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Beta Chemokine expression in an in vitro model of the human blood-brain barrier Chui, Ray 2004

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BETA CHEMOKINE EXPRESSION IN AN IN VITRO MODEL OF THE HUMAN BLOOD-BRAIN BARRIER by RAY CHUI B.Sc. (Honours, Biochemistry), The University of British Columbia A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE D E G R E E OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Pathology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December 2004 ABSTRACT It is now well established that the interactions between endothelium and circulating white blood cells are of critical importance in the evolution of an inflammatory response. Chemokines are small chemoattractant cytokines with the unique ability to stimulate and guide the movement of specific classes of inflammatory cells to sites of inflammation. The role of chemokines in C N S inflammation has not been fully elucidated. Over the last decade, various studies have suggested that CCL2(MCP-1) and C C L 3 (MIP-1a) are major players in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalitis (EAE). The aim of the present study is to investigate the expression and cytokine upregulation of C C L 2 and C C L 3 by human brain microvessel endothelial cells (HBMEC), an in vitro model of the human blood-brain barrier. Primary cultures of H B M E C grown to confluence were stimulated with TNFoc, IFNy, IL-1(3 or LPS for 24-72 h. RNA expression was confirmed by R T - P C R and intracellular protein expression was detected with immunogold silver staining (IGSS). Protein release was quantitated with ELISA. CCL2 RNA levels were similar in both unstimulated and cytokine-treated cells. C C L 3 RNA was upregulated upon cytokine stimulation. IGSS confirmed chemokine expression in both unstimulated and cytokine-treated cells. The basal release of CCL2 by resting H B M E C was significantly upregulated after cytokine treatment in a concentration and time-dependent manner. ELISA of the supernatants showed CCL3 release only after stimulation. Immuno-electron microscopy studies show chemokine binding to the apical and basal surfaces of HBMEC. These studies suggest that the cerebral endothelium may play an active role in the initiation, selective recruitment and activation of circulating lymphocytes and monocytes during C N S inflammation. TABLE OF CONTENTS Abstract " Table of Contents List of Tables v i List of Figures v l List of Abbreviations v i i Acknowledgements i x Body 1 References 80 CHAPTER 1: INTRODUCTION 1.1 Inflammation 1 1.2 Chemokines 2 1.2.1 Nomenclature and Structure 2 Nomenclature 2 Structure 3 Interaction with Extracellular Matrix Components 7 1.2.2 Chemokine Receptors 9 Organization 10 Structure 10 Interaction with Ligands 11 Heterotrimeric G Proteins 12 Receptor Activation 13 Cellular Effects 16 1.2.3 Chemokines and Their Receptors: Role in Disease 17 Inflammation 18 Cancer 20 Infectious Diseases 22 1.3 The Central Nervous System (CNS) 24 1.3.1 The Blood-Brain Barrier (BBB) 24 1.3.2 Inflammation in the C N S 26 1.3.3 C N S Cells Participating in Inflammation 27 Astrocytes 27 Microglia 27 Perivascular Macrophages/Dendritic cells 28 iii Endothelial Cells 31 1.3.4 Lymphocyte Trafficking in the C N S 32 Lymphocytes 32 Cell Adhesion Molecules 33 1.3.5 Chemokines and Chemokines Receptors 33 Expression by C N S Resident Cells 33 Role in C N S Inflammation 34 CCL2 and CCL3 and Their Receptors 34 CCR11 38 1.3.6 Cytokines in C N S Inflammation 39 Tumour Necrosis Factor (TNF)-a 39 Interferon (IFN)-y 40 lnterleukin(IL)-1|3 40 Lipopolysaccharide (LPS) 41 1.3.7 Immunopathogenesis of CNS Autoimmune Disorders 41 1.4 Human Brain Microvessel Endothelial Cells (HBMEC) 44 1.4.1 Endothelial Cell Heterogeneity 44 1.4.2 In vitro Model of the Human Blood-Brain Barrier 45 1.5 Objective and Specific Aims 46 1.5.1 Hypothesis 46 1.5.2 Specific Aims 46 CHAPTER 2: MATERIALS AND METHODS 2.1 Endothelial Cell Cultures 48 2.1.1 Isolation of Human Brain Microvessel Endothelial Cells (HBMEC) 48 2.1.2 Isolation of Human Umbilical Vein Endothelial Cells (HUVEC) 49 2.1.3 Culture Conditions 49 2.2 Cytokines and LPS 50 2.3 Antibodies 50 2.4 Semi-quantitative Reverse Transcription PCR (RT-PCR) 51 2.5 Immunocytochemistry 53 2.5.1 Immunogold Silver Staining 53 Surface Localization 53 Intracellular Localization 53 2.5.2 Immunoperoxidase Staining 54 2.6 Immunogold Electron Microscopy 54 2.7 Enzyme-linked Immunosorbent Assay (ELISA) 55 2.8 Immunohistochemistry 56 2.9 Data Collection and Statistics 56 CHAPTER 3: RESULTS 3.1 Human Brain Microvessel Endothelial Cells (HBMEC) 57 3.2 C C L 2 Expression by HBMEC 57 3.2.1 RNA Expression Detected by Semi-quantitative R T - P C R 57 iv 3.2.2 Intracellular CCL2 Protein Expression by H B M E C 58 3.2.3 CCL2 Surface Localization in HBMEC by IEM 58 3.2.4 Secretion of CCL2 by HBMEC Detected by Sandwich ELISA 59 3.3 CCL3 Expression by HBMEC 60 3.3.1 RNA Expression Detected by Semi-quantitative R T - P C R 60 3.3.2 C C L 3 Intracellular Protein Expression by H B M E C 61 3.3.3 Surface Localization of C C L 3 in HBMEC by IEM 61 3.3.4 Protein Release of CCL3 Determined by Sandwich ELISA 62 3.4 Expression of CCL2 and CCL3 in Acute MS 62 3.5 Expression of p-Chemokine Receptors 63 3.1.1 RNA Expression Detected by RT-PCR 63 3.2.2 Immunocytochemistry 63 CHAPTER 4: DISCUSSION 4.1 HBMEC as an in vitro Model of the BBB 65 4.2 CCL2 and CCL3 Expression by HBMEC 67 4.3 CCL2 and CCL3 Expression in Acute MS 72 4.4 Expression of p-Chemokine Receptors by HBMEC 73 CHAPTER 5: CONCLUSIONS 5.1 Summary and Significance 75 5.2 Future Directions 78 REFERENCES 80 v LIST OF TABLES Table 1 Chemokine Nomenclature Table 2 P C R Primer Sequences LIST OF FIGURES Figure 1 Primary cultures of human brain microvessel endothelial cells Figure 2 RNA expression of CCL2 by HBMEC and H U V E C Figure 3 Intracellular localization of CCL2 in H B M E C Figure 4 Surface localization of CCL2 on H B M E C Figure 5 CCL2 release under simulated inflammatory conditions Figure 6 RNA expression of CCL3 by HBMEC and H U V E C Figure 7 Intracellular localization of C C L 3 in H B M E C Figure 8 Surface localization of CCL3 on H B M E C Figure 9 C C L 3 release by HBMEC under simulated inflammatory conditions Figure 10 RNA and intracellular protein expression of the former CCR11 Figure 11 CCL2 and CCL3 expression in acute MS Figure 12 Proposed hypothesis for the role of chemokines in C N S inflammation vi ABBREVIATIONS Ab antibody A E C 3-amino-9-ethylcarbazole Ag antigen(s) ANOVA analysis of variance A P C antigen presenting cell BBB blood brain barrier CCL2 monocyte chemoattractant protein-1 (MCP-1) CCL3 macrophage inflammatory protein-1 a (MIP-1a) C N S central nervous system C S F cerebrospinal fluid DAB 3,3'-diaminobenzidine EAE experimental allergic encephalomyelitis EC endothelial cell(s) E C M extracellular matrix EM electron microscopy FN fibronectin G A G glycosaminoglycan(s) HBMEC human brain microvessel endothelial cell(s) HIV-1 human immunodeficiency virus type 1 HRP horseradish peroxidase HUVEC human umbilical vein endothelial cell ICAM-1 intercellular adhesion molecule - 1 vii IFNy interferon y IGSS immunogold silver staining IL-1p interleukin-1 beta IEM immunoelectron microscopy LFA-1 leukocyte function-associated antigen - 1 LPS lipopolysaccharide mAb monoclonal antibody MS multiple sclerosis PBMC peripheral blood mononuclear cell(s) PECAM-1 platelet/endothelial cell adhesion molecule - 1 RT-PCR reverse-transcriptase polymerase chain reaction TEM transmission electron microscopy TMD transmembrane domain TNFoc tumor necrosis factor alpha VCAM-1 vascular cell adhesion molecule - 1 VLA very late activation antigen viii ACKNOWLEDGEMENTS I would like to take this opportunity to thank a number of people: those who have supported me during all these years and those whose words and teachings will stay with me for the rest of my life. To my committee members: Drs. Chantler, Waterfield and Moore - a heartfelt thanks. To Reza: for all your countless hours in the darkroom; Esther - thanks for teaching me about life; Rukmini - you challenged me to be better, in many different ways; and Kakuri - thanks for being a great friend! And last, but not least, Dr. Zis - your guidance has helped me develop not only as a scientist, but also as an individual. For all of you, I will never forget the days that have gone by, because they have helped shape who I am - and for that, it has been my profound honour. November 2004 ix CHAPTER 1: INTRODUCTION 1.1 INFLAMMATION When an injurious agent, be it a microbe, toxin or trauma, affects the human body, a complex set of reactions within the local, vascularized tissue, known as inflammation, is initiated. Fundamentally, these reactions serve a protective role by destroying or neutralizing the stimulus and are closely linked to mechanisms that promote repair and recovery of the affected tissue. Historically, the concept of inflammation was described during the first century AD, when the four classical signs of inflammation were noted by the Romans: rubor (redness), tumor (swelling), calor (heat) and dolor (pain). However, symptoms had been previously described several millennia earlier by the ancient Egyptians. Rudolf Virchow added a fifth clinical sign during the nineteenth-century - functio laesa or loss of function. Presently, inflammation is subdivided into acute and chronic. Acute inflammation is the immediate and early response to a stimulus. It is characterized by: 1) edema secondary to vascular dilatation, increased permeability of the vascular wall and stasis and 2) leukocyte infiltration, consisting primarily of neutrophils. In contrast, chronic inflammation is more difficult to define. It is usually a prolonged reaction to persistent infections, chronic exposure to toxins or autoimmunity. Chronic inflammation is characterized by chronic inflammatory cell infiltrates (lymphocytes, macrophages and plasma cells) and tissue destruction mediated by these cellular infiltrates. 1 1.2 CHEMOKINES Within the last decade or so, a growing family of molecules has emerged characterized by their unique ability to both recruit and activate several cell types - the chemoattractant cytokine or chemokine family. In the years since Walz et al (1977) sequenced C X C L 4 (platelet factor-4/PF-4), the diversity of the chemokines and their roles in mediating normal physiological and pathological processes have increased significantly. To date, these proteins are associated with a broad range of biological processes (Rossi and Zlotnik 2000; Mackay 2001; Fernandez and Lolis 2002), which include angiogenesis (Keane and Strieter 1999), atherosclerosis/cardiovascular disease (Sasayama et al. 2000; Rollins 2001), cancer biology (Nomura and Hasegawa 2000; Murphy 2001), autoimmune diseases (Arimilli et al. 2000), nervous system inflammation (Huang et al. 2000), asthma (Lukacs 2001), and haematopoiesis, to mention a few. 1.2.1 Nomenclature and Structure Nomenclature To date, through traditional methods, as well as the development of EST (expressed sequence tag) databases and bioinformatics, there are about fifty known chemokine members (Tanase and Nomiyama 2001). These are divided into four sub-families by virtue of highly conserved N-terminal cysteine motifs (disulfide bonds) and the presence or absence of intervening amino acids. The two major sub-families consist of the a- (or C X C or SCYa (small secreted cytokine)) and B- (or C C or SCYb) chemokines, with the y- (or C or SCYc) and 8-chemokines (or C X 3 C or SCVcO having one representative each, lymphotactin (two isoforms) and fractalkine, respectively. 2 CXC-chemokines are further divided into ELR (Glu-Leu-Arg) and non-ELR containing constituents. A new nomenclature for chemokines was introduced in 1999 (Murphy et al. 2000; Zlotnik and Yoshie 2000). The new naming system is based on the receptor nomenclature in current use. For example, CXC-chemokines are named CXCL1 - 16; CC-chemokines, CCL1 - 28; C-chemokines, XCL1/2; and the only member of the CX 3 C-chemokine subfamily, CX 3 CL1 (Tanase and Nomiyama 2001). For reference, the following table is provided to relate the old nomenclature to the new. 1 2 . 1 2 Structure For the most part, chemokines are believed to have arisen from gene duplication events over the course of evolution. The general clustering of subfamily members within individual chromosomes reflects this. For example, the majority of CXC-chemokines are found in humans at 4q12-13, 21. The exceptions, CXCL12 (SDF-1 a/p) and CXCL16 (SR-PSOX) are found at 10q 11.1 and on 17, respectively. CC-chemokines are mostly found at 17q11.2 with some members residing on chromosomes 2, 5, 7, 9, 16 and 19 (Zlotnik and Yoshie 2000; Tanase and Nomiyama 2001). XCL1/2 (C-chemokine/lymphotactin a/p) is located on chromosome 1 and CX 3 CL1 (CX 3C-chemokine/fractalkine) on chromosome 16 (Keane and Strieter 2000). In general, chemokine genes code for proteins of 92 - 125 amino acids with short signal peptides of 20 - 25 amino acids (Baggiolini et al. 1997). The secreted molecule itself is fairly small, on the order of 8 - 12 kDa. As mentioned earlier (section, N-terminal cysteine residues and the presence or absence of intervening amino acids are the main structural characteristics of chemokines. As a general rule, 3 TABLE 1: Chemokine Nomenclature (adapted from Murphy et al (2000)) Fam ily New Nam ing Conventional Naming C X C L 1 G R O a ; M S G A ; N 5 1 / K C ; M I P - 2 C X C L 2 Gro(3; M I P - 2 a C X C L 3 G r o y ; M I P - 2 p C X C L 4 P F - 4 C X C L 5 E N A - 7 8 C X C L 6 G C P - 2 C X C (a) C X C L 7 C X C L 8 P B P = > C T A P - l l l = > p - T G = > N A P - 2 IL-8 C X C L 9 M I G C X C L 1 0 y l P - 1 0 ; C R G - 2 C X C L 1 1 l - T A C ; P - R 1 ; I P 9 ; H 1 7 4 C X C L 1 2 S D F - 1 a ; S D F - i p ; P B S F C X C L 1 3 B C A - 1 ; B L C C X C L 1 4 B R A K ; b o l e k i n e C C L 1 I-309; T C A - 3 C C L 2 M C P - 1 ; M C A F ; J E C C L 3 M I P - 1 a ; M I P - 1 a S ; L D 7 8 a N A L D 7 8 P ; M I P - 1 a P C C L 4 MIP - 1 P C C L 5 R A N T E S C C L 6 C 1 0 ; M R P - 1 C C L 7 M C P - 3 C C L 8 M C P - 2 C C L 9 M R P - 2 ; M I P - 1 y C C L 1 0 r e s e r v e d C C L 1 1 E o t a x i n C C L 1 2 M C P - 5 cc (p) C C L 1 3 C C L 1 4 M C P - 4 C C - 1 ; H C C - 1 ; N C C - 2 ; C C C K - 1 / C C C K - 3 ; c k p i ; M C I F C C L 1 5 H C C - 2 ; l e u k o t a c t i n - 1 ; M I P - 5 ; C C 2 ; N C C - 3 ; M I P - 1 6 C C L 1 6 H C C - 4 ; L E C ; N C C - 4 ; L M C ; m o n o t a c t i n - 1 ; L C C - 1 ; I L I N C K C C L 1 7 T A R C C C L 1 8 D C - C K - 1 ; P A R C ; M I P - 4 ; A M A C - 1 ; c k p 7 C C L 1 9 M I P - 3 P ; E L C ; e x o d u s - 3 ; c k p 1 1 C C L 2 0 M I P - 3 a ; L A R C ; e x o d u s - 1 ; S T 3 8 C C L 2 1 6 c k i n e ; S L C ; e x o d u s - 2 ; T C A 4 ; C K P 9 C C L 2 2 M D C ; d c / p - c k ; a b c d - 1 C C L 2 3 M P I F - 1 ; M I P - 3 ; c k p 8 - 1 C C L 2 4 M P I F - 2 ; e o t a x i n - 2 ; c k p 6 C C L 2 5 T E C K ; c k 1 5 C C L 2 6 E o t a x i n - 3 ; M I P - 4 a C C L 2 7 E S k i n e ; C T A C K ; I L C ; A L P ; s k i n k i n e c (y) X C L 1 L y m p h o t a c t i n a; S C M - 1 a ; A T A C X C L 2 L y m p h o t a c t i n P; S C M - 1 P ; A T A C C X 3 C (6) C X 3 C L 1 F r a c t a l k i n e ; n e u r o t a c t i n 4 the first cysteine residue forms a covalent bond with the third cysteine residue, and the second with the fourth, with the exception of lymphotactin that lacks the first and third cysteine residues (Kelner et al. 1994). For the most part, chemokines are secreted molecules, with the exception of C X 3 C L I and CXCL16. C X 3 C L 1 exists in both a membrane-bound form, consisting of a transmembrane segment and a chemokine domain suspended by a mucin-like stalk, and in a secreted form (Bazan et al. 1997; Pan et al. 1997). CXCL16 , is similarly expressed in membrane-bound and secreted forms, induces chemotaxis via C X C R 6 (orphan receptor STRL33/Bonzo/TYMSTR) and localizes to chromosome 17, where most CC-chemokine genes are situated (Matloubian et al. 2000; Wilbanks et al. 2001). Wilbanks et al (2001) suggest that the unique characteristics of CXCL16, despite its designation, may make it a member of a completely novel class of chemokine. The secondary structure of chemokines is not remarkable. An unstructured, extended N-terminus precedes the first set of cysteine(s), followed by an approximately ten residue loop (termed N-loop). This is often followed by a 3-io helix. A B-pleated sheet follows, consisting of three antiparallel p-strands linked by flexible loops. Disulfide bridges and hydrophobic interactions further stabilize the central core, though this is by no means universal as chemical modification, site-directed mutagenesis or disulfide reduction do not always affect biological activity (Fernandez and Lolis 2002). The N-loop, due to its flexibility, may be important in receptor interaction/activation (Crump et al. 1999; Ye et al. 2000), though the intervening segment (termed the 30s loop) between the first and second p-strands may have some function as well (Clark-Lewis et al. 1994). 5 A multitude of studies have investigated the possibility that chemokine quaternary structure plays a role in biological functions and receptor binding dynamics. To date, biochemical evidence suggests that these proteins can form homodimers, heterodimers and even higher order structures. The physiological relevance of these structures is still relatively unclear. Structural analyses, such as X-ray crystallography and nuclear magnetic resonance, require much higher concentrations than would be present under physiological circumstances and as a result, may account for the presence of multimeric structures (Fernandez and Lolis 2002). In general, dimerization of CC-chemokines is mediated by interaction of N-terminal residues (Baysal and Atilgan 2001), which results in an elongated structure. C X C and C X 3 C -chemokines tend to form globular dimers through the interaction of B-strands (Fernandez and Lolis 2002). Higher order structures, such as the crystallized, tetrameric form of NAP-2 (neutrophil-activating peptide) (Rajarathnam et al. 1997), have been shown to exist. However, the micromolar dissociation constants of these multimeric clusters, along with mutation and truncation studies suggest that biological activity is mediated by monomers (Avalos et al. 1994; Burrows et al. 1994; Rajarathnam et al. 1994; Clark-Lewis et al. 1995; Paavola et al. 1998; Ali et al. 2001), though this has not been entirely resolved (Zhang and Rollins 1995; Laurence et al. 1998). Furthermore, Guan et al (2001) provide evidence suggesting that heterodimers are secreted at nanomolar concentrations, under physiological conditions, by activated human monocytes and peripheral blood lymphocytes. 