell adhesion molecule-1  Man  mannose  MCP-1  macrophage chemotactic protein-1  M-CSF  macrophage colony stimulating factor  MIP-1  macrophage inflammatory protein-1  Xlll  MMP-9  matrix metalloproteinase-9  NDST-1  heparan sulfate JV-deacetylase/JV-sulfotransferase-l  NeuAc  neuraminic acid (sialic acid)  PAPS  3' -phosphoadenosine 5' -phosphosulfate  PB  3'-phosphate binding site  PBM  peripheral blood monocytes  PBMC  peripheral blood mononuclear cells  PBS  phosphate-buffered saline  PE  phycoerythrin  PECAM  platelet-endothelial cell adhesion molecule  PLN  peripheral lymph node  PMA  phorbol 12-myristate 13-acetate  PMSF  phenylmethylsulfonyl fluoride  PNAd  peripheral lymph node addresin  PNGase F  Peptide JV:Glycosidase F  PRR  pattern recognition receptors  PSB  5'-phosphosulfate binding site  PSGL-1  P-selectin glycoprotein ligand-1  PVDF  polyvinylidine difluoride  RANTES  regulated upon activation, normal T cell expressed and secreted  RHAMM  receptor for hyaluronic acid-mediate motility  RNI  reactive nitrogen intermediate  ROI  reactive oxygen intermediate  RPMI  Roswell Park Memorial Institute 1640 medium  RT-PCR  reverse transcriptase polymerase chain reaction  SDS-PAGE  sodium dodecyl sulfate-polyacrylamide gel electrophoresis  Ser  serine  TBST  Tris-buffered saline with Tween 20  TCR  T cell receptor  TGF0  transforming growth factor P  TGN  trans-Golgi network  xiv  Thr  threonine  TLR  Toll-like receptor  TNFa  tumour necrosis factor a  TSG-6  TNFa-stimulated gene-6  VCAM-1  vascular cell adhesion molecule-1  VE-cadherin  vascular endothelial cell specific-cadherin  VLA-4  very late antigen-4  xv  LIST OF PUBLICATIONS  Tjew, S.L., Brown, K . L . , Kannagi, R., and Johnson, P. 2005. Expression of Nacetylglucosamine 6-0 sulfotransferases (GlcNAc6STs)-l and -4 in human monocytes: GlcNAc6ST-l is implicated in the generation of the 6-sulfo N-acetyllactosamine/Lewis x epitope on CD44 and is induced by T N F a . Glycobiology, 15:7-13C.  xvi  ACKNOWLEDGEMENTS  I would like to thank my supervisor, Dr. Pauline Johnson, for allowing me the opportunity to pursue my research interests in her lab, my committee members, Dr. Francois Jean and Dr. Hermann Ziltener for their helpful input, and Dr. Alice Mui for agreeing to serve as the external examiner. I am grateful to past and present members of the Johnson lab for their assistance over the years and words of wisdom. Finally, I would like to thank my family and friends, without whom this journey would not have been possible.  xvii  CHAPTER 1 Introduction  1  1.1  Overview of inflammation The classical symptoms of inflammation described by Celsus in the first century  A D were redness, heat, pain and swelling (1). Inflammation has since been recognized as a major host defense mechanism against tissue damage caused by invading microorganisms, physical trauma, chemical agents or autoimmune responses (2-5). It is a complex and highly regulated process that can be acute or chronic in nature. The principal features of acute inflammation are the influx of fluid, plasma proteins and leukocytes, primarily neutrophils and monocytes, into the site of injury that generally last for several hours to a few days. Chronic inflammation is associated with the presence of macrophages and lymphocytes, proliferation of blood vessels, fibrosis and tissue necrosis. It results from the persistence of inflammatory stimuli that prevents the normal and beneficial progression towards resolution and wound healing. Chronic inflammation has been implicated as the cause of, or contributing factor to a stunning number of pathological conditions including arthritis, atherosclerosis, asthma and cancer (3). Clearly, a deeper understanding of the interplay between cellular and molecular components of the inflammatory response can yield enormous therapeutic benefits.  1.2  Role of macrophages in inflammation Macrophages play important roles in the initiation, maintenance and resolution of  inflammation. These roles are manifest by the ability of macrophages to present antigens, recognize and destroy certain microbes, phagocytose dead cells and debris, and release soluble mediators involved in recruiting cells to sites of inflammation (5-7).  2  1.2.1  Development of macrophages Myeloid precursors in the adult bone marrow develop into monocytes that travel  via the peripheral blood to various tissues where they mature into macrophages or dendritic cells. The microenvironment specific to each tissue can have a profound impact on the functional and phenotypic characteristics of macrophages (6). For example, alveolar macrophages are I F N Y ' macrophages are IFNy 1.2.2  111811  0 W  and poor at phagocytosis while elicited peritoneal  and highly phagocytic (8).  Initiation of inflammation The initial inflammatory stimulus results in the release of many pro-inflammatory  mediators such as interleukin (IL)-l, IL-6, IL-8, granulocyte-macrophage colony stimulating factor (GM-CSF) and tumour necrosis factor (TNF)-a by local cells including tissue-resident macrophages (5). These molecules mitigate crucial inflammatory responses such as increased vascular permeability and upregulated expression of adhesion molecules on the luminal side of the endothelium for recruiting leukocytes. It has been well established that macrophages express pattern recognition receptors (PRR) including members of the infamous Toll-like receptor (TLR) family (9). Pathogens often provide the initial inflammatory stimulus when they are recognized by PRR on tissueresident macrophages. Therefore, tissue-resident macrophages play a key role in the initiation of inflammation. 1.2.3  Macrophage activation/polarization Mature tissue-resident macrophages are initially in a resting state and conversion  to an activated state is important for inflammation. Macrophages are primed for activation by interferon (IFN)-y, which is produced by T helper (Th)-1 cells and natural  3  killer cells (10). Classical macrophage activation occurs after it receives a second signal provided by T N F a , and results in an increased capacity to kill microbes (11). However, it has recently become apparent that macrophages can be driven towards several distinct activation programs (reviewed in 7, 12, 13). In the first such account, macrophages treated with IL-4 or glucocorticoids were found to upregulate certain phagocytic receptors but were poor at killing intracellular pathogens and did not induce production of T N F a (14). These were termed alternatively activated macrophages, and in hindsight was a rather inappropriate designation as subsequent studies revealed other activation profiles. Most notably, type II-activated macrophages, named for their role in Th2 responses (15) were generated after exposure to immune complexes in the presence of TLRligands(16). Under a new nomenclature recently proposed (13, 17), classically activated macrophages would be termed M l macrophages and all other types of activated macrophages would be termed M2 macrophages (see Table 1.1). The M1/M2 nomenclature reflects the role of these macrophages in T h l and Th2 responses, respectively. Moreover, M2 macrophages share other common characteristics, such as the production of substantial amounts of IL-10, a cytokine with potent anti-inflammatory properties. It should be noted that the aforementioned properties of macrophage activation pertains to the mouse, although studies using human macrophages have produced similar results. However, important differences do exist. For example, in vitro culture of human peripheral blood monocytes (PBM) in the presence of GM-CSF or M CSF was sufficient to generate macrophages with M l and M2 properties, respectively (18).  4  Table 1.1. Comparison of activated macrophages. Summary of different forms of macrophage activation induced by different environmental signals. This table was adapted from (13).  Ml  M2a  M2b  (Classical)  (Alternative)  Type II)  Activating signals  IFNy + LPS or T N F a  IL-4, IL-13, glucocorticoids  IgG complexes, T L R ligations  IL-10  Secretory  RNI, ROI TTNFa, i IL-10 t IL-12, IL-23 IL-l,IL-6  IL-10 TIL-IRa  T N F a , t IL-10  IL-10 TGFp  Biological markers  | M H C II TCD86 IMR  JCD14 |MR, |SR  Functional  Thl responses,  Th2 response,  activities  Type I  Type II  inflammation  inflammation,  DTH, killing  killing of  intracellular,  parasites  products  M2c  1IL-12 IL-1, IL-6  t M H C II tCD86  •  MR SLAM  Th2 activation, immunoregulation, immunoregulation matrix deposition, tissue remodeling  pathogens, tumour resistance  Abbreviations: D T H , delayed-type hypersensitivity; IgG, immunoglobulin G; IL-IRa, IL-1 receptor antagonist; M R , mannose receptor; ROI, reactive oxygen intermediates; RNI, reactive nitrogen intermediates; S L A M , signaling lymphocytic activation molecule; SR, scavenger receptor  5  1.2.4  Resolution of inflammation Resolution of inflammation requires the removal or deactivation of inflammatory  cells and mediators followed by repair of damaged tissues. Macrophages have a significant impact on the resolution of inflammation. First, pro-inflammatory M l macrophages need to be deactivated or removed. This may be accomplished in part by the arrival of, or conversion to M2 macrophages that secrete IL-10 a cytokine with potent anti-inflammatory properties (19-21). Inhibitors of proteases are known to promote resolution of inflammation. Macrophages exposed to IL-10 (M2c macrophages) secrete transforming growth factor (TGF)-p\ which in turn induces production of inhibitors of matrix metalloproteinases (22). Macrophages are renowned for their healthy appetite for apoptotic cells, a function that is crucial to resolving inflammation as a failure to clear apoptotic cells in a timely manner can lead to an unremitting inflammatory response (23).  1.3  Recruitment of leukocytes Recruitment of leukocytes from the peripheral blood is no simple task as they are  subjected to large shear forces. Accordingly, a highly co-ordinated system involving adhesion molecules, cytokines and chemokines has evolved in post-capillary venules to efficiently capture leukocytes. While the specifics of leukocyte recruitment can differ depending on the tissue, leukocyte subset, and whether it occurs under healthy or diseased conditions, it follows a general pattern of rolling, firm adhesion and diapedesis (see Figure 1.1), with each step being a prerequisite for the next (24, 25).  6  CZ5  Rolling  Firm adhesion  Diapedesis  Chemotactic gradient  t2  Figure 1.1. Overview of leukocyte recruitment. Leukocytes under shear flow roll via selectins, and in some cases, a4 integrins. During rolling, leukocytes are exposed to inflammatory stimuli such as chemokines (represented by •) that convert integrins into a high affinity binding state that then mediates firm adhesion. Leukocytes transmigrate across the vascular endothelium by squeezing between endothelial cells and migrate along a chemotactic gradient towards the source of inflammation. CD44 has been implicated in rolling and adhesion of activated T cells and in adhesion and diapedesis of neutrophils. Extravasated monocytes mature into macrophages with different functional phenotypes depending on the environmental signals present in the tissue. Constitutive lymphocyte homing to peripheral lymph nodes occurs in a similar pattern.  7  1.3.1 Rolling Studies in mice lacking E-, P- and/or L-selectin expression have underscored the major role these adhesion molecules play in recruiting leukocytes under non-pathologic and inflammatory conditions (26-29). The selectins are calcium-dependent (C-type) lectins that are present on the surface of leukocytes (L-selectin), endothelial cells (E- and P-selectin) and platelets (P-selectin). Rolling is mediated by selectin-carbohydrate interactions that are weak and transient (30). Although the sialyl Lewis x (NeuAca2^3Gal(31^4[Fucal-*3]GlcNAcpi->R) moiety is recognized by each selectin, the preferred ligand for L-selectin is sialyl 6-sulfo Lewis x (addition of a sulfate group at the 6-0 position of GlcNAc) and optimal binding to P-selectin is observed when proximal tyrosine residues in the glycoprotein bearing the sialyl Lewis x epitope are sulfated (see Table 1.2)(31, 32). P-selectin glycoprotein ligand-1 (PSGL-1) carries the appropriate carbohydrate structures and modifications that allow it to serve as a ligand not only for P-selectin, as its name suggests, but for E- and L-selectin as well (31). Similarly, ligands for E-selectin have been identified and include E-selectin ligand-1 (ESL-1) and CD44, at least for neutrophils (33). The constitutive homing of naive lymphocytes to secondary lymphoid organs is critically dependent on L-selectin mediated rolling (34). Numerous studies have revealed that a set of mucin-like glycoproteins including glycosylated cell adhesion molecule ( G l y C A M ) - l , CD34, and mucosal vascular addressin cell adhesion molecule ( M A d C A M ) - l present on high-walled endothelial venules (HEV) carry the L-selectin ligand, sialyl 6-sulfo Lewis x (reviewed in 35, 36). Other studies suggest that L-selectin mediated rolling is also inducible under various inflammation-associated diseases  8  Table 1.2. Structure of the major carbohydrate determinant recognized by selectins and their physiological ligands. Recognized  Physiological  Carbohydrate  ligands  Determinant r~  (preferred)  P-selectin  Sialyl Lewis x, including sulfated Tyrosine residues on carrier protein  PSGL-1  L-selectin  Sialyl 6-sulfo Lewis x present on extended core 1 and core 2 O-linked glycans  PSGL-1 GlyCAM-l,CD34 M A d C A M - 1 , Sgp200 podocalyxin, CD44?  E-selectin  Sialyl Lewis x  PSGL-1 ESL-1 CD44  Abbreviations: ESL-1, E-selectin ligand-1; G l y C A M - 1 , glycosylated cell adhesion molecule-1; M A d C A M - 1 , mucosal vascular addressin cell adhesion molecule-1; PSGL-1, P-selectin glycoprotein ligand-1.  9  because sialyl 6-sulfo Lewis x determinants were upregulated in certain tissues (37). Selectin-independent rolling has also been documented. Although integrins play a larger role in firm adhesion, it has been well established that a4 integrins (CD49d) can mediate rolling of lymphocytes and eosinophils (38-40), and for neutrophils, but only under chronic inflammatory conditions in rodents (39, 41-46). Although monocytes also roll via a4-integrin, its contribution is minor compared to the selectins (47). Interactions between CD44 on lymphocytes and hyaluronic acid (HA) on the surface of activated endothelial cells are also capable of mediating rolling (48, 49). 1.3.2  Firm Adhesion Adhesion must follow rolling in a timely matter or else the leukocyte will bypass  its intended area or else risk being swept back into the mainstream of blood. As leukocytes roll, they encounter chemokines and other stimuli present on the surface of endothelial cells that activate integrins on the leukocyte to bind with high affinity to their counter-receptors. Leukocyte integrins lymphocyte function associated antigen-1 (LFA1, CD1 la/CD18 or aL|32) and Mac-1 (CD1 lb/CD 18 or <xM|32) bind to intercellular adhesion molecule-1 (ICAM-1) and ICAM-2 while very late antigen-4 (VLA-4, CD49d/CD29 or a4|31) binds to vascular cell adhesion molecule-1 (VCAM-1) on the endothelial cells (50). Although these integrins are expressed on most leukocytes, each subset differs in its abilty to be activated by a varying array of chemotactic factors. This allows the endothelium exquisite control over the makeup of the recruited cells. For example, monocyte chemotactic protein-1 (MCP-1) and IL-8 preferentially recruits monocytes and neutrophils to sites of inflammation, respectively (51).  10  1.3.3  Diapedesis Leukocytes adhered to the vascular endothelium transmigrate between the borders  of tightly apposed endothelial cells in an amoeba-like fashion. This step in leukocyte recruitment is the least understood but is known to involve a rapid reorganization of the cytoskeleton and is mediated by another set of adhesion molecules including plateletendothelial cell adhesion molecule (PECAM), CD99, vascular endothelial cell specific cadherin (VE-cadherin), and junctional adhesion molecule (JAM)-A, -B, and -C (25). Although considered rare, leukocytes have also been reported to pass directly through the endothelial cells (52).  1.4  CD44  1.4.1  Structure CD44 represents a diverse family of type I transmembrane glycoproteins 80-250  kDa in size that are expressed on almost every cell type (reviewed in 53, 54). A number of variant isoforms of CD44 (CD44v) are generated by the alternative splicing of at least 10 exons (see Figure 1.2)(55). The main form of CD44, referred to as CD44 hematopoetic (CD44H) or CD44 standard (CD44s), does not contain any of the alternatively spliced exons and is 85-90 kDa. CD44H consists of an extracellular domain of -270 amino acids (aa), a transmembrane domain of 21 aa and a cytosplasmic domain of 72 aa. The amino terminal -180 aa of the extracellular domain is highly conserved (-85% homology) among mammals. This conserved sequence contains a region with -35% homology to the Link module, a protein domain common to a family of proteins  11  -<3 N-linked glycan ®  - ©  Phosphorylation  SaaS  O-linked glycan ^  # # GAG  Sulfation  Sulfate bond  Figure 1.2. Schematic diagram of C D 4 4 . Structural features of CD44. Amino acid residues are numbered according to (56). Abbreviations: E R M , ezrin/radixin/moeisin; G A G , glycosaminoglycan.  12  that bind hyaluronic acid (HA)(56, 57). Two B X 7 B motifs, which are implicated in H A binding (58), are present in the conserved sequence, with one of the motifs overlapping the region containing the Link module. B X 7 B are basic clusters where B represents a basic aa and X is a neutral or basic aa. Kohda and colleagues (59) revealed that the Link module present in TNF-stimulated gene (TSG)-6 had a structure similar to that of C-type lectins. Most cells, including leukocytes and many mesenchymal cells, express CD44H. Less common isoforms of CD44 are mainly restricted to epithelial cells, activated T lymphocytes and some cancer cells (60). CD44 also has multiple sites for the addition of N- and 0-linked glycans and glycosaminoglycans (GAG), resulting in many glycoforms of CD44. The carbohydrate component of CD44 is quite significant as the core protein is only 37 kDa. 1.4.2 Ligands CD44 can bind to serglycin (61, 62), osteopontin (63) and chondroitin (CS)- and heparan sulfate (HS)-modified forms of CD44 have been implicated in binding macrophage inflammatory protein (MIP)-ip (64), matrix metalloproteinase (MMP)-9 . (65), fibroblast growth factor (FGF)-2, -4 and -8 (66) and components of the E C M , such as fibronectin (67) and collagen XIV (68). Certain human hematopoietic cell lines express a novel glycoform of CD44 that could bind E- and L-selectin via jV-linked glycans elaborated with sialylated and fucosylated determinants (69-72). More recently, Katayama and colleagues (33) were able to demonstrate that CD44 was a physiological E-selectin ligand on neutrophils isolated from humans and mice. E-selectin ligand activity was also dependent on sialylated and fucosylated AMinked glycans present on  13  CD44. However, the most characterized ligand of CD44 is hyaluronic acid (HA), a ubiquitous component of the E C M that is also present on the pericellular matrix. 