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Expression and distribution of Endoglycan on B cells Lam, Mindy Ching Wan 2005

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Expression and Distribution of Endoglycan on B cells by MINDY CHING WAN LAM B.Sc. (H), The University of British Columbia, 2002 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in  THE FACULTY OF GRADUATE STUDIES (Genetics)  THE UNIVERSITY OF BRITISH COLUMBIA July 2005 © Mindy Lam, 2005  ABSTRACT  Endoglycan is the third and newest member of the CD34-family (CD34, Podocalyxin and Endoglycan) of sialomucins.  Although all three molecules are markers of early  hematopoietic progenitors and vascular associated tissues, they also have additional unique distribution patterns. In most tissues, CD34 and Podocalyxin have been shown to act as anti-adhesives, most likely due to the strong negative charges conferred by terminal sialic acid residues on their highly glycosylated mucin domains (Doyonnas et al., 2001 and unpublished data).  We have demonstrated that ectopic expression of  CD34 on murine mast cells, or Podocalyxin in breast cancer cells, induces a profound reduction in homotypic aggregation/adhesion.  Surprisingly, ectopic expression of  Endoglycan in similar assays does not decrease aggregation, suggesting that it may have a different function. In addition, most mucins (including CD34 and Podocalyxin) do not show a high degree of interspecific sequence conservation. However, Endoglycan shows a strikingly high degree of conservation across species.  Therefore, the  conservation in the Endoglycan sequence may reflect the maintenance of a specific binding domain for extracellular ligand(s). Hence, we propose that Endoglycan has a pro-adhesive function and act as an antagonist of Podocalyxin and CD34. Using an anti-Endoglycan monoclonal antibody (F4B10) generated in our lab, along with RT-PCR data, we determined the general distribution of Endoglycan. In addition to early hematopoietic progenitors and vascular associated tissues, Endoglycan is expressed on macrophages, thymocytes and lipopolysaccharide (LPS)-stimulated B cells. A survey of splenic B cells and immature B cells showed that Endoglycan is expressed on l med/hi gM  |gD  io  CD11b  med  CD21  med/hi  CD22  hi  CD23  io  s u b s e t  o f  S  plenocytes. These cells are  characteristic of marginal zone B cells, which have the phenotype: lgM , lgD'°, CD21 , hl  CD22 , CD23' and CD1 . hi  0  hi  hl  Furthermore, I did not detect Endoglycan expression on  immature or pre-B cells. My results show that Endoglycan is up-regulated on L P S stimulated B220-positive splenocytes. In these cultures, Endoglycan is expressed on a subset of plasma cells (as indicated by the marker syndecan-1 (SDC1)), and all Endoglycan-positive cells express pi-integrin.  We have evaluated the time course of  Endoglycan induction by LPS using FACS and RT-PCR analysis, cell surface expression peaks at 48hr and Endoglycan transcript levels peaks at 24hr.  The induction of  Endoglycan is specific to TLR-stimulations and is not induced by other B cell activators, such as anti-IgM, anti-CD40 or B A F F .  Since T cell cytokines play a large role in the regulation of B cell activation and maturation, I examined the effect of T cell secreted cytokines on Endoglycan expression. I found that IL-2, IL-4, IL-5 and T G F p reduce the up-regulation of Endolgycan by L P S on splenic B cells.  On the other hand, IL-ip increases Endoglycan expression.  The  regulated expression of Endoglycan suggests that this molecule may serve an important role on activated B cells.  The data in this thesis provide the first description of Endoglycan expression and regulation on B cells. Endoglycan is a highly specific marker for TLR-activated B cells. Expression of pi-integrin on these Endoglycan-positive plasma cells suggests that these cells may home to specific niches for the establishment of long-term antibody repertoire. Therefore, Endoglycan may have a role in homing and migration of these cells  TABLE OF CONTENTS  Page ABSTRACT  ii  TABLE OF CONTENTS.!  iv  LIST O F T A B L E S  '.  vii  LIST O F F I G U R E S  viii  LIST O F A B B R E V I A T I O N S ;  x  ACKNOWLEDGMENTS  xiii  1. I N T R O D U C T I O N  1  1.1 C D 3 4 Family  1  1.1.1 Overview of C D 3 4 and Podocalyxin  3  1.1.2 Overview of Endoglycan  4  1.2 B cell Development in Bone Marrow  11  1.3 Peripheral B cells  15  1.4 Mechanisms for Localisation and Migration of B cells in Lymphoid Organs  18  1.5 B cell Activation and Inflammatory Response  20  1.5.1 T cell-Dependent Activation  20  1.5.2 T cell-Independent Activation  20  1.5.3 T L R s and B cell Activation  21  1.5.4 Memory B and Plasma cells Formation  23  1.6 Mechanism for Localisation and Migration of Memory and Plasma cells  24  1.7 Cytokines and Their Effect on B cells 1.8 T L R Signaling  26 — 28  1.9 Thesis Objectives  .'30  2. M A T E R I A L S A N D M E T H O D S  34  2.1 Mouse Strains......  34  2.2 Primary Cell Culture  34  2.3 Cell Lines  .-.  35  2.4 Expression of mouse Endoglycan (mEndoglycan) by Retroviral Infection  35  iv  2.6 Anti-mEndoglycan Antibody (F4B10) Production  36  2.7 Antibodies  36  :  2.8 Flow Cytometry  37  2.9 Bone Marrow-Derived Mast Cell Aggregation Assays  37  2.10 L P S Administration  38  2.11 Semi-quantitative Reverse-Transcriptase Polymerase Chain Reaction ( R T - P C R )  38  3. R E S U L T S  39  3.1 Endoglycan Expression by B cells  39  3.1.1 Endoglycan is selectively expressed on splenic B cells with a marginal zone B phenotype and is not expressed on immature B cells  39  3.2 Endoglycan Expression on Activated B cells  42  3.2.1 Endoglycan surface expression and transcript levels increase upon LPS-stimulation  42  3.2.2 CD1 mice are defective for LPS-induced Endoglycan expression on B cells  45  3.2.3 Endoglycan is expressed on a subset of plasma cells and these are (31-integrin positive  46  3.2.4 Endoglycan expression by B splenocytes is increased upon LPS-administration in vivo  48  3.2.5 Endoglycan expression is selectively upregulated by T L R agonists  50  3.3 LPS-induced Endoglycan Expression is Suppressed by T 2-type Cytokines and the Anti-Inflammatory Factor, T G F p H  4. D I S C U S S I O N  52  56  4.1 Endoglycan is on splenic B cells with MZ B phenotype  56  4.2 Endoglycan is expressed on B cells activated via T L R  56  4.3 LPS-induced Endoglycan expression on a subset of plasma cells and this expression is downregulated by T 2-type H  cytokines and T G F p  57  v  4.4 Differences in the effect of IL-4 and IL-13 on Endoglycan expression  •  59  4.5 Differences in LPS-induced expression of Endoglycan between CD1 and other mouse strains  60  4.6 Endoglycan expression increases upon LPS-administration in the spleen and B M .  61  5. C O N C L U S I O N  62  5.1 Endoglycan on MZ B cells  62  5.2 A possible role for Endoglycan in homing and migration during inflammation?  62  5.3 Concluding Remark  63  6. B I B L I O G R A P H Y  64  vi  LIST O F T A B L E S Page Table 1.1: Tissue distribution of the C D 3 4 family  10  Table 1.2: Toll-like receptors and their ligands  22  Table 4.1: Transgenic mice with alterations in M Z B cells  vii  56  LIST O F FIGURES  Page Fig. 1.1: CD34 family  2  Fig. 1.2: CD34 family domain structure and alternative splicing  3  Fig. 1.3: Amino acid sequence of human Endoglycan  5  Fig. 1.4A: Mouse and human amino acid alignment of Endoglycan  6  Fig. 1.4B: Mouse and human amino acid alignment of Podocalyxin  7  Fig. 1.4C: Mouse and human amino acid alignment of CD34  8  Fig. 1.5: Predicted transcription factor binding sites in the Endoglycan promoter  10  Fig. 1.6: Hematopoiesis  12  Fig. 1.7: Molecular markers and B cell lymphopoiesis....  13  Fig. 1.8: Maturation of transitional B cells  16  Fig. 1.9: Microvascularture of the spleen  19  Fig. 1.10: Molecular interaction for retention of plasma cells in BM  25  Fig. 1.11: MyD88-dependent TLR signalling pathway  29  Fig. 1.12: MyD88-independent TLR signalling pathway  30  Fig. 1.13: Bone marrow-derived mast cell aggregation assay  31  Fig. 1.14: Expression levels of Podocalyxin and Endoglycan on breast cancer cell lines  33  Fig. 3.1: Endoglycan is expressed on cells with a MZ B phenotype  39  Fig. 3.2: B1a B cell deficient mice show a normal frequency of Endoglycan-positive B cells  41  Fig. 3.3: Endoglycan is not expressed on immature or pre-B cells in BM  42  Fig. 3.4: Endoglycan expression on B cells induced by LPS treatment and is TLR4 dependent  43  Fig. 3.5: Endoglycan induction by LPS is blocked by IL-4 treatment and is regulated by transcriptional and post-transcriptional mechanisms Fig. 3.6: Endoglycan expression by various inbred and KO mouse strains  44 45  Fig. 3.7: Endoglycan is upregulated on a subset of plasma cells expressing |31-integrin  47  Fig. 3.8: Expression of Endoglycan on hematopoietic tissues after LPS-administration at 6 and 24hr  48  Fig. 3.9: Endoglycan expression is upregulated by TLR2, 4 and 9 agonists viii  50  Fig. 3.10: Endoglycan expression on splenocytes is not affected by stimulation with anti-IgM, anti-CD40, B A F F , T N F a a n d IFNy.... Fig. 3.11: Effect of T 1 and T 2 cytokines on Endoglycan expression H  H  51 53  Fig. 3.12: IL-4 treatment after 24hr of LPS-stimulation blocks Endoglycan expression on splenocytes Fig. 3.13: Effect of IL-1(3 and TGF(3 on Endoglycan expression  ix  53 55  LIST O F ABBREVIATIONS P-Me  beta-mercaptoethanol  Ab  antibody  BAFF  B cell activating factor belonging to the T N F family  BCR  B cell receptor  Blimp-1  B-lymphocyte-induced maturation protein 1  BM  bone marrow  bp  base pair  C2GnTI  core2 p(1,6)-N-acetylglucosaminyltransferase I  CD40L  clustered domain 40 ligand  cDNA  complement deoxyribonucleic acid  CK2  casein kinase II  CLP  common lymphoid progenitor  Co-Smad  common mediator Smad  DMEM  Dulbecco's Modified Eagle Medium  DNA  deoxyribonucleic acid  DTHL  aspartic acid, threonine, histidine, leucine  ELISA  enzyme-linked immunosorbent assay  ' ERM  ezrin/radixin/moesin  EST  expressed sequence tag  FACS  fluorescence activated cell sorter  FBS  fetal bovine serum  FDC  follicular dendritic cell  FITC  fluorescein isothiocyanate  FO  follicular  Fr.  fractions  FTVII  a(1,3)-fucosyltransferase Vll  GADD45  growth arrest and DNA-damage-inducible 45  HAT  hypoxanthine, aminopterin, thymidine  HBSS  Hank's balanced saline solution  HSA  heat-stable antigen  HSC  hematopoietic stem cell  ICOS  inducible T-cell co-stimulator  IFNy  interferon Y x  ig igH  immunoglobulin  IL-1(3  interleukin ip  IL-10  interleukin 10  IL-13  interleukin 13  IL-2  interleukin 2  IL-3  interleukin 3  IL-4  interleukin 4  IL-5  interleukin 5  IL-6  interleukin 6  IL-7Roc  IL-7 receptor a chain  IRAK  IL-1 receptor-associated kinases  IRF  interferon-regulatory factor  kb  kilobase  kDa  kiloDalton  KO  knockout  LN  lymph node  LPS  lipopolysaccharide  MARCO  macrophage receptor with collagenous structure  MFI  mean fluorescence intensity  MLP  multilineage progenitor  MR  mature recirculating  MyD  myeloid differentiation protein  MZ  marginal zone  MZM  marginal zone macrophage  NF  newly formed  NHERF-1  Na+/H+ exchanger regulatory factor-1  NK  natural killer  OD  optical density  v-PALS  immunoglobulin heavy  periarteriolar lymphoid sheath  PAMPS  pathogen-associated molecular patterns  PBS  phosphate buffered saline  PCR  polymerase chain reaction  PDZ  PSD-95/Drosophila disk large (Dlg)-1/ZO-1 xi  PE  phycoerythrin  PKC  protein kinase C  PRR  pattern recognition receptors  PSGL-1  P-selectin glycoprotein ligand-1  PW  peritoneal wash  RAG  recombination activating gene  RHIM  RIP homotypic interaction motif  RPMI  Roswell Park Memorial Institute  R-Smad  receptor-activated Smad  RT-PCR  reverse transcriptase polymerase chain reaction  S1P  lysophospholipid sphingosine-1 phosphate  SCID  severe combined immunodeficiency  SDC1  syndecan-1  SLC  surrogate light chain  STAT  signal transducers and activators of transcription  T(3RI  type I T G F p receptor  TpRII  type II T G F p receptor  T1  transitional stage 1  T2  transitional stage 2  T3  transitional stage 3  TAK1  T G F p activated kinase 1  TGFp  transforming growth factor p  TI-2  T-cell independent type 2  TIR  Toll-interleukin 1 receptor  TLR  toll-like receptor  TNFa  tumour necrosis factor a  TRAF  TNF-receptor associated factor  VCAM-1  vascular cell-adhesion molecule 1  xii  A C K N O W L E D G E M E N T S I would like to thank all the individuals who have provided me guidance and assistance throughout the course of my studies.  My supervisor, Dr. Kelly McNagny, provided constant  support, encouragement and mentorship. study in his lab.  I am grateful to him for giving me the opportunity to  I would like to thank members of the McNagny lab, both past and present,  especially Helen Merkens, Shierley Chelliah, Erin Drew, Sebastian Furness and P o h Tan. They have not only provided training and technical assistance, but also for their support, stimulating discussions and friendship.  In addition, thanks to the members of my committee, Dr. Michael  Gold and Dr. Fabio Rossi, for their suggestions and mentorship at multiple committee meetings.  Over the years, I have had the opportunity to be involved in various collaborations. I would like to thank Muriel David (Schrader Lab), Kevin Lin (Gold Lab) and Arthur Sampaio (Underhill Lab) for their help and guidance; Dr. Mansoor Husain (University of Toronto) for providing the M O V A S cell line; and Dr. Dietmar Vestweber (University of Munster) for supernatant.  providing the anti-PSGL-1  As well, I have received advice or reagents from various people both inside and  outside of the Biomedical Research Centre (BRC).  I would like to acknowledge Nelson W o n g  (Johnson Lab), Jason Grant (Jefferies Lab), Caylib Durand (Gold Lab), Jeff Duenas (Rossi Lab), Doug Carlow (Ziltener Lab), Jasmeen Merzaban (Ziltener lab), Jennifer Bishop (Finlay Lab) and Brian Ruffell (Johnson Lab).  The  B R C provided an enriching environment that fostered cooperation, interaction and scientific  discussions and I would like to thank everyone for creating such a wonderful environment.  learning  However, this superb environment would not have been possible without core  staffs of the BRC.  I would like to thank Lea W o n g and S a m Hoium for their management of the  animal facility.  A s well, I would like to thank Nicole Voglmaier and L e s Rollins for their  preparation of various reagents for this institute; Takahide Murakami for managing the B R C Genotyping Facility; and Andy Johnson for managing the B R C F A C S Facility.  I would like to extend thanks to my family and friends who have been so understanding and encouraging. In particular, I would like to thank Florence L a m , Samantha Mak, David W o n g , Eric Yee,  Yanni W a n g , Jacqueline Lai, Nina Maeshima, Eunice Y a o , Dennis Lefevbre and Sie-Lung  Tjew for their continuous support.  Lastly, research was made possible through funding from  Heart and Stroke Foundation of Canada, Stem Cell Network and C I H R / M S F H R Transplantation Research.  xiii  1.  INTRODUCTION  1.1  CD34 Family  The CD34 family includes three molecules, CD34, Podocalyxin and Endoglycan. The term was coined by Sassetti and colleagues (Sassetti et al., 2000) when they identified human Endoglycan as the third member of this family. These molecules are classified by similar domain structures, as well as similar exon and intron organization (Doyonnas et al., 2001). The most studied member of the CD34-family is CD34. Although little is known about the function of this molecule, the use of C D 3 4 as a marker for the enrichment of hematopoietic stem cells (HSC) began shortly after its discovery in 1988 (Berenson et al., 1988; Sutherland et al., 1996). The other two members, Podocalyxin and Endoglycan, are relatively new in comparison to C D 3 4 . However, despite their later discovery, much progress has been made to understanding the function of these molecules in recent years.  Members of the CD34 family are type-I transmembrane sialomucins (Fig. 1.1).  