6 Interaction with Extracellular Matrix Components Chemokines, as mentioned earlier, in general, are secreted molecules. Therefore, in order to maintain their presence at sites of inflammation, it is intuitive to assume that some sort of sequestration occurs. There is abundant evidence to suggest that secreted chemokines bind to glycosaminoglycans (GAG) in the extracellular matrix (ECM) and that these structures are physiologically relevant. The question is whether chemokines require G A G s in order to exert their cellular effects. G A G s are long linear polysaccharide chains made up of disaccharide subunits added to proteins at serine residues, forming complexes known as proteoglycans (Tanaka et al. 1993). The available evidence points to the critical role played by chemokine-GAG binding, especially on endothelial surfaces, in chemokine presentation to high affinity chemokine receptors, sequestration, and protection from degradation. Almost all chemokines have the ability to bind heparan sulfate (HS), a member of the G A G family. However, Ali et al (2000) showed that cell surface G A G expression was not necessary for the biological activity of C C L 3 (macrophage inflammatory protein-1 a/MIP-1 a), C C L 5 (regulated on activation normal T cell expressed and secreted/RANTES), or CCL4 (MIP-1B). Instead, G A G s served to enhance chemokine activity at low concentrations via a mechanism that appears to involve sequestration. In most cases, the HS-interacting region is found on the C-terminal a-helix or in loops facing the N-terminus and thus, is distinct from the receptor-interacting surface (Lortat-Jacob et al. 2002). Hoogewerf et al (1997) demonstrated that cell surface G A G s induce oligomerization of chemokines, thereby increasing the local concentrations and enhancing their effects on high-affinity receptors. Moreover, 7 thermal denaturation of C X C L 8 (interleukin-8/lL-8) is prevented in the presence of HS or heparin, indicating structural stabilization. As such, the biological effect of the chemokine may be prolonged (Goger et al. 2002). Furthermore, Kuschert et al (1999) showed that chemokines can discriminate between and selectively interact with both cell surface and soluble G A G s , though these interactions appear to have different biological functions. Soluble GAG-chemokine complexes cannot bind to the high-affinity receptor, a possible result of electrostatic effects mediated by the acidic nature of both the G A G and the N-terminus of the chemokine receptor. Apparently contradictory evidence was presented by Burns et al (1999) who demonstrated that soluble complexes of C C L 5 and G A G chains can bind to chemokine receptors and suppress macrophage-tropic HIV-1 activity, but failed to elicit intracellular Ca2+ mobilization in peripheral blood mononuclear cells. The authors speculate that soluble G A G s may induce structural changes in C C L 5 that differ from those induced by cell surface G A G s , translating into differential cellular effects. Similar results were seen with respect to CCL21 (secondary lymphoid tissue chemokine/6Ckine/ S L C ). Hirose et al (2001) showed that CCL21, when bound to versican, a chondroitin sulfate proteoglycan, is unable to initiate intracellular C a 2 + flux via interaction with C C R 7 . They suggest that the C C L 2 1 - G A G complex can bind to the receptor, but is unable to activate it. The volume of in vitro data regarding chemokine-GAG interactions is enormous. However, the physiological relevance of such interactions is less clear. To address these concerns, Wagner et al (1998) demonstrated that CD8+ cytotoxic T lymphocytes (CTL), following Ag-specific activation, secrete CCL5 , C C L 3 , and CCL4 , together as a macromolecular complex containing granzyme A and sulfated proteoglycans. HS was 8 also found to help facilitate C C L 5 inhibition of HIV-1 infection of monocytes. The combination of in vitro and in vivo data suggests that chemokine-GAG interactions may be critical. 1.2.2 Chemokine Receptors Chemokine receptors are membrane-bound components responsible for the activation of signal transduction pathways upon chemokine ligation. Interest in the receptor biology field boomed with the discovery that isolates of HIV-1 (human immunodeficiency virus type 1) used C C R 5 and C X C R 4 as cofactors for cellular entry. Discovery of HIV-1 resistance in certain high-risk populations intensified the scrutiny on the physiological functions of these receptors (Berger et al. 1999; Horuk 1999). In addition, the knowledge that certain wild type and modified chemokines had the ability to block HIV-1 entry resulted in the pharmaceutical industry becoming heavily involved in such research. The possible therapeutic value of such data is being explored at this point. Chemokine receptors belong to the rhodopsin family of seven-transmembrane domain (7TMD), heterotrimeric G (guanine nucleotide binding) protein coupled receptors (GPCR). Receptor-like sequences have been documented in mammals, birds and fish, but not in invertebrates, suggesting a rather recent evolutionary origin. G P C R s , as a superfamily, are an incredibly diverse group. In vertebrates, there are 1000 - 2000 members within this superfamily, which accounts for >1% of the genome. Light, odorants, peptides, nucleotides, and proteins account for some of the messages that G P C R s can respond to. This response manifests itself in the activation of 9 heterotrimeric G proteins and a variety of downstream signal transduction pathways (Bockaertand Pin 1999). Organization Chemokine receptors are organized in a systematic manner - on the basis of their ability to signal upon binding to one or more members of the same subfamily. At present, there are 18 receptors that correspond to this definition. They are designated CXCR1 - 6 (a-chemokine family), CCR1 - 11 (P), XCR1 (y), and C X 3 C R 1 (5). In addition, D6 and the DARC (Duffy antigen receptor for chemokines) bind ligands belonging to both C C and C X C subfamilies. However, they do not appear to signal and therefore, are not designated as chemokine receptors in the new nomenclature (Murphy et al. 2000). Studies have suggested that chemokines and their receptors have been subjects of divergent evolution and also co-evolution, in order to maintain ligand specificities (Goh et al. 2000). 12.2.2 Structure In general, chemokine receptors, like most 7TMD receptors, are encoded within a single exon. On the protein level, the receptors share a number of common elements: seven-transmembrane a-helical domains; conserved structure with 25 -80% amino acid identity; and interaction with Gj class of GTP-binding proteins (G proteins) (Murphy et al. 2000). In general, these proteins are 350 amino acids in length, have a short, acidic N-terminus and contain N-linked glycosylation sites. The cytoplasmic C-terminus contains serine and threonine phosphorylation sites for receptor regulation, as well as interaction sites for G proteins. There are three 10 extracellular and three intracellular loops connecting the transmembrane segments, with a disulfide bridge connecting highly conserved cysteine residues in extracellular loops 1 and 2. Intracellular loop 3 is also a possible G protein interaction site (Murdoch and Finn 2000). The conserved DRYLAIV motif, unique to chemokine receptors, in the second intracellular loop is important for signalling. Functional differences between the receptors, within and across groups, may have arisen from rapid gene duplication events (Shields 2000). At present, none of the chemokine receptors have had their structures elucidated. However, the crystal structure of bovine rhodopsin, a member of the G P C R superfamily, has been determined to 2.8 angstroms resolution (Palczewski et al. 2000; Okada and Palczewski 2001) and further refined (Teller et al. 2001). Rhodopsin, an integral membrane protein found in the retina, behaves in a manner typical of G P C R s . Interaction with Ligands Chemokine receptors interact with their ligands via the N-terminus and the extracellular loops (ECL). Some receptors bind to a single chemokine, whereas others have the ability to bind multiple ligands that belong to the same subfamily. On the chemokine level, some can mediate their effects via a number of different receptors while others have single receptor specificity. The general model of ligand-receptor interaction has the N-loop of the chemokine making the initial contact with the receptor, allowing for subsequent activation by the N-terminus of the chemokine. There is some variation with this general model when applied to individual chemokine-receptor pairs. For example, 11 CCR2b binds CCL2 and C C L 7 with its N-terminus, but signals through interaction of the bound chemokine with the receptor's ECL. CXCL12 (stromal cell-derived fac tor -1/ SDF-1) interacts with C X C R 4 in a manner similar to C C L 2 and CCL7 . CCL3 binds and signals through ECL3 of C C R 1 . ECL2 of C C R 5 is the critical loop in the binding and activation by C C L 3 , CCL4 and CCL5 (Loetscher and Clark-Lewis 2001; Stantchev and Broder2001). Heterotrimeric G Proteins The G P C R superfamily encompasses greater than 1000 identified members grouped into numerous functional subfamilies. Aside from common structural similarities, G P C R s share one very important characteristic - the ability to activate heterotrimeric G proteins in response to ligand/receptor interaction. G proteins, with their characteristic heterotrimeric structure, are made up of a, (3, and y subunits. Each G protein subunit shows great diversity - there are at least 20 a, 6 p, and 12 y genes known. Four main classes of G a proteins exist: G| ( a n , a i 2 , a i 3 , a 0 i , a 0 2 , ocz, a t , a g ) ! G s ( a s , a 0 i f) ; G q (a q, a n , a n , a i 5 , a-i6); and G 1 2 (0,12, a i 3 ) . Some of these are ubiquitous ( a n , a i2 , a i 3 , a s , a q , a-12, oci 3 ), whereas others are cell-type specific. The functional roles of the various p and y proteins seem to be more limited than their a counterparts (Hamm 1998; Stantchev and Broder 2001). The inactive heterotrimer consists of a guanosine diphosphate (GDP) bound to the a subunit, which is, in turn, associated with the Py subunits. Ligand binding induces a conformational change in the receptor as a result of a shift in the relative orientation of transmembrane helices 3 and 6. This affects the intracellular loops (ICL) that interact with G proteins and uncovers additional binding 12 sites. The activated receptor acts as a guanine nucleotide exchange factor (GEF) when the G D P bound form of the aBy heterotrimer interacts with these new binding sites. Receptor-mediated release of G D P from the G a subunit and subsequent binding of GTP results in the dissociation of the G a subunit from the G P y subunits. Each group is capable of activating a number of different effector pathways (Hamm 1998; Keane and Strieter 2000; Stantchev and Broder 2001). All chemokine receptors can signal through the G| class of G a proteins, which accounts for the general sensitivity of chemokine receptors to Bordetella pertussis toxin. However, a number of groups have shown that chemokine receptors can also signal through the G q class of G a proteins, in a pertussis toxin-resistant manner. Furthermore, the cell type determines exactly which members of the G q class are involved in signalling (Arai and Charo 1996; Kuang et al. 1996). Receptor Activation Interaction between a specific chemokine and its receptor results in conformational change in the receptor, dimerization, recruitment of effector molecules, and the activation of diverse and complex signalling pathways. The active receptor mediates the binding of G T P to the G a subunit and its dissociation from the G P y subunit (Forse 2000). The subunits of the dissociated heterotrimer and receptor dimerization activate different effector molecules and will be examined briefly. The signalling pathways elicited by ligand binding are not necessarily unique to each receptor, but may be the result of "crosstalk" between the various receptors activated. The formation of homodimers and/or heterodimers, upon chemokine binding, functions to activate the JAK/STAT (Janus kinase/signal transducers and 13 activators of transcription) pathway. Homodimerization has been shown to be important for signalling in C C R 2 , C C R 5 and C X C R 4 . However, sequence homology and the presence of the conserved DRY motif suggests that receptor dimerization is not simply restricted to the aforementioned chemokine receptors, but may be a common feature (Mellado et al. 2001). Mellado et al (2001) demonstrated that heterodimerization between C C R 2 and C C R 5 activated distinct signalling pathways. It was shown that a n , of the G q class, was active in mediating pertussis toxin-resistant calcium flux and triggered cell adhesion instead of chemotaxis. Rodriguez-Frade et al (2001) suggest that homodimerization and heterodimerization may be a functional response to the degree of chemokine availability. The cumulative effects of ligand-receptor interaction are multivariate. Dimerization allows the recruitment and coupling of JAK to the receptor, followed by tyrosine phosphorylation of both the receptor and JAK - pertussis toxin-independent events. JAK association initiates G protein signalling and the recruitment and activation of STAT transcription factors (Keane and Strieter 2000; Mellado et al. 2001; Mellado et al. 2001). GTP-Gj remains associated with the activated receptor and has been implicated in activating a number of different intracellular effectors, such as Ras and Rho, phospholipase A2, phosphatidylinositol-3-kinase, tyrosine kinases, and the MAP (mitogen-activated protein) kinase pathway. G P y activates P L C - B 2 (phospholipase Cp 2 ) and PLC-p 3 , which cleaves phosphatidylinositol 4,5-biphosphate (PIP 2) to form inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP 3 binds to a specific receptor on the endoplasmic reticulum, leading to an intracellular C a 2 + mobilization, which combined with DAG activates various P K C (protein kinase C) isoforms. P K C , in turn, activates a cascade of signal transduction mechanisms in the 14 cytoplasm and in the nucleus (Keane and Strieter 2000; Mellado et al. 2001; Thelen 2001). The proper regulation of cellular responses to a chemotactic gradient is critical. With respect to chemokines and their receptors, there are a number of different pathways that mediate the desensitization and recycling/degradation, hence recovery, of activated receptors. Hydrolysis of GTP to G D P by the GTPase activity of the G e t subunit results in reversion to an inactive status and reassociation with Gp y. To enable recovery of the signalling system, the R G S (regulator of G protein signalling) family is critical to accelerating G T P hydrolysis (Sowa et al. 2000). In addition, the G P y subunit acts as an adapter for many signalling pathways, including those for receptor desensitization (Wu et al. 1998). G R K (G protein receptor kinases) proteins, a family of serine/threonine kinases, are recruited from the cytosol to the membrane and phsophorylates the receptor at serine and/or threonine residues on the carboxyl terminal domain (Franci et al. 1996; Aragay et al. 1998; Aragay et al. 1998; Mellado et al. 1998; Yang et al. 1999; Keane and Strieter 2000). Furthermore, G R K activity appears to be greatly enhanced by binding to an activated receptor. The GRK-phosphorylated receptor has a high affinity for p-arrestin, which is presumed to disrupt interactions between the receptor and G proteins. Binding of p-arrestin results in the dynamin-mediated sequestration of the receptor to clathrin-coated pits, leading to the internalization of the receptor to the endosome. The internalized receptor can be dephosphorylated and recycled to the surface or degraded, possibly via the ubiquitin pathway, as in the case of C X C R 4 (Bohm et al. 1997; Koenig and Edwardson 1997; Yang et al. 1999; Marchese and Benovic 2001; Sauty et al. 2001). Mueller et al (2002) demonstrated that C C R 5 desensitization was affected by inhibitors to both caveolae-15 dependent internalization and clathrin-mediated endocytosis, suggesting possibly redundant and/or complementary pathways for receptor regulation. There is some suggestion that other molecules may be involved in the internalization of chemokine receptors. Fan et al (2001) demonstrated, using a mutant that no longer undergoes agonist-induced phosphorylation, that adaptin 2 (adaptor protein-2/assembly protein-2/AP-2) may function in the endocytosis of C X C R 2 . The AP-2 protein complex is involved in the assembly of clathrin-coated pits and functions as an adaptor by linking receptors to the clathrin lattice. They have been shown to be important in G P C R endocytosis (Schmid 1997; Laporte et al. 1999). To confuse things further, a recent study by Fernandis et al (2002) was able to show that C X C R 4 and C C R 5 could be down-modulated by the proteosomal pathway, by receptor association with a particular catalytic particle of the 26S proteosome. Finally, resensitization has been shown by different groups to be a relevant mechanism in receptor dynamics. At present, this process is poorly understood, especially with respect to chemokine receptors. An earlier study by Krueger et al (1997) showed that the acid environment of the endosome may be responsible for regulating dephosphorylation and resensitization of the p2-adrenergic receptor, a member of the G P C R superfamily. Recently, Zaslaver et al (2001) demonstrated that functionally intact actin filaments were required for both the endocytosis and resensitization of CXCR1 and C X C R 2 . 