1.4.3  Function in inflammation CD44 has been implicated in a variety of biological functions including  embryogenesis (73), hematopoiesis (74), and tumour metastasis (75). However, CD44deficient mice survived and showed no abnormalities in development, T cell priming or migration to inflammatory sites (76). Only a subtle difference in myeloid progenitor egress from the bone marrow alongside an increased granuloma response to an intracellular bacteria and an inhibitory effect on tumour growth was observed. Other molecules with similar functions to CD44 might be upregulated and compensate for its absence in CD44-deficient mice. For example, several proteins are capable of binding H A , such as TSG-6 and receptor for HA-mediated motility (RHAMM)(77). Despite this caveat, the role of CD44 in the inflammatory process has been well established and continues to be an area of intense research. A number of studies reported that anti-CD44 antibodies inhibited inflammation in murine models of experimental autoimmune encephalomyelitis (78), inflammatory bowel disease, and collagen- and proteoglycan-induced arthritis (79). Meanwhile, other studies have shed light on the molecular mechanism through which CD44 influences inflammation. CD44 on activated T lymphocytes was able to mediate rolling on H A and resulted in the recruitment of these cells to sites of inflammation (48, 49). CD44 did not mediate rolling on H A in neutrophils, but CD44-HA interactions were important for adhesion and extravasation (80). Because CD44 can also bind many components of the E C M it may also play a role in the retention and localization of extravasated leukocytes. In macrophages, binding of  14  H A to CD44 can result in secretion of pro-inflammatory mediators such as T N F a , IL-1 and IL-12 (81-84). Activation of macrophages mediated by CD44 also induces production of reactive oxygen and nitrogen species, MMPs, cytokines and chemokines. However, CD44 does not always play a pro-inflammatory role. In a bleomycin-induced model of lung inflammation, CD44-deficient mice succumbed to an unremitting inflammatory response (23). Reconstitution with C D 4 4  +ve  bone marrow cells partially  reversed this phenotype and was correlated with the clearance of apoptotic cells. Several studies have implicated CD44 on macrophages in the phagocytic uptake and removal of apoptotic neutrophils (85, 86). Apoptotic cells, if not removed in a timely manner, progress towards necrosis, a process that releases many pro-inflammatory mediators. Alternatively, CD44 may promote the resolution of inflammation by binding to MMP-9, a protease that cleaves and activates TGF-p* (87), a potent down-regulator of the inflammatory response (88). In addition, CD44 has been implicated in tissue H A turnover (89-92). Although H A normally exists as a high molecular weight (HMW) polymer (>10 Da) in healthy tissues, it gets broken down into low molecular (LMW) 6  weight fragments during inflammation. Several studies have demonstrated that L M W H A can act as a pro-inflammatory signal (93-95). CD44 also participates in another aspect important to resolving the inflammatory response and that is the proliferation and migration of fibroblasts, endothelial cells and epithelial cells associated with tissue remodeling (84, 96). These and other studies highlight the importance of CD44 in mediating both pro- and anti-inflammatory processes, in particular, CD44-HA interactions. Consequently, CD44-HA binding is a tightly regulated process.  15  1.4.4  Regulation of CD44-mediated hyaluronic acid binding Although many cell types express CD44, not all are able to bind to HA. While  some cells bind H A constitutively, others can be induced to bind H A (97). Reported activators of H A binding in T lymphocytes include IL-2, T N F a , MIP-lp\ IL-8 and regulated upon activation, normal T cell expressed and secreted (RANTES) and ligation of T cell receptor (TCR)(98). In human peripheral blood monocytes (PBM), proinflammatory mediators such as IL-1, IL-3, GM-CSF, IFNy and lipopolysaccharide (LPS) can induce H A binding in human P B M , but primarily via a TNFa-dependent pathway (99-103). IL-4 and IL-13 are well-documented inhibitors of H A binding (99). IL-10 inhibits IL-1-induced H A binding, but can also induce H A binding when added alone to human P B M (99). The primary mechanism for regulating CD44-mediated H A binding is glycosylation. Although increased CD44 levels promote H A binding, it alone is not sufficient to induce H A binding. N- and O-linked glycans can inhibit H A binding while TV-linked glycans can sometimes have the opposite effect (104, 105). The addition of CS to CD44 was found to inhibit H A binding, at least in a murine fibroblast cell line (106). Terminal sialic acid residues present on CD44 negatively regulate H A binding (105). Treatment of human P B M with LPS resulted in enhanced H A binding that was associated with increased sialidase activity (103). The diversity and flexibility of glycosylation makes it well suited for this cell type- and activation state-specific process. Previous studies in our laboratory on a myeloid cell line and in human P B M suggest that T N F a induced CD44-mediated H A binding may also be regulated by sulfation (100, 107).  16  1.5  Glycosylation Carbohydrate structures range in size from a single monosaccharide to polymers  exceeding one hundred monosaccharides and can be found as free molecules or linked to proteins and lipids in a post-translational process called glycosylation. Complex carbohydrates, termed glycans, are constituents of the E C M and are found on the surface of all mammalian cells. The potential for variation in glycan structures far exceeds that for nucleic acids and proteins since the monosaccharide building blocks can be added using different linkages and can also be connected to form branched structures. For example, a simple tetrasaccharide can, in theory, be assembled in over 15 million different ways. The biological roles of glycosylation are as varied and diverse as the carbohydrate structures themselves; it has been implicated in the development, growth and survival of an organism (reviewed in 108). Glycosylation can affect the structure, function and stability of proteins and can also provide ligands for specific binding events that mediate protein targeting, cell-cell or cell-ECM interactions. Human and murine CD44 can be modified by the addition of N- and O-linked glycans as well as glycosaminoglycans (GAGs). AMinked protein glycosylation begins in the endoplasmic reticulum (ER) with the co-translational transfer of a common lipidlinked oligosaccharide precursor (Glc3Man GlcNAc2) to an asparagine (Asn) residue 9  within the consensus sequence Asn-X-Serine (Ser) or Asn-X-Threonine (Thr) (109), where X can be any amino acid except proline (110). This oligosaccharide precursor is then trimmed to Man3GlcNAc2 by specific glucosidases and mannosidases in the E R and Golgi and serves as the common core for all AMinked glycans. Further processing and trimming in the Golgi produces AMinked glycans that have been classified into 3  17  Complex type iV-glycan  Hybrid type TV-glycan  • TV-acetvlslucosamine  O Mannose  •  6-0 sulfated /V-acetvlglucosamine  A Fucose  •  Galactose  • Sialic acid  J TV-acetvllactosamine unit  High mannose type TV-glycan  A Glucose 0 Growing polypeptide chain  Figure 1.3. Biosynthesis of TV-linked glycans. (111) A common oligosaccharide precursor is transferred to the amino group of asparagine residues with the Asn-XSer/Thr (where X is any amino acid except proline) consensus sequence in growing polypeptides chains in the ER. (b) Extensive trimming reduces the original oligosaccharide to the trimannosyl (Man3GlcNAc2) common to all A^-linked glycans. (c) Sulfation of terminal GlcNAc residues by trans-Golgi- and TGN-resident sulfotransferases such as GlcNAc6ST-l (146). (d) Further elaboration with other glycosyltransferases yields a sialyl 6-sulfo Lewis x epitope (structure within oval). The three subgroups of TV-linked glycans are complex type, hybrid type and high mannose type. Abbreviations: Asn, asparagine; ER, endoplasmic reticulum; Ser, serine; T G N , trans-Golgi network; Thr, threonine.  18  subgroups (complex, high mannose and hybrid) based on complexity and content of mannose residues and is illustrated in Figure 1.3. The most common type of 0-linked glycans are those linked to Ser or Thr residues in the protein backbone via a GalNAc residue. The biosynthesis of these mucintype 0-linked glycans also occurs along the ER-Golgi pathway and are further characterized depending on its core structure (Figure 1.4). GAGs consist of 1-25000 repeats of a characteristic disaccharide subunit (see Figure 1.5 for structure). With the exception of H A and heparin, GAGs are covalently linked to a core protein in the Golgi and the resulting molecule is termed a proteoglycan. A l l GAGs, except HA, are subject to extensive microheterogeneities due to epimerization and differential acetylation and sulfation of each disaccharide subunit. H A is composed only of repeating units of glucuronic acid and Af-acetylglucosamine ((31^4GlcA|31-»3GlcNAc)„ disaccharides, is not modified by sulfation and is synthesized on the plasma membrane (reviewed in 112).  1.6  Sulfation  Sulfation is a form of glycosylation that can occur on carbohydrates, proteins and lipids (113). Sulfated compounds have been reported in bacteria, plants, invertebrates and vertebrates (114, 115). Genetic defects affecting sulfation can have serious consequences, including loss of corneal transparency and embryonic dorsal/ventral polarity and skeletal underdevelopment (116, 117). 3'-phosphoadenosine 5'phosphosulfate (PAPS) is the universal sulfate donor and is generated in the cytosol by the sequential conversion of adenosine triphosphate (ATP) by A T P sulfurylase and adenosine phosphosulfate (APS) kinase. In mammals, PAPS synthetase provides both  19  Lumen ER  medial Golgi  Golgi  trans Golgi  TGN Cytosol  Core 1 O-glycan  Core 3 O-glycan  Core 2 O-glycan  Core 4 O-glycan  • N-acetylglucosamine  A Fucose  • 6-0 sulfated N-acetylglucosamine  •  •  Q Growing polypeptide chain  Galactose  • N-acetyllactosamine unit  Sialic acid  • /V-acetylgalactosamine  Figure 1.4. Biosynthesis of 0-linked glycans. ( I l l ) Addition of GalNAc to the hydroxyl (OH) group of serine or threonine residues in growing polypeptide chains in the ER. (b) Elaboration of the core 1 disaccharide, (c) the core 2 trisaccharide and extension of core 1 chain, (d) Sulfation of terminal GlcNAc residue on extended core 1 chain at the 6-0 position by medial-Go\%\ resident sulfotransferases such as GlcNAc6ST-2. (e) Sulfation of terminal GlcNAc residues present on core 2 chain at the 6-0 position by a trans-Go\g\- and TGN-resident sulfotransferases such as GlcNAc6ST-l (f) Further elaboration with other glycosyltransferases results in sialyl 6-sulfo Lewis x (structure within oval) at the terminal ends of both core 1 and core 2 chains. Four types of core structures found in O-linked glycans are also shown (boxed structure). Abbreviations: ER, endoplasmic reticulum; T G N , trans-Go\g\ network.  20  Hyaluronic acid  •4 GlcA (31^3 GlcNAc p l - »  ^ 4 GlcA p i - » 3 GlcNAc p l -  Chondroitin sulfate  •4 GlcA pi->3 GalNAc p i - *  o  o  Dermatan sulfate  -*4 IdoA a l - * 3 GalNAc p l -  o  Heparan sulfate  Heparin  •4 GlcA p i ^ 4 GlcNAc a l - *  ©  o  -»4 IdoA a l - * 4 GlcNAc a l -  ©  ©  ©  Keratan sulfate  0  0  ^ 3 Gal p i ^ 4 GlcNAc p i - *  0  0  ^ 3 Gal pi-*4 GlcNAc pi-  Figure 1.5 Classification of glycosaminoglycans and their microheterogeneities. Except for hyaluronic acid, epimerization at the C-5 position of uronic acids and N- and 0-sulfation (locations indicated by circled numbers) are the sources of microheterogeneities. Abbrevations: IdoA, iduronic acid; Gal, galactosamine; GalNAc, ^-acetylgalactosamine; GlcA, glucuronic acid; GlcNAc, JV-acetylglucosamine. • • • represents repeating disaccharide subunits.  21  enzymatic activities (118). Chlorate is a potent inhibitor of ATP sulfurylase and has been used in numerous studies to inhibit sulfation (119). Sulfotransferases are classified into two main groups based on cellular location. Sulfotransferases present in the cytosol are generally involved in the inactivation of small soluble molecules such as hormones and toxins (120). Golgi-associated sulfotransferases are membrane-bound and act on either carbohydrate or tyrosine residues present on glycoproteins, proteoglycans and glycolipids destined for the extracellular environment. The coordinated activities of sulfotransferases and other glycosyltransferases in the Golgi often generates unique sulfated structures that can be recognized by E C M proteins, cell surface receptors and viruses (114, 121-123).  1.7  A^acetylglucosamine 6-0 sulfotransferases (GlcNAc6STs) Earlier studies with chlorate had indicated that sulfate was an important structural  component of L-selectin ligands (124). After subsequent studies revealed that sulfation occurring at the 6-0 position of GlcNAc within the sialyl 6-sulfo Lewis x moiety was critical for optimal L-selectin ligand activity (36, 125-128), there was a tremendous amount of interest in identifying the carbohydrate sulfotransferase(s) responsible for this modification. To date, 5 GlcNAc6STs have been identified in humans along with 4 orthologues in the mouse (129). The chromosomal location, tissue distribution and protein length of the human GlcNAc6STs are summarized in Table 1.3. GlcNAc6ST-2 attracted the most attention since its expression was highly restricted to high-walled endothelial venules (HEV) of peripheral lymph nodes (PLN) among a limited number of other sites (130-133). In addition, GlcNAc6ST-2 was also induced at sites of chronic inflammation that develop HEV-like vessels (134) and its exogenous expression in  22  Table 1.3. Summary of cloned human 7V-acetylglucosamine 6-0 sulfotransferases (GlcNAc6STs). Chromosal location, tissue distribution, protein length and accession numbers of the five human GlcNAc6STs. Note that the tissue distribution of the four orthologues in the mouse is very similar to those in humans.  Name  Tissue expression  Protein  Chromosomal  Accession  length  location  Number  7q31  ABO14680  3q24  AF83066  (aa) GlcNAc6ST-l  530  Broad: PBL, heart, placenta, lung, spleen, brain, ovary, stomach, thymus, colon, etc  GlcNAc6ST-2  H E V , liver, pancreas, T and B lymphocytes  386  16q23.1-23.2  AF131235  GlcNAc6ST-3  Intestine, H E V ,  390  16q23.1-23.2  AF176838  486  Xp11  AB037187  T and B lymphocytes  GlcNAc6ST-4  Broad: PBL, kidney, brain, heart, spleen,  AF280089  liver, intestine, ovary,  AB014680  pancreas  GlcNAc6ST-5  396  Cornea of eye,  16q23.1-23.2  AF219990 AF280086  brain, lung  Abbreviations: aa, amino acid; H E V , high-walled endothelial venule; PBL, peripheral blood leukocyte.  23  Chinese Hamster ovary cells resulted in increased binding to an L-selectin/IgM chimera (131). Genetic deletion of GlcNAc6ST-2 in mice verified that this sulfotransferase was involved in the biosynthesis of HEV-associated L-selectin ligands in vivo (135) as lymphocyte homing to P L N was reduced by 50%. Transcripts for GlcNAc6ST-l are present in P L N , although they are also present in a broad range of tissues (136, 137). Lymphocyte homing to P L N was reduced 25% in GlcNAc6ST-l knockout mice (138) and 75% in mice deficient in GlcNAc6ST-l and GlcNAc6ST-2 (139, 140). The sialyl 6sulfo Lewis x moiety on O-linked glycans can serve as L-selectin ligands (141, 142) but there is no evidence that the same moiety on TV-linked glycans can (130). Although in vitro studies have shown that both GlcNAc6ST-l and GlcNAc6ST-2 can transfer sulfate to O-linked glycans (132, 143), GlcNAc6ST-l prefers to sulfate TV-linked glycans (130, 132, 144, 145) while the opposite is true for GlcNAc6ST-2. A recent study by De Graffenried and colleagues (145) suggests that this may be related to the distinct localization of GlcNAc6STs in the Golgi. GlcNAc6ST-4 along with GlcNAc6ST-l are the only GlcNAc6STs that are expressed in a wide variety of tissues, although there are differences in the expression pattern (144, 146, 147). A n initial study reported GlcNAc6ST-4 as a GalNAc 6-0 sulfotransferase (147), but this could not be verified in subsequent studies by other researchers (144, 146). No biological function has yet been attributed to the activity of GlcNAc6ST-4 in humans or mice. In humans, the expression of GlcNAc6ST-3 and -5 are highly restricted to the intestine, and to the brain and cornea of the eye (148), respectively. These isoenzymes are likely the result of a recent gene duplication event (occurring after the divergence of  24  GlcHRcEST-1 GlcHRcGST-4 GlcNRc£ST-2 GlcHflcEST-3 GlcHRcEST-5  70  50  10  BO  90  100 — I  rBRSPWWU'rTOLPI^aRnPRMflPm HKGRRRRRREYCK-FflLLLVLYTLVLLLVPSVUJGGRDGDKGREHCPELQRSL  S ' - P S B 101 GlcHRcEST-1 GlcNRcEST-4 GlcHReGST-2 GlcNRcEST-3 GlcRflcGST-5  GlcHRcEST-1 GlcHBcEST-4 GlcNflcEST-2 GlcHBcEST-3 GlcHRcEST-5  110  120  130  140  6VUSLEH  HHHCEREqGSEHBHHEEGEHNqSPRFPSNLSGRVGEH  llHLPRFSSKTVTVUlHQTTCUJjnSRPIiPS-SPRBGEDRVHVLVLSSHKSUSSFLGgLFSUHPIJVFYLribPfiUHV HRLPRVSSTnVTnUXWF-LUJT_VS^^ 230 240 201 210 220 I 1 • * 1 MOKLYPUO V S QG ft - L S R L R U I L S V F U L S P H b S S B R U0M.Tl»6flr€SLfl6i«JWrlLRSLFRCDFSVUlYI»^ MWTFlCOSTflHHI HMflVRni I R f l ? F L f . l ) r 1 S V F D n Y l C P G P R HTTl^QGSnRTlHnflVRni HRSIFLCDtlDVFDRYH-PQSR MTTLSQGSHRT1HJ1HVRI1I VRS¥I L CIIHOVF UH>1_-J*MRR  310  250  410 ,  260  330  340  420 «.  280  290  300  + 1 N L T T L G I SHH!NKVV S L PHY—RKEWGLVOOR K K - PPORLHRF P L PGflF KHFflEVGLVEDXR ERS P P V R I R H L RQSSLFRHERSRfiLCSaPflCBnPlBE nPRRHCRLLCSBOPFEW N L 3 H F MHRTSRflLI >Pr R r S f l F P R C T ISKQDVr.KTL TRQPrSLfl HLSDI I QMHVSRfll PIRISflFPRGB I S S E R V i KPI RRUS! T L B •  •  1  350  360  370  380  3'-PB 320  270  1  EEECRICYim.VIKGVRVFOmLBPLLRUPRLDL^ E R E r R K Y P W ' / I K D V R L L n i G7I V P I I RDPGI Ml KVVQI FWIPKHVUMSRLKSRIJGI F J C R r R S Y S H V i L K E V R F F N L Q S I YPI1 KDPSI HI HTVHI VRnPRflVFRfiRERTKGOl IB^T«YSHTVlJ<EVWFNLaVLYPLLSUPHLHLIClVHLVKUPRH*»lJ<S h f l R G P I R F R r . R S Y S H W L K E V R F F N L Q V I Y P I I S f i r f l l HI RTVHI V R n r R f i V L R n R E Q T R K f l  401 I  330  400  L R F S I D V L R T R S R E O R F M t V L I RHGVGHRPGGQSRRLPRflPRf) HinSRTVhGQHEOKLnCDQPYY RK N G I . L G T H B W V E R - D P H - R RR NETVI G T H E T W E n - D P G L R  430  440  450  460  470  4B0  490  •  ,  ,  H  ,  1  H  500 1  DYlW^Mtmi.llSIMttLaTR-LBPPDMLQGI^ DTTLTM-EYICERHLroLLFR-RSflPRMLRIKY RHLfifl tRFH^rfrRXflflYrjB-RP HL5RR0DREHVHRHR VWQV'I i Q S g L E T Y t C T H S L P K f i - l Q E R Y L L V R Y F O ! R R f l P V f l Q T S R r t Y E F f G l E F L P W QTHVHHITRfJffiirjD—HRFHTHRRnflLNVSqfiHR LHcF>fSSHVRIREflRTLKPPPFI RGRYRLVRFFm RREPLflEIRRLYflrTGlTLTPQI FRHIHHITH&SGIGRPIEflrHTSSRHRRNVSflfiHR V V 1 E 7 I . 1 K H V R I R E R R T I K P P P F I RGRYR1 V R F F n i flREPLREIRHL Yfll TGI S L T P q i I RHIHHITHTiSGPGflRRERI K T S S R H H L H V S H R H R  501 GlcHRcEST-1 GlcNHcEST-4 GlcNflcEST-2 GlcHHcEST-3 GlcNRcGST-5  V S R E K f l H [ Y V H R T M R T X i S S F U i F l FNQHPriVFYI TEPIIHHL  mPKKMIOXLFLVSIimiU^rTHHV^NISr^^  CSB  GlcHflcGST-1 GlcHflcBST-4 GlcHRcGST-2 r.lcMRcGST-3 GlcHRcEST-5  200  G3«cPEiHTBB»HqWRLnLRTPYRPPI^  301 GlcHHcEST-1 GlcMRcEST-4 GleNlcGST-2 GlcHHcBSI-3 GlcHRcEST-5  190  170  160  150  510  520  T i l T F Q Q K Q V L F Y Q P RV ERI S R £ Q D t ) V VR H P f l ' R l MS P Y E X S R L K f l GDfl Ml HHLPF T K 1 L R V U E V C H G H L f l L L H M PFRKTRRVQELCflGAl Q L I  530  540  550  561  G ERVN5PE-VIC iLSKTLLRKPRL R PRSEEFGfWQPREGETPLEIflnDGHT G RHVRSFO OR 1 LI ni L S TMTVPEDIR Ii T RPV YS W O O K U L T L D L V I J ' R G P O H F S U R S P D GYRPVYSEDrO^NLRlDlVI-PFGLHfiFTHRSSTflSHPRN  Figure 1.6. Sequence alignment of the five cloned human /V-acetylglucosamine 6-0 sulfotransferases. Red and blue letters indicate that 100% and >50% of the residues at that position are identical, respectively. 