Their  extracellular region contains a mucin-like domain that is decorated by sialylated O-linked carbohydrate chains. The densely glycosylated domain not only provides an area of high negative charge; it also promotes an extended rod-like structure, allowing these molecules to protrude pass most cell surface molecules. In the extracellular membrane proximal region is a cysteine-rich domain, presumed to fold into immunoglobulin-like structures through disulfide bonds of paired cysteines. The cytoplasmic domain of these molecules is 73-80 amino acids long and is highly conserved through evolution. Moreover, this is also the region with the highest homology between the three family members.  The cytoplasmic tail contains potential phosphorylation sites, as well as a  potential docking site at the C-terminus for PDZ-containing_proteins. All three members are encoded by eight exons, which are alternatively spliced to produce transcripts encoding a protein with either a full length or a cytoplasmically truncated tail (Fig. 1.2, (Doyonnas et al., 2001; Suda et al., 1992).  These proteins are expressed on early  hematopoietic progenitors and vascular associated tissues; however, each protein also has additional unique distribution patterns.  The following two sections will present a  brief review of the family members.  l  OTEL  mCD34  mPodocalyxin (mMEP21)  mEndoglycan  mCD43  Fig. 1.1: C D 3 4 family. Illustration of the predicted murine structure of C D 4 3 and members of the C D 3 4 family (indicated by black box). Bars, putative O l i n k e d carbohydrates; Black circles, putative N-linked carbohydrates; Triangles, potential sialic acid motifs; Blue boxes; mucin domains; Green boxes, cysteine-rich domain; Pink boxes, glutamate-rich region; P K C , C K 2 and TK, potential phosphorylation sites; D T E L or DTHL, potential PDZ-docking sites. Adapted from Doyonnas et al., 2001.  2  endgl 5'UTR&  Signal peptide  B  stop Long forms 8a  stop Truncated forms 8b  F i g . 1.2: C D 3 4 family domain structure and alternative splicing. A. Genomic organization of murine cd34, podocalyxin (podxf) and endoglycan (endgl). Boxes indicate the eight exons and dotted regions indicate introns, the size of each intron is indicated in kbs. Light purple boxes, 5' UTR; Purple boxes, signal peptide; Blue boxes, mucin domain; Pink box, glutamate-rich region; Green boxes, cysteine-rich dromain; Yellow boxes, stalk domain; Orange boxes, transmembrane (TM) domain; Red boxes, cytoplasmic tail. B. Schematic of the naturally occurring alternative splicing of the cytoplasmic tail. Adapted from Doyonnas et al., 2001.  1.1.1  Overview of C D 3 4 and Podocalyxin  Expression of the CD34-related molecules on hematopoietic progenitors has led to the clinical use of C D 3 4 in the isolation of H S C ; however, these molecules are also expressed on distinct mature cell populations (Table 1.1). CD34, for example, is found on murine mast cells (Drew et al., 2002) while Podocalyxin is expressed by platelets (McNagny et al., 1997; Miettinen et al., 1999), embryonic and anemic erythroid cells (Doyonnas et al., 2005; McNagny et al., 1992) and by non-hematopoietic cells like podocytes of the kidney glomerulus  (Kershaw et al., 1995).  Due to the  bulky,  glycosylated extracellular domain of these molecules, it has been proposed that they have anti-adhesive properties due to charge repulsion. It has been demonstrated that the lack of CD34 on murine mast cells leads to homotypic aggregation, and that, this molecule is required for the repopulation of peritoneal mast cells (Drew et al., 2005). Similarly, Podocalyxin-deficient mice die shortly after birth from renal defect due to the lost of filtration slits on foot processes of podocytes (Doyonnas et al., 2001). 3  Both molecules, when expressed on high endothelial venules (HEV) function as Lselectin ligands (Baumhueter et al., 1994; Sassetti et al., 1998). Thus, there is evidence for both pro- and anti-adhesion functions for these molecules.  Interestingly,  CD34-  deficient mice show no difference in neutrophil extravasation in inflammatory response (Cheng et al., 1996) but reduction in eosinophil migration to the lungs in allergic response (Suzuki et al., 1996). These opposing results may be explained by functional compensation between members of the C D 3 4 family.  1.1.2  Overview of E n d o g l y c a n  Endoglycan is the third and newest member of the CD34 family. The human form of this molecule was isolated by searching for homology to the cytoplasmic domain of Podocalyxin using GenBank expressed sequence tag (EST).  The full length human  c D N A was then cloned via P C R using primers designed based on the available mouse sequence (Fig. 1.3, Sassetti et al., 2000). The mouse form of Endoglycan was cloned at the same time as the human form in our laboratory. Through homology to Podocalyxin cytoplasmic tail, an E S T clone (accession # W13047) was identified and obtained from IMAGE consortium. This 348bp sequence was labelled with mouse c D N A XExlox phage library (Novagen).  3 2  P for screening of a 16d  Clones were isolated, sequenced and  aligned resulting in the identification of a 2092bp (605 amino acids) mouse full length Endoglycan sequence.  Despite similar domain structures to C D 3 4 and Podocalyxin, Endoglycan possesses a unique N-terminal acidic domain of 161 amino acids. polyglutamate tracts of 5-11 residues each.  This region contains three  Moreover, the cysteine-rich domain of  Endoglycan contains three cysteines, whereas CD34 and Podocalyxin have six and four cysteines  respectively,  therefore  the  unpaired  cysteine  in  Endoglycan  permits  homodimerization (Sassetti et al., 2000).  The cytoplasmic domain consists of several potential phosphorylation sites for casein kinase II (CK2) and protein kinase C (PKC).  The C-terminal amino acid sequence of  Endoglycan is DTHL (aspartic acid, threonine, histidine, leucine), which is a potential PDZ-domain docking sequence. By using a ^.phage screen, our laboratory has isolated Na+/H+ exchanger regulatory factor-1 (NHERF-1) as a potential intracellular binding 4  partner of Endoglycan. NHERF-1 contains tandem PDZ-domains and an ERM-domain. ERM-domains have been shown to associate with the actin cytoskeleton, therefore, this molecule is an important adaptor for the targeting and trafficking of PDZ-binding proteins (Fanning and Anderson, 1998; Shenolikar and Weinman, 2001). I  CCA<7rrcCGGCACOAGGACCATaG<^CGCT<^  1 101 28  H  G  ft  100  L  A  A  R  L  P  g  L  L  S  P  L  I  ^  L  L  L  V  G  C  A  f  l  J  21  G G OTQCC T G T G T G C C T G G G T C T G A T G A G C C T G C C C C AGAGGC C C T C A C C T C C A C C T C C C T G C T A G A C CTC C T G C T G C C C A C T G G C T T G C A O C C ACTOGAC G A C V A G 3 D B P G F B G L T S T S L L D L L L P T G L E p L D  200 60  201 T C AG AGG A G C C T A C T G AGACCATGGGCCTGGGACCTGGGCTGCG A G C C C C T G G C T C A G G C T T C C C C A C C G A A G A G A A T O A A G A G T C T C G G A T T C T C C A G C filSEEPSETMGLGAGLGAP-G S C r p S E B S E E S f t l L Q P  300 94  301 95  C A C C A C A G T A C T T C T G G G A A G A C G A C G A A G A G C T G A A T G A C T C AAGTCTGGACCTGGGACCCACTGC A G A T T A T G T T T T T C C T C A C T T A A C T C A G A A C C C P Q ^ F W E H S K E L j « D S S L D L Q P T A D ^ V P P D L T E K A  400 X27  401 129  A G G T T C C A T T G A A G AC A C T A G C C A G C C T C AAG AGCTGCC AAAC C T C C C C T C T C C C T T G C C C A A G A T G A A T C T G G T T G A G C C T C CCTGGC A T A T G C C T C C C G 5 Q A Q S L P N L P 5 P L P K H H L V E P P W H M P P  500 ISO  501 151  AGAGAGG AGGAAGAAGAGGAAGAGGAAGAGGAGGAGAGGGAGAACGAAGAGGTAGAGAAACAAGA R E E E E S E B E B B R R E K S E V E K O . E  600 194  801  CATCCCAAGAAGAAC^CAAGCCTCAGGTCCGTGACTrTTC^  I B D T S  135  5  Q  E  E  A  K  P  O  .  V  R  D  P  S  I  H  S  E  GAGGAGC T G C T C C C T G T G A A T G K E L L P V S C  E  700  .  T  S  5  S  Q  T  P  G  A  T  K  S  R  H  E  D  S ^ G  0  701 128  C C AGGCCTC ATC AGG TGTGGAGCTGGAGAGCAGC ATGGGGCC C A G C T T G C T G C T t K C T T C A C T C A C C C C A A C T A C A G T G A C T C CGGGGG A C C AGGACTC C O A S Q S M G P S t L L P S V T P T T V T P G D Q D S U  801  AC C AG CC AAGAGCCAGAGGCCACAGTGCTGC C A G C T G C AGGCCTTGGGGT AGAGTTVGACCC*TCC*TC AGGAAGC AAGC G AGGAAGCC A C T G C A G G A G C A G  2 901 295  S  S  l  T  S  O  E  V  3  G  800 260  V B V B S  A  E  A  T  V  L  ;  CTGGTTTGTCT3GCCAGCACGAGGAGGTGCC G  227  O  .  H  E  E  V  P  P  A  A  G  L  G  V  E  r  E  A  P  0  E  A  S  E  E  A  T  A  G  A  900  A  294  G G C C T T G C C T T C A T T C C C T C A A A C CA C A G C T C CC AGTGGGGCCGAGC A C C C A Q A T C A A G A T C C C C T T C O A  L  P  S  P  P  O  .  T  T  A  P  3  G  A  S  B  P  D  E  D  P  L  1000  G  32 7  10 01 328  C T C T A G A A C C T C A G C C T C T T C C C C A C T G G C C C C T G G A G A C ATGGAACTGAC A C C T T C C T C T < J C T ACCTTGGGAC AAG AAG A T C T C A A C C A G C A G C T C C T A S B T S A S S P L A P G O H E I > T P S S A T L G O . E D L « Q , Q I . L  1100 360  1101 361  G AAGGGC A G G C A G C T C A A G C T C A A T C C A G G A T A C C C T G C G A T T C T A C G C A G G T C A T C J K C A A G G A C T G C A G C A A T C T C G C T G G G A A A A A C T A C A T C A T T C E G Q A A S A O, S R 1 P H D S T Q V I (c) K D W 3 N L A G K H V I Z L  1200 394  |t  M  1201 395  T G A A C A 7 G A C A G A G A A C A T A G A C J G T G A G G T G T T C C GGCAGCAC CGGG GGCCA CAGCTC CTGGCCCTGGTGGAAG A G G T G C T C C C C C G C C A T G G C AGTGG H M T E H I D ( C J E V P R Q H R G P O L L A L V S B V L P R H G S _G  1300 427  1301 428  C C A C C A T G G G G C CTG C C ^ C A T C T C T C T G A G C A A G C C C A G C G A G A A G C A G C A G C A C C T T C T C A T C A C A C T G G T G C G C C A G C A G G G G G T ^ H H G A W H I S L E K P S E K E O . B L L H T L V G B O . G V V P  AA  14 0 0 4tf0  U  G A T G T C C T T T C C A T G C T G G G T C A C A T C C G C A G G A C C CTGGAGG A G A T T GGC A T C C A G A A C T A T T C C A C A A C C A G C A G C T G C CAGCCGCGGGCCAGC CAGG D V L S H L G D I R R S L E E I G I O . H Y S T T S s ( c ) o , A R A S O. V  1500 494  01 461  1501  495  T  O  .  TGCGCAGCGACTACGGCACGCTCTTCGTGGTGCTGGTGGTCATT^  R  S  D  Y  C  T  ^  ^  J  J  —j- _ _  V  nr  V n  . *—r ^  1500  *— L-  SL X  mL mj ml mj mJ'mJ^^mmhmm 1  I .  8.  L  S  Q  R  5  2  7  1501 538  C C G C C T C C C C A A G C T C A A G C A C C T C T C C C A C G O C G A G G A G C T G C G C T T CGTGCAGAACGGCTGC C A C GACA A C C C C A C GCTGGACGTGGC C A G C G A C A G C R L P K L K H V S H G E E L R r v E S C C « D H P T L D V A S O S  1700 560  1701 551  C A G T C G G A G A T G C A G G A G A A G C ACCCC A G C C TGAACCGC GGC GGGGC C C T C A A C GGCCCGGGGAGCTGGG GGGCGCTCATGCGGGGCAAGCGGGACCCC G Q S E H Q E K H P S L K G G G A L M G P G S W G A C K G G K R D P E  1800 594  18 01 595  A G G A C T C GG A C G T G T T C G A G G A G G AC A C G C A C C T G T G A G CGC A G C G A G GCGCAGGCCGAGTGGGCC GCC AGG A C C AAGCG AGGTGCACCC C G A A A C G G A C D S D V F B E D T H L *  1900  1901  GGCCCGG AG C C C GC A C C A G C CC CGC GCCT AC CCGGC C G C C C C C GCG C CTGG CC CTCGGCGCGGGCTC C T T C C CGC T T C C C C C G A C T T C A C A C C G C G C C T T  2 001  CGGACCAACTCCCTCACTCCCGCCCGAGGGGCAGGCCTCAAAGCCM  2100  2101  C T T C C T C C G C C C C G G G A C T C A A T T AAAC C C GCCC GGAGAC CAC G C C ^ G C C C A G C G A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A  2300  2291  AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA  2000  22$9  Fig. 1.3: Amino acid sequence of human Endoglycan. Underline, signal peptide; Double underline, potential sites for glycosaminoglycan addition; Triangle, potential N linked carbohydrate addition site; Asterisk, potential tyrosine sulfation site; Circle, cysteines in cysteine-rich region; Dotted underline, predicted transmembrane domain. Adapted from Sassetti et al., 2000.  Alignment of the mouse and human orthologs of the CD34 family members shows that Endoglycan contains high amino acid conservation in the extracellular domain (73%) as compared to CD34 and Podocalyxin (55% and 30% respectively, Fig. 1.4A-C). Such a high degree of identity in mucin domains is rare. Glycosylations are added on serine, threonine or asparagine residues by glycotransferases. These transferases recognize short consensus sequences; therefore, if the purpose of the mucin domain is to provide  5  general bulk and charge to a molecule, conservation of these short consensus sequences is not necessary. Therefore, the conservation of this extracellular domain across species suggests that there  maybe  an extracellular  binding  partner  for  Endoglycan.  mEndoglycan hEndoglycan '  MARPLRAARLPP PliliLAAGASLGAYAVGVDEPGPEGLTSTSLLDLIiPTDFEPLD MrtBT.T.RAagT.PPT.T.sPT.T.T.T.T.vmarT/iArvafignEPfiPErtT.TgTgT.T.nT.r.r.PTgT.Epm  * * ********  ******  ** **  * *********************  57 60  ****  SEEPSE AMGLDAGL-APGSGFPSEDSEESRLLQPPQYFWEEEE-LNGSSIJ3LGPTADYVF SEEPSE1MGLGAGLGAPGSGFP3EENEESRILQPPQYFWEEEEELNDSSLDLGPTADYVF * * * * * * .*** *** * * * * * * * * * ****' * * * * * * * * * * * * ** *************  115 120  PDLTEKVASMEDPGO^PDLP^PSILPKMDLAEPPWHMPLQEEEEEEEEEEEEREEEERE PDLTEKAGSIEXIT3QA0EI.PNI.PSPLPKMNLVEPPWHMPPREEEEEEEEEEE-REKEEVE ****** * ** ** ****** **** * ******* *********** ** ** *  175 179  KEAEEEEEKGKSIXPVNRVPKEPPAQAHAPSPSTSSSTSSQSPGATRHRQEDSGDQATSG  235  KQEEEEEEE-—li.PVNGSQEEAKPOVRDFSI.TSSS  QTPGATKSRHEDSGDQASSG  232  *  * ****  *****  *****  *  *  *  **  * *******  **  MEVESSVKPTLSVPSVTPSTVAPGVQN-YSQESGGTEWPTGGLGVQSEVPQGAGEGATVG VEVESSMGPSIiLPSVTPTTVTPGDQDSTSQEAEATVI.PAAGLGVEFEAPQEASEEATAG ***** * * * * * * * ** ** * *** * * **** * * * * * * * *  294 292  AADFDGQQGALPSS-SLPQTVPPSGTEVPSEGPLYPRIPDSLPPGPQDTESTPSSATWGQ AAGLSGQHEEVPAI.PSFPQTTAPSGAEHPDEDPLGSRTSASSPLAPGDME1.TPSSATLGQ ** ** * * *** *** * * * * * * * * * * * ****** **  353 352  V  Y  EGLSEQPLEGQAAEAHSLTPWDSTQVICKDWSNLAGKSYIILNMTENIDCEVFRRHRGLR  413  EDI^O^IXEGQAAEAQSRIPWDSTQVICKDWSNLAGKNYIILNMTENIDCEVFRQHRGPQ  412  * *  * ******** *  ******************  ****************  ***  1XAI.VEEV1.PRHRSGHRGDWHISLSKPSEKEQHIXLMTI.VGEQGVVPTQDVLSMI.SGIRRS LLALVEEVLPRHGSGHHGAWHISLSKPSEKEQHIjIi^TIjVGEQGWPTQDVLSMLGDIRRS ************ *** * *********************************** ****  473 472  Y I£EIGIOtreSTTSSCQAPATQVRSDYGTI.FVVLVITGVICFIIIVI^IiYNCWQRRMPKL IXEIGIOXYSTTSSCO^UlA^O^^DYGTLFAArLWIGAICIIIIAIfilXYNC^RRLPKL ******************* * * * * * * * * * * * * * * :r.:* *•-?.** * *.* v:*:*.** ******* ***  533 532  KHVSHGEELRFVENGCHDNPTLDVASDSQSEMQEKQPSIUGG-AINGPSSWSALMGSKRD  592  KHVSHGEEIJlHVENGCHDNPTIJDVASDSQSEMQEKHPSLNGGGAIilGPGSWGALMGGKRD  592  ***********************************  ******  *  ***  **  ****  ***  PEDSDVFEEDTHL  605  PEDSDVFEEDTHL  605  % A m i n o A c i d Identity  Fig.  1.4A:  Overall  76%  Extracellular D o m a i n  72%  Cytoplasmic Domain  92%  M o u s e and human amino acid alignment of E n d o g l y c a n .  Amino acid  sequence alignment of the mouse and human forms of Endoglycan using SIM Binary Alignment from E x P A S y Proteomics Tools (www.expasy.org, 2003). Stars indicate identical amino acids. Triangles indicate cysteines for disulfide bonding. The shaded region indicate the transmembrane domain. The overall sequence identity is 76%, the extracellular and cytoplasmic regions contain 72% and 92% identity respectively.  6  m P o d o c a l y x i n  M P P T T A L S A T . T . T J . T . T . S P  h P o d o c a l y x i n  h l R C J U ^ A L S A L I i l ^ T P P I i P S S P S P S P S P S P S Q N A T Q T T T D S S N K T A P T P A S S V T I M A T  6 0  A S T L A S T Q P S  1 0 0  *  A S H S H N G N - E T S T S A I K S S T V Q S H Q S A T T S T E V T T G H P V  **********  * *  *  N P T P F T T S T Q S  P  D T A Q Q S T V P T S K A N E I L A S V K A T T L G V S S D S *  *  *  *  *  *  * *  **  5 5  *  S M P T S T P N P T S N Q S G G N L T S  S V S  P G T T T L A Q Q V S G P V N T T V A R G G G S G N P T T  *  *  *  *  *  *  *  1 2 0  .  