12.2.6 Cellular Effects The activation of multiple signal transduction pathways, upon chemokine binding, results in a number of cellular effects. These include: actin-dependent cellular changes; ion mobilization; upregulation of adhesion molecules; release of the contents 16 of cytoplasmic granules, such as proteases from neutrophils and monocytes, histamine from basophils, and cytotoxic proteins from eosinophils; oxidative burst; phagocytosis; lipid mediator synthesis; and the formation of focal adhesions which underlie chemotaxis, to mention a few (Baggiolini 1998; Horuk 2001). In the context of inflammation, the critical role of chemokines and receptor activation is well established. At sites of inflammation or injury, the presence of proinflammatory cytokines activates the endothelium to express adhesion molecules and chemokines that enable the recruitment of circulating leukocytes (Ransohoff 1999; Brown 2001; Omari et al. 2004). The prevailing theory is that blood-borne inflammatory cells attach to the luminal surface of the endothelium in postcapillary venules, where the shear stress is lowest. Transendothelial migration is a multistep process where the initial tethering and rolling is mediated by selectins. Chemokines binding to their receptors activates integrins, and other cell-specific processes, on the leukocytes, enabling firm attachment to EC. The presence of stable chemotactic gradients allows for the transendothelial migration (TEM) of recruited cells, though there is evidence that other factors such as shear stress may also be critical for successful T E M (Cinamon et al. 2001). Furthermore, the expression of functional chemokine receptors on endothelium may play a role in endothelial activation. 1.2.3 Chemokines and Their Receptors: Role in Disease Under physiological conditions, chemokine and chemokine receptor expression is finely controlled in both a spatial and temporal manner. This allows for the exquisite control of many biological processes. However, there is considerable evidence that these molecules are also major factors induced in response to injury and inflammation. 17 The desire to understand this area is driven by the need to discover viable therapies for diseases where the chemokine/chemokine receptor system has been shown to play a role. Inflammation There is a great deal of interest in the role of chemokines and chemokine receptors in inflammation. They have been implicated in allergy, atherosclerosis, and autoimmune disorders, such as arthritis and multiple sclerosis (MS). One of the clearest indications that chemokines/receptors are important in inflammatory responses is provided with the observation that intradermal injection of chemokines, results in the accumulation of mast cells and basophils to the site of injection (Conti et al. 1997). Well-studied models of allergy are asthma in humans and murine allergic airway disease. The concept of temporal and spatial distribution of chemokines controlling the recruitment of different leukocyte subpopulations in allergic responses is supported by studies involving allergic tissue reactions in human skin (Ying et al. 1999). Lung parenchymal cells, including airway epithelial cells, EC, alveolar macrophages, smooth muscle cells, and fibroblasts, express numerous chemokines that act in the recruitment and infiltration of leukocytes, particularly eosinophils. For example, CCL11 and CCL13 mRNA levels are substantially elevated in the lungs of asthma patient, correlating with the levels of infiltrating eosinophils (Taha et al. 1999). Furthermore, in patients with asthma, there appears to be an increased bone marrow pool of CCR3+ mature and immature eosinophils available for rapid mobilization (Zeibecoglou et al. 1999). Other chemokines have been postulated to play roles in the pathogenesis and progression associated with allergic reactions. There is evidence to 18 suggest that C C L 2 , acting on a variety of leukocyte populations, may have a role in driving the early phases of the asthmatic response (Lloyd 2002). Panina-Bordignon et al (2001) provide evidence that T cells infiltrating the airway mucosa of atopic asthmatics exhibited a Th2 phenotype and expressed C C R 4 and, to a lesser extent, C C R 8 , with their numbers increasing upon allergen challenge. Also, the authors found a correlation between the number of CCR8+ T cells and the severity of asthmatic response upon allergen challenge. The ligands for C C R 4 , CCL17 and CCL22, were also found to be upregulated in airway epithelial cells following allergen challenge. The role of macrophages in the development of the atherosclerotic lesion has long been recognized. Studies by Nelken et al (1991) showed that CCL2 was abundantly expressed in macrophage rich areas bordering the lipid core, suggesting that this particular chemokine may be an important factor in lesion development. Further evidence using the human apolipoprotein (apo) B expressing/CCL2-deficient transgenic mouse has provided compelling evidence that C C L 2 does play a critical role in the initiation of atherosclerosis. CCL2 deficiency reduces the susceptibility to lesion formation in these mice, even when fed a high fat, high cholesterol diet (Gu et al. 1998; Gosling et al. 1999). Boring et al (1998) generated mice that lacked CCR2 , the receptor for CCL2 , and crossed them with apolipoprotein E deficient mice, which are prone to developing severe atherosclerosis. The resultant C C R 2 -/-, apo E -/-mice have significantly decreased lesion formation. Autoimmunity is the vigorous response generated by the immune recognition of some self-antigens resulting in significant leukocyte activation. The role of chemokines and their receptors has been well established in various animal models and, to a lesser extent, in humans. Rheumatoid arthritis is a chronic systemic 19 inflammatory disorder, of unknown etiology, that principally attacks the joints. The molecular events that underlie this disorder lead to a chronic synovitis and ultimately to the destruction of articular cartilage and bone. Elevated levels of both C C and C X C -chemokines, enrichment for certain leukocyte subpopulations expressing particular chemokine receptors, lymphoid neogenesis, as well as expression of particular chemokines by synovial cells strongly suggest that chemokines/receptors play a critical role in pathogenesis (Godessart and Kunkel 2001). The role of chemokines/receptors in MS and its animal model, EAE, has been well documented. MS is an autoimmune C N S disorder characterized by demyelination, inflammation and axonal injury resulting in neurological deficits. Environmental, genetic, and immune factors have been implicated in the etiology and progression of MS. There are a number of correlative studies involving MS patients, as well as studies involving animals that suggest a causative role for chemokines/receptors in disease development. These will be discussed in more detail in later sections (sections 1.3.5 and 1.3.7). Cancer Metastasis is the ability of cancer cells to move through tissue, into the blood or the lymphatic circulation, cross through the vascular barrier and invade normal tissues. Muller et al (2001) demonstrated that C X C R 4 and C C R 7 are highly expressed on breast cancer cells, malignant breast tumours and metastases. In addition, malignant melanoma cells show high levels of expression of not only C X C R 4 and C C R 7 , but of CCR10 as well. In vivo experiments using Ab blockade of C X C R 4 resulted in significant suppression of breast tumour metastases to the lung and the lymph nodes 20 in immunodeficient mice. The authors suggest that this finding, combined with their other results, provides compelling evidence that the ligand for C X C R 4 , CXCL12 (SDF-1), may be a factor in attracting metastatic cancer cells to specific organs. These results may be a step towards understanding the bias for specific organs by various cancers (Liotta 2001; Murphy 2001). The putative role of chemokines/chemokine receptors in cancer is not simply restricted to metastases. There is considerable evidence that CXC-chemokines are major factors in both angiogenesis and angiostasis in tumour biology (Dias et al. 2001). The angiogenic potential of ELR CXC-chemokines have resulted in them being considered important mediators of tumourigenesis. In melanoma, C X C L 1 , 2, and 3 (GRO (growth-related oncogene)-a, -p, and -y, respectively) have been shown to act both as autocrine growth factors and as paracrine mediators for angiogenesis. Ovarian carcinoma, prostate cancer and non-small cell lung carcinoma (NSCLC) appear to be dependent on CXCL8 for neovascularization and/or metastases. There is also some correlation between the expression of C X C L 5 (epithelial cell-derived neutrophil-activating factor, 78 amino acids/ENA-78) and the tumourigenesis of N S C L C (Belperio et al. 2000; Rossi and Zlotnik 2000). The angiostatic/antitumour potential of the non-ELR CXC-chemokines, CXCL9 and CXCL10, was demonstrated in the context of Burkitt's lymphoma in nude mice. Both C X C L 9 and CXCL10 levels were higher in regressing than in progressive forms of the lymphoma. Furthermore, intra-tumour inoculation with C X C L 9 or CXCL10 led to extensive necrosis and capillary damage (Sgadari et al. 1996; Sgadari et al. 1997; Teruya-Feldstein et al. 1997). Strieter et al (1995) demonstrated that the non-ELR CXC-chemokines, CXCL4 , C X C L 9, and CXCL10, are potent angiostatic regulators of 21 neovascularization. In fact, their data suggests that the balance between angiogenic and angiostatic CXC-chemokines may be a factor in overall angiogenesis. The demonstration by various groups that chemokines have some role in determining the fate of certain tumours may have profound implications in the development of future cancer therapies. However, the study of cancer biology is not restricted to chemoattraction/migration of cancer cells, but encompasses the molecules involved in adhesion and migration events at metastatic sites. Infectious Diseases Chemokines and chemokine receptors play prominent roles in two major diseases: malaria and infection with the human immunodeficiency virus (HIV). Malaria is a disease transmitted by female anopheline mosquitoes infected with Plasmodium protozoa - a single celled parasite. Upon infection, these parasites invade and develop within hepatocytes. When released, these parasites bind to and enter erythrocytes, adopting a life cycle that ultimately leads to cell lysis and further invasion (Newton and White 1999). It has been shown that both P. vivax and P. knowlesi gain entry to erythrocytes via specific binding to the promiscuous chemokine receptor DARC (Duffy antigen receptor for chemokines) (Hadley and Peiper 1997; Murdoch and Finn 2000). HIV is similar to malaria in that the infectious agent enters the target cell via chemokine receptors. This disease has received a tremendous amount of attention due to the discovery that certain chemokines can block HIV-1 infection and that specific chemokine receptors act as cofactors for HIV-1 and HIV-2 entry. Several chemokine receptors (CCR2b, C C R 3 , C C R 5 , C C R 8 , C C R 9 , C X C R 4 , and CX 3 CR1) 22 and chemokine receptor-like orphans (STRL33/Bonzo/TYMSTR (now designated CXCR6) , GPR15 /BOB and Apj) have been shown to act in concert with CD4 (cluster of differentiation antigen 4) to allow HIV entry (Horuk et al. 1998; Berger et al. 1999; Stantchev and Broder 2001). However, C C R 5 and C X C R 4 remain the principal coreceptors for R5 (macrophage (M)-tropic, utilizing CCR5) , X4 (T cell-tropic, CXCR4) or R5X4 (dual tropic) clinical isolates of HIV. There is evidence that HIV-2 can enter cells via a CD4 - independent pathway (Clapham et al. 1992). However, clinically relevant strains of HIV-1 have a critical requirement for the CD4 molecule. From an evolutionary perspective, there is some suggestion that chemokine receptors were initially the "primary" receptors for HIV and that CD4 is a more recent adaptation (Dimitrov eta l . 1998). The discovery of certain people "resistant" to HIV infection was met with a great deal of excitement. Numerous studies have proposed that some chemokines, CCL3 , CCL4, and C C L 5 (Cocchi et al. 1995; Paxton et al. 1996; Zagury et al. 1998), and CXCL12 (Oberlin et al. 1996) play a protective role against HIV. In vitro experiments involving the homodimerization of C C R 5 by a monoclonal antibody suggests a mechanism by which chemokines can block HIV-1 infection (Vila-Coro et al. 2000). Dean et al (1996) and Michael et al (1997) report that in their AIDS (acquired immunodeficiency syndrome) cohorts, there exists individuals both homozygous and heterozygous for a 32 base pair deletion in the C C R 5 gene (known as CCR5A32). This deletion is found in the region encoding the second ECL, resulting in a frameshift, the introduction of a premature stop codon in transmembrane domain 5, and a truncated protein that is not expressed on the cell surface (Liu et al. 1996; Berger et al. 1999). Those homozygous for CCR5A32 were initially thought to be highly resistant to 23 infection by M-tropic HIV-1, though exceptions have been noted (Biti et al. 1997; O'Brien et al. 1997; Theodorou et al. 1997). Heterozygous individuals may have a delayed disease progression, but no obvious protective benefit. Lack of C C R 5 expression does not result in any observable health problems, which may suggest redundancy in function. Subsequently, other genetic polymorphisms have been suggested to have some role in delaying the progression to AIDS: CCR2-V64I (valine to isoleucine at position 64 in the first TMD of CCR2) ; C C R 5 59029 G/A (G versus A single nucleotide polymorphism at base pair 59029 of the C C R 5 promoter); and SDF-1 3'A (G to A transition at base pair 809 of the 3' untranslated region of the mRNA encoding the SDF-1 B isoform) (Berger eta l . 1999). 1.3 THE CENTRAL NERVOUS SYSTEM (CNS) 1.3.1 The Blood-Brain Barrier (BBB) The concept of the blood-brain barrier (BBB) first arose during the late 19 t h century, when the German bacteriologist Paul Ehrlich observed that dyes injected into the systemic circulation stained all the organs of small animals, but curiously, were excluded from the brain (1885), indicating that the brain had a different, lower affinity for the dyes. His student Edwin E. Goldmann further showed that trypan blue injected directly into the brain stained C N S tissues, but not peripheral organs (1909; 1913). In the 1960's, Reese and Karnovsky (1967) performed ultrastructural studies using exogenous horseradish peroxidase that showed conclusively that the cerebral EC in adult mice were the anatomical basis of the BBB. Over the years, a clearer understanding of the BBB and its functional properties has been achieved. The main function of the BBB is to provide a stable chemical 24 environment for the brain, thereby ensuring optimal neuronal activity. It also serves to regulate the passage of drugs and inflammatory cells from the blood to the brain. The BBB is present by the end of the first trimester and there is some evidence that astrocytes may play some role in the maturation of this barrier (Janzer and Raff 1987). The BBB is found in almost every region in the brain, with the exception of the choroid plexus, pituitary, area postrema, pineal gland, and hypothalamus. At these sites, fenestrations allow for the freer movement of solutes and hormones. This is of critical importance for the maintenance of peripheral homeostasis (Rapoport 1976). The fundamental structural feature of the BBB is the presence of tight junctions between adjacent EC cells. In vivo experiments on frog and rat brains have demonstrated that cerebral microvessels have a transendothelial electrical resistance on the order of 1000 - 2000 Q .cm 2 , akin to tight epithelial barriers. This is in comparison to peripheral endothelium, which is significantly lower, at approximately 10 Q .cm 2 (Crone and Olesen 1982; Butt et al. 1990). Pinocytotic vesicles are rare or absent in cerebral endothelium, unlike peripheral EC (Rapoport 1976). Essential metabolic substances, such as glucose, amino acids, sodium and potassium are transported across the EC by specific transporters (Rapoport 1976; Betz 1992; Stewart 2000). In addition, tight junctions and the lack of fenestrations enable cerebral EC to have exquisite control over the macromolecular and cellular exchange between blood and brain. Tight junctions reduce the interendothelial spaces to 12 angstroms, far smaller than most proteins (Rapoport 1976). Junctional complexes are found in both the apical and basolateral regions of the endothelium and are often referred to as the 'gate' and 'fence' functions. 25 These serve to isolate the apical and basolateral domains and are the molecular basis for the "tightness" of the junctions (Gloor et al. 2001). 1.3.2 Inflammation in the CNS Brain inflammation is the culmination of numerous processes. For a long time, it was believed that the C N S was an immunologically privileged site, due to the presence of the BBB. The question of whether the C N S had a true lymphatic system was not clear. Furthermore, transplanted tissue in the C N S did not seem to elicit the same immune rejection, as did transplantation in the periphery. Later studies showed that rejection did indeed occur in the C N S , albeit at a much slower rate (Poltorak and Freed 1997). Anatomically, a classical lymphatic drainage system does not exist in the C N S . Despite this, it appears that the movement of cerebrospinal fluid (CSF) and brain extracellular fluid allows for drainage of possible antigenic materials into identifiable lymphatics, which are found under the cribriform plate and along cranial and spinal nerve roots (Cserr et al. 1992; Knopf et al. 1995; Cserr and Knopf 1997; Hickey 2001) and into the cervical lymph nodes. Lymphocyte traffic also appears to have some role in immune surveillance. The data suggest that both activated T and B cells have the ability to enter the brain in a more or less random manner (Hickey 1999; Hickey 2001). Furthermore, there is evidence that perivascular cells are fully capable of presenting C N S antigens to T lymphocytes (Hickey and Kimura 1988; Hickey et al. 1992). It appears that the idea of complete immunologic isolation of the brain is no longer valid. The evidence suggests that immune surveillance does indeed occur, albeit at a slower and more limited rate than in the periphery. 