5'-PSB corresponds to the 5'-phosphosulfate binding site, 3'-PB corresponds to the 3'-phosphate binding site and CSB represents the putative carbohydrate substrate binding site.  25  rodents and primates because in mice only one form of this sulfotransferase, called GlcNAc6ST-3, is expressed in all the same tissues as observed in humans) as they are located within 50 kilobases on chromosome 16q23.1-23.2 and are 85.6% identical at the amino acid level. Although GlcNAc6ST-3 was found to have a preference for sulfating O-linked glycans, it was not involved in the synthesis of L-selectin ligands (143). However, mucins (heavily O-glycosylated proteins) are abundant in the intestine, many of which are sulfated. Mutations in the GlcNAc6ST-5 gene are associated with macular corneal dystrophy, an autosomal recessive hereditary disease wherein the hyposulfation of keratan sulfate in the cornea results in corneal opacity eventually leading to loss of vision (117, 149-153). GlcNAc6STs are type II single-span transmembrane proteins that have their Cterminal catalytic domain within the lumen of the Golgi. They are >40% similar at the amino acid level within their catalytic domains (see Figure 1.6). X-ray crystal structures exist for several cytosolic sulfotransferases (154-158) and the Golgi-associated sulfotransferase, heparan sulfate A^-deacetylase/A^-sulfotransfefase-l (NDST-1) (113, 159, 160), but none are available for any of the GlcNAc6STs. However, the GlcNac6STs have two regions with homology to the 5'-phosphosulfate (PSB) and 3'-phosphate (PB) binding sites (161). A n additional region with homology among the sulfotransferases has been speculated to be the carbohydrate substrate binding site (CSB). In vitro studies revealed that the GlcNAc6STs had greatly increased activity towards carbohydrate structures with terminal GlcNAc residues over those with internal GlcNAc residues (146, 162), although GlcNAc6ST-3 was found to have substantial activity towards a sialylated JV-acetyllactosamine (LacNAc) substrate in one study (162). This indicated that GlcNAc  26  6-0 sulfation occurred as a relatively early committed step in glycan biosynthesis and as a consequence, i) each sulfotransferase may be involved in the synthesis of multiple sulfated moieties and ii) the final structure of those sulfated moieties are dependent on the cell type and the full complement of glycosyltransferases present in each.  1.8  Thesis Objectives The sulfation of proteoglycans and glycoproteins has been implicated in many  biological processes including cell-cell adhesion, cell proliferation through the capture of soluble growth factors and chemokines and in viral and bacterial invasion (114, 163). CD44 is sulfated in human P B M (100) and in the human myeloid cell line, SR91 (107, 164). In both cases, CD44 sulfation was increased upon exposure to the proinflammatory cytokine, T N F a . Investigation into the sulfation of CD44 in SR91 cells revealed a complex pattern of chondroitin and glycan sulfation that was altered upon T N F a stimulation (164). Experiments involving a series of monoclonal antibodies (mAbs) directed at different sulfated carbohydrate epitopes identified 6-sulfo Nacetyllactosamine (LacNAc)/Lewis x on CD44 that was unmasked after removal of terminal sialic acid residues with neuraminidase. The inflammatory response requires an intricate co-ordination of cell-cell and cell-ECM interactions. Failure can lead to a number of pathological conditions. The family of CD44 adhesion molecules bind to many components of the E C M , including its most characterized ligand, HA. CD44-HA interactions are implicated in a number of inflammatory processes, such as recruitment of neutrophils (80) and activated T lymphocytes to sites of inflammation (49). Consequently, CD44-mediated H A binding is  27  a tightly regulated process that is cell type- and activation state-specific. In monocytes and macrophages, changes in N- and Olinked glycans and G A G addition are known to modulate CD44-HA binding (103, 105, 165). Sodium chlorate, an inhibitor of sulfation, reduced the level of TNFa-induced H A binding in SR91 cells (107) and human P B M (100). Macrophages are derived from P B M and differentiate into a number of functionally distinct states depending on the milieu of factors present in each tissue. Since the inflammatory process involves the release of a multitude of cytokines, chemokines and other soluble products, it can have a profound effect on the activation state of macrophages. In general, macrophages with pro-inflammatory attributes and involved in T h l responses are termed M l , and those with anti-inflammatory attributes or involved in Th2 responses and wound healing are termed M2.  These observations, along with others, have led to the hypotheses that the induction of the A G 107 epitope on CD44 is associated with increased expression of a specific iV-acetylglucosamine 6-0 sulfotransferase (GlcNAc6ST) and that the A G 107 epitope is involved in the inflammatory process by mediating CD44-ligand interactions on monocytes and macrophages. There were five specific objectives for this thesis: 1. To determine if the expression of any of the GlcNAc6STs are induced by T N F a and responsible for increased carbohydrate sulfation of CD44, in particular, the 6-sulfo LacNAc/Lewis x epitope, defined by mAb A G 107. 2. To determine if the A G 107 epitope is the sulfate-bearing determinant involved in the regulation of CD44-mediated H A binding in SR91 cells and human P B M .  28  3. To ascertain whether the A G 107 epitope can mediate leukocyte rolling by serving as an L-selectin ligand. 4. To evaluate the expression of GlcNAc6STs in human macrophages with M l and M2 properties to determine which facet of inflammation these sulfotransferases may be involved in. 5. To evaluate the expression of GlcNAc6STs in mouse macrophages to determine the validity of using mouse models to study the role of GlcNAc6STs in inflammation.  29  CHAPTER 2 Materials and Methods  30  2.1  Cell lines The human myeloid cell line, SR91 (166) and monocytic cell lines, THP-1 (167)  and U937 (168), and the murine BW5174 T cell line (169) were available from American Type Culture Collection (ATCC) and were maintained in Roswell Park Memorial Institute 1640 medium (RPMI; Invitrogen) supplemented with 10% fetal calf serum (FCS; from Invitrogen or HyClone), 2 m M glutamine and 1 m M sodium pyruvate. The medium for U937 cells was also supplemented with 10 m M 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES). SR91 cells resuspended at 1 X 10 cells/ml and 6  THP-1 and U937 cells resuspended at 5 X 10 cells/ml were incubated with or without 10 5  ng/ml recombinant human T N F a (R&D Systems) for 24 hr prior to analysis by flow cytometry and isolation of total R N A . THP-1 and U937 cells were differentiated into adherent, macrophage-like cells by culturing in the presence of 100 ng/ml phorbol 12myristate 13-acetate (PMA; Sigma) for 96 hr (170, 171) or 10 ng/ml P M A for 48 hr, respectively (172, 173). For these experiments involving P M A , T N F a was added at a final concentration of 10 ng/ml for the final 24 hr period. ECV304 (174), ECV304 cells transfected with GlcNAc6ST-l, Fuc-T VII, or both (175) were all provided by R. Kannagi and were maintained in Dulbecco's Modified Eagle Medium ( D M E M ; Invitrogen) supplemented with 10% FCS, 2 m M glutamine and 1 m M sodium pyruvate with 6 U/ml hygromycin B (Calbiochem) for GlcNAc6ST-l transfectants, or 1 mg/ml active Geneticin (Invitrogen) for Fuc-T VII transfectants. The monkey fibroblast cell line, COS-7 (176), and murine fibroblast cell line, L929 (177), were available from A T C C and were maintained in D M E M or RPMI, respectively, supplemented with 10% FCS, 2 m M glutamine and 1 m M sodium pyruvate.  31  Primary cells  2.2 Human 2.2.1  Peripheral blood monocytes Whole blood (20-100 ml) was collected from healthy volunteers by venous  puncture into vacuum-sealed tubes containing sodium heparin (Becton Dickinson), then diluted with Hank's balanced salt solution (HBSS; Invitrogen) and centrifuged over a Ficoll-Paque Plus (Amersham) density gradient. The buffy coat consisting of peripheral blood mononuclear cells (PBMC) was isolated with a glass pipette and the contaminating red blood cells were lysed with 0.15 M NH C1 for 5 min on ice. The P B M C were 4  resuspended in RPMI medium containing 10% FCS, 2 m M glutamine and 1 m M sodium pyruvate and incubated in 6-well tissue-culture treated plates (Becton Dickinson) at 2.5 X 10 cells/ml with or without 20 ng/ml recombinant human T N F a (R&D Systems) for 72 6  hr. P B M C were then washed with PBS to remove CD 14 in the tissue culture supernatant and resuspended in ice-cold PBS/2% FCS at 1 X 10 cells/ml. Anti-CD 14 magnetic 7  Dynabeads (Dynal) were added (25 ul per 1 X 10 cells) and rotated end over end for 1 hr 7  at 4°C. The labeled monocytes were then immobilized on a magnet (Dynal), the CD14"  ve  P B M C were removed by washing with PBS/2% FCS, then used for flow cytometric analysis or isolation of total R N A . 2.2.2  Peripheral blood monocyte-derived macrophages P B M C were isolated as described above, resuspended in M A C S buffer (PBS,  0.5% bovine serum albumin [BSA; Sigma], 2 m M ethylenediaminetetraacetic acid [EDTA], pH 7.2) and C D 1 4  +ve  monocytes were isolated using anti-human CD 14-  conjugated magnetic beads (Miltenyi) according to the manufacturer's instructions. The  32  monocytes were resuspended in RPMI medium containing 10% FCS, 2 m M glutamine, 1 m M sodium pyruvate and I X penicillin/streptomycin and incubated in 6-well tissueculture treated plates (Becton Dickinson) at a density of 7-9 X 10 cells/ml in the 5  presence of 50 ng/ml recombinant human M-CSF or 0.5-20 ng/ml recombinant GM-CSF (both from R & D Systems) for 6 to 7 days before the addition of 500 U/ml recombinant human interferon (IFN)-y (R&D Systems), 1 ng/ml LPS (Sigma) or 20 ng/ml recombinant human T N F a (R&D Systems) for 24 hr. The adherent macrophages were lysed directly on the plate for isolation of total R N A or else, were removed by repeatedly incubating with 2X Versene (1.17 g N a H P 0 , 0.18 g K H P 0 , 0.2 g E D T A , 8 g NaCl, 2  4  2  4  0.2 g KC1 per L, p H 7.4) for 10 min at 37°C followed by vigorous pipetting for analysis by flow cytometry. The macrophages were visualized using phase contrast microscopy on an inverted microscope (Nikon) and images were captured at 100, 200 and 400X magnification using a digital camera (Nikon) attached to the microscope. Mouse Mouse strains C57B1/6 and Balb/c were purchased from Charles River Laboratories and used for experiments according to the protocols described below. A l l mice were sacrificed by C O 2 asphyxiation followed by cervical dislocation according to animal care regulations adopted at the University of British Columbia. 2.2.3  Bone-marrow derived macrophages (BMDM) The femurs and tibia were isolated and the bone marrow was flushed out with  PBS by puncturing the head of the bone with a 26-gauge, '/z-inch needle attached to a 10 ml syringe. The bone marrow was resuspended in D M E M medium containing 20% FCS, 2.5% L cell conditioned medium (LCCM), 2 m M glutamine, 1 m M sodium pyruvate, I X  33  penicillin/streptomycin and added to 10 cm petri dishes (Fischer Scientific) at a concentration of 1 X 10 cells per dish in a final volume of 8 ml for 4 days. The adherent 7  cells were then removed by incubating with 2 X Versene for 10 min at 37°C and re-plated at 1:4 dilution in D M E M medium containing 10% FCS, 1% L C C M , 2 m M glutamine, 1 m M sodium pyruvate, I X penicillin/streptomycin in a final volume of 6 ml with or without 20 ng/ml recombinant murine T N F a (R&D Systems) for the next 3 days. The adherent macrophages were again removed with 2X Versene prior to analysis by flow cytometry or isolation of total R N A . 2.2.4  Thioglycollate-elicitedperitoneal macrophages Mice were injected with 1 ml of 3% Brewer's thioglycollate (Difco) using a 26-  gauge, Vi-inch needle attached to a 5 ml syringe. Negative control mice were injected with 1 ml distilled water. The mice were sacrificed 5 days later and the abdominal skin was retracted to expose the peritoneal wall. With a 22-gauge, 1-inch needle, 10 ml of ice-cold PBS was injected into the peritoneal cavity then withdrawn after the abdomen was gently massaged (~8 ml was consistently recovered). The exudates were washed with PBS then resuspended in 0.15 M NH4CI for 5 min on ice to lyse red blood cells. The remaining leukocytes were washed with PBS, resuspended in RPMI medium containing 10% FCS, 2 m M glutamine, 1 m M sodium pyruvate, I X penicillin/streptomycin and plated at 3 X 10 cell/ml in a 24-well tissue culture-treated 5  plate (Becton Dickinson) for 2 hr to allow macrophages to adhere, then washed 2 times with PBS to remove non-adherent leukocytes. The adherent macrophages were then incubated with or without 20 ng/ml rmTNFa (R&D Systems) for 24 hr prior to flow cytometric analysis and isolation of total R N A .  34  2.2.5  Splenic macrophages Three days after Listeria monocytogenes (1 X 10 colony forming units; strain 5  10403s) was injected into the lateral tail vein, the mice were sacrificed, the spleen was isolated and homogenized by passing over a metal mesh with a plastic pestle into a dish containing RPMI medium supplemented with 10% FCS, 2 m M glutamine, 1 m M sodium pyruvate and I X penicillin/streptomycin. After the isolated cells were spun down, contaminating red blood cells were lysed by incubating with 0.15 M N H 4 C I for 5 min on ice. The remaining leukocytes were then resuspended in M A C S buffer (PBS, 0.5% B S A , 2 m M E D T A , pH 7.2) and CD1 l b  + v e  splenic macrophages were isolated by using anti-  mouse CD1 lb-conjugated magnetic beads (Miltenyi) according to the manufacturer's instructions and used for flow cytometry or isolation of total R N A . 2.2.6  Brain and kidney After the mouse was sacrificed, the brain and kidneys were isolated and  homogenized by passing the organs over a metal mesh with a plastic pestle into a dish containing PBS. The cells were spun down and contaminating red blood cells were lysed by incubating with 0.15 M NH4CI for 5 min on ice before total R N A was isolated.  2.3  Reagents Hyaluronic acid (HA) isolated from rooster comb (Sigma) was conjugated to  fluorescein as described by de Belder and Wik (178). L C C M is a source of murine M CSF and was generated by concentrating the tissue culture supernatant of mouse L929 cells. Purified IM7.8.1 was coupled to CNBr-activated Sepharose CL-4B beads (Sigma) to yield a final concentration of approximately 4 mg/ml and was stored as a 50% slurry in  35  PBS/0.02% sodium azide. Plasmid encoding L-selectin/IgM (L-sel/IgM) was a kind gift from S. Rosen.  2.4  Antibodies The rat anti-mouse/human CD44 mAb, IM7.8.1 (TIB-235), and mouse anti-  human CD44 mAb, Hermes-3 (HB-9480), were available from A T T C . Purified IM7.8.1 and mouse anti-human CD44, 3G12, was kindly provided by B. Hyman and G. Dougherty, respectively. Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgM and goat anti-rat Abs and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and goat anti-mouse IgM Abs were purchased from Jackson ImmunoResearch Laboratories. FITC-conjugated IM7.8.1, F4/80, rat IgG2b and phycoerythrin (PE)conjugated anti-mouse CD1 lb was from eBioscience. PE-conjugated mouse anti-human CD3, CD14, CD19 and PE-conjugated mouse IgGl and IgG2a were from Caltag Laboratories. The following are antibodies that recognize carbohydrate determinants (see Table 2.1 for structures): the mouse IgM mAbs, 2F3, anti-sialyl Lewis x/sialyl 6-sulfo Lewis x (179), AG107, anti-6-sulfo LacNAc/Lewis x (180), AG223, anti-6-sulfo Lewis x (181), G72, anti-sialyl 6-sulfo LacNAc/Lewis x (181) and G152, anti-sialyl 6-sulfo Lewis x (181) were kindly provided by R. Kannagi and DD-2, anti-6-sulfo LacNAc (182), was from P. Rieber.  2.5  Flow cytometry andfluorescence-associatedcell sorting Cells were resuspended in ice-cold F A C S buffer (PBS, 2% horse serum, 5 m M  EDTA) and aliquoted into a 96-well round bottom plate at 2 X 10 cells per well. A l l 5  36  Table 2.1. Carbohydrate determinants recognized by monoclonal antibodies used in this study. m Ab  Determinant recognized  2F3  Sialyl Lewis x  Structure  ~1  ./V-acetylglucosamine 6-0 sulfated Af-acetylglucosamine  Sialyl 6-sulfo Lewis x A G 107  Galactose  6-sulfo LacNAc  LacNAc  6-sulfo Lewis x AG223  6-sulfo Lewis x  G72  Sialyl 6-sulfo LacNAc  •  Sialic acid  A Fucose  •••  Sialyl 6-sulfo Lewis x G152  Sialyl 6-sulfo Lewis x  DD-2  6-sulfo LacNAc  Abbreviations: LacNAc, A -acetyllactosamine; mAb, monoclonal antibody. r  37  subsequent incubations were performed on ice. To block Fc receptors, human P B M and macrophages were incubated with 200 ul heat-inactivated human plasma for 10 min while mouse B M D M and macrophages were incubated with 100 ul 2.4G2 hybridoma supernatant for 10 min. Cells were then incubated for 20 min with the following primary antibodies: 50 ul of 2F3 (1:16 dilution), AG107 (1:8, 1:16 or 1:32 dilution; 1:4 dilution was used for human macrophages), AG223 (1:16 dilution), G72, G152, DD-2 (1:20 dilution) hybridoma supernatant, 100 ul of IM7 or 3G12 hybridoma supernatant. The cells were washed with FACS buffer and incubated for 20 min with the following appropriate secondary antibodies [100 ul of goat anti-rat FITC (1:200 dilution), goat antimouse FITC, IgM specific (1:200 dilution)], directly conjugated antibodies [100 ul of anti-human CD14-PE (1:500 dilution), anti-human CD3-PE (1:200 dilution), anti-human CD19-PE (1:200 dilution), anti-mouse CD1 lb-PE (1:400 dilution), F4/80-PE (1:100 dilution), IM7-FITC (1:100 dilution)], isotype controls [mouse IgGl-PE (1:50 dilution), mouse IgG2a-PE (1:50 dilution), rat IgG2b-FITC (1:100 dilution)] or 100 ul of F L - H A diluted to 5 ug/ml. The labeled cells were then washed two times and resuspended in F A C S buffer with 2 [xg/ml propidium iodide to stain for dead cells. A minimum of 4000 live SR91, THP-1, U937, ECV304 cells or B M D M , splenic macrophages or 2000 live human P B M or macrophages were collected on a FACScan flow cytometer (Becton Dickinson) and analyzed using CellQuest software (Becton Dickinson). T N F a stimulated SR91 cells labeled with F L - H A or mAb A G 107 were sorted (bottom and top 10% based on signal from FL-1 channel) on a FACSVantage flow cytometer (Becton Dickinson).  38  2.6  Neuraminidase treatment Cells were resuspended at 1 X 10 cells/ml in 1:1 RPMI/PBS and incubated with 7  0.05 U/ml Vibrio cholerae neuraminidase (Roche Diagnostics) at 37°C for 60-75 min to remove terminal sialic acid residues prior to flow cytometric analysis.  2.7  Immunoprecipitation, PNGase F digestion and western blot analysis Human P B M , M-CSF-cultured macrophages, ECV304 and SR91 cells were lysed  in 10 m M Tris, pH 7.2, containing 140 m M KC1, 1% Triton X-100, 1 m M phenylmethylsulfonyl fluoride (PMSF), 1 [xg/ml leupeptin, 1 u.g/ml aprotinin and 1 u.g/ml pepstatin. CD44 was immunoprecipitated with 40 ul of 50% slurry of IM7.8.1conjugated Sepharose beads (4 mg/ml) per 10 cell equivalents, rotated end over end for 7  2 hr at 4°C then treated overnight with or without 500 U of Peptide MGlycosidase F (PNGase F; New England Biolabs) in 20 ul 50 m M sodium phosphate buffer, pH 7.5, containing 0.5% SDS, 1% NP-40 and 1% (3-mercaptoethanol. The samples were washed 2 times with acetone and incubated at -20°C for 1 hr with 800 ul acetone, centrifuged, resuspended in reducing sample buffer, separated by 7.5% SDS-PAGE and transferred to PVDF membrane. The dried membranes were incubated with hybridoma supernatant of mAbs 2F3, AG107 or AG223 diluted 1:10 with 3% BSA/TBST (20 m M Tris pH 7.5, 150 m M NaCl, 0.1% Tween 20; 500-600 ul total volume in a sealed plastic bag) overnight at 4°C. The blots were washed 3 X 5 min with TBST and incubated with 1:5000 HRPconjugated goat anti-mouse IgM for 1 hr at room temperature and washed 3 X 5 min with TBST followed with one final wash for at least 1 hr prior to detection with E C L (Amersham Biosciences). Chemiluminescent bands were captured and quantified using  39  the VersaDoc imaging system (Bio-Rad) or by exposure to Kodak BioMax film (Kodak), conversion to digital images and quantified using Image J software (NIH Image). Membranes were stripped by incubating with 100 m M p-mercaptoethanol, 2% SDS, 62.5 m M Tris, pH 6.7 for 90 min at 70°C, dried, then incubated for 1 hr at room temperature with Hermes-3 hybridoma supernatant diluted 1:50 with TBST containing 3% milk powder, washed 3 X 5 min with TBST, followed by 1:5000 HRP-conjugated goat antimouse IgG for 1 hr at room temperature and detected with E C L (Amersham Biosciences).  