E V D K T K T S S P S S T A F T S S S G Q T A S S G G K S G D S F T T A P T T T L G L I N V S S Q P T D I i N - T T S K L  1 5 9  T 1 E S P K S T K S A D T T T V A T S T A T A K P N T T S S Q - N G A E D T T N S G G K S S H S V T T D L T S T K A E H  1 7 9  *  *  *  * *  *  * *  *  *  *  *  *  *  L S T P T T D N T T S P Q Q P V D S S P S T A S H P V G Q Q T P A A V P S S S G S T P S T D N S T L T W K — P T T H K  2 1 7  L T T P H P T S P I ^ P R Q P T L T H P V A T P T S S G H D H L M K I S S S S - S T V A I P G Y T F T S P G M T T T L P  2 3 8  * **  * * * * *  *  *** **  * *  **  P L G T S E A T Q P L T S Q T P G I T T L P V S — T L Q Q S M A S T V G T T T E E F T H L . I S N G T P V A P P G — P  2 7 4  S S V I S Q R T Q Q T S S Q M P A S S T A P S S Q E T V Q P T S P A T A L R T P T L P E ' m S S S P T A A S T T H R Y P  2 9 8  *  *  *  *  *  *  *  *  *  *  *  *  *  *  *  *  y S T P S P I W A F G N Y Q L N C E P P I R P D — E E L L I L N L T R A S L C E R S L L D E K E K L V E L L . C H S V K A  3 3 1  K T P S P T V A H E S N W A K C E D I ^ T Q T Q S E K Q L V L N L T G N T L C A G G A S D E — K L I S L I C R A V K A  3 5 6  * * * *  *  * *  *  *  *  *  *  *  * *  * *  * *  *  *  *  *  *  y S F K P A E D L C T L H V A P I L D N O A V A V K R I I I E T K L S P K A V Y E L L K D R W D D L T E A G V S D M K L G  3 9 1  T F N P A C O K C G I R L A S V P G S Q T W V K E I T I H T K L P A K D V Y E R L K D K W D E L K E A G V S D M K L G  4 1 6  *  *  *  *  *  .  *  *  *  *  *  *  *  *  *  *  *  *  *  *  *  *  *  * *  *  * * * * * * * * * *  K E G P P E W E D R F S L P L I I T r V C M A S F L L L V A A L Y G C C H Q R I S Q R K D Q Q R L T E E L Q T V E N G  4 5 1  D Q G P P E E A E D R F S M P I i l l T I V G M A S F l i l ^ V A A L Y G C C H Q R L S Q R K D Q Q R L T E E L Q T V E N G  4 7 6  ****  *****  **************************  *******************  YHDNPTLEVMETPSEMQEKK\A/NI^GELGDSWIVPLDNLTKDDIJDEEEDTHL  503  Y H D N P T I J ^ / M E T S S E M Q E K K V V S L N G E L G D S W I V P L D N L T K D D L D E E E D T H L  52  ************  *********  8  *****************************  % Amino Acid Identity Overall  43%  Extracellular Domain  30%  Cytoplasmic Domain  96%  F i g . 1 . 4 B : M o u s e a n d h u m a n a m i n o a c i d a l i g n m e n t o f P o d o c a l y x i n . Amino acid sequence alignment of the mouse and human forms of Endoglycan using SIM Binary Alignment from E x P A S y Proteomics Tools (www.expasy.org, 2003). Stars indicate identical amino acids. Triangles indicate cysteines for disulfide bonding. The shaded region indicate the transmembrane domain. The overall sequence identity is 4 3 % , the extracellular and cytoplasmic regions contain 30% and 96% identity respectively. )  7  mCD34 hCD34  MQVHRDTRAGLLLPWRWVALCLMSLLH LNNLTSATTETSTQGISPSVPTNESVEE MPRGWTALCIiSIXPSGFMSI^NNGTATPELPTQGTFSNVSTNVSYQE  *  * **** ***  * *  ** *  ***  * ** *  55 48  *  NITSSIPGSTSHYLIYQDSSKTTPAISETMVNFTVTSGIPSGSGTPHTFSQPQTSPTGIL  115  TTTPSTLGSTSLHPVSQHGNEATTNITETTVKFTSTSVTTSVYGNTNSSVQSQTSVISTV * * **** * * * * * * * * * * * * * * * * *  108  PTTSDSISTSEMTWKSSLPSINVSDYSPNNSSFEMTSPTEPYAYTSSSAPSAIKGEIKCS FTTPANVSTPETTI^SLSPGWSDLSTTSTSLAT-SPTKPYT-SSSPILSDIKAEIKCS  175 166  **  * * * * * * *  ****  *  *  * * *  **  **  *  **  *****  V GIREViaAQGICT^LSEASSCEEFKKEKGEDLIQII^EKEEAEADAGASVCSLLLAQSEV GIREVKLTQGICI^QNKTSSCAEFKKDRGEGLARVT^GEEQADADAGA^ *****  *  ******  * * *  ****  **  *  **  *  *  *****  235 226  * * * * * * * * * * *  V RPECIJ^lVXANSTELPSKI^IJMEKHQ  295  RPQCI^LVLANRTEISSKIJQLMKKH **  * * *  ****  **  ******  286  ******  ****  *  **  ******  * * * * * * * * * *  VLIAILGTTGYFIJ4NRRSWSPTGERIJGEDPYYTENGGGC)GYSSGPGASPETQGKANVTRG  355  ALIA'VXGITGYFIMNRRSWSPTGERIjGEDPYYTENGGGQGYSSGPGTSPEAQGKASViroG  346  ***  **  * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *  * * *  ****  **  382 373  AQENGTGQATSRNGHSARQHWADTEL AQKNGTGQATSRNGHSARQHWADTEL **  *  * * * * * * * * * * * * * * * * * * * * * * * *  % A m i n o A c i d Identity Overall  65%  Extracellular Domain  55%  Cytoplasmic Domain  93%  Fig. 1.4C: M o u s e a n d human amino acid alignment of CD34. Amino acid sequence  alignment of the mouse and human forms of Endoglycan using SIM Binary Alignment from E x P A S y Proteomics Tools (www.expasv.org. 2003). Stars indicate identical amino acids. Triangles indicate cysteines for disulfide bonding. The shaded region indicate the transmembrane domain. The overall sequence identity is 65%, the extracellular and cytoplasmic regions contain 55% and 9 3 % identity respectively.  In a recent study (Fieger et al., 2003), similarities were drawn between the structure and sequence of Endoglycan and P-selectin glycoprotein ligand-1 (PSGL-1).  These two  proteins have homologies in residues important for PSGL-1 ligand function, such as sulfated tyrosine residues and sLex modified threonine residues.  8  In addition, both  P S G L - 1 and Endoglycan have an unpaired cysteine in the membrane proximal region that permits dimerization.  Through the use of recombinant Endoglycan chimeras and  exogeneous post-translational modification enzymes, such as oc(1,3)-fucosyltransferase Vll (FTVII), Endoglycan was shown to bind L-selectin under flow conditions.  Initial analysis of Endoglycan m R N A expression in various human tissues by Sassetti and colleagues showed that there is broad expression pattern of the transcript, which includes brain, pancreas, kidney, liver and all hematopoietic tissues (Sassetti et al., 2000). This broad distribution of Endoglycan is likely to reflect its vascular endothelial expression. In sharp contrast, both monoclonal rat anti-mEndoglycan antibody staining (F4B10, generated in our laboratory), as well as R T - P C R analysis, failed to show the endothelial expression of mouse Endoglycan in primary tissue or cell lines. However, we did detect expression of Endoglycan on closely-associated vascular smooth muscle, and in macrophages, thymocytes and activated B cells (Table 1.1).  To get a hint at the transcriptional regulation of Endoglycan, the databases T S S G , T S S W and Neural Network Promoter Prediction was used to screen the promoter of Endoglycan for potential transcriptional factor binding sites. Our analysis predicts that there  is an overlapping  Stat5b/6 and N F - K B  transcriptional start site respectively.  site, 247bp and 243bp from the  This may indicate competitive regulation of  Endoglycan expression whereby N F - K B enhances transcription and Stat5b/6 represses. Moreover, potential binding site for the transcriptional activator, IRF, was found (Fig. 1.5).  The signaling pathways leading to the activation of these potential transcription  factors will be described in later sections.  9  Tissue/Cells  Endoglycan  Podocalyxin  CD34  Multipotent hematopoietic precursors Adult  + +  Embryo Monopotent precursors Erythroid  +  Thrombocytic  ?  +  Myeloid Lymphoid (subset of thymocytes)  +  Mature hematopoietic cells B Cells (LPS activated) T Cells Macrophages Granulocytes Eosinophils Mast Cells Erythrocytes  +  Platelets  +* +  Vessels Vascular endothelial Vascular smooth muscle  +  Intestinal Epithelial  +  Podocytes  +/+  Brain (Neurons) Boundary Elements (mesothelial)  * embryonic erythrocytes only ** ependymal layer only  Table 1.1: T i s s u e distribution of the C D 3 4 family. Table indicating th edistribution of Endoglycan, Podocalyxin and C D 3 4 in murine tissue. Undetermined expression indicated by (?); Controversy between human and mouse data indicated by (+/-). Data courtesy of Helen Merkens.  IRF3/7  -300  -200  -100  -243 -237  -120  ATG start  bp -86  -73  # TSS = Transcription Start Site Promoter predicted using TSSGJSSW and Neural Network Promoter Prediction, February 2005.  F i g . 1.5: Predicted transcription factor binding sites in the E n d o g l y c a n promoter. Schematic of potential transcription factor binding sites on the Endoglycan promoter. The binding sites for Stat6/5B overlaps with NFKB. Stars, potential transcription start sites; Black arrowhead, A T G start site.  to  1.2  B cell Development in B o n e Marrow  B cells are generated in the liver during mid- to late-stage fetal development and in the bone marrow (BM) thereafter. progenitors ( C L P , Fig. 1.6).  These cells are derivative of the common lymphoid  There are five stages in B cell development in the bone  marrow and they are characterised by various cell surface markers (Fig. 1.7) and rearrangement  and  expression of  immunoglobulin  (Ig)  genes.  B cell  lineage  commitment is distinguished by the expression of the B220 isoform of C D 4 5 (CD45R). Through the use of surface markers, six different factions (Fr.) representing various stages of B cell development have been identified (Fr. A, Fr. B/C, Fr. C , Fr. D and Fr. E) in the bone marrow. There are two different nomenclatures, Philadelphia and Basel, for naming these various B cell fractions and this is summarised in Fig. 1.7. For simplicity, the following description of B cell development will use the Philadelphia nomenclature (Hardy and Hayakawa, 2001).  11  ' Erythrocyte  Fig. 1.6: Hematopoiesis. Schematic of hematopoiesis. H S C , hematopoietic stem cell; M L P , multi-lineage progenitor; C L P , common lymphoid progenitor; C M P , common myeloid progenitor; GMP, granulocyte/macrophage progenitor; MEP, megakaryocyte/eryhroid progenitor. Adapted from (Wagers et al., 2002).  12  0>  a u  1 i i I  2  Z — 0) 0)  a  m  o  0)  o  =  CM OQ  "D <B  .c a.  <5  ^  a  Q O  < W X  „ Q_ CD  6  cc =!  03  0 2  Q  O  o  s  o cc  rr  a. CO  F i g . 1.7: Molecular markers and B cell l y m p h o p o i e s i s . Table showing markers expressed through B cell lymphopoiesis. Adapted from Hardy and Hayakawa, 2001.  13  C L P are cells that give rise to B, T and natural killer (NK) cells. They lack the expression of lineage markers including B220/CD45R (B lineage marker), CD11b/Mac-1 and Gr-1 (myeloid/granulocyte/macrophage  lineage markers)  and Ter119 (erythroid  lineage  marker). However, they are characterised by the expression of c-kit and IL-7 receptor a chain (IL-7Roc).  Immediately following C L P stage and B lineage commitment is the pre-pro-B stage characterised by the expression of B220/CD45R, AA4.1 and low levels of heat-stable antigen (CD24/HSA). AA4.1 is an antigen expressed on cells from early hematopoietic progenitors to immature B cell stage. Pre-pro-B cells have little or no Ig rearrangement and low levels of IL-7Ra. Although the low level of IL-7Ra expression has been under controversy in recent years (Tudor et al., 2000), it has been suggested through propidium iodide analysis of D N A content that this diminished expression is due, in part, to cell arrest in GJG,  stage (Hardy and Hayakawa, 2001). Therefore, suggesting that  these cells are quiescent and IL-7Rtx has a higher turnover rate than other surface molecules such as B220 or A A 4 . 1 .  Hence, the use of IL-7Ra as an identifier and  molecular handle for the isolation of B cells should be used with caution as the proliferative phase of cells can alter its expression.  Pro-B cells are defined as a stage when immunoglobulin  heavy (IgH)  locus is  undergoing rearrangement for production of the immunoglobulin p heavy chain. These cells are characterised by C D 1 9 and high level of IL-7Ra expression. Moreover, Pro-B cells express the surrogate light chain (SLC), as well as the B cell receptor (BCR)associated molecules Iga and lgp\ The S L C (comprised of two proteins, VpreB and X.5) together with Igji and the signaling heterodimer Iga/lgp" will form the pre-BCR in early pre-B cells. Transgenic mice have been generated that targeted proteins responsible for Ig rearrangement,  such as the severe combined immunodeficiency  recombination activating gene (RAG)-deficient mice.  (SCID)  and  These mice fail to generate the  pre-BCR leading to a block of B cell development at the early pro-B stage (Blunt et al., 1995; Oettinger et al., 1990; Reichman-Fried et al., 1990; Shinkai et al., 1992). Similarly, elimination of components of the pre-BCR, such as X5 or IgP also blocks development at this stage (Gong and Nussenzweig, 1996; Kitamura et al., 1992).  14  Early pre-B cells are marked by a rapid proliferation and are characterised by the expression of the pre-BCR, BP-1 and C D 2 5 .  A conceptual model for this proliferative  burst is that it is an Igu-dependent quality assurance step for the future production of stable BCR.  The S L C acts as a template and failure of the Igu. to interact with the S L C  to form a properly signaling pre-BCR will be negatively selected (Karasuyama et al., 1996;  Meffre and Nussenzweig, 2002). Upon exiting this proliferative phase, cells enter  the late pre-B stage. At this stage, cells are undergoing immunoglobulin light chain (IgL) locus rearrangement for the production of the immunoglobulin K or A, light chain. Late pre-B cells no longer express c-kit, C D 4 3 or  SLC.  Cell surface expression of the B C R (which consists of Igp heavy chain, K or X light chain and  Igct/lgB) is a hallmark of new or immature B cells. Cells at this stage of maturation  no longer express C D 2 5 , IL-7Ra or BP-1,  and also at this stage, selection against self-  reactive B cells occurs. One of three events may follow during this selection process: 1. Apoptosis (clonal deletion); 2. Anergy; or 3. Receptor editing. Ligation of an antigen with a B C R that produces a strong signal leads to apoptosis. This is a negative selection process to prevent autoreactive B cells from leaving the bone marrow. Of the - 2 x 1 0  7  immature B cells produced in the B M of mice daily, only - 1 0 % exit to the periphery (Chung et al., 2003; Freitas et al., 1986). Lower affinity interactions will result in anergy, which are nonresponsive short-lived B cells, or editing, whereby cells revise their B C R to eliminate self-reactivity (Benschop et al., 1999; Goodnow et al., 1990; Hartley et al., 1991;  1.3  Hartley and Goodnow, 1994).  Peripheral B cells  There are seven subsets of peripheral B cells and they differ by expression of cell surface markers (Fig. 1.8), reactivity and location. However, it is important to note that the use of surface markers for characterisation of peripheral B cells must be interpreted with caution as maturation may lead to transient expression of precursor-like molecules, and  activation may alter the expression of these molecules. Transitional B cells mark  the link between immature and mature B cells; these cells express AA4.1 and high levels of CD24/HSA.  The ability for these cells to edit their receptor is still a matter of  controversy with studies of R A G expression in these cells. Suggesting that selection and  receptor editing may take place in the periphery prior to B cell activation (Monroe et  15  al., 1999; Y u et al., 1999). Transitional B cells migrate to the spleen upon entering the open circulation from the B M , however, they are excluded from the lymph nodes.  Red pulp outer P A L S IgMhi  IgDIo C023C021lo CDIdio  16  The tissue specific homing phenomenon may be due, in part, to the lack of C D 6 2 L or fibronectin expression on these cells (Flaishon et al., 2000; Loder et al., 1999). There are three types of transitional B cells, Transitional stage 1 (T1), transitional stage 2 (T2) and transitional stage 3 (T3) B cells. T1 B cells, also known as newly formed (NF) B cells, phenotypically resemble immature B cells in their cell surface markers and susceptibility to negative selection (Allman et al., 1992; Norvell and Monroe, 1996). They are lgM lgD CD21 CD23"CD1d'° and are found in the red pulp outer periarteriolar hi  l0  lo  lymphoid sheath ( P A L S , Liu, 1997). T2 B cells can be delineated from T1 B cells by their expression of CD23, CD21 and IgD.  These cells are detected in splenic B cell  follicles (Chung et al., 2002; Loder et al., 1999; Schiemann et al., 2001), a migrational process that is dependent on the expression of the chemokine receptor C X C R 5 (Forster et al., 1996). There are two types of T2 B cells: T2 follicular precursors (T2-FP) and T2 marginal zone precursor (T2-MZP). T 2 - F P B cells are recirculating B cells that reside primarily in follicles and are CD1d'° (Pillai et al., 2005).  T 2 - M Z P B cells are C D 1 d  hi  cycling cells found only in the spleen and not in the B M , blood or lymph nodes (Loder et al., 1999; Saito et al., 2003). The third transitional subset, T3 B cells, are newly matured follicular (FO) B cells found in the spleen or lymph node that are phenotypically similar to T2 B cells with the exception of lower surface IgM expression (Allman et al., 2001). The sequential maturation of transitional B cells is summarised in Fig. 1.8.  