26 1.3.3 Cells Participating in CNS Inflammation Astrocytes Astrocytes are glial cells that have a major role in the maintenance of C N S homeostasis. Astrocytes help regulate neuronal function and development, play a role in neurotransmitter metabolism, and regulate extracellular pH and ion levels. Furthermore, there is evidence to suggest that astrocytes increase the number of mature, functional synapses on C N S neurons and are required for synaptic maintenance in vitro (Ullian et al. 2001). In addition to homeostatic functions, these cells contribute to the maintenance of the BBB through the close interaction of perivascular astrocytic endfeet and cerebral endothelium (Kacem et al. 1998). In the context of C N S damage, astrocytes can play a similar role to fibroblasts in the periphery by undergoing proliferation in a process termed astrogliosis. The role of astrocytes in C N S immune function is relatively unclear. There is evidence that these cells are capable of producing specific cytokines and chemokines under inflammatory conditions. Their role as antigen presenting cells (APC) in the C N S is less well established. The expression of class II MHC (major histocompatibility complex) molecules is debatable and the expression of various costimulatory molecules (CD40, CD80, and CD86) is still controversial (Dong and Benveniste 2001). Microglia Microglia keep a rather quiescent profile in the normal C N S even though they share the same myeloid progenitor with macrophages in the periphery. In response to physiological or stress stimuli, these cells undergo a maturation process or activation, 27 which results in the acquisition of various macrophage properties. Activated microglia express a number of pattern recognition receptors; Fc and complement receptors; cytokines and their receptors; chemokines and their receptors; and prostanoids and prostanoid receptors. Furthermore, during C N S inflammation, activated microglia express class II MHC molecules and essential adhesion/costimulatory molecules (such as CD11a, CD40, CD54, CD58, CD80, and CD86) (Stoll and Jander 1999; Aloisi 2001). There is much evidence that microglial activation plays a major role in C N S pathophysiology. Perivascular Macrophages/Dendritic Cells Perivascular macrophages, also known as the perivascular microglial cell, fluorescent granular perihelial cell or 'Mato' cell, are bone marrow-derived and continuously enter the C N S as part of normal physiology and may serve as the principal antigen-presenting cell in the C N S (Hickey and Kimura 1988; Hickey et al. 1992). There is some evidence that this cell type returns to the lymph nodes and spleen with C N S material (Broadwell et al. 1994). However, other studies contradict this view, as deposition within the C N S of the particulate material collected by macrophages fails to elicit an immune response against the antigen (Matyszak and Perry 1998). The concept of the critical nature of the perivascular macrophage is appealing, however definitive evidence for this role has been somewhat lacking. Dendritic cells (DC) are derived from haematopoietic stem cells in the bone marrow and are found as immature cells in virtually all tissues. DC readily uptake, process and present antigen, thereby leading to their maturation and concurrent migration to lymphoid organs, where they interact with naive T cells. Their maturation 28 results in an alteration in their complement of surface receptors and secreted molecules, including chemokines. DC have recently been identified in experimental C N S inflammation and in MS, though previously, there had been a lack of clarity on this issue (Fischer et al. 2000; Serafini et al. 2000; Fischer and Reichmann 2001; Pashenkov et al. 2001; Pashenkov et al. 2002). However, the question remains as to their presence in the normal CNS. Matyszak and Perry (1996) were able to observe a small number of cells in the choroid plexus and meninges. McMenamin et al (1999) demonstrated DC in the dura mater, leptomeninges and choroid plexus. Endothelial Cells Over the years, numerous studies have strongly suggested that the endothelium plays an active role in inflammation. Some have looked at the potential of EC to function as antigen-presenting cells (APC) by the expression of MHC class II and costimulatory molecules (McCarron et al. 1991; Huynh et al. 1995; Omari and Dorovini-Zis 2001). Others have demonstrated the expression of chemokines in the active recruitment of circulating inflammatory cells. Chemokine expression by human peripheral EC has been fairly well documented in a number of disease states. However, in the human CNS, this is less clear as fewer studies have attempted to address this. Shukaliak and Dorovini-Zis demonstrated CCL4 and C C L 5 expression under stimulated inflammatory conditions (2000). Prat et al (2001) were able to show CCL2 and C X C L 8 mRNA expression in normal brain EC with upregulation upon addition of glial cell conditioned media, but were unable to confirm this at the protein level. Zhang et al (1999) were able to show 29 the upregulation of C C L 2 and CXCL8 RNA and the increased release of protein in experimental ischemia. Many studies have used H U V E C as a model system. Of note are two studies; one by Wolff et al (1998) which showed that E C were capable of storing C X C L 8 in Weibel-Palade bodies following an inflammatory insult, enabling the cells to mobilize these stores immediately without de novo protein synthesis. The other study of note demonstrated that E C can internalize C X C L 8 on the abluminal surface which is then transcytosed and presented on the luminal surface (Middleton et al. 1997). 1.3.4 Lymphocyte Trafficking in the CNS Lymphocytes There has been considerable interest on the role lymphocytes, particularly T cells, play in C N S inflammation, under a variety of conditions. Much of this is rationalized by the postulated role of T cells in the pathophysiology of multiple sclerosis (MS), viral encephalitides, and experimental diseases, especially EAE . It is now well established that activated, but not resting, T cells, irrespective of their antigen specificity, can cross the BBB (Wekerle et al. 1986; Wekerle et al. 1987). Hickey et al (1991) demonstrated that T lymphoblasts enter the C N S and other tissues in an apparently random manner while T cells not in the blast phase were excluded. They found that the activation state appears to be the major determining factor in the ability to enter the C N S . Furthermore, the retention of activated lymphocytes in the C N S is dependent on major histocompatibility complex (MHC)-restricted antigen recognition (Hickeyetal . 1991). 30 Many groups have shown that the C N S is under constant immune surveillance, but the question remains as to the degree of surveillance. The traditional belief that the CNS is an immunologically privileged site lends credence to the idea that there is not a significant amount of leukocyte traffic. It has been shown that the concentration of T cells is not evenly distributed among all organs. In one study, using 1 1 1 ln-labelled activated T cells in in vivo trafficking studies, it was found that the amount of traffic of these cells is relatively lower in the CNS than in other tissues. Furthermore, there are distinct differences in lymphocyte tissue concentration in various areas of the C N S (Yeager et al. 2000). The molecular basis of this is not known. In addition to T cells, natural killer (NK) and B cells have been implicated in C N S inflammation, though their roles are less clearly defined. NK T cells have been shown to be an integral part of the inflammatory infiltrate. In acute E A E , depletion of NK cells results in increased severity of disease, suggesting a possible moderating or suppressive role (Zhang et al. 1997; Matsumoto et al. 1998). Hickey (1999) noted a number of curious features about NK cell entry into the C N S : 1) in the rat E A E model, they are detectable within a day after the first T cells arrive; and 2) entry does not occur unless the T cells have located their antigen and the C N S is primed for inflammation. The mechanisms by which T cells can elicit the entry of NK cells are currently unknown, but the presence of NK cells signifies the commitment of the CNS to the initiation of the inflammatory cascade. Furthermore, NK cells have been implicated in the immunopathogenesis of MS (Kastrukoff et al. 1998; Kastrukoff et al. 1999), though the exact nature of their role is presently unknown. There is uncertainty regarding the role of B cells in C N S inflammation. The cerebrospinal fluid (CSF) levels of immunoglobulins are approximately 0.2 - 0.4% of 31 their plasma levels (Rapoport 1976). The presence of oligoclonal bands in some CNS diseases suggests the presence and clonal expansion of plasma cells behind the BBB. Knopf et al (1998) were able to demonstrate the apparent ability of B cells to seek their antigen in the C N S and to produce oligoclonal IgG bands against that antigen. Like other lymphocytic types, it is unclear what signals are responsible for B cell entry into the CNS. Cell Adhesion Molecules A central question regarding the entry of activated T lymphocytes into the CNS relates to the mechanisms of entry and the interactions between T cells and the endothelium. Since resting, naive, unactivated T cells cannot enter the C N S , the complement of surface molecules on activated T cells must dictate their ability to cross the BBB (Hickey 1999). Lymphocyte emigration is a multi-step process whereby initial interactions are mediated by selectin/carbohydrate binding between lymphocytes and the EC during the 'rolling' stage. Firm adhesion is mediated by the interaction between the cellular adhesion molecules, (intercellular adhesion molecule - 1 (ICAM-1) and vascular cell adhesion molecule - 1 (VCAM-1)), on EC and their ligands expressed by lymphocytes, lymphocyte function associated antigen - 1 (LFA-1) and very late antigen - 4 (VLA-4), respectively. In vitro studies have shown that treatment of brain endothelial cultures with cytokines results in the upregulation of ICAM-1, VCAM-1 and E-selectin (Wong and Dorovini-Zis 1992; Wong and Dorovini-Zis 1995; Stanimirovic et al. 1997; Stins et al. 32 1997). It was later shown that ICAM-1/LFA-1 interactions were critical for T cell adhesion and transmigration (Wong et al. 1999). In MS, Bo et al (1996) demonstrated that endothelial ICAM-1 was significantly increased in lesions. In fact, 81% of vessels in MS lesions showed ICAM-1 immunoreactivity whereas only 37% of vessels were immunoreactive in non-lesion areas. Furthermore, LFA-1 was detected in the vast majority of infiltrating lymphocytes and monocytes in and near MS lesions. In addition, a number of other studies have demonstrated that VCAM-1A/LA-4 interactions may be important in MS. VCAM-1-immunoreactivity has been observed in some microglia/monocytes localized predominantly to the edges of chronic-active and active foci of demyelination (Ransohoff 1999). In E A E , the expression of VLA-4 by lymphocytes has been linked to disease pathogenesis (Yednock et al. 1992; Baron et al. 1993; Kuchroo et al. 1993). 1.3.5 Chemokines and Chemokine Receptors Expression by CNS Resident Cells In the C N S , chemokines and chemokine receptors are expressed by microglia, astrocytes, oligodendrocytes, and neurons under a variety of normal physiological conditions, with increased expression after induction with inflammatory mediators and other stimuli. These proteins have been implicated in a number of C N S disorders. Chemokines and their receptors have also been shown to have roles in neuroprotection, neurodevelopment, and in the modulation of neurotransmission (Biberetal . 2002). 33 Role in CNS Inflammation In the C N S , chemokines and their receptors are critical mediators of immune responses. The action of these proteins has been implicated in almost all CNS diseases by activating glial elements and recruiting local immune cells and/or blood-borne leukocyte subsets. The expression of these molecules has been documented extensively in trauma, Alzheimer's disease, Behcet's disease, HIV-1 encephalitis and dementia, MS, meningitis, myelopathy, and spinal cord contusion injury (Biber et al. 2002). CCL2 and CCL3 and Their Receptors CCL2 and C C L 3 , and their receptors C C R 2 and C C R 1 / C C R 5 , respectively, are expressed by resident C N S cells and by infiltrating leukocytes, under a variety of pathological conditions, including mechanical injury (Glabinski et al. 1996), stab wound injury (Ghirnikar et al. 1996), cerebral trauma (Hausmann et al. 1998), cerebral ischemia (Kim et al. 1995; Takami et al. 1997), and in bacterial and aseptic meningitis (Sprenger et al. 1996; Spanaus et al. 1997). For brevity, only their roles in MS and its animal model, E A E , will be discussed in detail. In MS, the role of chemokines and their receptors has not been clearly established. Numerous studies have shown the presence of C C L 2 and/or CCL3 , and C C R 1 , C C R 2 and C C R 5 , but a causative role has been difficult to demonstrate. In acute MS lesions, C C L 2 , C C L 3 and CCL4 were selectively expressed by astrocytes, macrophages, and microglia in the lesion centre and in the surrounding white matter. EC , perivascular cells and surrounding astrocytes in actively demyelinating plaques, expressed C C L 5 as well (Simpson et al. 1998). McManus et al (1998) used in situ 34 hybridization and immunostaining to demonstrate the presence of C C L 2 , CCL7 and CCL8 in acute and chronic active MS lesions, primarily in hypertrophic astrocytes and, to a variable degree, in inflammatory cells. Van Der Voorn et al (1999) showed similar findings with respect to C C L 2 , with localization to reactive hypertrophic astrocytes in both active demyelinating and chronic active MS lesions. C C L 2 and CXCL10 were studied by Franciotta et al (2001) in acute and stable MS. They showed that C S F CCL2 levels were significantly lower in acute MS than in stable M S and that current MS therapies do not modify circulating levels of C C L 2 or C X C L 1 0 . They also suggest that CCL2 may be constitutively produced in the brain. In addition, C C L 3 was found to be strongly associated with microglia/macrophages in affected white matter of MS patients (Balashov et al. 1999). In E A E , the animal model of MS, the contributions of C C L 2 and C C L 3 towards disease pathogenesis are more clearly defined. Early studies by Ransohoff et al (1993) demonstrated the accumulation of CXCL10 and C C L 2 in close relation to the onset of histologic and clinical EAE in the SJL /J mouse. Hulkower et al (1993), using the Lewis rat, showed that CCL2 mRNA was elevated before onset of clinical signs, peaked with height of clinical disease, and declined with resolution. Glabinski et al (1995) demonstrated that the C N S mRNA expression of C C L 2 and CXCL10 was closely related to the onset of clinical disease in the murine model of EAE. Furthermore, they found that chemokine mRNAs accumulated within astrocytes, near inflammatory infiltrates. Using the rat model of E A E , Berman et al (1996) demonstrated that C C L 2 expression was detected in lymphocytes and EC in subarachnoid locations, at the onset of inflammation, prior to clinical disease. The levels of C C L 2 correlated with disease activity. Using immunohistochemistry and in 35 situ hybridization, lymphocytes, macrophages, astrocytes, and E C were identified as the cellular sources of C C L 2 . In the murine model of E A E , Godiska et al (1995) demonstrated C N S mRNA expression of CCL2 , C C L 3 , CCL4 , C C L 5 , CXCL10, TCA-3, KC and M A R C were induced in the spinal cord 1-2 days prior to clinical symptoms. In chronic, relapsing E A E , Glabinski et al (1997) observed increased expression of CCL2 , CCL3 , C C L 5 , CXCL10 and KC during clinical relapse. C C L 2 , CXCL10, and KC were produced by astrocytes, and infiltrating leukocytes were the sources of CCL3 and CCL5. Using the SJL / J mouse, the critical nature of C C L 2 and CCL3 in the pathogenesis of E A E was clarified. In the C N S , the expression of C C L 3 correlated with the onset of clinical signs. Administration of anti-CCL3 prevented the development of both acute and relapsing disease as well as the infiltration of mononuclear cells into the C N S initiated by the transfer of neuroantigen peptide-activated T cells (Karpus et al. 1995). The same group later showed that acute and relapsing E A E are regulated by the differential expression of C C L 2 and CCL3 . CCL2 and C C L 3 expression patterns, combined with antibody therapy studies, suggested that C C L 3 controls mononuclear cell accumulation during acute E A E , whereas CCL2 controls cellular infiltration during relapsing disease (Kennedy et al. 1998). Furthermore, these chemokines could drive Th1/Th2 lymphocyte differentiation (Karpus and Kennedy 1997). Youssef et al (1998) vaccinated Lewis rats with naked DNA encoding CC-chemokines and found that CCL2 and C C L 3 vaccines prevented E A E . These data suggest that C C L 2 and C C L 3 have critical and non-redundant roles in the pathogenesis of E A E . 