2.8  [ S] Sulfate labeling 35  ECV304 cells were seeded onto 60 mm plates in the presence or absence of 100 u.Ci/ml N a  3 5 2  S 0 (ICN Biochemicals) such that they were -80% confluent the next day. 4  CD44 from ECV304 cells was immunoprecipitated, subjected to SDS-PAGE and transferred to PVDF membrane and exposed to film with an intensifying screen at -80°C for 24 hr before Western blotting with mAb Hermes-3 as described above. M-CSFcultured macrophages at approximately 80% confluency were incubated overnight in the presence or absence of 100 uCi/ml Na SC>4. CD44 was immunoprecipitated and the 35  2  majority of each sample was subjected to SDS-PAGE and transferred to PVDF membrane. Membranes with the radioactive samples were exposed to film with an intensifying screen at -80°C for at least 24 hr and membranes with the non-radioactive samples were blotted with the mAb A G 107. The remaining sample of immunoprecipitated CD44 was diluted 10-fold, subjected to SDS-PAGE, transferred to PVDF membrane and blotted with mAb Hermes-3 as described above.  40  2.9  Semi-quantitative reverse-transcriptase polymerase chain reaction (RT-  PCR) Total R N A was isolated using RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Five u.g of total R N A from SR91, ECV304, THP-1, U937, B M D M or 0.6-4.0 p,g of total R N A from human P B M or human and mouse macrophages was reverse transcribed with Superscript II (Invitrogen), according to manufacturers' instructions. A n aliquot was then subjected to PCR with Platinum Pjx polymerase (Invitrogen) in total volume of 50 ul under the following conditions: 1 cycle at 94°C for 5 min; 30-35 cycles at 94°C for 30 sec, 49-55°C (see below for specific annealing temperatures) for 30 sec and 68°C for 60 sec; 1 cycle at 68°C for 10 min. Transcripts were monitored over several cycles and determined to be within the linear range when amplified for 21-23 cycles for p-actin and 30-35 cycles for sulfotransferases. To verify that the transcripts were amplified from cDNA derived from messenger R N A , parallel reactions were carried out in the absence of Superscript II. Twenty ul of the PCR product was electrophoresed in 1.0% agarose gel, stained with ethidium bromide or S Y B R Safe (Invitrogen), visualized under ultraviolet light and analyzed using Alpha Imager E C software (Alpha Innotech). Sulfotransferase transcripts were normalized to P-actin transcripts generated in the same PCR reaction tube. When no PCR product was detected after 40 cycles, the efficacy of that primer set was tested against genomic D N A . Amplified transcripts for human GlcNAc6ST-l and GlcNAc6ST-4 were excised from the agarose gel and purified using the QIAquick Gel Extraction Kit (Qiagen), according to the manufacturer's instructions, and verified by nucleotide sequencing (Nucleic Acid Protein Services Unit at the University of British Columbia). The following primer sets  41  were used to amplify each of the following human or mouse sulfotransferase and (3-actin cDNAs. The annealing temperature used during PCR and fragment size is as indicated: Human GlcNAc6ST-l, 847 bp, 51°C: 5 ' - G A G G T G T T C T T T C T C T A C G A G C C - 3 '  and  5 ' - C C A C G A A A G G C T T G G A G G A G G - 3 ' (183) GlcNAc6ST-2, 742 bp, 53°C: 5 ' - T G A C A T C A T C C C A C A A G A T G A A A T C A T C C C - 3 ' and 5' - G A T T T G C T C A G G G A C A G T C C A G G T - 3 ' GlcNAc6ST-3, 844 bp, 53°C: 5 ' - A C C A T C A G C A A G C A G G A C G T A T - 3 ' and 5' - T C T A A G G C C C A G A G T T C T C A G T C A - 3 ' GlcNAc6ST-4, 793 bp, 51-55°C: 5 ' - G A A C C A G T C T C C T C G G T T C C C A A G - 3 ' and 5' - A C C G G T C A G G A A G A A A T C G G C G C G - 3 ' GlcNAc6ST-5, 851 bp, 51°C: 5'-CGTGTTTGATGCCTATCTGCCTTG-3'  and  5' - T G C G A G G C G G T G G A T G A T - 3 ' C6ST-1, 834 bp, 49°C: 5 ' - A A G G G T C T C A G A C A A G C T G A A G C A - 3 ' and 5' - C A C T T C T T C C A G G T C T T A T A C T T G C C G - 3 ' KSGal6ST, 877 bp, 49° C: 5 ' - T T C A C C G C C A A G T C C T T T C A C A - 3 ' and 5' - C C G T A G A T C T C C T C G G T C T T C T T C A T - 3 ' p-actin, 512 bp, 49-55°C: 5 ' G A C T A C C T C A T G A A G A T C C T - 3 ' and 5' - A T C C A C A T C T G C T G G A A G G T - 3 ' Mouse GlcNAc6ST-l, 847 bp, 51°C: 5 ' - G A G G T G T T C T T C C T C T A T G A G C C - 3 ' 5'-CC A C G A A A G G C T T G G A G G A G G - 3 '  42  and  GlcNAc6ST-2, 922 bp, 47-50°C: 5 ' - C C A G G T C A T C G T T G T A G C T C T - 3 ' and 5'CGTTCCTGGCGTTAGTATGGAA-3' GlcNAc6ST-3, 733 bp, 50-51°C: 5 ' - A T C T C T T C C A G T G G G C G G T G - 3 ' and 5'CGAATGCACAGACCGGTAACC-3' GlcNAc6ST-4, 735 bp, 53°C: 5 ' - A C C C A G G A A A A G C A A C A C A T C T A T G - 3 ' and 5'GGTTAAGAAGAAATC AGCGCGTGG-3' C4ST, 375 bp, 53°C: 5 ' - T G C C T A C C G C A A C A A G T T C A C G - 3 ' and 5' - A A G A A C T C C G T G G T C A T C T C G T C G - 3 ' C6ST-1, 928 bp, 50-51°C: 5' - G G A C C T T G T A C A C A G C C T A A A G A T T C G - 3 ' and 5'CTCGGACAGCCACTTCTTCCA-3' KSGal6ST, 934 bp, 50°C: 5'- A G T A C A C A G C C A T C C G C A C T T - 3 ' and 5'TGTGCCACGTGACTGTCCA-3' P-actin: Same primer set for human and mouse 2.10  Isolation of genomic DNA Genomic D N A was isolated from SR91 and BW5174 cells with DNeasy Tissue  Kit (Qiagen) according to the manufacturer's instructions and used to test the efficacy of certain human and murine primer sets for PCR, respectively.  2.11  Production of L-selectin/IgM tissue culture supernatant Plasmid encoding L-selectin/IgM (L-sel/IgM) was transfected into COS-7 cells  using Lipofectamine 2000 (Invitrogen) with slight modifications from the manufacturer's instructions. O f note, 12 u.g of L-sel/IgM plasmid was complexed with 36 ul of Lipofectamine 2000 in Opti-MEM (Invitrogen) after an initial incubation period at room  43  temperature, then added dropwise to COS-7 cells at 80-100% confluency in a 10 cm tissue culture-treated plate (Becton Dickinson). Six hr later, the culture was replaced with fresh Opti-MEM medium and 24 to 48 after transfection the tissue culture supernatant (TCS) was collected, centrifuged to remove cells and cellular debris, and stored at 4°C when not being used as a source of L-sel/IgM in parallel plate flow chamber experiments, as described below.  2.12  Parallel plateflowchamber After diluting in 0.1 M N a H C 0 , 100 ul of L-sel/IgM (1:5) and/or F L - H A (2.5 3  mg/ml) was immobilized to the center of a 35 mm tissue culture-treated plate (Corning) after incubation at 4°C overnight; the outline of the liquid drop was marked for future reference. The plate was then was washed and blocked with PBS/1 % B S A for 1 hr at 37°C before use. A polycarbonate chamber was fitted into the coated plate with parallel plate geometry, as described previously (184, 185). SR91 cells were perfused across the plate surface containing the immobilized L-sel/IgM and/or F L - H A at a constant rate to mimic physiological shear forces encountered in the vasculature (1 or 2 dyne/cm ) by 2  using a syringe pump (Harvard Apparatus). The flow chamber was placed onto the stage of an inverted microscope (Zeiss), and after allowing 5 min for the flow rate to equilibrate, leukocyte/substrate interactions were visualized at 100X magnification using phase contrast and recorded with a digital video camera (Hitachi) attached to the microscope for later analysis. Rolling cells were defined as those traveling slower than free-flowing cells. The number of rolling cells was calculated by counting the number of cells that rolled past the field of view over a time period of 15 sec. Adherent cells were  44  defined as those remaining stationary for 10 sec or more. A minimum of 10 fields of view were observed for each sample and used to determine the average number of rolling/interacting cells per minute. For statistical analysis, the mean number of rolling cells in each sample was normalized to that of the unstimulated control sample within the same experiment. Means were determined to be significantly different when P < 0.05, as calculated using Student's t test. SR91 cells were stimulated with T N F a and treated with neuraminidase as described earlier, then resuspended at 1 X 10 cells/ml in RPMI medium containing 10% 6  FCS, 2 m M glutamine, 1 m M sodium pyruvate with or without 10 m M E D T A and maintained at 37°C throughout the rolling experiment. To reduce cell surface expression of CD44 on SR91 cells, purified IM7.8.1 was diluted in PBS (25 Lig/ml) and immobilized on a 6-well tissue culture-treated plate (Becton Dickinson) overnight at 4°C. SR91 cells were then incubated on IM7.8.1-coated plates during the 24 hr used for T N F a stimulation. Reduction of cell surface CD44 expression was verified by flow cytometry using the anti-CD44 mAb, 3 G12.  2.13  Enzyme-linked immunosorbant assay (ELISA) Tissue culture supernatant was collected from Day 7 or 8 unstimulated and  stimulated GM-CSF- or M-CSF-cultured human macrophages and stored at -20°C. Samples were later thawed and assayed in triplicate for concentrations of T N F a and IL10 using Ready-SET-Go! ELISA kits (eBioscience) according to the manufacturer's instructions.  45  CHAPTER 3 Results  46  3.1  GlcNAc6ST-l is upregulated in the SR91 myeloid cell line by TNFa Through the use of monoclonal antibodies (mAbs) that recognize specific sulfated  carbohydrate determinants, it was established in a previous study that expression of the mAb AG107-defined epitope, 6-sulfo #-acetyllactosamine[LacNAc]/Lewis x, was induced on AMinked, and to a lesser extent on 0-linked glycans, of CD44 by T N F a in the human myeloid cell line, SR91 (164). The AG107 epitope is sulfated at the 6-0 position of the Af-acetylglucosamine (GlcNAc) carbohydrate residue within the LacNAc moiety. To determine if T N F a upregulated the expression of the A G 107 epitope by inducing the expression of a specific sulfotransferase, transcript levels of all known GlcNAc 6-0 sulfotransferases (GlcNAc6STs) were evaluated by semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR). For all subsequent RT-PCR experiments, GlcNAc6ST transcript levels were normalized with that of p-actin transcript levels amplified in the same tube. GlcNAc6ST-4 transcripts were detected at low levels in SR91 cells and did not change upon stimulation with 10 ng/ml T N F a for 24 hr (Figure 3.1). In contrast, GlcNAc6ST-l transcripts were not readily detectable in unstimulated SR91 cells, but were induced significantly upon T N F a stimulation (11.7 ± 5.1 fold). Transcripts for GlcNAc6ST-2, -3, or -5 were not detected or detected at low levels and were not increased upon T N F a stimulation. This was also found to be the case for the galactose and galactose/A -acetylgalactosamine (Gal/GalNAc) 6-0 sulfotransferases, 7  KSGal6ST and C6ST-1, respectively (data not shown). A time course revealed that increases in GlcNAc6ST-l transcripts occurred as early as 1 hr after the addition of T N F a . This suggests that the expression of the AG107 epitope, 6-sulfo LacNAc/Lewis x, on CD44 may be regulated by the activity of GlcNAc6ST-l in SR91 cells.  47  GlcNAc6ST TNF-a  -1  p-actin  GlcNAc6ST TNF-a  -5  -2  31  P-actin C  1  J  B 20-,  y-  it  »  e 5 » Itf  15  • 10  " " _3 n  b = .=  c £ W o  GlcNAc6ST-1 |}-actin TNF-a Time (hrs)  Figure 3.1. Effect of TNFa on the expression of JV-acetylglucosamine 6-0 sulfotransferase (GlcNAc6ST) transcripts in SR91 cells. (A) Total RNA amounting  to 5-1 Oug was isolated from SR91 cells cultured with or without TNFa for 24 hr. Semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) was performed for 30-32 cycles for GlcNAc6ST (1-5) and compared with p-actin transcripts amplified for 21 cycles in the same tube. This is one representative experiment repeated at least 3 times. (B) Graphical representation of the fold increase in GlcNAc6ST-1 and GlcNAc6ST-4 in TNFa-stimulated versus unstimulated cells. Values represent the average from at least 3 separate experiments with error bars representing standard deviation. (C) RT-PCR for GlcNAc6ST-1 as described in (A) except that total RNA was isolated from SR91 cells cultured with or without TNFa over various time points. This is one representative experiment repeated 3 times.  48  3.2  TNFa upregulates expression of the AG107 epitope on CD44 in human  peripheral blood monocytes Sulfation of CD44 was shown to increase in human P B M in response to T N F a  v  (100). To determine if the increased sulfation of CD44 observed in human P B M was associated with increased expression of the A G 107 epitope in response to T N F a , as had been observed for SR91 cells (164), unstimulated and TNFa-stimulated human P B M (CD14  +ve  peripheral blood mononuclear cells [PBMC]) were treated with neuraminidase  and incubated with the mAb AG107. Flow cytometry of P B M demonstrated that unstimulated P B M expressed low levels of the AG107 epitope after neuraminidase treatment and that this was increased after T N F a stimulation (Figure 3.2A). A western blot with mAb A G 107 of immunoprecipitated CD44 from unstimulated and T N F a stimulated P B M established that the sulfated determinant detected by mAb A G 107 was present on CD44 (Figure 3.2B). The expression of CD44 and the AG107 epitope increased upon T N F a stimulation. On average, the AG107 epitope increased 5.9 ± 2.9 fold per cell and 2.0 ± 0.5 fold per CD44 molecule. CD44 isolated from unstimulated and TNFa-stimulated CD14" P B M C did not bind the A G 107 mAb, even after treatment ve  with neuraminidase (data not shown). This indicated that the AG107 epitope, 6-sulfo LacNAc/Lewis x, was upregulated in human P B M in response to T N F a and was present on CD44. The lack of mAb A G 107 reactivity before neuraminidase treatment indicated that the determinant present on human P B M was masked by terminal sialic acid residues* suggesting that the sulfated epitope expressed on CD44 is sialyl 6-sulfo LacNAc/Lewis x.  49  A  -TNFa  +TNFa Neuraminidase  A G 107  ST  - v e control  10'  A. 10 10 2  3  10  1  10 10 2  a £ CD  3  -•-Neuraminidase A G 107  f^frTTTTirtfrTr  -ve control  B  10  1  10 10 1 0' 1 0 F l u o r e s c e n c e Intensity 2  TNFa  -  Neuraminidase  —  3  2  O  10  3  A G 107  CD44  m  10 8  ra £- 6  <B o  go  4 2 0  Figure 3 . 2 Effect of TNFa on the expression of the AG107 epitope (6-sulfo Nacetyllactosamine [LacNAc]/Lewis x) on human peripheral blood monocytes (PBM) and on  CD44. Peripheral blood mononuclear cells (PBMC) were separated from whole blood by centrifugation over a Ficoll-Paque Plus density gradient and then incubated for 72 hr in the presence of absence of 20 ng/ml T N F a . (A) Flow cytometric of cells gated for C D 1 4 expression (PBM). Cells were treated with or without 0.05U/ml neuraminidase for 1 hr at 37°C and then labeled with the AG107 monoclonal antibody (mAb) orwith the secondary A b alone (-ve control). Fluorescence intensity is represented on the x-axis and cell number on the y-axis. (B) AG107 western blot of CD44 immunoprecipitated from P B M isolated using anti-CDI4-conjugated magnetic beads and treated as indicated (upper panel). The membrane was stripped and reprobed with the C O M mAb Hermes-3 (lower panel). Molecular mass markers are indicated at the right in kDa. (C) Graphical representation of the relative increase of A G 1 0 7 bound to TNFastimulated versus unstimulated P B M and to C D 4 4 from TNFa-stimulated versus unstimulated P B M . Values represent the average from 3 separate experiments with error bars representing standard deviation.  50  3.3  GlcNAc6ST-l and GlcNAc6ST-4 were upregulated in human peripheral  blood monocytes by TNFa To determine i f T N F a upregulated the expression of the AG107 epitope by inducing the expression of a specific sulfotransferase in human P B M as in SR91 cells, transcript levels of GlcNAc6ST 1-5 were evaluated by RT-PCR. Similar to SR91 cells, transcripts for GlcNAc6ST-2, -3 and -5 were either not detected or present at very low levels and were not increased with T N F a stimulation in human P B M (Figure 3.3). However, in contrast to SR91 cells, GlcNAc6ST-l and GlcNAc6ST-4 transcripts were present at low levels and increased 2.0 ± 0.2 fold and 2.0 ± 0.6 fold, respectively. These increases were specific to human P B M as transcript levels for GlcNAc6ST-l and GlcNAc6ST-4 were not increased with T N F a stimulation in CD14" P B M C (data not ve  shown). This data suggests that the expression of the A G 107 epitope, 6-sulfo LacNAc/Lewis x, on CD44 may be regulated by the activity of GlcNAc6ST-l, GlcNAc6ST-4, or both, in human P B M ,  3.4  GlcNAc6ST-l is implicated in the generation of the AG107 epitope on CD44  3.4.1  Expression of GlcNAc6ST-l leads to increased sulfation of CD44 To ascertain whether GlcNAc6ST-l and/or GlcNAc6ST-4 can contribute towards  the generation of the AG107 epitope on CD44, ECV304 cells or ECV304 cells transfected with GlcNAc6ST-l (175) were examined. RT-PCR revealed that ECV304 cells expressed significant levels of transcripts for GlcNAc6ST-4 but did not express significant levels of transcript for GlcNAc6ST-l, -2, -3, and -5 (Figure 3.4 and data not shown). As expected, ECV304 cells transfected with GlcNAc6ST-l expressed readily  51  GlcNAc6ST TNF-a p—actin  GlcNAc6ST TNF-a  -2  P-actin  B  in o  J  1  &  Figure 3.3. Effect of TNFa on the expression of W-acetylglucosamine 6-0  sulfotransferase (GlcNAc6ST) transcripts. Peripheral blood mononuclear cells (PBMC) were separated from whole blood by centrifugation over a Ficoll-Paque Plus density gradient. CD14  + v e  peripheral blood monocytes (PBM) were isolated from PBMC with anti-CD14-  conjugated magnetic beads. (A) Total RNA amounting to 0.6-2.5 ug was isolated from -1X10 CD14 PBM cultured with or without 20ng/ml T N F a for 72 hr. Semiquantitative reversetranscriptase polymerase chain reaction (RT-PCR) was performed for 32-35 cycles for GlcNAc6ST (1-5) and compared with b-actin transcripts amplified for 23 cycles in the same tube. All the above data are from one representative experiment repeated at least 3 times. (B) Graphical representation of the fold increase in GlcNAc6ST-1 and GlcNAc6ST-4 in TNFastimulated versus unstimulated cells. Values represent the average from 5 separate experiments with error bars representing standard deviation. + v e  52  6  A GlcNAc6ST-1 P-actin GlcNAc6ST-4 P-actin  B [ S]sulfate 35  CD44  ? c 8 o ^  u-  p.  <0 <4J  4  2  ^o _f_  Figure 3.4. Effect of exogenous expression of W-acetylglucosamine 6-0 sulfotransferase-1 (GlcNAc6ST-1) on [ S]sulfate incorporation into CD44 from ECV304 cells. (A) Total RNA 35  amounting to 5u,g was isolated from 5 X 1 0 ^ nontransfected ECV304 cells (-) and from GlcNAc6ST-1 transfected ECV304 cells (G1). Semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) was performed for 30-32 cycles for GlcNAc6ST-1 or GlcNAc6ST-4 and compared with p-actin transcripts amplifiedfor21 cycles in the same tube. This is one representative experiment repeated 4 times. (B) CD44 was immunprecipitated from nontransfected (-) and GlcNAc6ST-1 transfected (G1) ECV304 cells cultured overnight in the presence of 100 uCi/ml N a 2 S 0 . Upper panel indicates [ S]sulfate incorporation detected by autoradiography and lower panel indicates CD44 expression determined by western blotting with monoclonal antibody Hermes-3. Molecular mass markers are indicated at the right in kDa. (C) Graphical representation of (B). Values represent the average of 3 separate experiments with error bars representing standard deviation. 