Accumulating evidence has demonstrated  the  role of positive selection on  recruitment of transitional B cells to the mature B cell pool.  the  In germ-free mice, the  number of mature B cells is reduced without a reduction in the number of T2 B cells (Loder et al., 1999). Moreover, in in vitro analyses, B C R engagement has been shown to positively select T2 B cells for differentiation into mature B cells (Su and Rawlings, 2002). Although other studies have also shown that T1 and T2 B cells are sensitive to deletion by antigen stimulation, apoptosis in these cells can be rescued by helper T cells (Allman et al., 2001; Chung et al., 2002).  Aside from antigen-dependent selection on  transitional B cells, studies are beginning to show the importance of non-antigen receptor-mediated selection processes. The absence of B A F F (B cell activating factor belonging to the tumour necrosis factor family) will block the maturation progression from T1 to T2 B cells (Gross et al., 2001; Schiemann et al., 2001).  17  There are five mature B cell subsets. F O B cells are lgM lgD CD21 CD23 CD1d'° and lo  hi  int  +  are found in follicles of spleen and lymph nodes. Marginal zone (MZ) B cells carry an activated phenotype ( l g M l g D C D 2 1 C D 1 d ) . They are found in the marginal zone of hi  l0  hi  hi  the spleen and represent - 5 % of the splenic B cell population. Mature recirculating (MR) or B-2 B cells lack AA4.1 and C D 5 expression. B-1a B cells also lack AA4.1 expression, but do express C D 5 . B-1b B cells are similar to B-1a B cells with the exception of no C D 5 expression.  Both B-1 B cell subpopulations are self-renewing and they have  preferential localisation in the peritoneal, pleural cavities and gut lamina propria. These cells express CD11b/Mac-1 in peritoneal or pleural cavities, however, CD11b/Mac-1 expression is absent in the spleen.  1.4  M e c h a n i s m s for Localisation a n d Migration of B cells in Lymphoid  As described previously, different  subsets of mature  B cells exhibit  Organs  preferential  localisation in different regions within lymphoid organs (Fig. 1.9). For example, the only subsets found in the follicles of spleen are F O , T2-FP and T2-MZP B cells. Conversely, MZ B cells are only found in the marginal zone. The following will explore some of the mechanisms that play a role in retaining MZ B cells in the marginal zone.  The entry of lymphocytes into the spleen is mediated by the interaction between LFA-1 and a4p1 integrins on lymphocytes and ICAM-1 and vascular cell-adhesion molecule 1 (VCAM-1) on splenic radioresistant cells, respectively (Lo et al., 2003). This adhesion event is likely to be dependent on a chemokine that is yet to be defined. Once in the spleen, B cells may be drawn into the follicles by a C X C L 1 3 chemokine gradient. C X C R 5 is the receptor found on B cells for C X C L 1 3 . However, MZ B cells are able to evade this gradient via high expression of  and S 1 P ' which are receptors for 3  lysophospholipid sphingosine-1 phosphate (S1P). SIP-deficiency represents the only genetic model in which MZ B cells are formed but are mislocalised in follicles (Cinamon et al., 2004). Therefore, C X C L 1 3 and S 1 P act in an opposing manner to manage the localisation of M Z B cells. downregulation of S1 P  1 t  Activation of M Z B cells by antigen or L P S leads to a  thus allowing for M Z B cells to migrate into follicles.  The mechanism by which M Z B cells are retained in the marginal zone is poorly understood.  It has been postulated that interaction between M Z B cells and marginal  zone macrophages (MZM) plays an important role in this phenomenon. One possible 18  molecular player for the interaction between MZ B cells and M Z M s is the scavenger receptor, macrophage receptor with collagenous structure  (MARCO), on  MZMs.  However, the biochemical basis of this interaction remains unknown (Karlsson et al., 2003; Kraal, 1992). Another possible mechanism in the retention of MZ B cells is the interaction between integrins on B cells and integrin ligands on stromal cells.  Both  integrins, LFA-1 and a4pi integrin, are expressed at higher levels on M Z B cells than F O B cells. Interactions between LFA-1 with ICAM-1, and a4pi integrin with VCAM-1 and fibronectin may be responsible for the adhesion of MZ B cells in marginal zone. Similar to S I P L these integrins are downregulated upon activation (Lu and Cyster, 2002).  RecJpulp Follicle  Fig. 1.9: Microvasculature of the spleen. Diagram showing the microvascular organisation of the spleen. Newly-formed B cells exit the artery into the periarteriolar lymphoid sheath (PALS) where they migrate to the follicle and marginal zone as they mature. Pillai et al., 2005.  1.5  B cell Activation and Inflammatory R e s p o n s e  1.5.1  T cell-Dependent Activation  Upon binding of the B C R on mature recirculating (MR) or F O B cells to an antigen, these cells migrate to the T cell zone where they receive co-stimulation by C D 4 0 ligand (CD40L) and cytokines, primarily IL-4, from helper T cells (Cyster et al., 1999; Gold and Matsuuchi, 1995).  If B cells fail to receive co-stimulatory signals, they will become  anergic and undergo apoptosis (Gold, 2002). On the other hand, if both B C R and costimulatory signals are present, B cells become activated, proliferate and migrate to the lymphoid follicles. Germinal centres (GC) are established in the follicles, and these are specialised compartments where activated B cells undergo a proliferative burst, affinity maturation and immunoglobulin class-switch recombination.  Affinity maturation is a  process where somatic hypermutation of Ig genes will refine the B C R to bind the stimulating antigen at the highest affinity, whereas class-switch recombination is a process where the Ig heavy chain (usually u.) is replaced by a 3' gene segment (y, e or a). Both antigen-specific T helper cells and follicular dendritic cells (FDC) are crucial for this response in the G C (Manser, 2004).  Helper T cells provide co-stimulation to the  activated B cells by the interaction of CD40L and inducible T-cell co-stimulator (ICOS) on T cells with C D 4 0 and ICOS ligand on B cells respectively. F D G s are responsible for sequestering antigen in germinal centres. Activity in germinal centres peak at 10 to 14 days post-immunization, when plasma cells and memory B cells exit the germinal centre.  1.5.2  T cell-Independent Activation  B-1 and MZ B cells have been described as cells with "natural memory" because they have a repertoire that recognises T-cell independent type 2 (TI-2) antigens.  TI-2  antigens contain multiple identical epitopes that are capable of cross-linking B C R s .  B-1  and MZ B cells are the first response to foreign antigens.  Upon activation, these cells  migrate to the red pulp of the spleen and form foci of plasmablasts (Lopes-Carvalho and Kearney, 2004), and mature into antibody-secreting plasma cells (Bikah et al., 1996; Hippen et al., 2000; Oliver et al., 1997).  The ability for these subsets of B cells to respond rapidly to multivalent antigens is due, in part, to the high expression of complement receptor 2 (CD21) that assists in the capture of complement coated polysaccharides (Guinamard et al., 2000). 20  Furthermore,  these cells have a lower threshold for activation in response to B C R or toll-like receptors (TLR) than MR and F O B cells. One reason for this lowered threshold is due to higher resting levels of B-lymphocyte-induced maturation protein 1 (Blimp-1), hence allowing these cells to differentiate quickly into plasma cells upon activation (Martin et al., 2001). Similar to T cell-dependent activation, B-1 and M Z B cells also require a second signal in addition to B C R ligation.  This signal is most likely provided by TLRs, however,  differentiation of these cells into plasma cells appears to require signals from bloodborne dendritic cells (Pillai et al., 2005). n  1.5.3  TLRs and B cell Activation  T L R s are a family of pattern recognition receptors (PRRs) that recognise pathogenassociated molecular patterns ( P A M P S ) . P A M P S are conserved molecular patterns that are unique to microorganisms (Janeway and Medzhitov, 1998). The number of T L R s varies between mammalian species; however, most have 10 to 15 T L R s . Different T L R s recognise different P A M P S (Table 1.2) and their specificity is regulated by location of expression.  For example, T L R 4 recgonises L P S , T L R 2 detects bacterial lipoproteins  and lipoteichoic acids and T L R 9 detects unmethylated C p G D N A of bacteria and viruses. A s shown in Table 1.4, T L R s 1, 2, 4, 5 and 6 are specific for the recognition of P A M P S unique to microorganisms. However, this self-non-self discrimination is not as evident in the ligands of T L R s 3, 7, 8 and 9. Therefore, specificity of these T L R s is controlled by intracellular localisation where they detect viral nucleic acids in late endosomes-lysosomes (Diebold et al., 2004; Heil et al., 2003; Lee et al., 2003; Lund et al., 2004; Matsumoto et al., 2003).  B-1 and MZ B cells are the most sensitive of all the B cells subsets to T L R stimulation in their TI-1 response. T L R stimulation in these cells leads to polyclonal activation and production of low affinity IgM antibodies. In the absence of B C R ligation, L P S alone is sufficient to induce expression of Blimp-1 and differentiation of B cells into plasma cells in murine splenocytes (Lin et al., 2002; Schliephake and Schimpl, 1996; Shaffer et al., 2002; Shaffer et al., 2004). Moreover, NF-KB-binding sites are found upstream of the gene encoding Blimp-1, Prdml. The murine 3T3 fibroblasts, which lack the p50 and p65  21  Receptor  Ligand  Origin of Egand  TLR1  Trlacyl lipopeptides Soluble factors  Neisseria meningitidis  Bacteria and  Lqxjproteirv^popepttdes Peptlctoglycan L|pot$tCT»ooc add  TUR2  various pathogens Gram-positive bacteria Gram-positive bacteria Mycobacteria \  L^parabfiTKinTarrian.  Phenol-soli^'mooulin GVcolniositc^rwsphQS^cte Glycolipids , Porins Atypical |xipolysaccharicte Atypical ^p^y^accnancte Zymosan Heat-shock protein 70* Double-stranded RNA  Trypemsoma cnjzl Treponema maltpphilum ..Neisseria"...  r  TLR3 TLR4  mycobacteria  ,.L  Leptospira interrogans Pofphyrornhms gingivalis  s s  Fungi Host  Viruses  L^potysacchatlcte TaxoS, Fuston protein Er^op© protein Heat^nock protein 60* Heat-^shock protein 70* Type III repeat extra domain A of fferonectir.* Oligosaccharides of hyaturanfc add* Polysaccharide fragments of heparan sulphate* fibrinogen*  Granvnegatlve bacteria Plants ^ Respiratory syncytial virus - " srvtoij^mamma^  Chlamydia pneumoniae Host ' Host.. .'. Host Host Host  TLRS  Rageliiri  Bacteria  TLR6  Diacyl ilpopeptides L^otefciwfc add Zymosan  Mycoplasma Gram-positive bacteria Fungi  TLR7  rrsdazoqucnoftne . Loxoribine Bropdrtrrtfie Singte-stranded RMA  Synthetic compounds Synthetic oompounrjs Synthetic compounds Viruses  TLRS  &rida2oqu^66ne Smgte-stranded RNA CpG-containing DNA  Synthetic compounds Viruses Bacteria and viruses  ND\i  ND  TUR9 TLR10 TLR 11  -V  . • ....  ^  -*  J '  N.D.  Uropathogenic bacteria T a b l e . 1.2: T o l l - l i k e r e c e p t o r s a n d t h e i r l i g a n d s . Table of various TLR, the potential ligands and the origins of those ligands. N . D . , not determined; TLR, toll-like receptor. Adapted from Akira and Takeda, 2004. subunits of NF-KB,. fail to elicit an induction of Blimp-1 upon T L R stimulation (ShapiroShelef and Calame, 2005). These results are consistent with the observation that T L R stimulation leads to B cell activation and plasma cell differentiation. In order to prevent autoreactivity, B cells expressing self-reactive B C R have induced tolerogenic signaling pathways that inhibit TLR-induced activation (Rui et al., 2003). However, in memory B  22  cells where specificity for antigens has already been selected for, T L R stimulation can induce polyclonal activation with cognate T cell help (Bernasconi et al.,  1.5.4  2002).  M e m o r y Band P l a s m a cells Formation  Memory B and plasma cells are products of germinal centres. Memory B cells retain their high affinity B C R and have an intrinsic ability to respond more quickly upon antigen stimulation than naive B cells (Tangye et al., 2003). These are lgP Syndecan-1(SDC1)~ +  cells that provide surveillance against secondary encounter of antigen and are not antibody-secreting cells. A s described previously, T L R stimulation combined with T cell help is suffice to induce the activation of memory B cells (Bernasconi et al., 2002). There are two subpopulations of memory B cells and they are delineated by their B220 expression. Through adoptive transfer and antigen recall experiments, it is believed that these two subsets are in a linear developmental path (Driver et al., 2001). The postgerminal centre memory B cells are B 2 2 0 and they differentiate into 'pre-plasma' +  memory cells which are B220". However, the existence of this B220" subpopulation has been challenged recently by the suggestion that these are non-B cells that captured B C R s via surface Fc receptors (Bell and Gray, 2003).  Plasma cells are S D C 1 \ terminally differentiated, non-dividing, antibody-secreting B cells. Their derivation is driven by the transcription factor Blimp-1 (Turner et al., 1994). Blimp-1 represses the expression of anti-apoptotic B C L - 2 family protein A1 and induces the expression of pro-apoptotic genes, such as growth arrest and DNA-damageinducible 45 (GADD45) and C / E B P homologous protein ( C H O P , Knodel et al., 1999; Shaffer et al., 2002). It has long been understood that, due, in part, to the expression of Blimp-1, plasma cells are short-lived.  In cultures, plasma cells stop dividing and die  within a few days of differentiation (Shapiro-Shelef and Calame, 2005). However, it has been demonstrated that adoptive transfer of virus-specific plasma cells into mice depleted of memory B cells leads to survival and antibody production for more than a year (Slifka et al., 1998). Therefore, although memory B cells have a role in long-term immunity, there is evidence for a new group of antibody-secreting B cells that provide persistent antibodies independent of antigen; these are called long-lived plasma cells. Long-lived plasma cells reside mainly in the B M ; however, some have been found to reside in the spleen (Sze et al., 2000).  The mechanism by which these two types of  plasma cells are derived is unknown. One possibility is that long-lived plasma cells arise 23  depending on the ability of plasma cells to find a niche in the B M for survival signals provided by B M stromal cells (Manz and Radbruch, 2002).  The relationship between memory B cells and plasma B cells is yet to be defined. There are two alternative hypotheses for these late stages of B cell differentiation: 1. Plasma cells differentiate from memory B cells in a linear pathway (Shapiro-Shelef et al., 2003), or 2. memory B cells and plasma cells are separate cell fates derived from germinal centres.  1.6  Mechanism for Localisation and Migration of Memory and Plasma cells  As described above, the migration and localisation of B cells in the marginal zone of lymphoid organs is highly specific and requires chemokine receptors, integrins and carbohydrates that bind selectins. The homing and migration of memory and plasma cells is similar and requires participation from each of these molecular players (Fig. 1.10).  By altering the expression levels of chemokine receptors, B cells are directed to different lymphoid regions and organs. Downregulation of C X C R 5 on post-germinal centre cells reduces signals from its ligand C X C L 1 3 , and allows these cells to exit from follicles (Hopken et al., 2004). Expression of C X C R 4 is critical for the migration of plasma cells to regions that express its ligand, C X C L 1 2 , such as splenic red pulp, lymph-node medullary cords and B M (Hargreaves et al., 2001). It has been shown that C X C R 3 and C X C R 6 with their ligands, C X C L 9 and C X C L 1 6 respectively, are important for plasma cell homing.  