36 In addition to chemokines, infiltrating and parenchymal cells express chemokine receptors under both physiological and pathological conditions. In EAE , the C C L 2 / C C R 2 relationship is rather intriguing. Fife et al (2001) showed that C C R 2 (-/-) mice did not develop clinical EAE or C N S histopathology associated with acute disease. Izikson et al (2000) confirmed these results using the same model. Furthermore, C N S levels of CCL2 , CCL5, CXCL10, C C R 1 , C C R 2 and C C R 5 did not increase with disease induction. After disease induction, CCR1 mRNA was differentially expressed by CD4+ T cells from the C N S and not from the spleen (Fife et al. 2001). Moreover, in vivo neutralization of the CCR1 ligand, C C L 3 , resulted in less encephalitogenic CD4+ T cell infiltration. In contrast, Tran et al (2000) found that C C L 3 (-/-) and C C R 5 (-/-) C57BL/6 mice were susceptible to the induction of EAE. The role of chemokine receptors in MS is less clear. The majority of studies have shown correlations between specific receptors - causation has been much more difficult to establish. For example, Zang et al (2000) demonstrated that T cells from MS patients had a significantly increased migratory rate towards C C L 3 and CCL5 in vitro as opposed to normal controls. In the blood of relapsing-remitting MS patients, Balashov et al (1999) found that CXCR3+ T cells were increased, whereas in progressive MS, both CCR5+ and CXCR3+ T cells were increased over normal controls. Among their findings, Sorensen et al (1999) detected C C R 5 on lymphocytes, macrophages, and microglia in actively demyelinating MS lesions. Furthermore, compared with circulating T cells, T cells in the cerebrospinal fluid (CSF) were enriched for C X C R 3 or C C R 5 . Studies of chronic active lesions by Simpson et al (2000) found C C R 2 , C C R 3 , and C C R 5 associated with foamy macrophages and activated microglia, with C C R 3 and C C R 5 on astrocytes in 5 of the 14 cases they 37 studied. The numbers of CCR5+ INFy and TNFa producing T cells were shown by Strunk et al (2000) to be significantly increased in the peripheral blood of MS patients. In addition, transcriptional profiling, using kinetic RT-PCR, revealed increased levels of several Th1 molecules, including CCL3 , as well as the differential expression of several chemokine receptors, including CCR1 and C C R 5 (Baranzini et al. 2000). Moreover, Trebst et al (2001) showed that CCR1+/CCR5+ mononuclear phagocytes were enriched in the C N S of MS patients. The critical role of chemokine receptors in mediating HIV-1 infection of CD4+ T cells is well characterized. In MS, it has been far more difficult to establish a clear relationship, as evidenced by the previously mentioned studies. To determine whether the CCR5A32 allele confers significant protection, as it does in HIV infection, a few studies have been conducted involving MS patients. Individuals heterozygous for the allele were not protected, but instead expressed a lower risk of recurrent clinical disease activity and delayed disease onset in family studies (Barcellos et al. 2000; Sellebjerg et al. 2000). CCR11 The CC-chemokine receptor 11, CCR11 , is one of the newest receptors in the (3-chemokine family. Two separate groups cloned it at around the same time, though the initial ligand designations for the new receptor were different. Gosling et al (2000) showed binding to S L C , ELC, and TECK, and designated the receptor CCR10 . The RNA of this receptor was detected in immature dendritic cells and primary T cells, and in the spleen and lymph node tissue. Non-lymphoid tissue expression included heart, kidney, placenta, trachea, and brain. Schweickart et al (2000) showed the ligand 38 specificities of CCR11 to be CCL2, CCL7, and CCL8 . CCR11 RNA was most abundantly expressed in the human heart, small intestine, and lung. A correction was later published showing agreement with Gosling et al, with the error attributed to contamination (Schweickart et al. 2001). In the C N S , there is little information available regarding CCR11 expression. Dorf et al (2000) demonstrated that murine astrocytes express numerous chemokine receptors, including the RNA of CCR11 . 1.3.6 Cytokines in CNS Inflammation A number of cytokines have been implicated in C N S inflammation, in that they have the ability to activate parenchymal and blood-borne components. Both CNS resident cells and infiltrating leukocytes, in a variety of normal physiological and pathogical circumstances, express cytokines. Furthermore, there is evidence that cytokines in the periphery may be able to activate the C N S , despite the formidable presence of the BBB. Brain E C may prove to be major factors in these interactions (Quan and Herkenham 2002). Tumour Necrosis Factor (TNF) a TNF-a exists both as a 157 amino acid, homotrimeric soluble protein and as a 233 amino acid type II transmembrane protein. Monocytes/macrophages are considered the major producers of TNFa. However, B and T lymphocytes, NK (natural killer) cells, DC, E C , osteoblasts, fibroblasts, basophils, mast cells, Kupffer cells, smooth muscle cells, and epidermal cells also have the ability to produce this cytokine. TNFa expression can be elicited by a number of different exogenous and endogenous mediators, including LPS , viruses, parasites, IL-1, IL-2, and the interferons, among 39 others. T N F a is an inflammatory cytokine that can induce monocytes and neutrophils to phagocytose, adhere to EC, and generate superoxide anion and hydrogen peroxide. TNFa can activate endothelium in a variety of ways: structural reorganization, upregulation of adhesion molecules, induction of chemokine expression and costimulatory molecules (Aggarwal et al. 2001). Within the C N S , T N F a is produced by astrocytes, microglia and neurons (Cannella and Raine 1995; Sei et al. 1995; Brosnan and Raine 1996; Morris and Esiri 1998). In acute M S lesions, Brosnan and Raine (1995) noted the occasional positivity of EC and hypertrophic astrocytes. Interferon (IFN) y IFNy, or type II or immune interferon, is a proinflammatory cytokine produced almost exclusively by NK cells and T lymphocytes, and has receptors on almost all cell types. Both endogenous and exogenous stimuli are capable of inducing IFNy expression. These include LPS , bacterial superantigens, certain lectins, "non-self substances, IL-12, and IL-18, to mention a few. IFNy is considered the main activator of macrophages and is also a strong stimulator of E C . IFNy, in addition to class II MHC and co-stimulatory molecules, also helps upregulate the expression of chemokines (Billiau and Vandenbroeck 2001). In the C N S , IFNy is expressed by microglia, astrocytes (Brosnan et al. 1995), and at low levels by endothelial cells (Morris and Esiri 1998). Interleukin (IL) - 1/3 IL-1B is an endogenous pyrogen and an inducer of the "acute phase response." It is synthesized as a precursor molecule, processed by the IL-1B-converting enzyme 40 (ICE/caspase I) and secreted. IL-ip is a proinflammatory cytokine induced by nearly all microbes and microbial products. The primary cellular sources of this cytokine are monocytes, macrophages and DC; although B lymphocytes and NK cells are capable of producing it as well (Dinarello 2001). In the brain, microglia are the main C N S resident producers of IL-ip in MS (Cannella and Raine 1995), acute trauma in mice (Herx et al. 2000), and in normal brain (Morris and Esiri 1998). E C have been documented to express low levels of IL-1p (Morris and Esiri 1998). Lipopolysaccharide (LPS) LPS , or endotoxin, is the major component of the outer membrane of Gram-negative bacteria. It elicits a broad, non-specific range of cellular events that mediates the secretion of a variety of inflammatory molecules produced primarily by activated macrophages and monocytes. In addition, L P S is capable of activating a multitude of different cell types, including neutrophils, E C , epithelial cells, fibroblasts, smooth muscle cells, DC, microglia and astrocytes. LPS , by itself or coupled to LPS-binding protein (LBP), mediates its actions via association with membrane-bound or soluble CD14. Intracellular signalling pathways are activated by the interaction of this complex with Toll-like receptor 4 (TLR4) (Kielian and Blecha 1995; Tobias et al. 1999; Takeuchi and Akira 2001). 1.3.7 Immunopathogenesis of CNS Autoimmune Disorders Multiple sclerosis (MS) is a human demyelinating autoimmune disease that is believed to result from T cells specific for one or more autoantigens in the CNS. Myelin destruction and axonal damage are the main features of this disease, resulting 41 in variable neurological deficits. Both genetic and environmental factors are believed to play a role in predisposing an individual to MS. Environmental causes have been difficult to establish, though geographical and temporal correlations have been noted (Noseworthy et al. 2000). The evidence for genetic factors playing a role in this disease is unequivocal. Sadovnick et al (1993) showed a concordance rate of 30.8% in monozygotic twins in comparison to 4.7% in dizygotic twins. A number of other pieces of evidence point to the role of genetics in MS: the increased susceptibility in individuals with the HLA-DR2 allele and other specific HLA-DR and DQ polymorphisms; low incidence of conjugal MS; and racial clustering (Noseworthy et al. 2000; Oksenberg and Barcellos 2000). To date, the mechanisms that underlie the pathogenesis of MS have not been elucidated. Human data, along with those derived from the animal model of MS, EAE, have resulted in the proposal of a general model to explain the molecular events that result in the development of demyelination (Noseworthy et al. 2000). Studies in rodents with E A E suggest that T cells of the CD4+ Th1 phenotype are critical in driving the immune response in MS. Numerous myelin and non-myelin proteins have been identified as targets for autoreactive T cells, including myelin basic protein (MBP), myelin-associated protein (MAG), myelin oligodendrocyte glycoprotein (MOG), proteolipid protein (PLP) (Stinissen et al. 1998; Al-Omaishi et al. 1999), aB-crystallin (van Noort et al. 1995; Bajramovic et al. 2000), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) (Walsh and Murray 1998) and S-100 protein (Schmidt et al. 1997). A number of different models have been proposed to explain the generation of autoimmune T cells, including molecular mimicry and the "mistaken self" models. The idea of molecular mimicry is twofold: that myelin-reactive T cells are activated by 42 bacterial or viral agents that "mimic" or share immunological epitopes with self antigens; or that T cells reactive to foreign antigens recognize self antigens (Wucherpfennig and Strominger 1995; Gran et al. 1999; Hafler 1999; Liblau and Gautam 2000). The concept of "mistaken self" is one that involves the de novo expression of aB-crystallin in infected lymphoid tissue. T cells, programmed for the absence of aB-crystallin in healthy lymphoid tissue, will treat the protein as foreign. Memory T cells may then recognize this protein on oligodendrocytes, leading to myelin damage (van Noort et al. 2000). CD4+ T cells are not the only cell type implicated in M S pathology, as several other cell types have been shown to play some role. It is believed that the entry of autoreactive CD4+ T cells, combined with the presentation of specific Ag, results in the activation of these cells. This activation promotes a proinflammatory milieu of increased cytokine and chemokine expression, which serves to recruit additional blood-borne cells as well as activate parenchymal cells. CD8+ T cells, y§ T cells, B cells, macrophages, chemokines, cytokines, and autoantibodies may play roles in pathogenesis (Al-Omaishi et al. 1999; Genain et al. 1999; Raine et al. 1999; Noseworthy et al. 2000; Trebst and Ransohoff 2001). The presence of these cells and the microenvironment they promote, results in significant myelin damage in MS. However, Chang et al (2002) showed that premyelinating oligodendrocytes were present in chronic MS lesions and that axons were not receptive to remyelination. The understanding of why remyelination fails may prove to be extremely valuable in therapeutic intervention. 43 1.4 HUMAN BRAIN MICROVESSEL ENDOTHELIAL C E L L S (HBMEC) 1.4.1 Endothelial Cell Heterogeneity Endothelial cells line the luminal surface of all the blood vessels in the body and thus, are major factors in many physiological and pathological processes, including homeostasis, coagulation, angiogenesis, and recruitment of inflammatory cells, to mention a few. The endothelium can be differentiated into two main groups: large vessel and microvascular endothelium. Large vessel endothelium forms the typical cobblestone appearance in vitro and in vivo and is responsible for maintaining vascular tone, blood pressure and other physiological parameters. In contrast, on the basis of morphology, the microvascular endothelium is divided into discontinuous, fenestrated and continuous phenotypes, corresponding to their vascular permeability. Discontinuous EC usually have clustered holes of 80 to 200 nm in diameter at the tapering edges of the cell, whereas fenestrated endothelium has similar pores with diaphragms. These types of endothelium are found where there is significant molecular, macromolecular, and/or cellular exchange: lymphoid organs (high endothelial venules in the lymph node); bone marrow (haematopoietic cells); spleen (culling of erythrocytes); choroid plexus (cerebrospinal fluid (CSF)); endocrine glands (hormones); liver (exchange of particles); gastrointestinal tract (absorption); and kidney glomerulus (filtration) (Risau 1995; Risau 1998). In continuous microvessels, the cytoplasm of the E C is continuous, with fusion of the lateral surfaces at tight junctions. The BBB consists of a continuous endothelium that is responsible for the maintenance of C N S homeostasis. In addition to the continuous nature of the endothelium, the BBB has a number of unique characteristics that allow for its specialization, such as tight junctions that account for 44 the high electrical resistance and paucity of pinocytotic vesicles. There is evidence that the BBB, similar to the endothelium within peripheral organ systems, may be influenced by the tissue environment. To this end, Janzer and Raff (1987) showed that astrocytes are capable of inducing BBB properties in non-neural E C in vivo. 1.4.2 In vitro Model of the Human Blood-Brain Barrier The E C of the BBB have many special properties that prevent the free movement of cells and molecules between blood and the C N S parenchyma. A number of in vitro models of the BBB have been established from different animals and humans. In our laboratory, a reproducible model of the human BBB has been developed. Thirteen years ago, human brain microvessel E C were first isolated from autopsy brains and temporal lobectomy specimens and subsequently established in culture (Dorovini-Zis et al, 1991). Over the years, cultured H B M E C have been shown to mirror their in vivo cerebral counterparts closely. Confluent monolayers form tight junctions of high electrical resistance, have few pinocytotic vesicles and have no fenestrations. Furthermore, HBMEC have characteristics that confirm their endothelial nature, such as Factor Vll l /Von Willebrand antigen, binding of Ulex europaeus agglutinin, and uptake of acetylated low density lipoprotein (Dil-Ac-LDL). Our laboratory has continued to study this in vitro model to further understand the role of the cerebral endothelium in the context of normal physiology, inflammation and disease. To address these issues, studies have looked at the upregulation of ICAM-1 (Wong and Dorovini-Zis 1992); effects of IFNy (Huynh and Dorovini-Zis 1993); expression and function of VCAM-1 (Wong and Dorovini-Zis 1995); E-selectin (Wong and Dorovini-Zis 1996); PECAM-1 (Wong and Dorovini-Zis 1996); LFA-3 (Omari and 45 Dorovini-Zis 1999); C C L 4 and C C L 5 (Shukaliak and Dorovini-Zis 2000); CD80 and CD86 (Omari and Dorovini-Zis 2001); and CD40 (Omari et al. 2004). 1.5 OBJECTIVE AND SPECIFIC AIMS 1.5.1 Hypothesis Historically, the dogma with respect to the C N S has been that it is an immunologically privileged site. The presence of the BBB has been shown to restrict the free movement of substances and cells under normal circumstances. Studies in both humans and animals have shown that this idea is not entirely tenable, yet the mechanisms by which cells can traverse the cerebral endothelium has not been clearly elucidated under either normal, physiological conditions or pathological ones. As EC are the first native cells of the C N S to encounter any blood-borne leukocytes, it is likely EC-leukocyte interactions are important in the recruitment of inflammatory cells across the BBB in C N S inflammation. The overall objective of this study is to test the hypothesis that human brain endothelial cells are capable of producing and releasing certain members of the C C -chemokine family, thereby enabling the recruitment, activation, adhesion and migration of specific subsets of lymphocytes during C N S inflammation. 1.5.2 Specific Aims This study will utilize a previously well-characterized in vitro model of the human blood-brain barrier to achieve the following specific aims: 46 1. To characterize the kinetics of expression, upregulation and release of the CC-chemokines, CCL2 and CCL3 by H B M E C following cytokine and LPS treatment. 2. To characterize the expression of the newest CC-chemokine receptor, C C R 1 1 , initially a putative M C P family receptor, by H B M E C . 3. To characterize the in vivo expression patterns of C C L 2 , C C L 3 and CCR11 in both normal brain and in a variety of C N S disorders. 47 CHAPTER 2: MATERIALS AND METHODS 2.1 ENDOTHELIAL CELL CULTURES 2.1.1 Isolation of Human Brain Microvessel Endothelial Cells (HBMEC) Human brain microvessel endothelial cells (HBMEC) were isolated from normal autopsy brains less than 12 hours post mortem. A review of the clinical history ensured that the brains were free of neurological disease. Briefly, the cerebral cortex was manually separated from underlying brain tissue and placed in M199 medium (Life Technologies Inc., Burlington, ON) containing antibiotics. Following the removal of the leptomeninges and superficial blood vessels, the cortex was cut into 1-2 mm cubes and incubated in 0.5% dispase (Life Technologies Inc.) for 3 hours in a 37°C shaking water bath. After incubation, the tissue was centrifuged for 10 minutes at 1000x g. The subsequent pellet was resuspended in 15% dextran (Sigma Chemical Co., St. Louis, MO) and centrifuged at 5800x g for 10 minutes. The resultant pellet, containing microvessels, basement membrane and pericytes, was suspended in M199 containing antibiotics and centrifuged at 100x g for 10 minutes. The pellets were then pooled and incubated overnight in 0.1% collagenase/dispase (Roche Diagnostics, Laval, QB) in a 37°C shaking water bath to remove the basement membrane and pericytes. The following day, 45% Percoll (Sigma Chemical Co.) was centrifuged at 26000x g for 1 hour to set up gradients. These allowed for the differential centrifugation of the microvessel pellet (1000x g for 10 minutes) resulting in the separation of red blood cells, basement membrane and pericytes from the EC. The E C band was aspirated, washed and plated on fibronectin-coated tissue culture plates (Corning Costar Corp., Cambridge, MA). 48 2.1.2 Isolation of Human Umbilical Vein Endothelial Cells (HUVEC) To allow for direct comparisons between brain microvessel and large vessel extracerebral endothelium, human umbilical vein endothelial cells (HUVEC) were isolated from umbilical cords obtained from term delivery placentas at the BC Women's and Children's Hospital, according to the method described by Jaffe and colleagues (1973). Briefly, cords were washed with Hank's balanced salt solution (Life Technologies Inc.), pH 7.4 and then infused with 0.1% (1 mg/ml) collagenase (Worthington Biochemical Corporation, Lakewood, NJ), incubated at 37°C for 15 minutes and flushed with warm M199. M199, containing 20% horse serum, was used to remove the EC. The cells were washed prior to plating. 