3 5  35  4  53  detectable levels of transcripts for both GlcNAc6ST-l and GlcNAc6ST-4. To distinguish which sulfotransferase was sufficient for CD44 sulfation, CD44 was immunoprecipitated from [ S]sulfate-labeled ECV304 and GlcNAc6ST-l transfected ECV304 cells. As 35  shown in Figure 3.4, CD44 from ECV304 cells incorporated very low levels of [ S]sulfate, whereas CD44 from GlcNAC6ST-l transfected ECV304 cells incorporated 35  significantly higher levels of [ S]sulfate (5.7 ± 1.7 fold). This demonstrated that CD44 35  was a substrate for GlcNAc6ST-l in vivo. 3.4.2  Expression of GlcNAc6ST-l leads to the expression of the AG107 epitope,  predominantly on the N-linked glycans of CD44 To determine if the increased sulfation of CD44 due to the expression of GlcNAc6ST-l in ECV304 cells resulted in the generation of the A G 107 epitope, flow cytometry was performed on these cells. Binding to mAb A G 107 was only observed with ECV304 cells that had been transfected with GlcNAc6ST-l or with GlcNAc6ST-l and a l , 3 fucosyltransferase (Fuc-T VII; Figure 3.5 and data not shown)(175). Binding to mAb AG107 was not observed with parental ECV304 cells or with ECV304 cells transfected with Fuc-T VII alone. CD44 was immunoprecipitated from ECV304 cells and ECV304 cells transfected with GlcNAc6ST-l and Fuc-T VII to determine if the AG107 epitope generated by GlcNAc6ST-l was present on CD44. The AG107 epitope was revealed only after neuraminidase treatment to remove terminal sialic acid residues and was present only on ECV304 expressing GlcNAc6ST-l. In addition, treatment of immunoprecipitated CD44 with Peptide MGlycosidase F (PNGase F) decreased the apparent molecular mass of CD44 and reduced the binding of mAb A G 107 to CD44 by 82 ± 17%. Although the possibility of GlcNAc6ST-4 contributing to CD44 carbohydrate  54  -  G1/F7  -(-Neuraminidase A G 107  A  0  -O  CD44 -ve control  £ z  I d  O  k lOMOMO 10'10 10 Fluorescence Intensity 3  2  B  3  G1/F7 Neuraminidase  PNGase F A G 107  CD44  G1/F7  SiNeuraminidase PNGase F  L  L  + -  + +  Figure 3.5. T h e e x p r e s s i o n of the A G 1 0 7 e p i t o p e (6-sulfo AV-acetyllactosamine [LacNAc]/Lewis x) o n C D 4 4 a n d o n /V-acetylglucosamine 6 - 0 sulfotransferase-1 (GlcNAc6ST-1) a n d a1,3 fucosy(transferase VII (Fuc-T VII) transfected E C V 3 0 4 c e l l s .  (A)  Flow cytometry of ECV304 cells (-) or GlcNAc6ST-1 / Fuc-T VII transfected ECV304 cells (G1/F7). Cells were treated with neuraminidase and then analyzed for binding to the AG107 monoclonal antibody (mAb). The negative control was secondary antibody (Ab) alone (-ve control). (B) Western blot with AG107 on CD44 immunoprecipitated from non-transfected (-) and GlcNAc6ST-1 / Fuc-T VII transfected (G1/F7) ECV304 cells and treated with or without neuraminidase and peptide A/-glycosidase F (PNGase F) as indicated (upper panel). The membrane was stripped and reprobed with the CD44 mAb Hermes-3 (lower panel). Molecular mass markers are indicated on the right in kDa. This is one representative experiment repeated at least 3 times. (C) Graphical representation of the relative amount of A G 107 mAb bound to CD44 when treated as indicated. Values represent the average from 3 separate experiments with error bars representing standard deviation.  55  sulfation cannot be excluded, these results implicated GlcNAc6ST-l in the synthesis of the 6-sulfo LacNAc/Lewis x epitope detected by mAb AG107 on CD44. Furthermore, this sulfated epitope is added predominantly to the AMinked glycans of CD44.  3.5  Expression of the A G 107 epitope did not correlate with TNFa-induced  hyaluronic acid binding In both SR91 cells and human P B M , increased expression of the A G 107 epitope on CD44 by T N F a correlated with induction of binding to FLA (100, 164). However, T N F a is a pleiotropic cytokine and may induce binding to H A through several mechanisms. For example, increased expression of CD44, which was observed in SR91 cells and human P B M , and alterations in N- and O-glycan addition have been reported to influence H A binding (105). To assess whether the addition of sulfated epitopes, specifically the A G 107 epitope, was involved in TNFa-induced H A binding, SR91 cells stimulated with 10 ng/ml T N F a for 24 hr were sorted for high or low expression levels of the A G 107 epitope. Since treatment with neuraminidase to expose the A G 107 epitope also induces binding to H A (105)(and our unpublished results) these cells were allowed ~ to recover for at least 1 week in culture. Subsequent analysis by flow cytometry revealed that these AG107-sorted cells had similar levels of H A binding when re-stimulated with T N F a (Figure 3.6). The dichotomy of AG107 expression in these cells was verified by treating a sample with neuraminidase and analyzing by flow cytometry for the level of A G 107 expression. It is possible that the mAb A G 107 is not sensitive enough to detect every A G 107 epitope present* and thus, the amount of A G 107 present in both A G 1 0 7  56  low  and A G 1 0 7  high  AG107  A G 1 0 7  AG107 9  | O W  HI  AG107 '9  iow  H  H  H  Figure 3.6. Induction of binding to fluoresceinated hyaluronic acid (FL-HA) in SR91 cells sorted for expression of the AG107 epitope (6-sulfo N-acetyllactosamine [LacNAc]/Lewis x). SR91 cells were stimulated with 10 ng/ml T N F a for 24 hr and then sorted by flow cytometry based on low or high expression level of the A G 107 epitope and then cultured separately for at least 1 week. The cells were then stimulated with 10 ng/ml T N F a for 24 hr and then assessed for expression of the AG107 epitope and binding to FL-HA by flow cytometry. Cells used for determining AG107 expression were incubated with 0.05 U/ml neuraminidase for 1 hr at 37°C to remove terminal sialic acid residues. (A) In the upper panel, histograms represent cells incubated with monoclonal antibody (mAb) AG107 (open) or secondary A b alone (filled). In the lower panel, histograms represent cells incubated with (open) or without (filled) FL-HA. Fluorescence intensity is represented on the x-axis and cell number on the y-axis. This is one representative experiment repeated 5 separate times. (B) Graphical representation of the expression of AG107 and binding to FL-HA in cells sorted for low or high expression of AG107. Values are the average from 5 separate experiments with error bars representing standard deviation.  57  cells may be sufficient to exert its maximum influence on H A binding. To rule out this possibility, TNFa-stimulated SR91 cells were sorted for low and high binding to H A and then treated with neuraminidase without further culturing to determine its level of A G 107 expression. As shown in Figure 3.7, low and high H A binding cells expressed similar levels of the A G 107 epitope when stimulated with T N F a . These results indicated that TNFa-induced binding to H A did not correlate with expression of the A G 107 epitope in SR91 cells.  3.6  Expression of the AG107 epitope did not lead to enhanced rolling on L -  selectin Recruitment of leukocytes to various tissues from the lymphatic or vascular system requires a cascade of interactions beginning with the initial capture or rolling phase. In peripheral lymph nodes, the rolling phase is mediated by L-selectin on naive T lymphocytes and glycoproteins on high-walled endothelial venules (HEV) bearing the sialyl 6-sulfo Lewis x carbohydrate moiety (35, 36). Since the AG107 epitope was only detected after the removal of terminal sialic acid residues, it indicated the presence of sialyl 6-sulfo LacNAc and/or sialyl 6-sulfo Lewis x epitopes on CD44. Therefore, it was assessed whether or not the AG107-defined determinant could mediate rolling on L selectin, and therefore, assist in the capture of monocytes from the peripheral blood. Using a parallel-plate flow chamber to mimic blood flow, unstimulated (AG107" ) and ve  TNFa-stimulated (AG107 ) SR91 cells were passed over immobilized L-selectin/IgM +ve  (L-sel/IgM) at a physiological flow rate of 1 dyne/cm . As shown in Figure 3.8, rolling 2  was essentially abolished when the cells were pre-treated with neuraminidase or  58  A  HA  kw  H A  high  Figure 3.7. Expression of the AG107 epitope (6-sulfo W-acetyllactosamine [LacNAc]/Lewis x) in TNFa-stimulated SR91 cells sorted for low and high binding to fluoresceinated hyaluronic acid (FL-HA). (A) SR91 cells were stimulated with 10 ng/ml T N F a for 24 hr and then sorted by flow cytometry based on low or high binding to FL-HA Sorted cells were then incubated with 0.05 U/ml neuraminidase for 1 hr at 37°C to remove terminal sialic acid residues and assessed for AG107 expression by flow cytometry. (A) In the upper panel, histograms represent cells incubated with (open) or without (filled) FL-HA. In the lower panel, histograms represent cells incubated with monoclonal antibody (mAb) AG107 (open) or secondary Ab alone (filled). Fluorescence intensity is represented on the x-axis and cell number on the y-axis. This is one representative experiment repeated 5 separate times. (B) Graphical representation of the binding to FL-HA and expression of AG107 in cells sorted for low or high FL-HA binding. Values are the average from 5 separate experiments with error bars representing standard deviation.  59  -TNF-o  +TNF-o  AG107  11  CD44 CD44  \  A,  A  tow  -ve control  10' 10 10 10' 10 10 Fluorescence Intensity 2  3  2  3  B  Mm  91  =  * 5 100  •3-S  70  !  60  "5 a  i  52  40  ^  •is  0  2&50  ° "8  40  U 30 10  | gTNFa 20 Neurarrinidase EDTA -  C  80  Q,£  0  + +  TNFa  +  + +  CQ^jlaw  +  CO  TNF-a CD44  tow  Neuraminidase EDTA  Figure 3.8. Effect of the AG107 epitope (6-sulfo .V-acetyllactosamine [LacNAc]/Lewis x) and CD44 on rolling on immobilized L-selectin/lmmunoglobulin M (L-sel/IgM) in SR91 cells. SR91 cells were incubated with or without 10 ng/ml TNFa for 24 hr. Cells co-incubated on an immobilized C O M Ab IM7.8.1 expressed reduced levels of CD44 on the cell surface (CD44 ). Cells used f a determining AG107 expression were incubated with 0.05 U/ml neuraminidase for 1 hrat 37°C to remove terminal sialic acid residues. (A) Flow cytometric analysis of CD44 (using mAb 3G12) and AG107 expression. The negative control was secondary Ab alone (-ve control). (B, C) For rolling experiments, cells were resuspended at 1X10 cells/ml in complete RPMI media with or without 10mM EDTAand perfused over immobilized L-sel/IgM in a parallel-plate flow chamber at a flow rate of 1 dyne/cm . This is one representative experiment repeated at least 3 times with error bars indicating standard deviation. (D) Graphical representation of relative number of rolling cells observed when treated as indicated compared to unstimulated SR91 cells set to a value of 1 and represented by the dotted line, values represent the average of at least 3 independent experiments with error bars indicating standard error of measurement. *, P < 0.05; **, P< 0.01 as determined by Student's f test. |0W  6  2  60  resuspended in the presence of 10 m M E D T A . This is consistent with reports that L selectin ligands are both sialylated and dependent on divalent cations. However, fewer TNFa-stimulated (AG107 ) cells rolled on immobilized L-sel/IgM compared to +ve  unstimulated (AG107" ) cells. This suggests that the A G 107 epitope or the increased ve  expression of CD44 may actually inhibit L-selectin mediated rolling. To address this issue, SR91 cells were grown on immobilized CD44 Ab to decrease surface expression of CD44. Normal unstimulated cells and unstimulated cells with decreased expression of CD44 (CD44 ) had similar number of cells roll on L-sel/IgM. However, more T N F a l0W  stimulated C D 4 4  low  cells rolled on L-sel/IgM when compared to TNFa-stimulated cells.  Moreover, the number of rolling cells in TNFa-stimulated C D 4 4  low  cells increased to the  level seen in unstimulated cells. Taken together, these observations indicated that i) the A G 107 epitope did not enhance and may actually inhibit rolling on L-selectin, ii) T N F a modified CD44 may inhibit rolling on L-selectin, and iii) CD44 was not a major L selectin ligand.  3.7  Expression of GlcNAc6STs, 6-sulfo LacNAc-containing epitopes and CD44  sulfation in human macrophages  After peripheral blood monocytes enter tissues they differentiate into distinct functional subsets of dendritic cells and macrophages depending on the signals received from the surrounding microenvironment. Studies in mice have shown that the classical macrophage, which is now also referred to as the M l macrophage, is induced by exposure to IFNy and LPS or T N F a alone and is characterized by high secretion levels of TNFa (TNFa  high  ) and low secretion levels of IL-10 (IL-10 ) (7, 12). While M2 low  61  macrophages represent a heterogeneous group, they have several features in common, such as being T N F a  l o w  and IL-10  hlgh  . Functionally, M l macrophages have been  implicated in Thl-mediated immune responses and are pro-inflammatory while M 2 macrophages have been implicated in Th2-mediated and anti-inflammatory responses as well as wound healing. A recent study by Verreck and colleagues (18) demonstrated that human peripheral blood monocytes were polarized into macrophages displaying M l and M2 properties after culture in media containing GM-CSF or M-CSF, respectively. To determine if GlcNAc6STs had a role in the functions associated with M l and M2 )  macrophages, it was important to assess i f GlcNAc6STs and 6-sulfo LacNAc-containing epitopes were expressed in these macrophage subsets. 3.7.1  Morphology and cytokine profile of human GM-CSF- and M-CSF-cultured  macrophages Human P B M were isolated from whole blood of healthy volunteers and cultured in media containing 10 ng/ml GM-CSF or 50 ng/ml M-CSF over 6-7 days to differentiate into macrophages with M l and M2 properties, respectively (18). Figure 3.9A shows that P B M (CD14  +ve  P B M C ) were effectively isolated from P B M C using anti-CD 14-  conjugated magnetic beads. Morphologically, GM-CSF-cultured macrophages were round while the majority of M-CSF-cultured macrophages were very long and thin (Figure 3.9B). However, most M-CSF-cultured macrophages adopted a circular morphology when incubated with 500 U/ml IFNy plus 10 ng/ml LPS or with 20 ng/ml T N F a for 24 hr. Analysis of the culture supernatant by an enzyme-linked immunosorbant assay (ELISA) confirmed that GM-CSF-cultured macrophages were  62  Figure 3.9. Isolation of human peripheral blood monocytes and morphology and cytokine profile of  GM-CSF (M1)- and M-CSF (M2)-cultured macrophages. Human peripheral blood mononuclear cells ( P B M C ) were separated from whole blood by centrifugation over a Ficoll-Paque Plus density gradient. CD14 peripheral blood monocytes ( P B M ) were isolated from P B M C using anti-CD14-conjugated magnetic beads. (A) Flow cytometry for C D 3 , C D 1 4 and C D 1 9 expression before and after separation with anti-CD14-conjugated magnetic beads. The negative control w a s an isotype matched control A b (Ab control). Fluorescence intensity is represented on the x-axis and cell number on the y-axis. C D 1 4 PBM were cultured for 6 or 7 days in the presence of 10 ng/ml G M - C S F or 50 ng/ml M - C S F to polarize cells into an M1 or M 2 macrophage phenotype, respectively. T h e s e cells were subsequently incubated with or without 500 U/ml IFNy plus 10 ng/ml L P S or with or without 20 ng/ml T N F a for an additional 24 hr before (B) digital images were obtained at a magnification of 200X and (C) the concentration of T N F a or IL-10 in the culture supernatant w a s determined by an enzyme-linked immunosorbant assay (ELISA). All the above data are representative of experiments repeated at least 3 times. + v e  + v e  63  TNFa  h i g h  and IL-10  low  when stimulated with IFNy and LPS and M-CSF-cultured  macrophages were T N F a  l o w  and IL-10  hlgh  when stimulated with IFNy and LPS.  Unstimulated M-CSF-cultured macrophages also secreted IL-10, but at lower levels compared to cells stimulated with IFNy and LPS. T N F a did not affect the amount of IL10 secreted by M-CSF-cultured macrophages. These results confirm that human G M CSF- and M-CSF-cultured macrophages displayed the T N F a and IL-10 secretion profile reported for human M l and M2 macrophages, respectively (18). 3.7.2  GlcNAc6ST-l and GlcNAc6ST-4 were upregulated by inflammatory  mediators in human M-CSF- but not GM-CSF-cultured macrophages To determine if GlcNAc6STs were expressed and regulated by inflammatory mediators total R N A was isolated from human GM-CSF- and M-CSF-cultured macrophages and subjected to semiquantitative RT-PCR. GlcNAc6ST-l and GlcNAc6ST-4 transcripts were detected in GM-CSF-cultured macrophages but both transcript levels did not increase (1.6+1.1 fold for GlcNAc6ST-l and 1.3 ± 0.6 fold for GlcNAc6ST-4) when incubated with 500 U/ml IFNy and 10 ng/ml LPS for 24 hr (Figure 3.10). In contrast GlcNAc6ST-l transcripts were increased in M-CSF-cultured macrophages incubated with IFNy plus LPS (2.6 ± 0.8 fold) or with T N F a (4.0 ± 1.9 fold). In addition, GlcNAc6ST-4 transcripts were increased in M-CSF-cultured macrophages incubated with IFNy plus LPS (2.0 ± 0.8 fold) and there was a tendency for GlcNAc6ST-4 transcripts to increase (1.7 ± 0.7 fold) when incubated with T N F a . Transcript levels for GlcNAc6ST-2, -3 and 5 were not detected or detected at low levels  64  M1  M2  I FN/, L P S  TNFa GcNac6ST-1|  |>acb'n | GcNAc6BT-4  p-actin  B  •  I 26  • GlcNAc6ST-4  i i si  "I II o>  GlcNAc6ST-1  n  14]  4  31  IFNy, LPS TNFa  M1  M2  Figure 3.10 Effect of IFNy plus LPS and TNFa on the expression of A/-acetylglucosamine 6-0 sulfotransferase (GlcNAc6ST)-1 and GlcNAc6ST-4 transcripts in human GM-CSF (M1)- and M-CSF (M2)-cultured macrophages. Human M1 and M2 macrophages were derived from C D 1 4 peripheral blood monocytes incubated with 10 ng/ml GM-CSF or 50 ng/ml M-CSF for 6-7 days, respectively. Total RNA amounting to 0.75-3.5 ng was isolated from M1 macrophages cultured with or without 500 U/ml IFNy and 10 ng/ml LPS and from M2 macrophages cultured with or without 500 U/ml IFNy and 10 ng/ml LPS or 20 ng/ml TNFa for 24 hr. (A) Semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) was performed for 32-35 cycles for GlcNAc6ST-1 or GlcNAc6ST-4 and compared with p-actin transcripts amplified for 23 cycles in the same tube. (B) Graphical representation of the relative fold increase in GlcNAc6ST-1 and GlcNAc6ST-4 in stimulated cells when compared to their respective unstimulated cells, which was assigned a value of 1 (dotted line). Values represent the average from at least 4 separate experiments with the error bars representing standard deviation. +ve  65  and were not increased upon stimulation with IFNy plus LPS or with T N F a in GM-CSFor M-CSF-cultured macrophages (data not shown). These results indicated that GlcNAc6ST-l and GlcNAc6ST-4 were upregulated in response to pro-inflammatory mediators IFNy, LPS and T N F a in human M-CSF- but not in GM-CSF-cultured macrophages. 3.7.3  No increase in CD44 sulfation in human M-CSF-cultured macrophages in  response to IFNy plus LPS or to TNFa To assess whether increased GlcNAc6ST-l and GlcNAc6ST-4 transcripts in M CSF-cultured macrophages stimulated with IFNy plus LPS or with T N F a resulted in increased sulfation of CD44 compared to unstimulated cells, CD44 was immunoprecipitated from these cells incubated in the presence of [ S]sulfate. As shown 35  in Figure 3.11, expression of CD44 was increased in M-CSF-cultured macrophages stimulated with IFNy plus LPS or with T N F a . However, the amount of [ S]sulfate 35  incorporated per CD44 molecule was not significantly increased in the stimulated M CSF-cultured macrophages. 