Similar to the expression of chemokine receptors, the expression of different integrins can alter homing of plasma cells. B M plasma cells express a 4 p i integrin, which binds V C A M - 1 at the surface of B M epithelial cells. However, plasma cells that express 0:4(37 integrin will bind mucosal vascular addressin cell-adhesion molecule 1 (MADCAM-1) on intestinal epithelial cells and thereby reside in mucosal sites (Kunkel and Butcher, 2003). The localisation of plasma cells in B M also involves interactions between endothelial-cell selectin (E-selectin) on B M epithelial cells and its polysaccharide ligand on plasma cells (Underhill et al., 2002).  24  F i g . 1.10: Molecular interaction for retention of p l a s m a c e l l s in B M . Illustration of the molecular interactions involved in retaining plasma cells in the B M niche. Adapted from Shapiro-Shelef and Calame, 2005.  Blimp-1, as described previously, is important in the differentiation of B cells into plasma cells. This transcription factor has been shown to repress expression of C X C R 5 (Shaffer et al., 2002) and induces expression of a4 integrin and C X C R 4 (Sciammas and Davis, 2004).  Although Blimp-1 is partially responsible for changes in expression levels of  25  these chemokine receptors and adhesion molecules, the regulatory mechanisms by which plasma cells determine their localisation is yet to be defined.  1.7  Cytokines and Their Effect on B cells  Cytokines secreted by dendritic cells or T cells have many roles in modulating B cell activation, development and survival. Cytokine receptors are heterodimers and usually consist of a long signaling chain and a shorter common chain. Different combinations of the various chains allow for cross-reactivity of cytokines.  The common chain, y , is c  shared in the receptor of IL-2, IL-4 IL-9, IL-7, IL-15 and IL-21.  However, receptor  combination is not restricted to the y chain. IL-4 and IL-13 share a common signaling c  chain, IL-4Ra. It was shown that IgM-specific antibodies inhibit the differentiation of splenic B cells into plasma cells by L P S (Schliephake and Schimpl, 1996). However, the presence of IL-2 and IL-5 can overcome the effects of IgM-specific antibodies and drive plasma cell differentiation (Schliephake and Schimpl, 1996). Moreover, CD40L, IL-4 and IL-5 along with multivalent B C R signals lead to generation of a much more pronounced plasma cell population than Ig-specific antibodies alone (Snapper et al., 1995). In studies with cell lines, IL-2 and IL-5 (Turner et al., 1994) or IL-6 alone (Piskurich et al., 2000) can induce Blimp-1 expression. In human centrocyte cultures, IL-10 was also able to induce Blimp-1 expression (Shapiro-Shelef and Calame, 2005).  Similarly, IL-21 can lead to  plasma cell formation in primary B cells (Ozaki et al., 2004). The signaling downstream of many interleukin  receptors involves the signal transducers and activators of  transcription (STAT) family of transcription factors. IL-4 and IL-5 induces the activation of STAT6 and STAT5b respectively.  Hence, showing the importance of these  transcription factors in the regulation of B cell development.  IL-1(3 is a unique cytokine because its receptor is a part of the IL-ip receptor/toll-like receptor superfamily. These receptors share similar signaling cytoplasmic domains with conserved regions called toll-interleukin 1 receptor (TIR) domain. Stimulation of the IL1P receptor will lead to expression of many pro-inflammatory genes; hence IL-ip is an important mediator of the inflammatory response (O'Neill, 2000).  The downstream  signaling from the receptor of this pro-inflammatory cytokine is similar to that of the T L R pathway which is described in detail below.  26  Conversely, transforming growth factor (3 (TGFP) is an anti-inflammatory cytokine. The role of this cytokine in early hematopoiesis has been shown to influence proliferation and differentiation (Jacobsen et al., 1991; Keller et al., 1991). The ability of this cytokine to inhibit cell cycle progression, along with endogenous production of T G F p by H S C , serves as a means for maintaining quiescence in these cells (Ducos et al., 2000; Hatzfeld et al., 1991; Pierelli et al., 2000). This is further demonstrated by studies using TGFp-deficient mice, where their number of circulating granulocytes, monocytes and platelets is significantly increased (Kulkarni et al., 1993; Shull et al., 1992). Moreover, the transcription of the stem cell antigen, C D 3 4 , is upregulated by T G F p (Batard et al., 2000; Marone et al., 2002).  In B cell lymphopoiesis, T G F p was shown to inhibit the proliferation and the expression of K light chain in B cell precursors (Lee et al., 1987). T G F p also inhibits proliferation of activated mature B cells (Armitage et al., 1993; Bouchard et al., 1994). Furthermore, the downmodulation of N F - K B activity by T G F p has been implicated in the induction of apoptosis of resting B cells (Lomo et al., 1995) and certain B cell lines (Arsura et al., 1996; Coffey et al., 1988; Fischer et al., 1994; Pietenpol et al., 1990). Aside from its antiproliferative and proapoptotic role, T G F p can also influence isotype switch in activated B cells. In contrast to IL-4, which promotes the secretion of IgE antibodies (Lebman and Coffman, 1988), T G F p antibodies (Kim and Kagnoff, 1990a).  induces isotype switching to secreted IgA It has also been observed that IL-2 and IL-5  increase this IgA-specificity effect (Coffman et al., 1989; Kim and Kagnoff, 1990a; Kim and Kagnoff, 1990b; Lebman et al., 1990; Sonoda et al., 1989).  T G F p signals through a two-part receptor, type I (TpRI) and type II (TpRII).  Upon  binding of T G F p to TpRII, TpRI is recruited and phosphorylated, forming the activated TGFp-receptor complex. TpRI then phosphorylates Smad2 and Smad3, both of which are receptor-activated Smad (R-Smad) proteins. Activated R-Smad forms a heteromeric complex with common mediator Smad (co-Smad) 4 and translocate into the nucleus where they act, directly or with coactivators or compressors, to regulate transcription (Hata, 2001; Wotton et al., 1999). This pathway can be modulated through inhibitorySmads, such as Smad6 and Smad7, which binds activated R-Smad to form a stable complex.  T G F p can also activate other signaling pathways including the PI3K and  27  M A P K pathways (Hartsough and Mulder, 1995; Kretzschmar et al., 1999; Sano et al., 1999; Shibuya et al., 1996; Yamaguchi et al., 1995; Y u e et al., 1999).  Signaling of these various cytokines is interwoven in a network and much crosstalk exists between the pathways.  Proinflammatory cytokines, such as IFNy, T N F a and IL-  1|3, inhibit the T G F P signaling pathway through expression of Smad7 (Bitzer et al., 2000; Topper et al., 1997; Ulloa et al., 1999). Similarly, the induction of proliferation by IL-ipinduced cytokines in H S C is inhibited by T G F P (Ruscetti et al., 1992).  Moreover, the  M A P K K K homologue, T G F p activated kinase 1 (TAK1, (Yamaguchi et al., 1995), is a mediator of both T G F p and IL-ip signaling cascades (Jiang et al., 2002; Ninomiya-Tsujj et al., 1999; Takaesu et al., 2000).  1.8  TLR  Signaling  All T L R share a common signaling cytoplasmic TIR domain.  The best-characterised  signaling pathway is the myeloid differentiation protein (MyD) 88-dependent pathway (Fig. 1.11).  MyD88 consists of two domains, a C-terminal TIR-binding domain that  interacts with T L R and an N-terminal death domain which recruits members of the IL-1 receptor-associated  kinases,  IRAK-1  and  IRAK-4.  These  kinases  undergo  autophosphorylation and associate with the TNF-receptor associated factor, T R A F 6 . Through unknown mechanisms, T R A F 6 activates M A P kinases, as well as the IKB kinases, IKKoc and IKKp.  Activation of these kinases leads to the activation of the  transcription factors AP-1 and NF-KB, and expression of proinflammatory genes.  However, emerging evidence is beginning to reveal the importance of another T L R signaling pathway, the MyD88-independent pathway (Fig. 1.12). Studies have shown that signals from T L R 3 and T L R 4 are not abolished in MyD88-deficient mice (Hoshino et al., 2002; Kawai et al., 2001). adaptor molecule, Trif.  This phenonmenon has been attributed to another TIR  Trif can associate directly with T L R 3 or indirectly with T L R 4  through another TIR adaptor molecule, T R A M . The importance of Trif in T L R signaling was demonstrated  through  Trif-MyD88  double-deficient  responses were abrogated (Yamamoto et al., 2003).  cells, whereby  all  TLR4  Trif associates with both the  transcription factor interferon regulatory factor 3 (IRF-3) and its activating kinase, TBK-1 (Fitzgerald et al., 2003; Sato et al., 2003; Sharma et al., 2003). Activation of IRF-3 will  28  TLR  Fig. 1.11: MyD88-dependent T L R s i g n a l i n g pathway. pathway upon T L R activation. Takeda and Akira, 2004.  MyD88-dependent signaling  lead to expression of type I interferons, such as IFNp\ Trif also associates with T R A F 6 , hence leading to downstream activation of M A P kinases and N F - K B , similar to the MyD88-dependent pathway.  More recently, Trif was shown to also associate with RIP1  and RIP3 through RIP homotypic interaction motif (RHIM) in the C-terminal region of Trif. Unlike RIP3, RIP1 contains a death domain capable of initiating downstream signaling to activate N F - K B .  Conversely, RIP3 acts as a negative regulator to RIP1 signaling by  preventing the association of RIP1 with Trif (Meylan et al., 2004).  29  IRF-3  NF-KB  MAPKS  Fig. 1.12: MyD88-independent T L R signaling pathway. MyD88-independent signaling pathway upon T L R activation. Barton and Medzhitov, 2004.  1.9  T h e s i s Objectives  Since the discovery of human Endoglycan by Sassetti and colleagues in 2000 and murine Endoglycan in our laboratory, little has been revealed about the regulation and function of this novel molecule. 30  Our initial attempt to determine the function of Endoglycan was to compare this molecule to its family members, CD34 and Podocalyxin. Our laboratory has demonstrated that ectopic expression of CD34 in CD34-deficient murine mast cells (Drew et al. 2005), or Podocalyxin in breast cancer cells, induces a profound  reduction  in  homotypic  aggregation/adhesion. Surprisingly, ectopic expression of Endoglycan in C D 3 4 / C D 4 3 deficient BM-derived mast cells did not decrease aggregation (Fig. 1.13).  Moreover,  higher levels of Endoglycan expression were observed in well-behaved highly polarized breast cancer cell lines (such as T47D and MCF-7) compared to aggressive lines (such as MDA-231), whereas the opposite is true of the anti-adhesin Podocalyxin (Fig. 1.14). These data suggest that Endoglycan may have a different function than C D 3 4 and Podocalyxin. Rather than a global anti-adhesive molecule, we propose that the highly conserved extracellular domain of Endoglycan confers a pro-adhesive function in this molecule.  Although Endoglycan has been demonstrated to bind L-selectin, these assays were performed in an in vitro and artificial environment (Fieger et al. 2003). Therefore, the function of. Endoglycan on primary cells remains largely unknown. tools available to analyse this novel molecule are also limited. generated extracellular  a rat monoclonal IgM antibody acidic  domain  of  Furthermore, the  Our laboratory has  against the region N-terminal  Endoglycan  using  the  to  sequence  the N-  V A S M E D P G Q A P D L P N L P S I L P K M D L A E P P W H M P L Q G G C - C . This antibody recognises both the mouse and human forms of Endoglycan; however, its utility is limited to flow cytometry. F i g . 1 . 1 3 : B o n e m a r r o w - d e r i v e d m a s t c e l l a g g r e g a t i o n a s s a y . Aggregation assay using BM-derived mast cells. A . Mast cells cultured from wild-type C57BI/6 B M . 3 ± 2% of these cells are in aggregates and the average number of cells per aggregate was 4 ± 2. B. Mast cells cultured from C D 3 4 and CD43-deficient C57BI/6 B M and infected with empty pMXpie vector. 94 ± 3% of these cells were in aggregates and the average number of cells per aggregate was 63 ± 8 cells. C . Mast cells cultured from C D 3 4 and CD43-deficient C57BI/6 B M and infected with pMXpie plasmid containing the full-length C D 3 4 . 41 ± 19% of these cells were in aggregates and the average number of cells per aggregate was 14 ± 5 cells. D. Mast cells cultured from C D 3 4 and CD43-deficient C57BI/6 B M and infected w/ cytoplasmically truncated CD34. 27 ± 2% of these cells were in aggregates and the average number of cells per aggregate was 14 ± 6. E. Mast cells cultured from CD34 and CD43-deficient C57BI/6 B M and infected with pMXpie plasmid containing full-length Endoglycan. 92 ± 1 % of these cells were in aggregates and the average number of cells per aggregate was 63 ± 8 cells. F. F A C S profiles 31  showing the expression of G F P and Endoglycan, represented by the x- and y-axis respectively, by the Endoglycan-expressing mast cells. Wildtype and CD34 data courtesy of Erin Drew (Drew et al., 2005).  A  B wildtype  30 -i  cd34^~ / cd43~^~ + p M X p i e  • % cells in aggregates • # cells/aggregate  20  10  T  IZZ n=8044  n=2240  C  D  cd34' ' / cd43' - + C D 3 4 /  /  E  cd34' - / cd43' + C D 3 4  F L  /  A  C T  cd34' - / cd43~ + E n d o g l y c a n /  n=8845  n=1838  n=5120  CO  o D>  O :  i  T3 C  LU .  GFP  GFP  32  A  A MDA-231  MCF-7  T47D  Endoglycan  H  Isotype Control  Podocalyxin  F i g . 1.14: E x p r e s s i o n levels of P o d o c a l y x i n and E n d o g l y c a n o n breast cancer cell lines. Expression of Endoglycan and Podocalyxin on three human breast carcinoma lines: MDA-231, M C F - 7 and T47D. A. F A C S profiles of Endoglycan expression on the three breast cancer cell lines. Endoglycan was not expressed on MDA-231. However, expression was found on both M C F - 7 and T47D. B. F A C S profiles of Podocalyxin on the breast cancer lines. Podocalyxin was highly expressed on MDA-231 and less on M C F - 7 . C. Western analysis showing the lack of Podocalyxin on T47D. Podocalyxin data courtesy of Julie Nielsen and (Doyonnas et al., 2001).  In order to study the function of Endoglycan in a natural setting, we looked to the naturally inducible expression of Endoglycan on primary splenocytes.  This regulated  expression of Endoglycan on activated B cells may be indicative of a specific function for Endoglycan in the inflammatory response. The results obtained in this thesis will provide understanding of the expression of Endoglycan on B cells and the regulation of its expression.  The function of Endoglycan still needs further exploration, however, by  understanding the regulation of this molecule in B cells, we provide here an in vivo functional model, as well as a system to further explore this field.  33  2.  MATERIALS AND METHODS  2.1  Mouse Strains  All mice were maintained at the Biomedical Research Centre (BRC) animal core facility. C57BI/6, C57BI/10, C B A / N , CBA/N-xid (Berning et al., 1980) and C57BI/10ScN (Poltorak et al., 2001) were all purchased originally from Jackson Laboratory (Bar Harbor, ME). Balb/c,  Balb/c-nu. and CD1-nu were  (Wilmington,  MA).  obtained  from  Charles River  Laboratory  CD1 mice were purchased from U B C Animal Care Centre  (Vancouver, B C ) . P-sel K O BI/6 mice (Dr. Richard Hynes, Massachusetts Institute of Technology) and PSGL-1 K O BI/6 mice (Bruce Furie, Harvard Medical School/Beth Israel Deaconess) were a kind gifts from Dr. Hermann Ziltener (Rossi et al., 2005). C2GnTI K O mice and PSGL-1/C2GnTI K O mice were a kind gifts from Dr. Hermann Ziltener (Merzaban et al., 2005).  C D 3 4 / C D 4 3 K O mice were generated in Ziltener  laboratory as described previously (Drew et al., 2005).  2.2  Primary Cell Culture  Primary B M cells were obtained by flushing the femur with H B S S supplemented with 2% heat-inactivated fetal bovine serum (FBS) using a 26 1/2 needle and homogenizing with G  a 22 1 1/2 needle. Red cells were lysed by treatment with 0.1 M NH CI for 3 min at 37°C G  4  and washed with equal volume of supplemented H B S S . B M cells were cultured in RPMI 1640 (Invitrogen Gibco, Burlington, ON) supplemented with 15% heat-inactivated F B S , 2mM glutamine, 2% IL-3 condition media ( J W W , B R C , Vancouver, BC), 15% stem cell 3  factor (SCF) condition media ( B R C , Vancouver, BC), 10ng/ml recombinant mouse IL-6 (R&D Systems, Minneapolis, MN), penicillin and streptomycin.  