2.1.3 Culture Conditions Both H B M E C and H U V E C cultures were grown under identical conditions on either fibronectin- or collagen-coated plates and maintained in M199 supplemented with 10% horse serum (Hyclone Laboratories, Logan, UT), 100 ag/ml heparin (Sigma Chemical Co.), 20 ag/ml endothelial growth supplement (Sigma Chemical Co.), 1% antibiotic/ antimycotic solution (Life Technologies Inc.) and 300 ucj/ml glutamine (Sigma Chemical Co.) (henceforth referred to as complete media). The cells were maintained at 37°C in a humidified 5% C02/95% air incubator and the culture media was changed every second day. EC reached confluence 7 to 10 days after plating. Confirmation of the purity and endothelial nature of E C cultures was determined by the positive staining for Factor VIIl-related antigen and Ulex europeaus I lectin binding. 49 2.2 CYTOKINES AND LPS TREATMENT Monolayers of H B M E C and HUVEC were grown to confluence in replicate wells and incubated with cytokines for 24, 48 and 72 hours. The cytokines used were tumour necrosis factor a (TNFa, 10-100 U/ml, Sigma Chemical Co.), interferon y (IFNy, 200-500 U/ml, NIAID, Bethesda, MD), interleukin-1 (3 (IL-1p, 10 U/ml, Genzyme, Cambridge, MA), bacterial liposaccharide (LPS, 5 ug/ml, Sigma Chemical Co.) and various combinations of the aforementioned cytokines. Supernatants of cytokine-treated monolayers were collected following the respective time points for detection of chemokine release by sandwich ELISA. 2.3 ANTIBODIES For immunocytochemistry and immunohistochemistry, the following primary antibodies (Abs) were used: monoclonal mouse anti-human C C L 2 ( 1 0 - 4 0 ug/ml) and CCL3 (10 - 50 pg/ml) were purchased from Pepro Tech (Rocky Hill, NJ) and Chemicon International Inc. (Temecula, CA); anti-Factor Vl l l related Ag and Ulex europeaus Ab were both purchased from Dako Diagnostics Canada Inc (Mississauga ON). Secondary Abs used for these studies included the following: 10 nm gold-conjugated goat anti-mouse IgG (1:40) and goat anti-rabbit IgG (1:40) (Auroprobe LM GAM IgG and Auroprobe LM GAR IgG, respectively, Cedarlane Laboratories Ltd, Hornby, ON); biotinylated goat anti-mouse IgG (Caltag Laboratories, Burlingame, CA); and biotinylated goat anti-rabbit IgG (Caltag Laboratories). The control Ab used were: mouse anti-human follicle stimulating hormone (FSH) (BioGenex Laboratories, San 50 Ramon, CA); normal mouse IgG (Cedarlane Laboratories Ltd, Hornby ON)); normal rabbit serum (Sigma Chemical Co.); and normal goat serum (Sigma Chemical Co.). 2.4 SEMI-QUANTITATIVE REVERSE TRANSCRIPTION PCR H B M E C and H U V E C cultures were grown to confluence on 100 mm diameter fibronectin-coated plates and collagen-coated plates, respectively. 24 hours after the addition of control media or cytokines (100 U/ml T N F a and 500 U/ml IFNy), the cells were removed using a rubber policeman, centrifuged and the cell pellets were frozen at -70°C until needed. Trizol (Life Technologies Inc.) was added to the frozen pellet, sonicated and the RNA extracted using chloroform. The RNA preparation was cleaned up using an RNeasy kit (Qiagen Inc., Mississauga, ON), according to the manufacturer's instructions. 2-5 \xg of total RNA was reverse transcribed using Moloney Murine Leukaemia Virus reverse transcriptase (MMLV-RT, USB Corp., Cleveland, OH) and random hexamer primers (Amersham Pharmacia Biotech Inc., Piscataway, NJ) occurred for 90 minutes at 37°C. P C R was performed using 2.5 U/25 uJ reaction AmpliTaq Gold (Applied Biosystems, Foster City, CA) with primers purchased from Life Technologies Inc. (Table 1) on 1-5 u1 of cDNA under the following conditions: one initial cycle with dissociation at 94°C for 8 minutes, annealing at 55-60°C for 30 seconds, extension at 72°C for 3 minutes; then cycling at 94°C for 1 minute, 55-60°C for 30 seconds and 72°C for 45 seconds on an Applied Biosystems GeneAmp P C R System 9700 thermalcycler. G A P D H was used as an internal standard. The following were used as positive controls: pBluescript-hCCL2 (kind gift from Dr. Teizo Yoshimura, NCI/NIH, MD); pBR322-hCCL3 (kind gift from Dr. Donald Forsdyke, Queen's 51 University, Kingston, ON); pBluescript-hCCR1, -hCCR2B and -hCCR5 (kind gifts from Dr. Philip Murphy, NIAID-NIH, Bethesda MD); and pcDNA3-hCCR11 (kind gift from Dr. Vicki Schweickart, ICOS Corporation, WA). P C R products were run on either a 6% polyacrylamide/TBE gel or a 2% agarose/TBE gels containing ethidium bromide (EtBr) and visualized under UV light. TABLE 2: PCR PRIMER SEQUENCES GENE PRIMER SEQUENCE CCL2 F: atg aaa gtc tct gcc gcc ctt ctg t R: agt ctt egg agt ttg ggt ttg ctt g CCL3 F: atg cag gtc tec act get gcc ctt R: gca etc age tec agg teg ctg aca t CCR1 F: cac cac aga gga eta tga cac gac cac aga R: cac caa aaa ccc agt cat cct tea act tgt CCR2A/B F: ggc ctg agt aac tgt gaa age ace agt caa c (A) R: gtc ctg aag act ggc ttc agg ggc tct (B) R: ate cac tgt etc cct gta gaa aac tgg aca ttg C C R 5 F: teg ata ggt acc tgg ctg teg tec tec atg c R: aag cct cac age cct gtg cct ctt ctt c CCR11 F: cgt cat tgg act tgc agg caa ttc cat g R: age aag atg gca gcc ate cag aca cag a GAPDH F: cca tgt teg tea tgg gtg tga acc a R: gcc agt aga ggc agg gat gat gtt c NOTES F: Forward/sense primer R: Reverse/anti-sense primer 52 2.5 IMMUNOCYTOCHEMISTRY 2.5.1 Immunogold Silver Staining Surface Localization Confluent monolayers were first washed with warm phosphate-buffered saline (PBS, pH 7.4) supplemented with 1% bovine serum albumin (BSA, Sigma Chemical Co.), 1% normal goat serum (NGS) and 0.05% sodium azide (NaN 3), and then incubated with primary Ab (10 ug/ml in carrier buffer consisting of P B S containing 5% BSA, 4% N G S and 0.05% NaN 3) for 40 minutes, washed again and then incubated with 5 nm gold-conjugated secondary Ab (1:40 in carrier buffer) for 1 hour. After additional washes, the cells were fixed for 30 seconds with buffered formaldehyde/acetone and washed with double distilled water. Silver enhancement solution (Amersham Life Sciences, Buckinghamshire, England) was applied and silver deposition was monitored for 22 - 26 minutes. To enhance the staining, a second silver enhancement step was applied in the same manner described. Nuclei were stained with Giemsa. Controls included cells incubated with carrier buffer, irrelevant primary Ab (anti-human FSH) or normal mouse IgG. The cells were examined under a Nikon Labophot light microscope. Intracellular Localization Confluent monolayers were washed with P B S and permeabilized using buffered formaldehyde/acetone containing 0.03% Triton X-100 for 10 minutes. The cultures were incubated with primary Ab (10 ug/ml made up in carrier buffer consisting of PBS containing 5% B S A and 4% NGS) for 1 hour at room temperature. A 10 nm gold 53 conjugated goat anti-mouse Ab (1:40 in carrier buffer) was used as a secondary Ab (Amersham International). Silver enhancement solution was applied and silver deposition was monitored. Nuclei were stained with Giemsa. Controls included cells incubated with carrier buffer, irrelevant primary antibody (anti-human FSH) or normal mouse IgG. The cells were examined under Nikon Labophot light microscope. 2.5.2 Immunoperoxidase Staining Cultured cells were washed with warm P B S and then fixed with acetone/ethanol (1:1) for 7 minutes at 4°C. The cells were washed with Tris-buffered saline containing 0.05% Tween 20 (Tris-Tween) and endogenous peroxidase activity was blocked with 0.75% H202/97.5% methanol for 30 minutes, followed by additional washes. Blocking with 1% bovine serum albumin (BSA) and 4% normal goat serum (NGS) for 30 minutes prevented non-specific binding. The samples were incubated with primary Ab (10 ug/ml in Tris-Tween buffer) for 60 minutes, washed and incubated with a biotinylated secondary Ab (5 ug/ml in Tris-Tween buffer) for 90 minutes. After several washes, avidin-HRP (Vector Laboratories, Burlingame CA) was added for 30 minutes. 3-amino-9-ethylcarbazole (AEC) was used as the chromogen for HRP and hematoxylin was used as a nuclear counterstain. Cells were subsequently coated with Crystal/Mount (Biomeda Corporation, Foster City, CA) 2.6 IMMUNOGOLD ELECTRON MICROSCOPY Cultured cells were washed with PBS containing 1% B S A and 20 mM NaN 3 , incubated with primary Ab (10 ug/ml in PBS containing 5% BSA, 4% NGS and 20 mM 54 NaNs) for 30 minutes, washed again and incubated with secondary Ab for 1 hour. Cells were then washed, fixed with cold 1/2-strength Karnovsky's fixative (2.5% glutaraldehyde and 2% paraformaldehyde in 0.2M sodium cacodylate buffer) for one hour at 4 °C. Following fixation, the cells were washed with 0.2M sodium cacodylate buffer and post-fixed with 1% osmium tetroxide for 1 hour at 4 °C. After block staining with uranyl magnesium acetate overnight at 4 °C, the cells were washed with sodium acetate buffer and dehydrated in ascending grades of methanol, embedded in Epon-Araldite and then re-embedded in English Araldite for cross staining. Thin sections were examined using a Zeiss EM 910 electron microscope. One hundred cells from each resting or treated culture were photographed under 25,000X magnification and the number of gold particles bound per micron of cell membrane on the apical or basal surface was quantitated, taking the magnification into account. 2.7 ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) H B M E C were grown to confluence on fibronectin-coated 96-well plates and subjected to various cytokine treatments for 24 - 72 hours in triplicate wells. The supernatants were collected and analyzed using ELISA kits purchased from R & D Systems (Minneapolis, MN), as per manufacturer's instructions. Briefly, culture supernatants and provided standards were added to plates containing the appropriate capture Ab. The application of detection Ab, along with substrate (TMB), allowed colour development, which was stopped with 2N sulfuric acid. Absorbance was read with an ELISA microtitire plate reader at a wavelength of 490 nm. The generation of a standard curve using chemokine standards allowed the calculation of the quantity of chemokines released by H B M E C in the culture supernatants. 55 2.8 IMMUNOHISTOCHEMISTRY 2.8.1 Frozen Sections Frozen brain sections were obtained from the Division of Neuropathology, V H H S C . Sections were allowed to dry at room temperature for 45 minutes and then fixed in acetone for 10 mins and air-dried. Slides were incubated with 0.5% H 2 0 2 in 100% methanol for 30 mins to block endogenous peroxidases, washed and then incubated with primary antibodies for 90 mins at room temperature. Controls included normal mouse immunoglobulin (IgG) and the absence of primary Ab. After the primary Ab was washed off, secondary antibodies were applied. A E C was used as the chromogen and the nuclei were counterstained with hematoxylin and base. 2.9 DATA COLLECTION AND STATISTICS Values derived from ELISA experiments were analyzed using analysis of variance (ANOVA) to determine if there were differences between different treatments. Student's t tests were used where differences were found. The Tukey test was also used as a multiple comparison method. Data that were not normally distributed were subjected to Kruskal-Wallis A N O V A on Ranks and Mann-Whitney U-tests. The chi-square test was used to analyze the immunoelectron data. Significant differences between cytokine treated cells and control groups are shown as (*) where p<0.05. 56 CHAPTER 3: RESULTS 3.1 HUMAN BRAIN MICROVESSEL ENDOTHELIAL CELLS (HBMEC) Confluent monolayers (Fig. 1a) of primary cultures of H B M E C were derived 8-11 days after plating on fibronectin-coated plastic wells. These cells showed strong positivity for markers indicative of their endothelial nature: Factor VIIIA/on Willebrand antigen (Fig. 1b) and binding of Ulex Europaeus agglutinin (Fig. 1c). Previous studies have shown that H B M E C are a suitable representation of the blood-brain barrier (BBB) in that they retain important in vivo characteristics of the BBB, such as paucity of pinocytotic vesicles and the presence of tight junctions that restrict the transendothelial passage of horseradish peroxidase (Dorovini-Zis et al. 1991). HUVEC were used to determine if there were differences between brain microvessel endothelium and that found in the periphery. These cells were isolated according to the method described by Jaffe et al (1973). The endothelial origin and purity of the preparations was determined as for H B M E C and H U V E C cultures were maintained under identical conditions as HBMEC cultures. As such, this allows for direct comparisons between brain microvessel and large extracerebral endothelium. 3.2 CCL2 EXPRESSION BY HBMEC 3.2.1 RNA Expression Detected by Semi-quantitative RT-PCR RNA extracted from confluent monolayers grown on 100 mm collagen (HUVEC) or fibronectin (HBMEC) coated plates was used to elucidate the expression pattern of CCL2 in both cultures. The RNA, from both unstimulated and cytokine-treated cells (100 U/ml T N F a and 500 U/ml IFNy), was extracted after a treatment period of 24 57 hours. The C C L 2 gene-specific primers (Table 2) used for these studies amplified a 298 base pair fragment. The expression of CCL2 RNA from both unstimulated and stimulated HBMEC and HUVEC were similar (Fig. 2). Though there does appear to be some upregulation of RNA expression following cytokine treatment, the nature of this type of R T - P C R did not allow for the quantitation of the relative RNA expression levels. C C L 2 cDNA cloned into pBluescript was used as the positive control in these experiments. 3.2.2 Intracellular CCL2 Protein Expression by HBMEC Immunogold silver staining was used to demonstrate intracellular C C L 2 protein expression by confluent monolayers of H B M E C under a variety of simulated inflammatory conditions. Unstimulated cells and cells treated for 24 to 72 hours with TNFa, IFNy, IL-1R and L P S , or combinations of these cytokines, were used for these studies. Virtually 100% of both untreated and treated cells stained positive for CCL2, with some variation in the intensity of staining among cells. Positive staining was shown in the form of fine, granular black deposits in the cell cytoplasm (Fig. 3 a-d). Treatment with cytokines and L P S resulted in significant increase in staining intensity in the majority of cells, as compared to untreated ones. Negative controls for these experiments showed no staining and included the use of normal mouse IgG, carrier buffer or irrelevant Ab (mouse anti-human FSH). 3.2.3 CCL2 Surface Localization in HBMEC by Immunoelectron Microscopy As chemokines, in general, are secreted molecules, they exert their actions via binding to cell surface glycosaminoglycans. Surface immunogold silver staining 58 resulted in a rather inconspicuous and weak surface staining (data not shown). In order to elucidate the binding patterns of C C L 2 to the cell membrane, an immunoelectron microscopic approach was employed. An additional advantage of this method is the ability to visualize both the apical and basal binding of the chemokine, a limitation of the immunogold silver staining technique. While subconfluent, EC were either left untreated (control) or stimulated for 24 hours with cytokines (100 U/ml TNFa + 500 U/ml IFNy). After processing, serial sections were visualized using TEM. CCL2 was localized in both apical and basal surfaces of cytokine-treated HBMEC. Only a small number of particles were associated with the cell surface of untreated HBMEC. Frequently, gold particles localized to the discontinuous, amorphous, basal lamina-like material underlying the basal cell surface. Gold particles were not observed along intercellular contacts. For each section, the number of gold particles, as well as the length of plasma membrane along which they were found, was quantitated. In control, unstimulated cells, CCL2 deposition on the apical side was found to be higher than that on the basal surface (Fig. 4). Upon cytokine treatment, there was a more favourable deposition on the basolateral surface as compared to the apical one, as demonstrated by the increased mean value. 3.2.4 Secretion of CCL2 by HBMEC Detected by Sandwich ELISA ELISA was used to determine the level of C C L 2 protein that was released by HBMEC confluent monolayers treated with TNFa, IFNy, IL-1p and LPS , or combination of these cytokines for 24 - 72 hours. The culture media, with or without cytokines, was removed at 24, 48 and 72 hours post stimulation and collected for ELISA analysis. 59 Unstimulated H B M E C released 1 0 - 2 1 ng/ml of C C L 2 over 24 - 72 hours with significant increase in release following cytokine treatment in a time-dependent fashion (Fig. 5). Treatment with T N F a induced the increase in C C L 2 release up to ~35 ng/ml at 24 hours and up to a maximum of ~63 ng/ml at 72 hours. This increase in CCL2 release was not concentration-dependent. Incubation with L P S or IL-1B resulted in, up to a maximum of, 124% and 156% increase (over control), respectively, in CCL2 release in a time-, but not concentration-, dependent manner. Incubation of HBMEC with IFNy alone resulted in much less dramatic upregulation of release that was similarly time-, but not concentration-, dependent. However, co-incubation with TNFa (100 U/ml) and IFNy (200 U/ml) augmented CCL2 release to levels comparable with TNFa treatment alone. 3.3 CCL3 EXPRESSION BY HBMEC 3.3.1 RNA Expression Detected by Semi-quantitative RT-PCR RNA extracted from confluent monolayers grown on 100 mm collagen (HUVEC) or fibronectin (HBMEC) coated plates was used to elucidate the expression pattern of CCL3 in both EC culture systems. The RNA, from both unstimulated and cytokine-treated cells (100 U/ml T N F a and 500 U/ml IFNy), was extracted after a period of 24 hours. The C C L 3 gene-specific primers (Table 2) used for these studies amplified a 274 base pair fragment. C C L 3 RNA was found in barely discernible levels in both unstimulated HBMEC and HUVEC (Fig. 6). Incubation with 100 U/ml T N F a and 500 U/ml IFNy for 24 hours resulted in increase in RNA expression. The control used for these experiments was CCL3 cDNA cloned into a pBluescript vector. 60 3.3.2 CCL3 Intracellular Protein Expression by HBMEC All unstimulated HBMEC cells showed relatively faint staining in the form of fine, black granular deposits of silver, over all time points investigated (Fig. 7). Incubation with TNFa , IFNy, IL-1p and LPS , or combination of T N F a and IFNy, resulted in the increased density of positive staining. Due to the presence of granular staining of virtually all cells, a quantitative difference in the absolute number of cells with positive staining was not found between unstimulated and cytokine-treated cells. 