3.7.4  Human M-CSF-cultured macrophages did not express the AG 107 epitope in  response to IFNy plus LPS or to TNFa Although no overall increase in CD44 sulfation was observed, it was still possible that certain sulfated carbohydrate structures were upregulated as long as there was a compensatory loss of other sulfate-bearing carbohydrate structures. Therefore,  66  IFN , LPS TNFa  B  Y  2 i  [ S] sulfate  E  a s  E  o  35  1.51 •  .1-3 "S  CD44  S • 0.5  IFN , LPS TNFa Y  Figure 3.11. Effect of IFNy plus LPS and TNFa on CD44 sulfation in human M-CSF-cultured  (M2) macrophages. Human M2 macrophages were derived from C D 1 4 peripheral blood monocytes incubated with 50 ng/ml M-CSF for 6-7 days before incubation with or without 500 U/ml IFNy plus 10 ng/ml LPS or 20 ng/ml TNFa for 24 hrinthe presence of 100 pCi/ml N a S 0 . (A) + v e  2  3 5  4  CD44 was immunoprecipitated and upper panel indicates [ S]sulfate incorporation as assessed by autoradiography and lower panel indicates CD44 expression as determined by western blotting with the monoclonal Ab (mAb) Hermes-3. Molecular mass markers are indicated at the right in kDa. (B) Graphical representation of relative amount of [ S]-sulfate incorporated per CD44 molecule in M2 macrophages when treated as indicated. Values are the average from 3 separate experiments with error bars representing standard deviation. 35  35  67  unstimulated and M-CSF-cultured macrophages incubated with IFNy plus LPS were analyzed by flow cytometry for the expression of the AG107 epitope. Binding to mAb A G 107 was not observed in unstimulated M-CSF-cultured macrophages and in these cells stimulated with IFNy and LPS when treated with neuraminidase and western blotting did not detect the AG107 epitope on CD44 (Figure 3.12). However, M-CSFcultured macrophages did express the DD-2 epitope (6-sulfo LacNAc), as determined by flow cytometry, but its expression was not upregulated by stimulation with IFNy and LPS. The lack of mAb DD-2 reactivity before neuraminidase treatment indicated that the determinant present on human M-CSE-cultured macrophages was sialyl 6-sulfo LacNAc.  3.8  Human M-CSF-cultured macrophages bind more HA and expressed higher  levels of CD14 than GM-CSF-cultured macrophages Similar to SR91 cells and human P B M , binding to F L - H A was low in unstimulated GM-CSF-cultured macrophages and was induced with pro-inflammatory mediators. By contrast, most unstimulated M-CSF-cultured macrophages were able to bind F L - H A and stimulation with IFNy and LPS resulted in all cells being able to bind even higher amounts of F L - H A . These macrophages also expressed high levels of C D 14 while GM-CSF-cultured macrophages had low to non-detectable levels of CD 14 (Figure 3.13).  3.9  Expression of other 6-sulfo A'-acetyllactosamine containing epitopes Numerous sulfated carbohydrate determinants have been identified on biological  compounds. A study of glycoproteins from the mucins of patients with cystic fibrosis  68  Unstimulated  +IFNy + L P S  -Neuraminidase DD-2  2F3  A,  ft i ^ 11 i  I.  A b control 10  1  10  2  10  10  3  1  10  2  10  3  9 .a  +Neuraminidase  DD-2  A G 107  A b control  5  o  1  10  1  10  2  10  10  3  1  10  2  10  3  F l u o r e s c e n c e intensity  B  IFNy, LPS TNFa  G1 *  AG  107  CD44  1 •  h175  M M l Ml  h83  Figure 3.12. Expression of 2F3, AG107 and DD-2 epitopes in human M-CSF-cultured (M2)  macrophages incubated with or without IFNy plus LPS. Human M2 macrophages were derived from C D 1 4 peripheral blood monocytes and incubated with 50ng/ml M-CSF for 6-7 days followed by incubation with or without 500U7ml IFNy and 10ng/ml LPS for 24 hr. (A) Cells were treated with or without 0.05U/ml neuraminidase for 1 hr at 3 7 ° C to remove terminal sialic acid residues and then analyzed for binding to the 2F3, A G 1 0 7 or DD-2 monoclonal Abs. The negative control was secondary Ab alone (Ab control). Fluorescence intensity is represented on the x-axis and cell number on the y-axis. This is one representative experiment repeated 2 times. (B) M2 macrophages were incubated with or without 500U7ml IFNy and 10ng/ml L P S or with 20ng/ml T N F a for 24 hr and treated with 0.05U/ml neuraminidase for 1 hr at 3 7 ° C . CD44 was immunoprecipitated and western blotted with mAb A G 107 (upper panel). The positive control was CD44 immunoprecipitated from ECV304 cells transfected with GlcNAc6ST-1 (G1). The lower panel indicates relative amounts of CD44 immunoprecipitated and used for western blotting in the upper panel. Molecular markers are indicated at the right in kDa. This is one representative experiment repeated 3 times. + v e  69  M1 macrophage Unstimulated  +IFNy+LPS  FL-HA -ve control CD14 Ab control 10  1  10  2  10 10 10 Fluorescence intensity 3  1  2  10  M2 macrophage Unstimulated  +IFNy +LPS  FL-HA  :z\:  -ve control CD14  J  Ab control 10  1  10  2  10 10 10 Fluorescence intensity 3  1  2  10  E  3 .C  "53 O  3  Figure 3.13. Effect of TNFa on binding to fluoresceinated hyaluronic acid (FL-HA) in human GM-CSF (M1)- and M-CSF (M2)-cultured macrophages. Human M1 and M2 macrophages were derived from CD14 peripheral blood monocytes incubated with 10 ng/ml GM-CSF or 50 ng/ml M-CSF for 6-7 days, respectively. Cells were then incubated with or without 500 U/ml IFNy and 10 ng/ml LPS for an additional 24 hr and analyzed by flow cytometry for CD14 expression and binding to FL-HA. The negative control for the anti-CD14 Ab was an isotype matched control Ab (Ab control). The negative control for FL-HA was cells alone (-ve control). Fluorescence intensity is represented on the x-axis and cell number on the y-axis. This is one representative experiments repeated at least 3 separate times. +ve  70  revealed an array of changes to the carbohydrate structures expressed, many of which contained 6-sulfo LacNAc moieties. The myeloid cell lines SR91, THP-1 and U937 were examined for binding to a range of mAbs specific for certain sulfated carbohydrate moieties, each containing a 6-sulfo LacNAc moiety. In addition, ECV304 and ECV304 cells transfected with GlcNAc6ST-l were examined to determine i f GlcNAc6ST-l was implicated in the synthesis any of these 6-sulfo LacNAc moieties. 3.9.1  Expression of other 6-sulfo V<-acetyllactosamine containing epitopes in SR91 cells SR91 cells were stimulated with T N F a and then treated with or without  neuraminidase. The cells were then analyzed for binding to mAbs that recognize a variety of 6-sulfo LacNAc-containing epitopes by flow cytometry. The lack of mAb A G 107 reactivity before neuraminidase treatment indicated that the determinant present on SR91 cells was masked by sialic acid, suggesting that the sulfated epitope was sialyl 6-sulfo LacNAc/Lewis x. However, of the 3 mAbs that can recognize these structures (2F3 [sialyl Lewis x/sialyl 6-sulfo Lewis x], G72 [sialyl 6-sulfo LacNAc/Lewis x], and G152 [sialyl 6-sulfo Lewis x]), only mAb 2F3 showed any reactivity (Figure 3.14). SR91 cells treated with neuraminidase did not bind mAb 2F3, which indicated that terminal sialic acid residues were effectively removed. The AG107 mAb is not able to distinguish between 6-sulfo LacNAc and 6-sulfo Lewis x. The fact that mAbs DD-2 (6-sulfo LacNAc) and AG223 (6-sulfo Lewis x) bound to neuraminidase-treated SR91 cells indicated that both sulfated moieties were present on TNFa-stimulated SR91 cells. Some binding to mAb DD-2 was observed in TNFa-stimulated SR91 cells not treated with neuraminidase. This revealed that not all 6-  71  2F3  AG223  DD-2  G72  G152  Figure 3.14. Expression of other 6-sulfo /V-acetyllactosamine (LacNAc)-containing epitopes in SR91 cells. SR91 cells were stimulated with or without 10 ng/ml TNF« for 24 hr and then incubated with or without 0.05 U/ml neuraminidase for 1 hr at 37°C. The cells were then analyzed for binding to monoclonal Abs specific for a variety of 6-sulfo LacNAc-containing epitopes by flow cytometry (open histograms). Filled histograms represent isotype matched control Ab. Fluorescence intensity is represented on the x-axis and cell number on the y-axis. All histograms are from one representative experiment repeated at least 3 times.  72  sulfo LacNAc epitopes were masked by terminal sialic acid residues. The inability of mAb A G 107 to detect these epitopes may be explained by a lower A b affinity or to differences in the underlying glycan structure that can sterically block access for the mAb AG107. 3.9.2  Expression of other 6-sulfo N-acetyllactosamine containing epitopes in THP-1  cells THP-1 is a monocytic cell line that can be differentiated with P M A to obtain characteristics of macrophages (170, 171). In the absence of P M A , GlcNAc6ST-l transcripts can be induced by incubation with T N F a (Figure 3.15). In addition, GlcNAc6ST-4 transcripts were present at a low level and were not upregulated by T N F a . Furthermore, binding to F L - H A was increased with T N F a stimulation. Therefore, monocytic THP-1 cells respond similarly to T N F a as SR91 cells with respect to the expression of GlcNAc6ST-l and GlcNAc6ST-4 and to F L - H A binding. However, the A G 107 and AG223 epitopes were not expressed at a level that could be detected by their respective mAbs. Upon differentiation with P M A into macrophage-like cells, both A G 107 and AG223 epitopes were detectable and their expression levels were increased slightly with T N F a stimulation. Paradoxically, while transcripts for GlcNAc6ST-l was induced by P M A and not further enhanced by T N F a in these differentiated cells, the levels present were below what was detected in undifferentiated cells stimulated with T N F a . This suggested that expression of GlcNAc6ST-l is not sufficient for the synthesis of the AG107 and AG223 epitopes in monocytic THP-1 cells. This is not too surprising since these epitopes would also require the presence and co-ordinated actions of the appropriate GlcNAc-, Gal- and Fucosyl-transferases. Despite the lower transcript levels  73  -PMA  A  GlcNAc6ST-1 TNFa  -  +~  +PMA  GlcNAc6ST-4  GlcNAc6ST-1  ~ -  ~  ~+~  ~+~  GlcNAc6ST-4 -  +  B  CD44  FL-HA  2F3  AG107  AG223  DD-2  G72  G152  Figure 3.15. Effect of TNFa on binding to fluoresceinated hyaluronic acid (FL-HA) and expression of C D 4 4 and 6-sulfo /V-acetyllactosamine (LacNAc)-containing epitopes in THP-1 cells incubated with or without PMA. (A) Total RNA amounting to 5 ug was isolated from THP-1 cells incubated with or without 10 ng/ml PMA for 24 hr followed by incubation with 10 ng/ml TNFa for an additional 24 hr. Semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) was performed for 35 cycles for GlcNAc6ST-1 or GlcNAc6ST-4 and compared with p-actin transcripts amplified for 23 cycles in the same tube. (B) Cells incubated with or without 0.05 U/ml neuraminidase for 1 hr at 37°C were analyzed for binding to CD44, FLHA and to monoclonal Abs specific for a variety of 6-sulfo LacNAc-containing epitopes by flow cytometry (open histograms). Filled histograms represent the negative control which was an isotype matched control Ab. The negative control for FL-HA was cells alone. Fluorescence intensity is represented on the x-axis and cell number on the y-axis. All histograms are from one representative experiment repeated at least 3 times.  74  of GlcNAc6ST-l, PMA-differentiated THP-1 cells were able to generate the A G 107 and AG223 epitopes. Moreover, a role for GlcNAc6ST-4 in the synthesis of these epitopes cannot be ruled out in these cells as P M A also upregulated the number of transcripts for GlcNAc6ST-4. Expression of the DD-2 epitope did correlate with the expression of GlcNAc6ST-l transcripts in undifferentiated and PMA-differentiated THP-1 cells. As in SR91 cells, the presence of the A G 107 and AG223 epitopes did not correspond with reactivity to mAbs G72 and G152. The inability of T N F a to upregulate the expression of GlcNAc6ST-l and GlcNAc6ST-4 in PMA-differentiated cells mirrors the results seen with human M l macrophages. The lack of A G 107 and AG223 expression argues that neither sulfated epitope, as defined by these mAbs, was involved in the TNFa-mediated upregulation of binding to F L - H A observed in monocytic THP-1 cells. 3.9.3  Expression of other 6-sulfo ~N-acetyllactosamine containing epitopes in U937  cells Similar to THP-1 cells, U937 is a monocytic cell line that can be differentiated with P M A to obtain macrophage characteristics (172, 173). In the absence of P M A , GlcNAc6ST-l and GlcNAc6ST-4 transcripts were present but were not induced with T N F a (Figure 3.16). Binding to F L - H A was only increased slightly with T N F a . Unlike undifferentiated THP-1 cells, undifferentiated U937 cells did express the A G 107 and AG223 epitopes. When incubated with P M A , expression of A G 107 and AG223 epitopes decreased. Conversely, transcript levels for GlcNAc6ST-l and GlcNAc6ST-4 were increased with P M A . In both cases, there was no further increase observed with T N F a stimulation. These results reinforce the notion that expression of GlcNAc6ST-l and  75  /  A  -PMA GlcNAc6ST-1 TNF«  -  +  +PMA  GlcNAc6ST-4 -  GlcNAc6ST-1  +  +  GlcNAc6ST-4 -  +  B  CD44  FL-HA  2F3  A G 107  AG223  DD-2  Figure 3.16. Expression of 6-sulfo /V-acetyllactosamine (LacNAc)-containing epitopes in U937 cells incubated with or without PMA. (A) Total R N A amounting to 5 ug was isolated from U937 cells incubated with or without 10 ng/ml P M A for 48 hr followed by incubation with 10 ng/ml T N F a for an additional 24 hr. Semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) was performed for 35 cycles for GlcNAc6ST-1 or GlcNAc6ST-4 and compared with p-actin transcripts amplified for 23 cycles in the same tube. (B) Cells incubated with or without 0.05 U/ml neuraminidase for 1 hr at 37°C were analyzed for binding to CD44, F L - H A and to monoclonal A b s specific for a variety of 6-sulfo LacNAc-containing epitopes by flow cytometry (open histograms). Filled histograms represent the negative control which was an isotype matched control Ab. The negative control for F L - H A w a s cells alone. Fluorescence intensity is represented on the x-axis and cell number on the y-axis. All histograms are from one representative experiment repeated at least 3 times.  76  GlcNAc6ST-4 alone are not reliable indicators for the expression of certain sulfated determininants, such as those defined by mAbs A G 107 and AG223. Clearly, other aspects of the cellular glycosylation machinery are important regulatory factors for the synthesis of these and other sulfated epitopes. 3.9.4  Effect of GlcNAc6ST-l on the expression of other 6-sulfo N-acetyllactosamine  containing epitopes in ECV304 cells  The expression of GlcNAc6ST-l in ECV304 cells resulted in the expression of the AG107 epitope. Therefore, these cells were used to determine i f GlcNAc6ST-l could also lead to the expression of other 6-sulfo LacNAc-containing epitopes. As shown in Figure 3.17, parental ECV304 cells did not express DD-2, G72 or G152 epitopes while ECV304 cells transfected with GlcNAc6ST-l did, thus, implicating GlcNAc6ST-l in their synthesis.  3.10  Expression of GlcNAc6ST-l and GlcNAc6ST-4 were not induced in mouse  macrophages under inflammatory conditions  ^  A mouse model system was sought to allow for studies aimed at determining i) the role of GlcNAc6STs in the inflammatory response and ii) the role of the corresponding 6-sulfo LacNAc-containing epitopes on CD44 in monocytes and macrophages. 3.10.1 Expression of GlcNAc6ST-l and GlcNAc6ST-4 were not induced while C6ST-1 was downregulated by TNFa in bone marrow-derived macrophages  Bone marrow was isolated from C57B1/6 mice and differentiated into macrophages by culturing with L-cell conditioned medium (LCCM), a source of M-CSF.  77  2F3  DD-2  G72  G152  Figure 3.17. Effect of expression of W-acetylglucosamine 6-0 sulfotransferase-1 (GlcNAc6ST-1) on the expression of other 6-sulfo W-acetyllactosamine (LacNAc)containing epitopes in ECV304 cells. ECV304 cells (-) and ECV304 expressing GlcNAc6ST-1 (+) were incubated with or without 0.05 U/ml neuraminidase for 1 hr at 37°C to remove terminal sialic acid residues. Cells were then analyzed for binding the the monoclonal Abs 2F3, DD-2, G72 and G152 by flow cytometry (open histograms). Filled histograms represent isotype matched control Ab. Fluorescence intensity is represented on the x-axis and cell number on the y-axis. All histograms are from one representative experiment repeated at least 3 times.  78  Unstimulated  +TNFa  F4/80 CD44 Ab control FL-HA -ve control  A 1  J  10  10  1  2  10  3  k 10  1  OJ  -O E  c "5  o  10  2  10  3  B BMDM TNF(/.  .  TNFK  +  GlcNAc6ST-1  GlcNAc6ST-4  /  BMDM .  +  I^ ' mmm  I ~~ r  TNFu  !  BMDM -  +  C6ST-1 p-actin  Figure 3.18. Effect of TNFa on binding to fluoresceinated hyaluronic acid (FL-HA) and the expression of A/-acetylglucosamine 6-0 sulfotransferase (GlcNAc6ST)-1, GlcNAc6ST-4 and chondroitin sulfotransferase-1 (C6ST-1) transcripts in mouse bone marrow-derived macrophages (BMDM). Mouse macrophages were derived from ceils isolated from the bone marrow and cultured for 4 days in media containing macrophage colony stimulating factor (MC S F ) before co-incubating with or without 20 ng/ml T N F a for an additional 3 days. (A) Cells were analyzed for binding to FL-HA and for expression of CD44 and the F4/80 epitope. T h e negative control for FL-HA was cells alone (-ve control). The negative control for the anti-CD44 and anti-F4/80 A b s was secondary Ab alone (Ab control). Fluorescence intensity is represented on the x-axis and cell number on the y-axis. This is one representative experiment repeated at least 10 times. (B) Total R N A amounting to 5 |ig was isolated and subjected to semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) for 4 0 cycles for GlcNAc6ST-1, GlcNAc6ST-4 or C6ST-1 and compared with p-actin transcripts amplified for 23 cycles in the same tube. R T - P C R was performed on R N A isolated from mouse brain and kidney to verify the specificity of the GlcNAc6ST-1 and GlcNAc6ST-4 primers, respectively. This is one representative experiment repeated at least 3 times.  79  As shown in Figure 3.18, these cells were positive for F4/80, a marker of mouse macrophages (186). Binding to F L - H A was upregulated in bone marrow-derived macrophages (BMDM) after stimulation with 20 ng/ml T N F a for 72 hr. However, transcripts for GlcNAc6ST-l and GlcNAc6ST-4 were not detected by RT-PCR in both unstimulated and TNFa-stimulated B M D M . This result was not specific to the strain of mice as no transcripts for GlcNAc6ST-l and GlcNAc6ST-4 were detected in B M D M from balb/c mice (data not shown). R N A isolated from tissues known to express GlcNAc6ST-l and GlcNAc6ST-4 was used to verify the specificity of the primers used to detect these transcripts. In a recent report, the removal of CS on CD44 results in increased binding to F L - H A (187). Additional studies in B M D M and T cells suggest that H A binding may be regulated by the addition and removal of CS in these cells (Johnson lab, unpublished results). Here, semiquantitative RT-PCR revealed that transcripts for Chondroitin 6-0 Sulfotransferase-1 (C6ST-1) were present in B M D M and were reduced to non-detectable levels when stimulated with T N F a . 3.10.2 No expression of 2F3, AG 107 and DD-2 epitopes in bone marrow-derived macrophages stimulated with or without TNFa Flow cytometry revealed that B M D M stimulated with or without 20 ng/ml T N F a for 72 hr did not express the 2F3, AG107 or DD-2 epitopes (Figure 3.19). 