B M mast cells were  derived by culturing B M cells in RPMI 1640 supplemented with 15% heat-inactivated F B S , 2mM glutamine, 4% J W W , penicillin and streptomycin. 3  Primary splenocytes were obtained through homogenizing whole spleen through 70pm nylon cell strainers (Becton Dickinson (BD) Biosciences Discovery Labware, Bedford, MA) in RPMI 1640 supplemented with 10% heat-inactivated F B S , 2 m M glutamine, penicillin and streptomycin. R e d cells were lysed with treated 0.1M NH CI for 3 min at 4  37°C and washed with equal volume of media. Primary cells were plated at 2x10 cells 6  per ml and cultured at 37°C.  In lipopolysaccharide (LPS) stimulations, cells were  stimulated with 15pg/ml of L P S , E. coli J 5 (EMD Biosciences, Inc., La Jolla, CA). Other T L R agonists tested include 5-15ug/ml L P S - E B ultrapure from E. coli 0111:B4 34  (Invivogen, San Diego, C A ) , 1LIM C p G D N A (Sigma-Genosys, The Woodlands, TX) containing the sequence 5 ' - T T C A T G A C p G T T C C T G A T G C T - 3 ' and 5ug/ml P a m 3 C S K 4 (Invivogen, S a n Diego, C A ) .  In cytokine stimulations, cells were stimulated with either 4% condition media ( B R C , Vancouver, BC) or 10ng/ml of recombinant cytokines, these includes: rmlL-1p, rmll_-3, rmll_-4, rmlL-6, rmll_-7 and rmlL-13  all purchased from R&D Systems (Minneapolis,  MN), rhulL-5 (Intergen, Purchase, NY) and rmll_-10 (StemCell Technologies, Vancouver, BC).  TNFoc (used at 10ng/ml), IFNy (used at 100ng/ml) and rmSDF-1 (used at  100ng/ml) were obtained from R&D Systems (Minneapolis, MN).  hTGFp-1 (used at  1ng/ml) was purchased from StemCell Technologies (Vancouver, B C V ) and S 1 P (used at 30LIM ) was obtained from Biomol (Plymouth Meeting, PA). antibody  Rat anti-mlgM-FITC  (BD Pharmingen, Mississauga, ON), at 5|iig/ml, was used to  co-treat  splenocytes stimulated with either 1 ug/ml anti-mCD40 Ab (a gift from Dr. Michael Gold, University of British Columbia) or 200ng/ml B A F F (Alexis Biochemicals, San Diego, CA).  2.3  Cell Lines  All cells were originally obtained from American Typed Culture Collection (ATCC). Y 3 rat B myeloma cells (Galfre et al., 1979) and their stable transfectants expressing Endoglycan were grown in RPMI 1640 supplemented with 10% heat-inactivated F B S , 2mM glutamine, 50pM p-ME, penicillin and streptomycin. A20 cells (Kim et al., 1979) were grown in RPMI 1640 supplemented with 10% heat-inactivated glutamine, 50uM P-ME,  1mM pyruvate, penicillin and streptomycin.  F B S , 2mM  B O S C - 1 cells  (Sena-Esteves et al., 1999) were grown in D M E M (Invitrogen Gibco, Burlington, ON) supplemented  with  10%  heat-inactivated  F B S , 2mM  glutamine,  penicillin  and  streptomycin.  2.4  E x p r e s s i o n of m o u s e Endoglycan (mEndoglycan) by Retroviral Infection  Full-length c D N A encoding mEndoglycan was cloned into T O P O vector (Invitrogen Life Technologies, Carlsbad, CA) and subsequently into pMXpie retroviral vector using BamHI and Xhol sites.  B O S C - 1 cells were transfected with 7ug of pMXpie vector or  pMXpie containing Endoglycan (pMXpie Endoglycan), along with 1ug of pclECO packaging plasmid using Lipofectamine P L U S (Invitrogen, Burlington, ON). Transfected  35  •  •  B O S C - 1 cells were cultured for two days and irradiated at 5000 rads. Infection of A20 cells or B M cells was achieved by co-culturing with transfected and irradiated B O S C - 1 cells supplemented with 6ug/ml polybrene (Drew et al., 2005). A 2 0 cells were selected in 0.25ug/ml puromycin (McLeod et al., 2004) and B M mast cells were selected in 0.8p.g/ml puromycin (Drew et al., 2005). Cells were sorted through FACSVantage ( B R C , Vancouver, B C ) after at least one week of puromycin-selection.  2.6  Anti-mEndoglycan Anitbody (F4B10) Production  Anti-mEndoglycan antibody was established in our laboratory by immunizing 10-12 weeks-old  Wistar  female  rats  with  a  synthetic  VASMEDPGQAPDLPNLPSILPKMDLAEPPWHMPLQGGC-C)  peptide encoding  (KM-5: the  Nnon-  glycosylated extracellular region of this molecule. Hybridomas were established by cell fusion of splenocytes from immunized rats and mouse X63-Ag8.653 myeloma according to Milstein et al. Hybridomas were selected with HAT medium and cultured in RPMI 1640 supplemented with 20% heat-inactivated F B S , 2% 3T3, 2mM glutamine, penicillin and streptomycin.  Antibodies produced by hybridoma clones were isotyped and  screened by reaction to KM-5 in enzyme-linked immunosorbent assay (ELISA) and to Y 3 rat myeloma expressing Endoglycan in flow cytometry. The anti-mEndoglycan antibody selected is the F4B10 hybridoma.  2.7  Antibodies (Ab)  Anti-mouse - B 2 2 0 / C D 4 5 R - P E (used at 0.7ug/ml), -CD138/SDC1 (used at 5ug/ml), CD29/(31-integrin (used at 5ug/ml), -IgM-PE (used at 5Lig/ml), - C D 1 1 b - P E (used at 5p.g/ml), -CD21-FITC (used at 5Lig/ml), -CD23-FITC (used at 5ug/ml), -CD19-FITC (used at 5ug/ml), -B220-PECy7 (used at 5ug/ml), -IgM-FITC (used at 5ug/ml) and -IgD-FITC (used at 5p,g/ml) were all purchased from BD Pharmingen (Mississauga, ON). Mouse anti-hamster-FITC (used at 5|ig/ml), purified hamster lgG2 (used at 5Lig/ml), biotinylated anti-rat IgM (used at 5pg/ml), streptavidin-PE (used at 2.5Lig/ml) and streptavidin-FITC (used at 2.5Lig/ml) were all purchased from BD Pharmingen (Mississauga, ON). Goat anti-rlgG-AlexaFlur488 used at 4p.g/ml was purchased from Invitrogen Molecular Probes (Burlington, ON). Biotechnology  Anti-mouse IgD-FITC used at 5p.g/ml was purchased at Southern  (Birmingham,  AL).  Anti-mouse  purchased from Chemicon (Temecula, C A ) .  36  CD22-FITC used at  5jng/ml  was  Anti-mlgG-FITC (used at 5ug/ml) and  purified rat IgM (used at 1ug/ml) were purchased from Cedarlane Laboratory (Hornby, ON).  Anti-mouse Endoglycan (F4B10, generated in our laboratory) supernatant was  used undiluted.  2.8  Flow  Cytometry  F A C S buffer used was P B S supplemented with 5% heat-inactivated F B S and 0.05% sodium azide. Mouse serum and goat serum were purchased from Cedarlane (Hornby, ON) and rat serum was purchased from StemCell Technology (Vancouver, B C ) . Serum was used at 2% diluted in F A C S buffer as blocking reagent prior to staining. Cells were harvested at 1200rpm for 3 minutes and stained in 96-well V-bottom plates (Nalge Nunc, Rochester, NY). Staining of cells with antibodies was performed on ice for at least 10 minutes and washed with 150pl F A C S buffer in between each staining interval. FACSCalibur or F A C S c a n (BD Biosciences, Franklin Lakes, NJ) was used to collect samples.  FlowJo version 4.5.5 (Tree Star, Ashland, OR) was used to analyse and  create all F A C S profiles. Relative fluorescence intensity was calculated by subtracting the mean fluorescence intensity of the background (isotype control) from staining. Secondary antibody used to detect F4B10, biotinylated anti-rat IgM, was pre-cleared before staining by incubating with 10% mouse serum on ice and centrifuged  at  13000rpm for 10 minutes before use.  2.9  Bone Marrow-Derived Mast Cell Aggregation A s s a y s  B M mast cells from wild-type C57BI/6 and CD34-/- CD43-/- C57BI/6 overexpressing either CD34 or mEndoglycan were derived as follows (Drew et al., 2005): Cells were plated at 5x10 cells/ml, 1ml in each well of a 6-well plate (BD Labware, Franklin Lakes, 5  NJ) and incubated at 37°C overnight.  Cells in groups of 3 or more were scored as  aggregates. The total cell number per field was estimated by determining the number of single suspension cells added to average number of cells in aggregates (counting the number of cells in three aggregates multiplied by the number of aggregates). Percent of cells in aggregates per field was calculated by determining the number of cells in aggregates and divided by the total number of cells.  37  2.10  LPS Administration  Mice were administered 0.5mg of L P S per kg intraperitoneally in 200ul volume with phosphate buffer saline (PBS).  Endoglycan expression on hematopoietic tissues were  analysed by F A C S at 6hr and 24hr.  2.11  Semi-quantitative Reverse-Transcriptase Polymerase Chain Reaction (RTPCR)  Cells were lysed in TRIzol (Invitrogen, Burlington, ON) reagent, and total R N A was precipitated with chloroform and isopropanol, followed by 7 5 % ethanol wash. Extracted R N A was quantitated at 260nm. Equal amounts of R N A was used to generate c D N A using oligo(dT) ON).  20  primers from Thermoscript R T - P C R System (Invitrogen, Burlington,  Primers  specific  for  ACCTGGCCGGGAAGAGCTACATC-3';  Endoglycan reverse:  transcript  (forward:  5'-  5'-AACACGTCCGAGTCCTCAG-3')  was used for the P C R reaction. P C R for Endoglycan was performed for 50 cycles of 94°C for 1min, 54°C for 1min and 72°C for 2min. Primers specific for H P R T (forward: 5'C T C G A A G T G T T G G A T A C A G G - 3 ' ; reverse: 5 - T G G C C T A T A G G C T C A T A G T G - 3 ' )  was  used as loading control. P C R for H P R T was performed for 30 cycles of 94°C for 1min, 55°C for 1min and 72°C for 2min. A20 cells overexpressing Endoglycan along with the vector  control  cells were  used as positive  and  negative  Endoglycan controls  respectively. R T - P C R products were visualised in 2% agarose gel containing ethidium bromide. Total R N A integrity was determined by running extracted R N A on 1 % agarose gel containing ethidium bromide. Densitometric analysis to determine the intensities of the ethidium bromide stained P C R products was carried out using the Alpha Imager System (Alpha Innotech Corporation, San Leandro, C A ) .  38  3.  RESULTS  3.1  Endoglycan Expression by B cells  3.1.1  Endoglycan is selectively expressed on splenic B cells with a marginal zone B phenotype and is not expressed on immature B cells  The expression of Endoglycan on resting splenocytes was investigated.  In order to  determine the subset of B cells that Endoglycan is expressed by these cells, we costained for various molecular markers indicative of discrete B cell subsets.  As  described previously, B cells can be categorised by the level of expression of surface IgM, IgD, C D 2 1 , CD22, C D 2 3 and CD11b. Endoglycan-positive B cells were found to be |  g  M  med/hi  |  g  D  io  C  D  1  1  b  med  C  D  2  1  med/hi  C  D  2  2  hi  C  D  2  3  io  T h j s  j g  characteristic of MZ B cells,  which are described as lgM , lgD'°, C D 2 1 , C D 2 2 , CD23'° and C D 1 hi  hi  hi  h i  (Fig. 3.1).  To  further confirm that Endoglycan is not expressed by the B-1 a subset of B cells, we examined CBA/N-xid mice carrying a defect for the development of B-1 a subset of B cells. W e observed no decrease in Endoglycan expression levels or the frequency of Endoglycan-positive cells in the spleen relative to B220 expression, suggesting that Endoglycan is not expressed on B-1 a B cells (Fig. 3.2).  To further characterise the distribution of Endoglycan on B lineage cells, the expression of this molecule was analysed on bone marrow cells, which contains the earliest committed B cell precursors, pre-B cells and immature, newly-formed B cells.  Co-  staining of bone marrow cells for Endoglycan and various early B cell markers (CD19, mlgM, B220) showed that Endoglycan is not expressed on immature B cells or pre-B cells (Fig. 3.3).  In summary, Endoglycan is expressed on a small population of primary splenocytes, and this population of cells are similar to marginal zone B cells. There was no evidence for the expression of this molecule on pre-B or immature B cells.  Moreover, analysis of  CBA/N-xid mice showed that Endoglycan expression is not compromised in these mice, hence suggesting that this molecule is not expressed on the B-1 a subset of B cells. Fig. 3 . 1 : Endoglycan is expressed on cells with a MZ B phenotype. Endoglycan is expressed by - 1 0 % of the resting splenocyte population. A. F A C S profile of staining of various B cell marker (x-axis) and the isotype control for Endoglycan (y-axis). B. Double staining of Endoglycan and B cell markers is represented by the y- and x-axis respectively. The levels of B cell marker expression by Endoglycan-positive cells are represented by coloured boxes. Endoglycan-positive resting splenocytes are |  g  M  med/hi|  g  D  lo  C  D  1  1  b  med  C  D  2  1  med/hi  C  D  2  2  hi  C  D  2  3  lo  39  40  A  F i g . 3.2: B1a B cell deficient m i c e s h o w a normal frequency of E n d o g l y c a n positive B c e l l s . A. F A C S profiles showing Endoglycan and B220 expression on 48hr LPS-stimulated splenocytes from C B A / N or CBA/N-xid mice. Gates are set with 2% background. B. Frequency of Endoglycan-positive B cells in C B A / N and CBA/N-xid mice relative to B220 expression. Representative of at least three experiments^ 41  A  IgM  IgD  F i g . 3.3: E n d o g l y c a n is not e x p r e s s e d on immature or pre-B cells in B M . A. F A C S profile of Endoglycan isotype control staining with B cell markers. B. Co-staining of B M cells with Endoglycan and B cell markers represented by the y- and x-axis respectively. Endoglycan-positive cells are not on immature or pre-B cells. Representative of at least three experiments.  3.2  E n d o g l y c a n E x p r e s s i o n o n Activated B cells  3.2.1  E n d o g l y c a n s u r f a c e e x p r e s s i o n a n d transcript levels increase u p o n L P S stimulation  Endoglycan expression on unstimulated splenocytes constitutes - 1 0 % of the entire splenic population (Fig. 3.1).  To test whether this reflected an activated subset of B  cells, splenocytes were stimulated with 5-15pg/ml L P S over 48hrs in culture. A s shown  42  in Fig. 3.5, L P S treatment led to a dramatic increase in Endoglycan expression in terms of both the level (mean fluorescence intensity (MFI) 3.8+3.0 before and 21 ±5.3 after treatment for 48hrs) and in terms of frequency (5.6±1.9% before and 35±2.0% after). The effect of L P S on splenocytes is specific as shown by L P S treatment on splenocyte cultures from G57BI/10ScN, which lacks the L P S receptor T L R 4 (Poltorak et al., 2001). C57BI/10ScN failed to show expression of Endoglycan in responses to L P S (MFI of 1.49+1.41; frequency of 4.17±2.12%). W e conclude that the induction of Endoglycan by L P S is T L R 4 dependent. The expression of Endoglycan is observed only on the B 2 2 0  +  (a pan-B cell marker) population (Fig. 3.4). Time course experiments revealed that Endoglycan expression peaked at 48hrs and sustained thereafter (Fig. 3.5). B  C57BI/10  C57BI/10SCN  w FSC  "  B220  "~  Fig. 3.4: Endoglycan expression on B cells is induced by LPS treatment and is TLR4 dependent. A. F A C S profiles showing Endoglycan expression after 48hr of L P S stimulation on splenocytes from C57BI/10 or C57BI/10ScN. The specificity of L P S induced Endoglycan upregulation is shown by the lack of LPS-effect on splenocytes from C57BI/10ScN, TLR4-deficient mice. B. F A C S profiles showing that Endoglycan is expressed by B220-positive splenocytes after 48hr of LPS-stimulation. Representative of at least three experiments.  In order to correlate the observed increase in surface expression of Endoglycan to transcriptional upregulation, a semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) was performed.  2pg of whole R N A extracts from LPS-stimulated  splenocytes at 0, 6, 12, 24, 48 and 72 hours post-stimulation was used in the R T - P C R reaction.  The "house keeping" transcript, hypoxanthine phdsphoribosyl transferase  (HPRT), was used as a loading control. A 2 0 cells transfected with pMXpie or pMXpie  43  containing Endoglycan were used as negative and positive controls for Endoglycan respectively. These cells express low levels of endogenous Endoglycan. A s shown in Fig.  3.5B, the band for the Endoglycan-positive control was higher than the other  samples, this is an anomaly due to overloading of DNA. Results by dot densitometry analysis revealed an increase in Endoglycan transcript over time that peaked at 48h (Fig.  3.5). 24hr  A  z  | I  6  6  12  12  48hr  24  24  48  72  48  72  LPS  6  6  12  12  72hr  24  48  24  48  72hr +  72hr  LPS/IL-4  Io z  F i g . 