3.3.3 Surface Localization of CCL3 in HBMEC by Immunoelectron Microscopy As with C C L 2 , light microscopic surface immunogold silver staining for CCL3 resulted in very weak staining of resting as well as cytokine-treated H B M E C (data not shown). We, therefore, used immunoelectron microscopy (IEM) to elucidate the binding patterns of C C L 3 to the cell membrane. An additional advantage of this method is the ability to visualize both the apical and basal binding of the chemokine, a limitation of the immunogold silver staining technique. H B M E C monolayers were used untreated (control) or following stimulation with cytokines (100 U/ml T N F a + 500 U/ml IFNy) for 24 hours. By immunoelectron microscopy, C C L 3 localized to both apical and basal cell surfaces and to the discontinuous, amorphous basal lamina-like material underlying the basal cell surface. Gold particles were not found along intercellular contacts. For each section, the number of gold particles, as well as the length of plasma membrane along which they were found, was quantitated. 61 In control, unstimulated cells, CCL3 deposition on the basal surface was found to be slightly higher than that on the apical surface (Fig. 8). Upon cytokine treatment, the mean number of gold particles binding to the apical surface was higher than that of the basal surface. 3.3.4 Protein Release of CCL3 by HBMEC Determined by Sandwich ELISA C C L 3 release by confluent monolayers of H B M E C was monitored at 24, 48 and 72 hour time points. Unstimulated cells did not release any detectable protein (Fig. 9). Stimulation with 100 U/ml T N F a resulted in small amounts of C C L 3 released within the first 24 hours and up to 100 pg/ml of CCL3 released over 48 and 72 hours. Lower concentrations of T N F a (10 U/ml), as well as IFNy at 200 and 500 U/ml, failed to induce C C L 3 release into the media. However, co-incubation of H B M E C with TNFa (100 U/ml) and IFNy (200 U/ml) augmented C C L 3 release to a maximum of 200 pg/ml in a time-dependent manner. Treatment of the monolayers with IL-1B or L P S similarly induced H B M E C to release approximately several hundred pg/ml of the chemokine. 3.4 EXPRESSION OF CCL2 AND CCL3 IN ACUTE MS The expression of CCL2 and CCL3 was determined in frozen brain sections from a patient diagnosed with acute MS. Strong staining was seen for both chemokines within active lesions (figure 11, panels A & B). The staining was found to colocalize to astrocytic processes, macrophages and in and around EC. In the immediate vicinity of the plaque centre, minimal staining was observed (data not shown). 62 3.5 EXPRESSION OF B-CHEMOKINE RECEPTORS 3.5.1 RNA Expression Detected by RT-PCR RNA extracted from confluent monolayers grown on 100 mm collagen (HUVEC) or fibronectin (HBMEC) coated plates was used to elucidate the expression pattern of (the former) CCR11 in both endothelial cell types. The RNA, from both unstimulated and cytokine-treated cells (100 U/ml TNFa and 500 U/ml IFNy), was extracted after a treatment period of 24 hours. The (former) CCR11 gene-specific primers (Table 1) used for these studies amplified a 348 base pair fragment. The R N A expression of (former) CCR11 (shown in Fig. 10A) in both HBMEC and HUVEC was approximately the same in both unstimulated and cytokine-treated cultures (100 U/ml T N F a and 500 U/ml IFNy for 24 hours) as determined by GAPDH expression. The positive control used for these experiments was human CCR11 cDNA inserted into a pcDNA3 vector. The RNA expression of the receptors for C C L 2 and C C L 3 , CCR2a/b and CCR1/5 , respectively, were negligible in both resting and cytokine-treated HBMEC and HUVEC (data not shown). 3.5.2 Immunocytochemistry The immunoperoxidase technique was used to determine the expression profile of CCR11 in confluent monolayers of HBMEC grown on fibronectin-coated 4-well plates. Unstimulated cells showed significant protein expression as determined by the strongly positive, granular red-orange perinuclear staining (Fig. 10B). Virtually all cells expressed high intracellular levels of this protein. Incubation of HBMEC with a combination of T N F a and IFNy (100 U/ml and 500 U/ml, respectively) for 24 hours 63 resulted in significant downregulation as determined by the marked decrease or loss of cytoplasmic staining in the majority of cells in culture (Fig. 10C). 64 CHAPTER 4: DISCUSSION 4.1 HBMEC AS AN IN VITRO MODEL OF THE BBB The BBB has for many years been somewhat of an enigma. The interest in the molecular interactions that occur at this interface between the C N S and the rest of the body has been wide-ranging, due to the influence this anatomic entity has on a large number of neurological disorders. Whether from a scientific and clinical perspective, in trying to understand the variables governing an inflammatory cascade, or from a drug discovery one, whereby one is interested in the permeability properties of small chemical compounds, the approaches to studying this system have been multi-factorial. From a drug discovery perspective, the concept of high throughput screening has resulted in the development of substitute systems to model the BBB. The use of the Madin-Darby canine kidney (MDCK) cell line, either in the presence or absence of astrocytic cell lines, is prevalent in the industry as a first pass diagnostic tool. Emerging lines of evidence suggesting that the p-glycoprotein (P-gp) molecule is responsible for the majority of small molecule efflux has resulted in the creation of a MDCK cell line expressing this protein. This model has, arguably, become the de rigeur model for the active efflux mechanisms present at the in vivo BBB. In addition, artificial membrane systems have been developed to model the passive diffusion characteristics of the barrier. From a purist perspective however, the development of such artificial models has obvious limitations, namely the absence of tissue-specific characteristics. To date, many investigators have utilized various in vitro BBB models, ranging from astrocytic co-culture systems (at times with peripheral endothelium or even cell lines) 65 to the use of glioma cell conditioned media, or even the addition of secondary messengers such as cyclic A M P (cAMP), to mediate and modulate the BBB phenotype. These manipulations, from both a purist view and a molecular standpoint, may prove to be obstacles in transitioning the in vitro results derived to the in vivo system. The use of primary cultures of HBMEC eliminates many of the limitations associated with the aforementioned models, thereby allowing direct study of the BBB, in a manner closely mimicking the in vivo state. This model retains several of the key phenotypic characteristics of the in vivo BBB endothelium, including tight junction formation, high electrical resistance, HRP impermeability and paucity of pinocytotic vesicles. Furthermore, as testimony to their endothelial origin, these cells demonstrate Factor V l l l : related antigen reactivity, Ulex europeaus I lectin binding, as well as retention of alkaline phosphatase activity. And finally, one other advantage of using primary cultures over cell lines, or even passaged primary cells, is the lack of phenotypic and genotypic drift, something that is often associated with the latter models. Over the years, the HBMEC model has proven to be very robust. It has allowed detailed study of adhesion molecules (Wong and Dorovini-Zis 1995; Wong and Dorovini-Zis 1996; Stins et al. 1997; Stins et al. 2003), T lymphocyte migration (Wong et al. 1999), expression of costimulatory molecules (Omari and Dorovini-Zis 2001) and expression of class II MHC molecules (Huynh et al. 1995), to mention a few. These studies have allowed a better understanding of the brain endothelium as a major contributor towards the initiation and perpetuation of C N S inflammation. 66 4.2 CCL2 AND CCL3 EXPRESSION BY HBMEC Chemokines have proven to be important instigators and propagators in many physiological and pathological processes. Their ability to recruit, activate and direct the migration of blood-bourne cells is paramount for the successful induction of the inflammatory response. Within the confines of the C N S , this important role is evident in a number of experimental models and in clinical disease. Since chemokines are produced in response to some initial inflammatory stimulus, the current studies were undertaken to investigate the expression and upregulation of C C L 2 and C C L 3 by human brain E C in an in vitro model of the human BBB under resting conditions and in an inflammatory milieu. On a RNA expression level, CCL2 is constitutively expressed by resting H B M E C with some upregulation upon treatment with pro-inflammatory cytokines. Intracellular protein expression was ascertained with IGSS. Unstimulated H B M E C showed relatively strong granular staining, with significantly increased staining upon stimulation with cytokines and LPS. ELISA on culture supernatants showed that the basal release of C C L 2 ranged from approximately 10 ng/ml (at 24 hours) to over 20 ng/ml (at 72 hours). Stimulation with TNFa alone or in combination with IFNy, as well as treatment with IL-1B or L P S resulted in significant, time-dependent increase in C C L 2 release, ranging from 30 ng/ml to greater than 50 ng/ml. Immunoelectron microscopic localization of constitutive C C L 2 showed preferential binding to the apical surface in resting HBMEC in comparison to the basal surface; this trend was reversed when the cells were treated with pro-inflammatory cytokines, with an increased propensity for basal surface binding. This differential partitioning of CCL2 induced by cytokine treatment may be a 67 mechanism for creating a chemotactic gradient for the recruitment of circulating lymphocytes. Numerous groups have published data suggesting that glial elements within the CNS are mainly responsible for the chemokine expression within the brain (Weiss et al. 1998; Van Der Voorn et al. 1999). Human cerebrovascular C C L 2 expression has not been fully characterized. RNA and protein expression have been shown in a number of experimental models: porcine cerebral endothelial cells cultured in endothelial cell growth factor and heparin-containing media, with upregulation upon stimulation with T N F a (Zach et al. 1997); basal protein levels present in and released by a rat brain (GP8/3.9) vascular endothelial cell line, with upregulation after stimulation with TNFa , IL-ip and IFNy (Harkness et al. 2003); and markedly expressed in mouse brain vascular endothelium upon stimulation with HIV Tat1-72 (Toborek et al. 2003). In humans, the picture is less clear. Some investigators believe that the astrocytes and microglial cells are the main producers of C C L 2 (Weiss et al. 1998) and potentially, this occurrence may be responsible for the opening of the BBB, by altering the expression of tight junction-associated proteins (Song and Pachter 2004). It is somewhat unclear as to the context in which human cerebral endothelium expresses CCL2. Weiss and colleagues (1998), in their previously mentioned work, found that CCL2 release by H U V E C grown alone on inserts, or in co-culture with human fetal astrocytes, was directional - more chemokine was released into the upper chamber (apical) than into the lower (basal) upon stimulation with T N F a or IL-1p. In fact, it was approximately 3 - 5 fold higher in the upper chamber than in the lower (150 ng/ml and 110 ng/ml vs. 30 ng/ml, for each of the respective cytokines). Frigerio et al (1998) demonstrated that C C L 2 mRNA could be detected in both resting and stimulated 68 (TNFa and IFNy) H U V E C and MS-HBEC, but in non-MS-HBEC, mRNA expression could only be detected after IFNy stimulation. Furthermore, the expression of CCL2 can be induced by endothelin-1 and ischemia in human brain-derived endothelial cells (Stanimirovic and Satoh 2000; Chen et al. 2001) and by certain Streptococcal strains (Vadeboncoeur et al. 2003). Our results suggest that there are small differences in C C L 2 RNA expression between cerebral microvascular and non-cerebral large vessel endothelial cells, since both cell types show constitutive, in vitro expression and similar upregulation upon treatment with cytokines. Though there is some discrepancy between the literature and our findings, our data, with respect to HBMEC, constitutes a more accurate, though somewhat novel, reflection of the cellular milieu found within the CNS. R T - P C R analysis of HBMEC shows minimal R N A expression in the resting state. Following T N F a and IFNy treatment, RNA expression was upregulated. Intracellular staining suggests some basal level of protein expression, with increased positive staining associated with combinations of T N F a and IFNy, IL-ip and LPS treatment. As chemokines exert their action as soluble molecules, ELISA studies of culture supernatants showed no basal release of C C L 3 . However, stimulation with 100 U/ml T N F a resulted in up to 100 pg/ml of protein released. Treatment with IL-1p, LPS or combination of T N F a and IFNy, resulted in a significant increase in the amount of CCL3 released. Binding of C C L 3 to the EC surface was minimal under resting conditions as determined by IEM, consistent with the ELISA data, showing no detectable protein release. Following cytokine stimulation, however, a greater number of CCL3 molecules preferentially bound to the apical cell surface. 69 The expression of C C L 3 by endothelium has been documented in a number of different clinical and experimental conditions. In animals, transection of the rat sciatic nerve resulted in increased expression of C C L 3 (as well as C C L 2 and CCL5) in the endothelium of both the epi- and endoneurium (Taskinen and Roytta 2000). Cheng et al (2002) were able to demonstrate CCL3 expression by murine bone marrow endothelial cells. Furthermore, Kobayashi et al (2003) demonstrated that alloantigen-primed T cells could induce the expression of this chemokine in a murine endothelial cell line. In humans, in disorders such as hemophagocytic syndrome, C C L 3 has been localized to, among others, EC of blood vessels and splenic sinusoids with evidence of phagocytic activity (Teruya-Feldstein et al. 1999). Using the H U V E C model, Cha et al (2000) demonstrated that activated platelets could induce the expression of several chemokines (including CCL3) . Furthermore, diamide (Yang et al. 2002; Yang et al. 2003) and L P S (Deng et al. 2003) could induce the expression of C C L 3 by HUVEC. In the human brain, the picture is less clear. There is very little evidence, to date, that clearly shows that brain endothelium is capable of synthesizing C C L 3 . RNA, protein expression and release patterns suggest potentially differential roles for C C L 2 and C C L 3 in the initiation of inflammation in the C N S . R T - P C R studies show that C C L 2 expression is constitutively highly expressed with some upregulation after cytokine treatment. CCL3 , however, differs in that the RNA expression levels are significantly lower in the resting state, with upregulation upon cytokine stimulation. These results are consistent with our ELISA data. C C L 2 release is typically two orders of magnitude greater than C C L 3 release under the same conditions. Under basal culture conditions, C C L 2 release ranges from 10 ng/ml to 20 ng/ml, whereas upon stimulation with various inflammatory cytokines or L P S , the release increases 70 significantly in a time-dependant manner. In contrast, C C L 3 release was only observed under stimulated conditions. As chemokine gradients are important for the directional migration of circulating lymphocytes, our surface binding data supports the initial assertion. Under basal culture conditions, C C L 2 preferentially localizes to the apical surface, whereas C C L 3 shows very minimal binding to the basolateral surface, which is consistent with our protein release results. In contrast, upon cytokine treatment, C C L 2 binding occurs preferentially on the basal surface and C C L 3 on the apical surface. One possibility suggested by these findings is that C C L 3 may be responsible for the initial recruitment and activation of C C R 1 and/or C C R 5 expressing cells, whereas C C L 2 plays a greater role in establishing the chemotactic gradients necessary for the directional cell migration into the brain parenchyma. Though a large number of the human data concerning these two molecules are correlatory in nature, studies with experimental animal models provides far more substantial evidence indicating their importance in the induction and propagation of the inflammatory cascade. Several groups have shown that C C L 2 and C C L 3 have differential roles in E A E . CCL2 correlates with relapsing disease, whereas CCL3 correlates with acute disease (Karpus and Kennedy 1997; Kennedy et al. 1998). Furthermore, antibody blocking studies support this assertion (Karpus and Kennedy 1997). Knockout models for CCL2 and C C R 2 both provide strong evidence of the importance of the C C L 2 - C C R 2 interaction (Izikson et al. 2000; Huang et al. 2001). In non-cerebral endothelium models, C C L 2 R N A transcripts have been demonstrated in human aortic, human pulmonary arterial and H U V E C cell cultures, as well as in freshly removed human arteries and veins (Li et al. 1993). Cai et al (1996) showed that C C L 2 was capable of stimulating T cell migration across microvascular 71 endothelium. Gertzen et al (1999) showed that CCL2 (among others) was capable of mediating the firm adhesion of monocytes under flow conditions. Finally, previous work in our laboratory demonstrated that CCL4 and C C L 5 could be expressed by both HBMEC and H U V E C (Shukaliak and Dorovini-Zis 2000) and that both chemokines enhance the adhesion of memory and recently activated CD4+ T cells to cytokine-treated HBMEC, thus indicating an important role for these chemokines in regulating the traffic of T cell subsets across the BBB (Quandt and Dorovini-Zis 2004). 