3.10.3 Expression of GlcNAc6ST-l and GlcNAc6ST-4 were not induced by TNFa in thioglycollate-elicitedperitoneal macrophages While B M D M can serve as a plentiful source of murine macrophages, they were differentiated in culture and do not have some of the properties and characteristics observed in tissue macrophages. As a result, B M D M may not have been exposed to  80  Unstimulated  -Neuraminidase  U DD-2 AG 107 2F3  +TNFa  7 y  A  / \  . -m  \  X  /  Ab control 10  1  10  2  10  10  3  10  1  2  10  3  3 C  ^Neuraminidase  0)  DD-2 AG 107 2F3  cu  s  o  \  \  \  II  Ab control 10  1  10  2  10  10  3  1  10  2  10  3  Fluorescence intensity  Figure 3.19. Expression of 6-sulfo /V-acetyllactosamine (LacNAc)-containing epitopes 2F3, AG107 and DD-2, in mouse bone marrow-derived macrophages (BMDM) stimulated with or without TNFa. Macrophages were derived from cells isolated from the bone marrow of C57BI/6 mice and cultured for 4 days in media containing M-CSF before co-incubating with or without 20 ng/ml TNFa for an additional 3 days. Cells were treated with or without 0.05 U/ml neuraminidase for 1 hr at 37°C to remove terminal sialic acid residues and analyzed for binding to the 2F3, AG107 or DD-2 monoclonal Abs by flow cytometry. The negative control was secondary Ab alone (Ab control). Fluorescence intensity is represented on the x-axis and cell number on the yaxis. This is one representative experiment repeated at least 3 times.  81  certain microenvironmental cues crucial to its full maturation into macrophages. Therefore, some responses may be deficient. In an attempt to obtain macrophages that are more physiologically-relevant, thioglycollate was injected into the peritoneal cavity of mice to promote the local recruitment of macrophages. However, transcripts for GlcNAc6ST-l and GlcNAc6ST-4 were not detectable by RT-PCR in thioglycollateelicited peritoneal macrophages incubated with or without T N F a for 24 hr (Figure 3.20). 3.10.4 Expression of GlcNAc6ST-l and GlcNAc6ST-4 transcripts was not induced in splenic macrophages from mice infected with Listeria monocytogenes T N F a stimulation alone may not be enough to induce an upregulation of GlcNAc6STs in ex vivo macrophages. Therefore, mice were injected with L. monocytogenes (1 X 10 colony forming units) and splenic macrophages were isolated 3 5  days later. These macrophages have matured in vivo and were exposed to the entire milieu of inflammatory cytokines and microenvironmental signals including high amounts of IFNy and T N F a , which are essential for primary defence against L. monocytogenes. As shown in Figure 3.21, splenic macrophages (CD1 l b  +ve  ) from mice  infected with L. monocytogenes contained no detectable GlcNAc6ST-l transcripts and a barely detectable amount of GlcNAc6ST-4 transcripts as determined by RT-PCR. Similar results were seen in the remaining population of leukocytes (CD1 lb" ) isolated ve  from the spleen of mice infected with L. monocytogenes.  82  Figure 3.20. Effect of TNFa on the expression of N-acetylglucosamine 6-0 sulfotransferase (GlcNAc6ST)-1 and GlcNAc6ST-4 transcripts in mouse thioglycollate-elicited peritoneal macrophages. Total RNA amounting to 2 ug was isolated from macrophages (M<j>) isolated from the peritoneal cavity of mice injected with 1 ml 3% thioglycollate for 4 days and then cultured with or without 20 ng/ml TNFa for 24 hr. Semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) was performed for 40 cycles for GlcNAc6ST-1 or GlcNAc6ST-4 and compared with p-actin transcripts amplified for 22 cycles in the same tube. RT-PCR was performed on RNA isolated from mouse brain and kidney to verify the specificity of the GlcNAc6ST-1 and GlcNAc6ST-4 primers, respectively. This is one representative experiment repeated 3 times.  83  CD11b-  ve  cells  CD11b  + v e  M<t>  Figure 3.21. E x p r e s s i o n of A/-acetylglucosamine 6-0 sulfotransferase (GlcNAc6ST)-1 a n d GlcNAc6ST-4 in splenic macrophages from mice infected with Listeria monocytogenes. L. monocytogenes (cone.) was injected injected into the tail vein of mice 3 days prior to isolation of the spleen. Leukocytes from the spleen were incubated with anti-CD11b-conjugated magnetic beads to isolate macrophages and then analyzed by flow cytometry to verify separation of macrophages ( C D 1 1 b M<j>) from other cells (CD11b" cells). CD11b expression is represented by the open histogram. The closed histogram represents the negative control which was an isotype matched Ab (-ve control). Fluorescence intensity is represented on the x-axis and cell number on the y-axis. Total RNA amounting to 2ug was subjected to semiquantitative reversetranscriptase polymerase chain reaction (RT-PCR) for 40 cycles for GlcNAc6ST-1 or GlcNAc6ST4 and compared with p-actin transcripts amplified for 23 cycles. RT-PCR was performed on R N A isolated from mouse brain and kidney to verify the specificity of the GlcNAc6ST-1 and GlcNAc6ST-4 primers, respectively. This is one representative experiment repeated at least 3 times. +ve  ve  84  CHAPTER 4 Discussion  85  4.1  GlcNAc6ST-l and GlcNAc6ST-4 expression was inducible in human  peripheral blood monocytes Several observations have suggested that expression of GlcNAc6STs may be altered under chronic inflammatory settings. Under these conditions, many vessels are capable of binding MECA-79, a monoclonal Ab used to detect a major class of sulfated L-selectin ligands.(188). Moreover, a majority of these vessels also adopt other features reminiscent of H E V of secondary lymphoid organs (189, 190), including expression of the HEV-restricted sulfotransferase GlcNAc6ST-2 (134). However, the expression of GlcNAc6ST-l was not determined. Other studies have revealed that mucins and glycoproteins, the main components of the respiratory mucus, are oversulfated in patients with cystic fibrosis (191-194). Structural analysis revealed an abundance of novel as well as sulfated carbohydrate structures, including sialyl 6-sulfo Lewis x (195). In another study, Delmotte and colleagues (196) were able to demonstrate that T N F a increases the activity of several glycosyl- and sulfo-transferases in human bronchial mucosa, including GlcNAc6ST-l and galactose 3-0 sulfotransferase. GlcNAc6ST-l was also found to be upregulated in human umbilical vein endothelial cells by another pro-inflammatory cytokine, IL-1 p (175). Taken together, these studies demonstrate that expression of GlcNAc6STs are often regulated and may contribute to the pathogenesis of many inflammatory disorders. However, expression of GlcNAc6STs has only been examined in endothelial cells and epithelial cells at present. Except for some initial observations in SR91 cells (107, 164) and human P B M (100), regulation of sulfation and GlcNAc6STs in cells of the hematopoietic system has not been extensively studied. Herein, semiquantitative RT-PCR revealed that transcripts for GlcNAc6ST-l were induced by  86  T N F a in SR91 cells, whereas transcripts for both GlcNAc6ST-l and GlcNAc6ST-4 were upregulated in human P B M . This demonstrated that expression of specific GlcNAc6STs could be regulated in myeloid cells under inflammatory conditions.  4.2  GlcNAc6ST-l was implicated in the synthesis of the AG107 epitope on CD44 The increased expression of both GlcNAc6ST-l and GlcNAc6ST-4 in human  P B M raised the possibility that either sulfotransferase could be involved in the synthesis of the AG107 epitope on CD44. Exogenous expression of GlcNAc6ST-l in ECV304 cells demonstrated that this sulfotransferase could utilize CD44 as a substrate. However, a role for GlcNAc6ST-4 in the sulfation of CD44 could not be ruled out even though very little GD44 was sulfated in parental ECV304 cells where much higher transcript levels for GlcNAc6ST-4 was detected compared to GlcNAc6ST-l. Moreover, RT-PCR may not accurately reflect actual protein levels as there may be differences in the rate of translation and amplification efficiency for different transcripts. As done for GlcNAc6ST-l, simply overexpressing GlcNAc6ST-4 may be sufficient to verify whether or not CD44 is a substrate for this enzyme. Parental ECV304 cells did not express the AG107 epitope on CD44, whereas cells transfected with GlcNAc6ST-l did. This indicated that GlcNAc6ST-l was involved in the synthesis of the A G 107 epitope on CD44 in human P B M . Once again, a role for GlcNAc6ST-4 in this process cannot be excluded at this time. In vitro studies have revealed that GlcNAc6STs prefer to sulfate terminal GlcNAc residues (162). Furthermore, GlcNAc6ST-l and GlcNAc6ST-4 preferred to sulfate T V linked glycans (130, 132, 144, 145) while GlcNAc6ST-2 showed a preference for O-  87  linked glycans (145). In addition to intrinsic substrate specificity, there is evidence that the localization of the sulfotransferse within the Golgi may also influence what glycans are sulfated. This is thought to reflect variances in the prevalence of terminal GlcNAc residues on TV- and O-linked glycans at different compartments within the Golgi. For instance, GlcNAc6ST-l is predominantly localized to the trans-Golgi network where TVlinked glycans are more likely to be elaborated with terminal GlcNAc residues (145). However, localization of GlcNAc6ST-4 within the Golgi was not examined in that study. GlcNAc6ST-l and GlcNAc6ST-4 share a preference for TV-linked glycans and are present in many of the same tissues. A comparison of the distribution of these sulfotransferases within the Golgi would determine if they were exposed to the same pool of carbohydrate structures and would provide a valuable insight into their potential for functional redundancy. Sulfation of CD44 in SR91 cells occurs on chondroitin sulfate as well as on both TV- and 0-linked glycans (164). While the overall sulfation of CD44 increases in these cells upon T N F a stimulation, the majority of the sulfation is added to TV-linked glycans, as was the A G 107 epitope. In this thesis, it was revealed that the A G 107 epitope was also present largely on TV-linked glycans of CD44 in ECV304 cells transfected with GlcNAc6ST-l. These results were consistent with previous studies that found GlcNAc6ST-l to have a preference for TV-linked glycans. Whether GlcNAc6ST-l also has a preference for synthesizing A G 107 epitopes on TV-linked glycans of CD44 in human human PBM remains to be determined.  88  4.3  Expression of the A G 1 0 7 epitope did not correlate with H A binding Previous results from our laboratory revealed an intriguing correlation between  increased sulfation of CD44 and H A binding in SR91 cells (107) and human P B M (100). However, because these studies used sodium chlorate, a general inhibitor of sulfation, it did not distinguish among sulfation from chondroitin sulfate or from N- or O-linked glycans. The identification of the A G 107 epitope allowed us to take a more specific approach: to ascertain whether this particular sulfated determinant was involved in upregulated H A binding induced by T N F a . Unfortunately, A G 107 could not be used as a blocking Ab because it required the removal of terminal sialic acid residues with neuraminidase, a process that induces H A binding beyond the levels observed in both unstimulated and TNFa-stimulated SR91 cells. Although not as powerful, sorting for cells based on high or low expression levels of the A G 107 epitope revealed that both populations were induced to bind H A to similar levels. Cells sorted based on high or low binding to H A expressed similar amounts of the A G 107 epitope. These results suggest that H A binding was not affected by the presence of 6-sulfo LacNAc/Lewis x on CD44 at the levels detectable by mAb A G 107. It remains possible that the maximal effect of the A G 107 epitope on H A binding can be accomplished by lower levels of the epitope, beyond the detection limit of the Ab. Several studies have indicated that changes in glycosylation affects H A binding in monocytes. Mutational analysis of murine CD44 revealed that the presence of TV-glycans at two of the five potential TV-glycosylation sites, Asn25 (NI) and Asnl20 (N5), has a strong inhibitory effect on H A binding (165). In addition, the enzymatic removal of terminal sialic acid residues from these TV-linked glycans by neuraminidase induces H A  89  binding (105). Other studies have extended these findings to human P B M , where increased binding to H A in response to T N F a or LPS was associated with increased endogenous sialidase activity (102, 103). It was apparent from X-ray crystallography and N M R spectroscopy how H A binding may be inhibited by charge repulsion from the addition of an TV-linked glycan bearing negatively charged sialic acid residues at TV1 (197). However, TV5 is located on the opposite side from where H A interacts with CD44, but near the binding sites for the anti-CD44 mAbs IRAWB14 and IRAWB26 (197, 198). These mAbs induce large increases in H A binding by clustering CD44 and thereby increasing avidity for H A (198, 199). It was postulated that sialic acid residues present on the TV5 glycan could interfere with this clustering (197). It seems counterintuitive to speculate that 6-sulfo LacNAc/Lewis x, with the addition of another negative charge from the sulfate group, would be involved in the induction of H A binding in T N F a stimulated SR91 cells given the polyanionic nature of H A and the inhibitory effects of sialic acid residues. However, it is possible that sulfation could interfere with the negative influence of sialic acid residues on H A binding, perhaps by inducing conformational changes. The study by Teriete and colleagues (197) determined that many of the residues within CD44 previously implicated in H A binding, including AsnlOO (TV3), were present on a single face of the H A binding domain. Moreover, "this region is surrounded on all sides by a shell of negative electrostatic charge that may help to guide the polyanionic H A toward its docking site via charge repulsion" (197). Perhaps A G 107 epitopes on TV-linked glycans at TV3 could facilitate H A binding in the same manner. Additional experiments need to be performed to conclude whether the A G 107 epitope is involved in inducing H A binding in human monocytes.  90  4.4  CD44 and AG107 did not enhance rolling on L-selectin It has been well established that sialyl 6-sulfo Lewis x on core 1 and core 2 O-  linked glycans contribute to the generation of the major class of L-selectin ligands on H E V (141, 142). On the other hand, very few studies have addressed whether sialyl 6sulfo Lewis x on TV-linked glycans can mediate rolling on L-selectin. Chinese Hamster Ovary cells transfected with GlcNAc6ST-l added sulfate largely to TV-linked glycans of a CD34/IgG chimera but this did not enhance rolling on L-selectin. However, this study did not confirm whether sialyl 6-sulfo Lewis x epitopes were actually present on the TVlinked glycans (130). Several reports have suggested that human hematopoietic progenitor cells express a glycoform of CD44, termed H C E L L , that presents ligands for L-selectin on TV-linked glycans (70-72, 200). Interestingly, ligand activity required sialylation and fucosylation but not sulfation. Whether H C E L L is present on monocytes and is capable of mediating leukocyte rolling remains to be determined. In vitro, it has been demonstrated that adherent neutrophils were able to capture other flowing neutrophils via PSGL-1 and L-selectin. However, the significance of leukocyte-assisted capture under physiological settings has been called into question (201, 202). A 6-sulfo LacNAc-containing epitope present on PSGL-1 on natural killer cells created a unique binding site for L-selectin (203). The mAb DD-2 detected its epitope, 6sulfo LacNAc, on a subset of human dendritic cells, although its function is not known (182). The 6-sulfo LacNAc and 6-sulfo Lewis x epitopes that can be detected by mAb AG107 did not enhance L-selectin mediated rolling. Low CD44 surface expression was observed in SR91 cells incubated in tissue culture plates coated with a CD44 Ab, IM7. While this procedure could have altered the  91  expression of other molecules or another aspect of cell biology, it can reasonably be inferred that CD44 was not an L-selectin ligand since rolling on L-selectin/IgM was not affected. Moreover, based on the results from TNFa-stimulated SR91 cells it is possible that expression of CD44 beyond a threshold level or TNFa-modified forms of CD44 can actually inhibit L-selectin mediated rolling. PSGL-1 can mediate rolling on all three selectins and is likely the glycoprotein mediating rolling in SR91 cells since rolling was observed in unstimulated and C D 4 4  low  cells and occurred over immobilized L -  selectin/IgM or immobilized P-selectin/IgG (data not shown). A greater number of rolling cells was observed over immobilized P-selectin/IgG compared to L-selectin/IgM. This observation is consistent with reports that PSGL-1 is a much better ligand for Pselectin than L-selectin. Likewise, if L-selectin preferred to interact with ligands on PSGL-1 over the AG107 epitope on CD44, then the effects of the latter on L-selectin mediated rolling may not be apparent. However, this hypothesis could not have been verified since no PSGL-1 null SR91 cells exist. In addition, the ability of anti-PSGL-1 Abs to block L-selectin mediated rolling has not been determined. Very few SR91 cells rolled under a flow rate of 1 dyne/cm over 2.5 mg/ml of 2  immobilized rooster comb H A (data not shown) in two independent experiments. In one of these trials an increased number of rolling cells was observed in unstimulated versus TNFa-stimulated SR91 cells (5.4 vs 14.4 cells/min). By contrast, an average of 82 unstimulated and 51 TNFa-stimulated SR91 cells per minute (n=5) rolled over immobilized L-selectin/IgM, with no prolonged stopping or adhesion observed. Conversely, the rolling velocity was much slower over immobilized H A compared to L selectin/IgM. Furthermore, rolling ceased within seconds and the cells remained  92  stationary for the rest of the experiment (usually 5 minutes). This indicated that CD44H A interactions were relatively poor at mediating rolling interactions and better at facilitating firm adhesion. This data contradicted other studies that have shown that CD44 and H A can mediate rolling, at least for lymphocytes (48, 204, 205). It is possible that this discrepancy is intrinsic to differences between lymphoid and myeloid CD44. When H A and L-selectin/IgM was immobilized together on the same plate no significant difference was observed between the number of rolling/adhered cells per minute in unstimulated and TNFa-stimulated SR91 cells, although there was a slight decrease in the latter (one experiment, data not shown). Only 38 rolling/adhered unstimulated cells per minute were observed over immobilized H A and L-selectin/IgM (compared to an average of 82 rolling cells per minute over immobilized L-selectin/IgM alone). Although rolling on L-selectin/IgM alone was not performed in the same experiment, it did suggest that the addition of H A did not augment rolling on L-selectin/IgM and may in fact have had an inhibitory effect.  