3.5: E n d o g l y c a n i n d u c t i o n b y L P S i s b l o c k e d b y I L - 4 t r e a t m e n t a n d i s regulated by transcriptional and post-transcriptional mechanisms. A. F A C S profiles showing Endoglycan expression on splenocytes over 72hr during L P S or LPS/IL4 stimulation. LPS-induced Endoglycan expression peaks at 48hr and IL-4 suppresses surface expression of Endoglycan as detected by F4B10 Ab. B. R T - P C R of R N A extracted from splenocytes stimulated with L P S or LPS/IL-4 over 72hrs. H P R T was 44  used as loading control and total R N A show the integrity of extracted R N A samples. C. Dot densitometry of Endoglycan m R N A normalised against H P R T mRNA. Representative of at least three experiments. R T - P C R data courtesy of Takahide Murakami.  3.2.2  C D 1 mice are defective for L P S - i n d u c e d E n d o g l y c a n expression o n B cells  The LPS-induced Endoglycan upregulation was not mouse-strain specific since we found that C57BI/6, C57BI/10, Balb/c, and C B A / N all responded with similar increases. As  well, splenocytes from various mutant and transgenic mice tested (Balb/c-nu C B A / N -  xid,  P-sel K O , P S G L - 1 K O , C2GnTI K O and PSGL-1/C2GnTI KO) responded to L P S  with increase in Endoglycan expression. The exception to this was CD1 mice. The CD1 strain seems to have a lower level of Endoglycan upregulation upon LPS-stimulation on splenocytes in culture.  Splenocytes from CD1 mice respond to C57BI/6 upon L P S -  stimulation with increase in B220 population.  However, CD1 splenocytes did not  upregulate Endoglycan upon L P S stimulation.  The possibility that this is a natural  occuring Endoglycan-deficient strain was eliminated since the thymic expression of Endoglycan in these mice was not compromised. Interestingly, Balb/c nude mice did not show a  reduced  Endoglycan upregulation  by  L P S stimulation  on  splenocytes.  Therefore, suggesting that Endoglycan expression is not T-cell dependent.  Moreover,  CBA/N-xid mice also did not show decreased level of Endoglycan expression upon L P S stimulation, further confirming Endoglycan is not expressed on B1a B cells (Fig. 3.6).  Endoglycan expression on LPS-treated splenocytes is T cell independent and is not on the B-1 a subset of B cells. Moreover, there are minimal strain differences in the levels of Endoglycan upregulation.  The variability observed in Endoglycan expression on CD1  splenocytes may explain differences in inflammatory response between these strains.  Fig. 3.6: E n d o g l y c a n e x p r e s s i o n  by various  inbred and K O mouse  strains.  A.  Table of the various mouse strains tested for LPS-induced Endoglycan expression on splenocytes. B. F A C S profiles showing Endoglycan and B220 expression, as represented by the y- and x-axis respectively, on 48hr LPS-stimulated splenocytes from CD1 and C57BI/6 mice. Gates were set with 2% background staining. C. F A C S profiles showing Endoglycan expression on resting thymocytes from CD1 and C57BI/6 mice. Representative of at least three experiments.  45  Splenic Endoglycan Expression with LPS  Phenotype  Strain C57BV6 C57BliO Balb/c Bafb/cnu CD1 CD1 -mi CBA/N CBA/N-xid P-sel KO PSGL-1KO C2GnTI KO PSGL-1/C2GnTI-KO C57Bl/10ScN  t a«hyrr*c; lack mature T ceHs X-Inked immune-deficient mice (reduced B-1 a B cell subset) lack P-setectn lack PSGL-1 lack C2GnTI enzyme lack PSGL-1 and C2GnTI enzyme lack TLR4  B  • Isotype - Endoglycan  M  4  ST  CS7BU6  CS7BWS  C01  C01  .»*  Mean Fluorescence Intensity  B22D  3.2.3  + * • + +  E n d o g l y c a n is e x p r e s s e d o n a s u b s e t of p l a s m a cells a n d t h e s e are  (31-  integrin positive It has been shown that L P S stimulation induces plasma cell differentiation in splenic B cells (Lin et al., 2002; Schliephake and Schimpl, 1996; Shaffer et al., 2002; Shaffer et al., 2004). Therefore, to confirm that Endolgycan is expressed on plasma cells, the plasma cell marker, C D 1 3 8 / S D C 1 , was used to co-stain L P S induced plasma cells. S D C 1 and Endoglycan co-staining shows that Endoglycan expressing cells are all SDC1-positive. However, not all SDC1-positive cells express Endolgycan.  Therefore, Endoglycan is  expressed on a subset of plasma cells. Moreover, co-staining of 48hr LPS-stimulated splenocytes for Endoglycan and (31-integrin show that these cultures are mostly (3146  integrin positive and that all Endoglycan-expressing cells express p"l-integrin as well (Fig. 3.7). A  2?  Endoglycan Isotype  Endoglycan Isotype  Endolgycan  Endolgycan  B 1.541  Beta-1 Isotype  Beta-1 Isotype  Beta-1  Beta-1  F i g . 3.7: E n d o g l y c a n i s u p r e g u l a t e d o n a s u b s e t o f p l a s m a c e l l s e x p e s s i n g p"li n t e g r i n . A. F A C S profiles of 48hr LPS-stimulated C57BI/6 splenocytes stained with isotype controls, S D C 1 and/or Endoglycan. B. F A C S profile of 48hr LPS-stimulated C57BI/6 splenocytes stained with isotype controls, [31-integrin and/or Endoglycan.  47  3.2.4  Endoglycan  administration  expression  in vivo  b yB s p l e n o c y t e s  i sincreased  upon  L P S -  In order to show that the LPS-induced upregulation of Endoglycan was not selective to in vitro cultured B cells, the expression of this molecule upon L P S stimulation was analysed in vivo. L P S was administered intraperitoneally into C57BI/6 or CD1 mice and hematopoietic tissues were analysed at 6 and 24hr. Since splenocytes from CD1 mice showed a reduced induction of Endoglycan expression by L P S in vitro, this strain of mice was used as a negative  control  for LPS-administration.  Although  Endoglycan  expression on CD1 splenocytes increases when activated in vivo, the intensity of upregulation is less than C57BI/6 splenocytes.  Endoglycan was upregulated in the  spleen as well as cells of the peritoneum of C57BI/6 mice after 6 and 24hr of L P S administration. However, the CD1 strain, in response to L P S in vivo, showed little or no increased induction of Endoglycan expression, similar to that observed in in vitro cultures (Fig. 3.8). The thymic expression of Endoglycan from CD1 mice is inherently higher than that of the C57BI/6. This difference in expression was shown using unstimulated thymocytes (Fig. 3.6C), as well, on thymocytes after LPS-administration from C57BI/6 and CD1 (Fig. 3.8B). 24hr after L P S administration in C57BI/6, there was a slight increase in Endoglycan expression in the B M . Further analysis is needed to determine if these are long-term plasma cells that have migrated to the B M niche or if these are B M cells that have induced Endoglycan expression upon L P S challenge.  Fig.  3.8:  Expression  o fEndoglycan  on  hematopoietic  tissues  after  L P S -  administration a t 6 and 24hr. F A C S profiles of Endoglycan expression on hematopoietic tissues/cells after LPS-administration. A . Endoglycan expression on C57BI/6 and CD1 splenocytes increases from 6hr to 24hr post L P S administration. B. Thymic expression of Endoglycan in C57BI/6 decreases from 6hr to 24hr post L P S administration. However, the Endoglycan expression on CD1 thymocytes increases from 6hr to 24hr post administration. C , D, E. Endoglycan expression in C57BI/6 B M (C), mesenteric lymph node (LN) (D) and blood (E) increases from 6hr to 24hr post L P S administration. The Endoglycan expression in CD1 of these tissues does not vary or increase to a slighter extent from 6hr to 24hr as compared to C57BI/6. F. Peritoneal wash (PW) show an increase in Endoglycan expressing cells from 6hr to 24hr post L P S administration in both mouse strains. However, whereas all B220 cells in C57BI/6 P W is Endoglycan-positive, B220 cells in CD1 P W are Endoglycan-negative.  48  3.2.5 The  E n d o g l y c a n e x p r e s s i o n is selectively u p r e g u l a t e d b y T L R expression of  Endoglycan on  splenocytes through  TLR4  agonists stimulation  was  consistently observed as described above. Therefore, to investigate if this upregulation is TLR4-specific, other TLR-agonists were surveyed for their ability to upregulate this molecule.  Stimulation of primary splenocyte cultures with 5pg/ml P a m 3 C S K 4 (TLR2  agonist) and 1uM C p G (TLR9 agonist), over 48hrs all induced the upregulation of Endoglycan. The lack of Endoglycan upregulation by L P S on C57BI/10ScN splenocytes demonstrates the specificity of LPS.  Therefore, stimulation of T L R 2 , 4 and 9 enhances  the expression of Endoglycan on splenocytes (Fig. 3.9). UltraPure L P S  Pam3CSK4  < CpG  C57BI/10  C57BI/10ScN  FSC  Fig. 3.9: E n d o g l y c a n e x p r e s s i o n is u p r e g u l a t e d b y T L R 2 , 4 a n d 9 agonists.  FACS  profiles showing Endoglycan expression on splenocytes from C57BI/10 or C57BI/10ScN induced by stimulation with UltraPure LPS, P a m 3 C S K 4 or CpG. Endoglycan expression on splenocytes was upregulated by stimulation with T L R 2 agonist, P a m 3 C S K 4 , T L R 4 agonist, UltraPure LPS, and T L R 9 agonist, C p G .  To TLR  test whether the increased Endoglycan expression on splenocytes was specific to agonists or could be induced by other B cell activators or mitogens, we treated  splenic B cells with anti-mlgM Ab (5ug/ml), B A F F (200ng/ml, with or without anti-mlgM costimulation), anti-mCD40 Ab (1pg/ml, with or without anti-mlgM costimulation), T N F a (10ng/ml) and IFNy (300ng/ml). Although these activatiors stimulated proliferation and 50  formation of blasts in culture, none led to the upregulation of Endoglycan on splenocytes over 48hr (Fig. 3.10).  W e conclude that the upregulation of Endoglycan on activated B cells is highly specific to TLR-stimulation and not achievable through other B cell signaling pathways. anti-IgM Ab  no anti-IgM Ab  anti-CD40 Ab  BAFF  FSC  B  IFNg  TNFa  FSC Fig. 3.10: E n d o g l y c a n e x p r e s s i o n o n s p l e n o c y t e s i snot affected b y stimulation with anti-IgM, anti-CD40, B A F F , T N F a a n d IFNy. A. F A C S profiles showing Endoglycan expression on splenocytes after 48hr stimulation with anti-CD40 Ab or B A F F , both with or without anti-mlgM Ab. Gates are set with 2 % background. B. F A C S profiles showing Endoglycan expression on splenocytes after stimulation with T N F a or IFNy for 48hr. Gates are set with 2% background.  51  3.3  L P S - i n d u c e d E n d o g l y c a n E x p r e s s i o n is S u p p r e s s e d b y T 2-type H  and the Anti-Inflammatory Factor,  Cytokines  T G F p  Cytokines have long been known to have a regulatory role in B cell development (Armitage et al., 1993; Bouchard et al., 1994; Ford et al., 1999; Ozaki et al., 2004). Therefore, the regulatory role of cytokines on Endoglycan expression induced by L P S stimulation was explored. 10ng/ml or 4 % condition media containing IL-1p\ IL-2, IL-3, IL4,  IL-5, IL-6, IL-7, IL-10 or IL-13 were used to co-stimulate splenic B cells with L P S and  Endoglycan expression was analysed by F A C S . LPS-induced Endoglycan upregulation.  IL-2, IL-4 and IL-5 suppressed the  Although not statistically significant, the data  suggest that combining IL-4 and IL-5 may have an additive affect (Fig. 3.11). The IL-4 suppressive effect was also observed when this cytokine was added at 24hr post L P S stimulation (at a time when Endoglycan m R N A levels have normally increase), suggesting the potential for IL-4 to suppress Endoglycan expression is a posttranscriptional-event.  The addition of IL-4 at 24hr suppressed the LPS-induced  upregulation of Endoglycan (Fig. 3.12: right panel), similar to that observed when both reagents L P S and IL-4 were added at time Ohr (Fig. 3.12: centre panel). However, this data does not rule out the possibility that IL-4 may also repress Endoglycan expression (Fig.  3.12).  52  100  *P  - 0.05;  LPS  ** P = 0.06; *** 0.07 < P < 0.10  Fig. 3.11: Effect o f T 1  and  H  T 2 H  cytokines  on  Endoglycan  expression.  Graph  showing the expression of Endoglycan on 48hr-stimulated splenocytes. Endoglycan expression is represented by relative fluorescence intensity (after reduction of bacground fluorescence). Gates are set with 2% background. The shaded area represent the expression of Endoglycan induced by L P S . L P S treatment with IL-2, IL-4 and IL-5 (or these cytokines in combination with other cytokines) reduced Endoglycan expression compared to L P S treatment alone, resulting in P values below 0.10 in unpaired single-tail T-test. LPS  ~  LPS/IL-4  L P S (IL-4 at 24hr)  mean fluorescence intensity  Fig.  3.12:  IL-4  treatment  after  24hr  o fLPS-stimulation  blocks  Endoglycan  [ expression on splenocytes. F A C S profiles showing the expression of Endoglycan on splenocytes cultured for 48hrs. LPS-induced expression of Endoglycan on splenocytes is shown in the left panel. Addition of IL-4 after 24hr of LPS-stimulation (right panel). Stimulation with both L P S and IL-4 at time Ohr (centre panel). 53  Since receptors for IL-4 and IL-13 share the same IL-4Ra signaling chain (Rolling et al., 1996; Umeshita-Suyama et al., 2000), it was expected that IL-13 should have the same affect on LPS-stimulated splenocytes as IL-4. Strikingly, IL-13 was unable to suppress Enodglycan expression.  Both IL-4 and IL-5 are secreted by T 2 cells and promote the development of IgG, IgE or h  IgA-secreting plasma cells.  Therefore, the suppressive effect of these cytokines on  LPS-induced Endoglycan expression suggests that Endoglycan is expressed on a distinct population of activated B cells.  Thus far, Endoglycan has been shown to be a pro-inflammatory marker on B cells in response to T L R stimulation, however, its expression as an activation marker remains to be verified with other T L R agonists.  Co-stimulation of splenocytes with L P S and the  anti-inflammatory factor, T G F p , suppresses the expression of Endoglycan. Moreover, co-stimulation of splenocytes with the pro-inflammatory cytokine, IL-1p, induces a slight increase in Endoglycan expression (Fig. 3.13). Therefore, Endoglycan is a marker on B cell during inflammatory responses.  r  54  F i g . 3 . 1 3 : E f f e c t o f I L - 1 p a n d T G F p o n E n d o g l y c a n e x p r e s s i o n . A graph illustrating Endoglycan expression on 48hr-stimulated splenocytes. Endoglycan expression is represented by the relative fluorescent intensity (after reduction of background fluorescence). Reduced Endoglycan expression with IL-4 treatment is consistent with previous observations. In addition, IL-ip seem to enhance Endoglycan expression where as T G F p reduces Endoglycan expression. None of the stimulations in the absence of L P S induces Endoglycan expression. Representative of two individual data sets.  55  4.  DISCUSSION  4.1  Endoglycan is on splenic B cells with a MZ B phenotype  In naive splenocytes, only 2-10% of the cells expresses Endoglycan. By delineating this small population using various B cell markers, it was observed that these cells are |gM  med/h  igD'°CD11b  med  CD21  med/hi  CD22 CD23 ; hi  a phenotype characteristic of M Z B cells.  10  Moreover, Endoglycan was not observed on immature B cells and B cell precursors in the B M by co-staining for Endoglycan and various early B cell markers. These findings suggest that Endoglycan is a marker on a specific population of mature B cells.  Several transgenic mice have been reported to have lower or higher levels of or lack M Z B cells (Table 4.1). Analysing various transgenics and the expression of Endoglycan in the spleens of these mice may confirm the expression of Endoglycan on M Z B cells. Knockout and transgenic mice with alterations in MZ B cells Mouse System Observations L5-/-, IL7-/-, IL7ra-/-, Rag-/- conditional  Larger proportion of MZ and B 1 B cells and reduced follicular B cells  C019-/-  Lack of MZ B cells; defects iri TD and Tl responses  Baff-/-, Baffr-/-  Lack of B cells beyond NF/TR stage  Baff-transgenic  Increase in TR and MZ B cells, in particular; B-cell autoimmune manisfestation  Pyk2-A  Lack of MZ B cells; normal B1 cells; hyporesponsivity to chemokines  Table 4.1: Transgenic mice with alterations in MZ B cells. Adapted from Martin and Kearney, 2002.  4.2  Endoglycan is expressed on B cells activated via TLR  Endoglycan is a specific marker on B cells activated via TLR. The expression of this molecule was analysed through F A C S and confirmed by R T - P C R .  A time course  studying the expression of Endoglycan on LPS-activated splenocytes over 72hrs showed increase in transcript levels for Endoglycan with increasing surface expression of this molecule a s detected by the rat monoclonal antibody, F4B10.  T h e level of  Endoglycan protein expression post-stimulation peaked at 48hr. The specificity of this L P S effect was tested by stimulation of a TLR4-deficient mouse, C57BI/10ScN, where Endoglycan expression was not observed.  Agonists of T L R 2 , 4 and 9 were able to  induce the upregulation of Endoglycan on splenocytes. enhances the LPS-induced upregulation,  therefore,  Moreover, IL-1(3  slightly  suggesting that the signaling  pathway is through TIR-domains since both T L R and 1L.-1 (3 receptors are TIR-containing receptors. Consistent with this hypothesis, in silico promoter analysis revealed potential  56  binding sites for N F - K B and IRF, TIR domains activate downstream signaling that lead to the activation  of these transcriptional  activators.  The specificity of  Endoglycan  upregulation to this TLR-downstream event is shown by stimulation of splenocytes with various activating factors such as anti-IgM, B A F F (in the presence and absence of antiIgM) and CD40L (in the presence and absence of anti-IgM).  The distribution  of  Endoglycan suggests that this molecule serve a specific funciton on plasma cells (see below).  In order to confirm the effect of N F - K B and IRF on Endoglycan expression, promoter deletion experiments need to be performed.  The binding sites for these transcription  factors can be mutated or altered, and changes in Endoglycan expression upon L P S stimulation can be analysed. Hence, the upregulation of Endoglycan by T L R agonists should be reduced or abolished by dysfunctioning either N F - K B and/or IRF binding sites.  4.3  L P S - i n d u c e d E n d o g l y c a n expression o n a subset of p l a s m a cells and this expression is downregulated by T 2-type cytokines and T G F p H  L P S has been shown to induce the formation of plasma cells from B cells (Lin et al., 2002; Schliephake and Schimpl, 1996; Shaffer et al., 2002; Shaffer et al., 2004).  To  confirm that the LPS-stimulated splenocyte cultures used in this thesis produced plasma cells, these cultures were stained with the plasma cell marker, S D C 1 . W e found that majority of the cells were S D C 1 . +  The co-stimulation of splenocytes with L P S and IL-4 has been shown to promote the differentiation of B cells into IgG- and IgE-secreting plasma cells (Hasbold et al., 1998; Tangye and Hodgkin, 2004) and co-stimulation with IL-5 leds to IgA-secreting plasma cells (Sonoda et al., 1989). However, data presented here show that co-stimulation of L P S with IL-4 and/or IL-5 reduces the LPS-induced Endoglycan expression. A possible explanation for this effect is suggested by transcription factor binding sites in the Endoglycan promoter. The overlapping binding sites for S T A T and N F - K B may suggest that N F - K B competes for binding site with the transcriptional repressors, Stat5 and Stat6. The anti-inflammatory factor, T G F p , also suppressed the LPS-induced upregulation of Endoglycan.  57'  These observations, together with the specificity for the induction of this antigen, suggest that Endoglycan expression is highly regulated and its expression marks a subset of plasma cells. The expression of Endoglycan may allow cells to exit follicles of lymphoid organs, either through chemotactic signals, adhesive or anti-adhesive mechanisms. Therefore, by downregulating this molecule in the presence of cytokines produced by T cells, B cells may be retained in the follicles or these cells may migrate to sites different from Endoglycan-positive cells.  The signaling capabilities of Endoglycan have not been explored.  However, the  potential for this molecule to dimerize, as well as, be phosphorylated in the cytoplasmic domain, does not rule out a possible signaling function. Moreover, the high amino acid identity across species suggests that this molecule have an unidentified extracellular ligand. A s suggested by studies using human recombinant Endoglycan proteins, this molecule has the potential to bind L-selectin (Fieger et al., 2003).  Since Endoglycan-  expressing cells in the LPS-stimulated splenocyte cultures all express |31-integrin, these cells may be long-lived plasma cells that home to B M . Long-lived plasma cells are retained in the B M niche by various adhesion molecules, including E-selectin (Underhill et al., 2002). Therefore, it may be possible that Endoglycan is an E-selectin ligand when expressed on activated B cells and thereby aid retention of plasma cells in B M . Due to the similarity between Endoglycan and its family members, C D 3 4 and Podocalyxin, Endoglycan may also act as an anti-adhesive molecule through its highly negativecharged extracellular domain (Doyonnas et al., 2001; Drew et al., 2005).  Thereby  allowing plasma cells to migrate through the endosteum to their B M niche.  Similar to the promoter mutational analyses that are needed to confirm of the effect of NF-KB  and  IRF,  mutagenesis  experiments  are  also  needed  to  confirm  the  downregulatory effect of Stats and Smads. Moreover, in order to test the homing effect of Endoglycan, ex vivo short-term homing assays can be performed.  By using L P S -  stimulated splenocyte cultures and sorting for B220 Endoglycan and B220 Endoglycan" +  +  +  populations, we may label these cells with fluorescencent dyes such as Tracker Orange or C S F E and analyse the differential homing of these cells over 24hrs by F A C S (Drew et al., 2005).  To test the possibility that Endolgycan acts as a selectin ligand, static  selectin-binding can be performed using recombinant selectin proteins and staining L P S stimulated splenocytes with or without IL-4. However, in order to verify that the binding 58  is due to Endoglycan, anti-Endo F interactions.  a D  fragments must be created to block selectin-binding  A s shown in chapter 1, full-length  Endoglycan when expressed in  CD34/CD43-deficient BM-derived mast cells did not decrease homotypic aggregation of these cells (Fig. 1.13). However, B M mast cells also express N H E R F - 1 . Since N H E R F 1 has been shown to associate with actin cytoskeleton (Shenolikar and Weinman, 2001), it is possible that the lack of anti-adhesive effect is due to capping of Endoglycan by NHERF-1.  Therefore, to confirm that this phenomenon is not due to capping of  Enodglycan, a naturally truncated form of Endoglycan needs to be cloned and expressed in the B M mast cell assay.  4.4  Differences in the effect of IL-4 and IL-13 on Endoglycan expression  Since the IL-13 receptor share a common signaling chain, IL-4Ra, as IL-4 receptor, it was expected that these two cytokines would have similar effect on Endoglycan expression by LPS-stimulation. However, it was observed that IL-13 had no effect on Endoglycan expression, whereas  IL-4  suppressed the  LPS-induced Endoglycan  expression. Early studies in transgenic mouse models have demonstrated that IL-4 and IL-13 have nonreduntant roles in immunity (Barner et al., 1998; Kaplan et al., 1996; McKenzie et al., 1999; Noben-Trauth et al., 1997; Takeda et al., 1996; Urban et al., 1998).  Therefore, the effect of these cytokines on Endoglycan expression may be  attributed to these nonredundant functions.  IL-13 can bind receptors consisting of the  IL-13Roc and IL-4Ra, or IL-13Rcc alone, and IL-4 can bind receptors consisting of IL-4Ra and y , or IL-13Ra and IL-4Rot, or IL-4Roc alone. c  Therefore, the suppression of  Endoglycan in LPS-stimulated splenocytes by IL-4, but not IL-13, may be due to signaling from the y common chain. Moreover, it has been demonstrated in human c  lymphocytes that IL-13 responses decrease with maturation  (Ford et al., 1999).  Therefore, the difference in the effect of these cytokines may also be due to differences in the expression levels or the complement of receptor chains for these cytokine receptors.  To confirm the suppressive effect of IL-4 on Endoglycan expression, IL-4 treatment on LPS-stimulated splenocyte cultures from IL-4Rcc-deficient mice should show no effect. It is difficult to test the signalling effect of the y chain in the IL-4 receptor because mice c  with mutations in the IL2RG gene, which encodes the y chain, exhibit severe combined c  immunodeficiency (SCID) mice and are deficient in B cells. In order to investigate the 59  expression levels of IL-4 and IL-13 receptors, F A C S staining of for these receptors on LPS-stimulated splenocytes should be carried out. Moreover, testing various dilutions of these cytokines will show if the suppressive effect is due to different levels of IL-4 receptor compared to IL-13 receptor.  4.5  Differences in LPS-induced expression of Endoglycan between C D 1 and other mouse strains  L P S was found to induced the expression of Endoglycan by B cells in virtually every strain of mice tested. This includes: CBA/N-xid, which have reduced B-1 a subsets of B cells; C2GnTI-deficient mice, which lack the enzyme for adding O-linked glycosylations to glycoproteins; and Balb/c nude mice, which are athymic and lack mature T cells. These observations suggest that Endoglycan is not found on B-1 a subsets and its upregulation is independent of T cells. Moreover, data from the C2GnTI-deficient mice confirmed that reactivity of the anti-mouse Endoglycan monoclonal antibody is not dependent on glycosylation added by C2GnTI. The exceptions were CD1 and CD1 nude mice and data suggest that CD1 strains have an intrinsic deficiency in upregulating this molecule. Analyses of thymocytes from CD1 mice show an elevated Endoglycan expression profile.  These mice do not lack the  ability to express the molecule per se, but rather, have a defect in the ability to express it on B cells. Hence, eliminating the possibility of a natural occurring Endoglycan-deficient strain.  Moreover, L P S administration intraperitoneally into these mice showed lower  levels of Endoglycan upregulation in the spleen after 24hr as compared to C57BI/6 mice.  Studies have shown that there is variability in the level of lymphocyte between different inbred mouse strains during  inflammatory  recruitment  response.  It  was  demonstrated that under uniform stimulus, of the four strains tested, C57BI/6 has the highest level of cell recruitment, then Balb/c, followed by CD1 and lastly 129x1/SvJ (White et al., 2002). It is possible that the lack of Endoglycan upregulation in response to L P S in CD1 mice is related to their low levels of cell recruitment compared to C57BI/6 and Balb/c strains. Experiments using R T - P C R will alert us to possible differences in the transcriptional regulation of Endoglycan between these two strains of mice.  A s well,  looking at the differences in the Endoglycan promoter between these strains of mice  60  may provide an explanation for the differences in Endoglycan expression upon L P S stimulation between C57BI/6 and C D 1 .  To investigate the possible role of Endoglycan in B cell recruitment, the LPS-induced upregulation of Endoglycan on 129x1/SvJ splenocytes can be analysed for a correlation between the observed levels of Endoglycan and the levels of cell recruitment. H S C transplants between C57BI/6 and CD1 strains or C57BI/6 and 129x1/SvJ strains will determine if the differences in Endoglycan expression on B cells is due to intrinsic or extrinsic factors. 4.4  Endoglycan expression increases upon LPS-administration in the spleen and BM  The observed in vitro upregulation of Endoglycan by L P S was also observed in vivo when L P S was administered intraperitoneally  into C57BI/6.  There was increased  Endoglycan expression in the spleen, peritoneal cavity and B M , after 24hr of L P S administration.  The increased expression in the spleen was observed on the B cell  population, as seen in in vitro splenocyte cultures.  The increase expression on  peritoneal cavity was on B220", suggesting that these are most likely macrophages.  activated  The increased level of expression in the B M may be attributed to the  induction of Endoglycan expression on B M cells during inflammatory response, or these may be Endoglycan-expressing plasma cells from lymphoid organs that have migrated totheBM. To test whether the increased Endoglycan expression by B M is due to migration of Endoglycan-expressing plasma cells, we wilj survey B M cells after L P S administration for B220 and Endoglycan co-expression.  Moreover, the ex vivo short-term  homing  assay described above should answer questions regarding the possibility of Endoglycanpositive plasma cell homing to B M .  61  5.  C O N C L U S I O N  5.1 MZ  Endoglycan on M Z B cells B cells are the first line of defense against pathogens, they have a repertoire that  recognizes TI-2 antigens.  This is a T cell independent event where M Z B cells  proliferate and mature into antibody-secreting cells upon antigen encounter. M Z B cells are retained in a specific region in the spleen (peripheral to the periarteriolar lymphoid sheath) that allows these cells to be exposed to blood-borne antigens for quick immune response.  Transgenic mice that lack M Z B cells are more susceptible to bacterial  infections (Tanigaki et al., 2002). A s well, it has been shown that M Z B cells proliferate prior to pathogenic T cells in lupus-prone NZB/W mice (Wither et al., 2000). Studying the importance of M Z B cells in immunity and autoimmunity would be greatly facilitated by a molecule that can be used to identify and isolate these cells. W e propose that Endoglycan is such a marker for M Z B cells that it may be important as a target for therapeutic interventions.  5.2  A  possible  role  for  Endoglycan  i n homing  and  migration  during  inflammation? Long-lived plasma cells have been found to home to B M , mucosa or inflammatory sites (Kunkel and Butcher, 2003), however, the mechanism by which this homing occurs remains largely unknown.  The fluctuations in the expression of chemokine receptors  (Hargreaves et al., 2001; Hopken et al., 2004) and adhesion molecules (Kunkel and Butcher, 2003), such as integrins, has been shown to be important in this migratory process. It has been demonstrated that plasma cells that reside in the B M expresses oc4pi -integrin, whereas mucosal plasma cells express a4p7-integrin.  Moreover, B M  plasma cells express E-selectin ligands that aid in their retention in the B M niche (Underhill et al., 2002). The difference in the expression of these surface molecules helps plasma cells determine their site of retention. By understanding the expression of other homing and/or adhesion molecules, we may be able to delineate the different subsets  of  long-lived  plasma cells and  determine  their  migratory  destination.  Endoglycan, as shown in the results from this thesis, is expressed on a subset of plasma cells upon activation and all Endoglycan-expressing cells also express (31-integrin. The expression of Endoglycan is modulated by cytokines that promote certain paths of plasma cell differentiation. Therefore, this molecule may mark unique plasma cells that are destined to home to specific tissues, such as B M or other specific sites of 62  inflammation.  Endoglycan maybe a new homing and/or adhesion molecule that will  allow the delineation of plasma cells that are programmed to migrate to different niches. Therefore, it will be important to study the differences in homing of Endoglycan-positive and negative plasma cells.  5.3  Concluding Remark  Endoglycan is the newest member of the CD34 family of sialomucins.  Since its  discovery five years ago, little has been known about its regulation and function. The results presented in this thesis are key to understanding the regulation of this molecule in B cells. Moreover, this thesis proposes possible function for this novel molecule on B cells.  There is much still to clarify about Endoglycan, including its: 1. transcriptional  regulation (specifically promoter mutation analysis); 2. function, in terms of homing, adhesion or anti-adhesion;  and 3. biochemical properties, such as extracellular and  intracellular binding partners. A s well, due to its novelty, only limited analytical tools are available. Therefore, molecular, genetic and biochemical tools are needed to further the study this molecule. 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