4.5  Expression of GlcNAc6ST-l and GlcNAc6ST-4 in human GM-CSF- and M -  CSF-cultured macrophages GM-CSF (206) and M-CSF (207) play a central role in the maturation of monocytes into macrophages, and there is evidence that human P B M cultured in the presence of GM-CSF or M-CSF develop into macrophages with either M l or M2 properties, respectively (18, 208). Many studies have revealed the plasticity of macrophage activation and it has been postulated that this versatility is a reflection of the ability of macrophages to constantly adapt their roles in response to changing  93  microenvironmental signals (209, 210). The data presented in this chapter demonstrated that GM-CSF- and M-CSF-cultured human macrophages also had striking morphological differences. Interestingly, M-CSF-cultured macrophages stimulated with IFNy plus LPS or T N F a alone were similar in appearance to GM-CSF-cultured macrophages, raising the possibility that there may have been a switch towards other M l properties as well. However, further analysis revealed that this did not involve changes in T N F a and IL-10 secretion as the stimulated M-CSF-cultured macrophages remained T N F a  l o w  and IL-  10 ' , a cytokine profile common to M2 macrophages. h gh  Given that GM-CSF-cultured macrophages stimulated with IFNy and LPS were exposed to endogenous secretions of 20 ng/ml T N F a on average, it was clear that macrophages polarized towards an M l functional state did not upregulate GlcNAc6ST-l and GlcNAc6ST-4 transcripts. This was rather unexpected given the results in human P B M and the pro-inflammatory nature of M l macrophages. Instead, transcripts for GlcNAc6ST-l and GlcNAc6ST-4 were upregulated in the M-CSF-cultured macrophages. Although M2 macrophages are associated with anti-inflammatory functions, the data presented here do not suggest that GlcNAc6ST-l and GlcNAc6ST-4 were upregulated by IL-10, a cytokine with potent anti-inflammatory properties. For instance, the transcript levels for GlcNAc6ST-l and GlcNAc6ST-4 were similar between GM-CSF- and M-CSFcultured macrophages even though IL-10 was present in the culture supernatant of the latter and not the former. Moreover, the addition of T N F a did not increase IL-10 production in M-CSF-cultured macrophages but it did upregulate transcripts for GlcNAc6ST-l and GlcNAc6ST-4. While these results did not exclude a role for IL-10 in  94  upregulating these two sulfotransferases, it suggests that IL-10 alone was not sufficient to mediate this process. While it was interesting to observe that transcripts for GlcNAc6ST-l and GlcNAc6ST-4 increased in M-CSF-cultured macrophages after the addition of IFNy and LPS, these sulfotransferases may have been responding to the concomitant production of lower amounts of T N F a instead. While preliminary data suggest that the addition of IFNy was inconsequential the overall results revealed that GlcNAc6ST-l and GlcNAc6ST-4 were upregulated in M-CSF-cultured macrophages in response to 20 ng/ml T N F a and possibly also to LPS or lower concentrations of T N F a . Furthermore, the addition of LPS alone did increase the production of IL-10 in M-CSF-cultured macrophages (data not shown). This response was reminiscent of M2b macrophages characterized in the mouse. Mice are protected from LPS toxicity due to the potent antiinflammatory effects from M2b macrophages and their high production of IL-10 (16, 211). M2b macrophages are unique among M2 macrophages in that they retain certain qualities of the M l macrophage, such as secretion of high amounts of pro-inflammatory, cytokines, including T N F a . Taken together, the data indicated that T N F a and possibly LPS increased transcripts for GlcNAc6ST-l and GlcNAc6ST-4 in M-CSF-cultured macrophages, cells that displayed some similarities with M2b macrophages. While these results need to be verified by more quantitative methods such as real time RT-PCR, there are many interesting questions remaining for future studies to address. For example, what role does these sulfotransferases play in M2b macrophages and under what other conditions are the GlcNAc6STs also upregulated in macrophages?  95  4.6  CD44 sulfation and the AG107 epitope in human M-CSF-cultured  macrophages Increased transcripts for GlcNAc6ST-l and GlcNAc6ST-4 were concomitant with increased sulfation of CD44 in human P B M but this was not observed in M-CSF-cultured macrophages stimulated with IFNy and LPS or T N F a alone. However, it was still possible that A G 107 epitopes were being generated as long as there was a compensatory decrease in other aspects of sulfation on CD44. A previous study demonstrated that different forms of sulfation on CD44 responded differently to T N F a in the SR91 cell line (164). However, neither flow cytometry nor a western blot of immunoprecipitated CD44 was able to detect the A G 107 epitope on stimulated M-CSF-cultured macrophages. In addition, flow cytometry did not detect the 2F3 epitope, sialyl Lewis x/sialyl 6-sulfo Lewis x, in these cells. The 2F3 mAb was generated against sialylated versions of the carbohydrate determinant used to generate the AG107 mAb (179). Although mAb 2F3 cannot distinguish between sialyl Lewis x and sialyl 6-sulfo Lewis x, cells that bind to mAb 2F3 would be predicted to also bind mAb A G 107, as long as the carbohydrate moiety was sulfated at the 6-0 position of GlcNAc. In both SR91 and ECV304 cells, the 2F3 epitope was present and increased expression of GlcNAc6ST-l resulted in induction of the AG107 epitope. No reactivity to mAb 2F3 suggests that M-CSF-cultured macrophages did not express the sialyl Lewis x determinants associated with the sulfated carbohydrate structure that can be detected by mAb AG107. While these macrophages did express the DD-2 epitope, 6-sulfo LacNAc, it was not significantly increased in response to IFNy and LPS. Whether the DD-2 epitope is present on CD44 and whether mAb DD-2 recognizes 6-sulfo LacNAc epitopes distinct from mAb A G 107, or precursor  96  structures that can be modified by fucosylation to be detected by mAbs 2F3 and A G 107 remains to be determined. The DD-2 epitope has been detected on a subset of human dendritic cells, although its function there is not known (182).  4.7  H A binding in human G M - C S F - and M-CSF-cultured macrophages A difference in H A binding and the level of induction of H A binding was  observed between GM-CSF- and M-CSF-cultured macrophages. While the majority of GM-CSF-cultured macrophages did not bind any F L - H A , most of these macrophages were induced to bind high levels of F L - H A with IFNy and LPS. In contrast, M-CSFcultured macrophages already bound substantial amounts of F L - H A and the addition of IFNy and LPS only resulted in a comparatively modest increase. While these results suggest that the activation state of macrophages can influence its H A binding characteristics, it does not reveal what role this serves in the inflammatory response. CD44-mediated H A binding can facilitate leukocyte interactions with other leukocytes, endothelial cells or the E C M supporting both migration and adhesion (212, 213). Previous results from our laboratory suggest that the functional outcome of CD44H A interactions may depend, in part, on the strength of these interactions; strong H A binding favoured adhesion while weak H A binding promoted migration of myeloid cells through a 3D gel containing H A and other E C M components (K. Brown, PhD thesis). This may explain the low F L - H A binding capability of GM-CSF-cultured macrophages since macrophages derived from monocytes that have just extravasated through the endothelium still need to migrate towards the central site of inflammation. Once there, macrophages may be retained, in part, by pro-inflammatory mediators such as IFNy and  97  LPS that then induce high H A binding. Several observations suggest that GM-CSFcultured macrophages are more characteristic of the macrophages derived from the initial wave of monocytes to sites of inflammation than M-CSF-cultured macrophages. For example, GM-CSF is not normally present in the serum of healthy individuals but it is induced under inflammatory conditions and present at sites of inflammation (206) while M-CSF is present in the serum and in tissues under homeostatic conditions (207). In addition, the presence of GM-CSF in the serum increased the number of circulating monocytes. Moreover, GM-CSF-cultured macrophages display characteristics reminiscent of the classical M l macrophage that promotes the early inflammatory response. Given the plasticity of macrophages, it has been speculated that M l macrophages simply acquire more M2 properties as the inflammatory response progresses towards resolution or towards conditions not associated with classical activation. Since these macrophages have already migrated to the site of inflammation, it may explain why M-CSF-cultured macrophages bind high levels of F L - H A . Restoration of the E C M , in particular the H A component, is a key factor in resolving inflammation and wound healing (214). Lower molecular weight (LMW) fragments of H A accumulate after tissue injury and can act as a pro-inflammatory signal (93-95, 215). However, the turnover of H A is not well understood at sites of inflammation. Although several cell types including chondrocytes (91) and endothelial cells can internalize H A (216, 217), a study by Teder et al. (23) indicated that expression of CD44 on hematopoietic cells was crucial to the clearance of L M W H A fragments in the lungs during inflammation. Although the particular cell type was not identified, macrophages are a likely candidate. Here, both GM-CSF- and M-CSF-cultured  98  macrophages were capable of binding F L - H A , although it is important to note that the F L - H A used in these experiments was derived from a high molecular weight form. Whether macrophages with M l and M2 properties can bind and internalize L M W H A represents an interesting direction for future studies.  4.8  Expression of other 6-sulfo LacNAc-containing epitopes The recent cloning of a group of GlcNAc6STs have resulted in the recognition  that GlcNAc6ST-l and GlcNAc6ST-2 were involved in the synthesis of sialyl 6-sulfo Lewis x epitopes on several glycoproteins present on peripheral lymph nodes that were critical for L-selectin mediated lymphocyte homing. Other studies including the data presented in this thesis have implicated GlcNAc6ST-l in the generation of epitopes recognized by mAbs 2F3, AG107, AG223, G72 and G152. However, mAbs G72 and G152 (181), which were raised against sialylated versions of the epitopes recognized by mAbs A G 107 (180) and AG223 (181), respectively, did not bind to TNFa-stimulated SR91 cells even though they were A G 1 0 7 A G 2 2 3 +ve  +ve  after treatment with  neuraminidase (164). Discrepancies between the expression of AG223 and G152 had . also been noted in an earlier study (218). In the myeloid cell lines examined here, there was no clear correlation between the reactivity of mAbs A G 107 and D D T 2 , which suggest that these mAbs may be recognizing distinct glycans even though they can both bind to the 6-sulfo LacNAc moiety. Results from the THP-1 and U937 cell lines further illustrate that there was no simple relationship between the expression of GlcNAc6ST-l and GlcNAc6ST-4 and the sulfated epitopes defined by this set of mAbs.  99  Differentiation of THP-1 and U937 cells with P M A also had a significant effect on the expression of 2F3, AG107, AG223 and DD-2 epitopes. While AG107 and AG223 epitopes were present in PMA-differentiated THP-1 cells, they were not detected in TNFa-stimulated THP-1 cells despite the increased level of GlcNAc6ST-l transcripts. This suggested that P M A had effects on other aspects of glycosylation that favoured the synthesis of A G 107 and AG223 epitopes. It is also possible that T N F a upregulated an isoform of GlcNAc6ST-l not capable of generating these sulfated epitopes, at least in monocytic THP-1 cells. Based on its nucleotide sequence, an alternate form of GlcNAc6ST-l with a long cytoplasmic tail may exist. However, the primers used to amplify GlcNAc6ST-l in these experiments would not have been able to discriminate between these two isoforms. The localization of the putative form of GlcNAc6ST-l may be quite different given that i) the cytoplasmic portion of GlcNAc6STs has been shown to affect its localization within the Golgi and that ii) (31-4 galactosyltransferase with a short or long cytoplasmic tail is found in the Golgi or on the cell surface, respectively (219). The existence of an extracellular GlcNAc6ST-l, although tantalizing, awaits verification. The results from the SR91, THP-1, U937 and ECV304 cell lines suggest that GlcNAc6ST-l can be involved in, but is not sufficient for the synthesis of several 6-sulfo LacNAc-containing epitopes including those defined by mAbs A G 107, AG223, G72 and G152. In addition, GlcNAc6ST-l may not be necessary for the synthesis of the DD-2 epitope. The structural diversity of carbohydrates and the preference of GlcNAc6STs for terminal GlcNAc residues suggest that the assortment of 6-sulfo LacNAc-containing epitopes generated by these sulfotransferases may be quite extensive. The limitation of using mAbs to identify sulfated epitopes is that they do not reveal the underlying glycan  100  structure. Clearly, the overall glycan structure is important for its function given that 6sulfo LacNAc when expressed on extended core 1 and core 2 0-glycan structures of PNAds can serve as an L-selectin ligand while the same sulfated moiety on TV-linked glycans of CD44 cannot. Thus, it may well be possible that AG223, or some other sulfated structure, is responsible for the upregulation of CD44-mediated H A binding in human P B M . The identification of 6-sulfo LacNAc-containing structures is only the beginning. Elucidating the overall glycan structure would be instrumental towards revealing the full spectrum of sulfated carbohydrate structures generated by each GlcNAc6ST and more importantly, towards assigning a function to each.  4.9  Expression of G l c N A c 6 S T - l and GlcNAc6ST-4 in mouse macrophages Although transcripts for GlcNAc6ST-l and GlcNAc6ST-4 were found to be  present in a broad range of tissues, there were differences in the expression pattern of both sulfotransferases between human and mice. For example, Northern blot analysis revealed an abundance of GlcNAc6ST-l transcripts in the spleen and bone marrow of humans but not in mice (136, 137). Conversely, transcripts for GlcNAc6ST-4 were readily detectable in kidneys from mice but not from humans (144). GlcNAc6ST-l and GlcNAc6ST-4 transcripts were known to be expressed in human peripheral blood leukocytes (PBL)(137, 144, 147) and results from this body of work indicated that they were also present in human P B M and monocyte-derived macrophages. However, the expression of GlcNAc6ST-l and GlcNAc6ST-4 in mouse leukocytes was not examined in previous studies. Here, transcripts for GlcNAc6ST-l and GlcNAc6ST-4 were not detected in B M D M or thioglycollate-elicited macrophages stimulated with or without  101  T N F a . Splenic macrophages that had matured in vivo and thus, exposed to the entire milieu of mediators present at the height of an inflammatory response initiated by L. monocytogenes also did not express or induce expression of GlcNAc6ST-l or GlcNAc6ST-4. Whether the absence of expression of these sulfotransferases was specific to these macrophages and conditions tested, or whether they are simply not expressed in mouse macrophages in general remains to be determined.  102  CHAPTER 5 Conclusion  103  5.1  Conclusion The main goals of this research project were to identify the sulfotransferase(s)  responsible for generating the A G 107 epitope, 6-sulfo LacNAc/Lewis x, on CD44 and to determine the role of this sulfated epitope in the inflammatory response. The results presented in this body of work demonstrated that the A G 107 epitope was expressed on CD44 in human P B M and was upregulated in response to T N F a . SR91 cells upregulated transcripts for GlcNAc6ST-l while human P B M upregulated transcripts for GlcNAc6ST-l and GlcNAc6ST-4 in response to T N F a , as determined by semi-quantitative RT-PCR. Furthermore, GlcNAc6ST-l was implicated in the synthesis of the A G 107 epitope on CD44. However, the role of GlcNAc6ST-4 in this process cannot be excluded at this time. The induction of the AG107 epitope on human P B M in response to T N F a suggests a potential role in the inflammatory response, but the results presented herein indicated that they were not involved in CD44-mediated H A binding or L-selectin mediated rolling, both processes that could recruit these cells to sites of inflammation. Interesting questions to address in future experiments would be the aspects of CD44 carbohydrate sulfation that are important for TNFa-induced H A binding and whether the A G 107 epitope can regulate cell-cell or cell-ECM interactions that are important for monocytes i) during development in and exit from the bone marrow or ii) for motility once they have extravasated into inflamed tissues. Transcripts for GlcNAc6ST-l and GlcNAc6ST-4 were upregulated in human monocyte-derived macrophages cultured in M-CSF (macrophages implicated in Th2mediated responses, resolution of inflammation and wound healing) but not in GM-CSF (macrophages implicated in promoting the inflammatory response and in Thl-mediated  104  immunity). For studies investigating the role of these sulfotransferases in the inflammatory response a mouse model system would be invaluable. To this end, mice deficient in GlcNAc6ST-l (138), GlcNAc6ST-2 (135) or both are available (139, 140). However, in experiments with mouse bone marrow-derived macrophages, thioglycollatei  elicited peritoneal macrophages, and splenic macrophages, the amount of GlcNAc6ST transcripts detected were very low or none at all, even after exposure to pro-inflammatory mediators. Therefore, the use of sulfotransferase-deflcient mice to study its role in inflammation may be limited if the GlcNAc6STs are not expressed in a similar manner in monocytes/macrophages from humans and mice. More types of murine macrophages need to be isolated and evaluated for expression of GlcNAc6STs to determine whether the absence of expression of these sulfotransferases in these cells is a general rule, or the exception. The panel of carbohydrate-binding mAbs used in this thesis recognized small structures, all containing the 6-sulfo LacNAc moiety. However, the reactivity of each mAb was mostly independent from the others. Although mAbs A G 107 and DD-2 were reported to recognize the 6-sulfo LacNAc moiety, the reactivity of these mAbs was inversely correlated between undifferentiated THP-1 and PMA-stimulated THP-1 cells. This, and other results presented in this thesis, underscore the importance of the underlying glycan structure not only in recognition by these mAbs, but more importantly, in its function. Sialyl 6-sulfo Lewis x, when present on O-linked glycans, serves as the preferred ligand for L-selectin (130). Although this structure contains the smaller 6-sulfo LacNAc and 6-sulfo Lewis x moieties, these epitopes when present on TV-linked glycans, as recognized by mAb AG107 in TNFa-stimulated SR91 cells did not mediate rolling on  105  L-selectin. Therefore, the number of functions associated with 6-sulfo LacNAc may be as vast as the number of structures containing this moiety. Indeed, 6-sulfo LacNAccontaining structures have been identified on respiratory mucins in patients with cystic fibrosis (195, 220), on PSGL-1 in natural killer cells and a subset of dendritic cells (182), and on HIV-1 gpl20 (221). Expression of GlcNAc6ST-l and GlcNAc6ST-2 has been shown to be regulated under several inflammatory conditions in endothelial and epithelial cells (134, 222). The results presented in thesis demonstrated that the expression of GlcNAc6ST-l and GlcNAc6ST-4 were also regulated in cells of the myeloid lineage, such as monocytes and macrophages. This may make GlcNAc6STs a very important target for the development of a host of new therapeutics. The greatest challenge to glycobiology has always been a technological one. Mass spectrometry, the main technology for analyzing carbohydrate structures, has improved in many important ways, including sensitivity and accuracy, and thus, has increased its accessibility and usefulness to the rest of the research community. Although sulfation of biological compounds was discovered well over 125 years ago (163), its biological functions have only recently begun to be unraveled. 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