4.3 CCL2 AND CCL3 EXPRESSION IN ACUTE MS Data pertaining to the in vivo expression of these chemokines would have been highly desirable, as it would have enabled us to bridge our in vitro results with their in vivo counterparts. Experiments performed using two sets of commercially available antibodies yielded unsatisfactory results when paraffin-embedded archival tissues were used. However, we were able to obtain frozen brain sections from one patient diagnosed with acute MS - obtaining frozen sections from additional cases proved difficult. Our results demonstrate strong staining for both C C L 2 and C C L 3 primarily in the lesion centre, with some, though minimal staining, in the periplaque regions. Glial cells show strong immunoreactivity. E C show strong staining on their cell surfaces, suggesting potential immobilization of the chemokines in the E C M . They also show variable cytoplasmic staining. With respect to C C L 2 , McManus and colleagues have shown, using both immunohistochemistry and in situ hybridization, that this chemokine (along with CCL7 and CCL8) are expressed prominently in acute and chronic-active MS lesions (1998). 72 Furthermore, C C L 2 is expressed predominantly by astrocytes (McManus et al. 1998; Simpson et al. 1998; Van Der Voorn et al. 1999). In addition, McManus et al (1998) found that E C were usually non-reactive, though C C L 7 immunostaining of the vasculature elements, particularly the E C M , was readily detectable. Simpson et al (1998) however, did show that EC were weakly immunoreactive for both CCL2 and C C L 3 in control C N S tissue. For the most part though, C C L 2 and CCL3 immunostaining was largely restricted to immune and glial elements. 4.4 EXPRESSION OF p-CHEMOKINE RECEPTORS BY HBMEC The RNA expression of the receptors for C C L 2 and C C L 3 (CCR2A/B and CCR1 & 5, respectively) was not detectable in HBMEC. The (former) CCR11 , on the other hand, was constitutively expressed in resting H B M E C , as well as in cytokine-treated H B M E C (100 U/ml T N F a and 500 U/ml IFNy, 24 hours) at the RNA level. With respect to the intracellular protein expression, it was found that the receptor was downregulated following cytokine activation. The expression of p-chemokine receptors on brain endothelium is not well documented. Berger and colleagues (1999) were able to use light and confocal microscopy to detect, albeit weakly, CCR2A and C C R 5 expression in human brain microvascular endothelial cells. They suggest that these, along with other chemokine receptors, may be involved in endothelial migration and repair. Andjelkovic and colleagues, in a number of publications (Andjelkovic et al. 1999; Andjelkovic and Pachter 2000), provide strong circumstantial evidence that isolated brain microvessels expressed binding sites for both CCL2 and C C L 3 . Initially, using biotinylated chemokines and a competition assay, they were able to show (Andjelkovic et al. 1999) 73 that these putative binding sites were saturable and that binding was independent of heparan sulfate, laminin and collagen in the subendothelial matrix. In a later publication, they showed that labelled chemokines could be internalized and that exposure to chemokines was accompanied by the initial loss and subsequent recovery of surface binding sites occurring on a time scale consistent with that of ligand-induced endocytosis and recycling (Andjelkovic and Pachter 2000). CCR11 was disqualified as a chemokine receptor at the XXXth International Union of Pharmacology (Murphy 2002), as a result of no discernible signalling response associated with this receptor. The fact that two independent groups confirmed the ability of this receptor to bind chemokines suggests that this protein may be capable of acting as a "sink," in the same manner as the Duffy antigen receptor for chemokines (DARC). This fact may be important, as this receptor may be responsible for regulating the amount of free chemokines under normal physiological conditions. However, in the presence of cytokines, removal of this safeguard ensures a vigourous immune response. 74 CHAPTER 5: CONCLUSIONS 5.1 SUMMARY AND SIGNIFICANCE The aim of this study was to investigate the differential expression, release and binding of CCL2 and C C L 3 to human brain microvessel endothelial cells (HBMEC) in primary culture. A secondary aim was to determine the expression of the receptors for these two B-chemokines by the BBB endothelium. Our initial hypothesis proposed that treatment with proinflammatory cytokines, including tumour necrosis factor alpha (TNFa), interferon gamma (IFNy), interleukin-1 beta (IL-1B) and lipopolysaccharide (LPS), could induce H B M E C to express and release these chemokines, which would then bind to the E C surface and subendothelial matrix. In effect, the presence of these immobilized molecules would be responsible for the establishment of chemotactic gradients at the level of the blood-brain barrier, which would be important in inducing lymphocyte entry across this barrier into the brain parenchyma during CNS inflammation. As summarized in figure 12, the process of inflammation in the C N S is complex. Traditionally, the dogma has been that the endothelium is a passive bystander, whereas the glial elements have played a more prominent role. Our results strongly suggest that this may not be the case. The experiments undertaken here show that HBMEC in vitro are capable of expressing both C C L 2 and C C L 3 RNA under both resting and cytokine-treated conditions. In both H B M E C and human umbilical vein endothelial cells (HUVEC), CCL2 RNA levels were found to be relatively similar, with slight upregulation after treatment with 100 U/ml T N F a and 500 U/ml IFNy for 24 hours. C C L 3 , on the other hand, in both cell types, showed very minimal, but 75 detectable, RNA expression in resting cells, with upregulation upon stimulation with 100 U/ml T N F a and 500 U/ml IFNy for 24 hours. Intracellular localization, as assayed by immunogold silver staining, showed that both CCL2 and C C L 3 were expressed in resting cells, whereas treatment with TNFa, IFNy, IL-1p or L P S resulted in upregulation of expression. Immunoelectron microscopy demonstrated that the surface binding patterns for CCL2 and CCL3 differed from each other. In resting cells, CCL2 was predominantly found on the apical surface, whereas C C L 3 was found only on the basal surface. Upon cytokine treatment, C C L 2 bound preferentially to the basal surface, whereas C C L 3 bound mainly to the apical surface. Chemokines are soluble signalling molecules released by the cell into the extracellular environment. As such, CCL2 protein release was quantitated using sandwich ELISA. Even under basal conditions, C C L 2 release by HBMEC varied between 10 and 21 ng/ml, in a time dependent manner. T N F a , at 10 and 100 U/ml, had the strongest effect on CCL2 release, ranging from 30+ to 60+ ng/ml. TNFa (100 U/ml) plus IFNy (200 U/ml), IL-ip (10 U/ml) and L P S (5 ug/ml) also showed similar levels of CCL2 release. IFNy (200 and 500 U/ml) alone resulted in smaller, but in general significant, amounts of chemokine release by H B M E C . In contrast, C C L 3 release was several orders of magnitude less than CCL2. Only T N F a (100 U/ml), T N F a (100 U/ml) plus IFNy (200 U/ml), IL-1p (10 U/ml) and L P S (5 ug/ml) induced any appreciable release of this chemokine. In the context of these results, and as illustrated in figure 12, with previous reports suggesting differential roles for CCL2 and C C L 3 in the regulation of CNS inflammation, as well as the monocyte specificity of C C L 2 in contrast to the 76 lymphocytic subset specificity of CCL3 , it is clear that the BBB does have some role in the initiation and propagation of the inflammatory response. Also, in considering that the EC is the first native C N S cell in contact with blood-bourne cells, it stands to reason that the endothelium does not simply act passively. Our results pertaining to acute MS supports this assertion, as there was strong immunoreactivity, for both chemokines, seen in the perivascular area (figure 11), as well as in glial and immune cell types. Furthermore, as mentioned previously, chemokines have the ability to activate lymphocytic subsets - in combination with the accepted knowledge that activated lymphocytes possess the ability to cross the BBB - this suggests that these molecules may play an important function in not only inflammation, but in normal, immune surveillance of the C N S as well. Studies to determine the expression profile of the receptors for CCL2 and C C L 3 were, for the most part, unsuccessful. The levels of RNA expression for C C R 1 , CCR2A/B and C C R 5 were undetectable in HBMEC. However, the RNA for the receptor, formerly known as CCR11 , was constitutively expressed in both resting and cytokine-treated cells (TNFa (100 U/ml) + IFNy (500 U/ml), 24 hours) and the protein was strongly expressed in unstimulated HBMEC. Interestingly, though RNA levels remained constant, cytokine-treatment resulted in significant downregulation of protein expression. Since, as demonstrated in the literature, this receptor does not possess downstream signalling pathways (and hence does not qualify for a true chemokine receptor), it is plausible that it may act as a "chemokine sink," analogous to the Duffy antigen receptor for chemokines (DARC). In the context of an inflammatory event, this may be critical. Though there is no direct evidence to suggest that this receptor can bind either C C L 2 or C C L 3 with high affinity, the downregulation of this protein in the 77 presence of cytokines may be indicative of the physiological removal of a regulatory element in the evolution of an inflammatory event, which makes intuitive sense. 5.2 FUTURE DIRECTIONS This study has addressed a number of issues pertaining to the role of the brain endothelium in the mediation of CNS inflammation. Additionally, it has raised a number of questions, the answers to which may serve to further clarify the significance of the endothelial cell in the context of initiating and propagating the inflammatory reaction in the in vivo C N S interface. First, using this in vitro model, the role of these two chemoattractant molecules could be determined in the context of recruiting and activating specific lymphocytic subsets and monocytes. Diffusion studies could be used to determine whether these molecules are capable of traversing the BBB, under both resting and stimulated ("inflammatory") conditions. This would further clarify the role of the BBB endothelium in initiating the inflammatory response. It may also grant a better understanding of the exact role the endothelium plays: be it as an initiator or as a propagator, or both. Second, as chemokines mediate their action via binding to proteoglycans and GAGs, it would be interesting to determine the molecular interactions involved in this binding at the level of the BBB. The use of heparinases would, in theory, minimize binding to G A G s . Competitive binding experiments would further elucidate the types of interactions required for chemokines to exert their actions. The third study would be to look at the possibility of autocrine function. As chemokines exert their function via G protein-coupled receptors, the use of both exogenous and endogenous chemokines in calcium mobilization experiments would 78 determine whether the specific signalling pathways are present in endothelial cells. Also, this raises the question that if endothelial cells can respond to chemokines, does this have an effect on BBB integrity? This has tremendous implications because if the BBB can both produce chemokines and activate itself in an autocrine manner, this may suggest a critical role for the endothelium in the initiation and propagation of an inflammatory reaction. The fourth study would further elucidate the role of the protein formerly known as CCR11 . At present, there is no clearly defined role for this molecule. If the hypothesis that this molecule is indeed a sink, this could suggest an important role in modulating the immune response. Finally, there are always limitations to in vitro assays in modelling the in vivo situation. Currently there are a number of animal models (such as the EAE model) that could assist in determining the relevance of in vitro data to its in vivo counterpart. Clearly, the full story of chemokines and the BBB has not been told. 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Mol Cell Biol 15(9): 4851-5. Zlotnik, A. and O. Yoshie (2000). "Chemokines: a new classification system and their role in immunity." Immunity 12(2): 121-7. 99 r A Figure 1. Primary cultures of human brain microvessel endothelial cells, an in vitro model of the human blood-brain barrier. Panel (a) shows a confluent monolayer under phase contrast. Panel (b) and (c) show strong positivity for Factor Vlll/von Willebrand antigen and Ulex europeaus lectin binding, respectively. These markers are indicative of the origin and retention of endothelial characteristics 100 U m b i l i c a l V e i n E n d o t h e l i a l C e l l s + RT | E c =3 £ -o .E a> 4-> 500. 400. 300. 200-300 , 200. 500 . 400 . 300 . 200 . 3 0 0 . 2 0 0 . CCL2 GAPDH CCL2 GAPDH Figure 2: RNA expression of C C L 2 (MCP-1) by H B M E C and HUVEC. The R N A expression of the p-chemokine CCL2 was determined by semi-quantitative RT-PCR. Increasing amounts of RNA (sequential two-fold serial dilutions with increasing concentrations from left to right, demonstrated in the "+RT" columns above) was used to show differential expression as a consequence of cytokine activation (100 U/ml T N F a and 500 U/ml IFNy for 24 hours). After amplification with gene-specific primers, C C L 2 and G A P D H P C R samples were run on a 2% agarose gel after 35 (annealing temperature = 60 °C) and 25 cycles (annealing temperature = 55 °C) respectively, under the following conditions: pre-PCR step (94 °C for 8 mins, 55-60 °C for 30 sees and 72 °C for 3 mins) and cycling (94 °C for 1 min, 55-60 °C for 30 sec and 72 °C for 45 sec). The data shown are representative of one of three experiments. 101 Figure 3: Intracellular localization of C C L 2 in HBMEC. Immuno-gold silver staining was used to demonstrate the basal intracellular expression levels of CCL2 (panel A), as well as those with T N F a (10 U/ml, 48 hours, panel B), 1 M B (10 U/ml, 72 hours, panel C) and L P S (5 ug/ml, 48 hours, panel D). Expression was determined by the fine, granular staining seen in all of the above panels. Control cultures incubated with secondary antibody only showed no staining (panel E). The cells were counterstained with Giemsa to visualize their nuclei. 102 A B Surface Localization of C C L 2 on HBMEC 0.10 H « c 2 0.08 S3 E i £ 0.06 c 3 4> Q. ai 0.04 H o TO Q. 0.02 •a •5 O 0.00 I Apical Basal Apical Basal Control (n = 88) Cytokine-treated (n = 108) Treatment Figure 4: Surface localization of CCL2 on HBMEC, as determined by immunoelectron microscopy. Serial sections of both control unstimulated (panel A) and cytokine-treated (100 U/ml T N F a + 500 U/ml IFNy, 24 hours) H B M E C (panel B) were analyzed and the number of gold particles (indicated with arrows in panels A and B) per unit membrane was determined (panel C). The basal cell surface is indicated with the arrowheads (A, B). Chi-square analysis did not show significant differences between the different groups. 103 70000 A ^ 60000 -I 50000 -I 0) V) « 40000 5 K CM 30000 20000 10000 ^ 0 unstim TNFa 10 U/ml TNFa 100 U/ml IFN-/ 200 U/ml IFN-y 500 U/ml Treatment TNFa 100 U/ml + IFN-y 200 U/ml IL-1P 10 U/ml LPS 5 ng/ml Figure 5: C C L 2 release under simulated inflammatory conditions. Chemokine release was determined by assaying the cell supernatants corresponding to the above treatments, at the indicated time intervals. Release was quantitated using a commercial ELISA kit purchased from R & D Systems. Values represent mean release (pg/ml) ± S E M (n = 3). Tukey test p < 0.001; * p < 0.05 as compared to unstimulated HBMEC. Values shown are the results of one of two independent, representative experiments. 104 200 1 E § c 3 Q T3 500 400 300 200 300 200 500 400 300 200 Figure 6: RNA expression of C C L 3 (MIP-1 a) by H B M E C and HUVEC. The R N A expression of the (3-chemokine C C L 3 was determined by semi-quantitative R T - P C R . Increasing amounts of RNA (sequential two-fold serial dilutions with increasing concentrations from left to right, demonstrated in the "+RT" columns above) was used to show differential expression as a consequence of cytokine activation (100 U/ml T N F a and 500 U/ml IFNy for 24 -hours). After amplification with gene-specific primers, C C L 3 and G A P D H P C R samples were run on a 2% agarose gel after 35 (annealing temperature = 60 °C) and 25 cycles (annealing temperature = 55 °C) respectively, under the following conditions: pre-PCR step (94 °C for 8 mins, 55-60 °C for 30 sees and 72 °C for 3 mins) and cycling (94 °C for 1 min, 55-60 °C for 30 sec and 72 °C for 45 sec). The data shown are representative of one of three experiments. 105 Figure 7: Intracellular localization of C C L 3 in HBMEC. Immuno-gold silver staining was used to demonstrate the basal intracellular expression levels of C C L 3 (panel A), as well as those with T N F a + IFNy (100 U/ml and 200 U/ml, respectively, 72 hours, panel B), IL-1B (10 U/ml, 72 hours, panel C) and L P S (5 ng/ml, 48 hours, panel D). Expression was determined by the fine, granular staining seen in all of the above panels. Control cultures incubated with secondary antibody only showed no staining (panel E). The cells were counterstained with Giemsa to visualize their nuclei. 106 c Surface Localization of CCL3 on HBMEC E 0 . 0 2 5 -C 1 jj 0 . 020 -E I 2 ~ 0 . 0 1 5 -I in 0 . 0 1 0 -4) O re Q- 0 . 0 0 5 -T3 J 1 L J | _ | ___Apjca[ Basal Apical Basal Control (n = 103) Cytokine-treated (n = 101) Treatment Figure 8: Surface localization of C C L 3 on HBMEC, as determined by Immunoelectron microscopy. Serial sections of both control unstimulated (panel A) and cytokine-treated (100 U/ml T N F a + 500 U/ml IFNy, 24 hours) H B M E C (panel B) were analyzed and the number of gold particles (indicated with arrows in panels A and B) per unit membrane was determined (panel C). The basal cell surface is indicated with the arrowheads (A, B). Chi-square analysis did not show significant differences between the different groups. 107 O) a 400 300 -J a (0 re a> © 200 O O 100 -I 24 hours 48 hours 72 hours unstim TNFa 10 U/ml TNFa 100 U/ml IFN^y 200 U/ml IFN-y 500 U/ml Treatment TNFa 100 U/ml + IFN-r 200 U/ml IL-1p 10 U/ml LPS 5 ^ /ml Figure 9: C C L 3 release by HBMEC under simulated inflammatory conditions. Chemokine release was determined by assaying the cell supernatants corresponding to the above treatments, at the indicated time intervals. Release was quantitated using a commercial ELISA kit purchased from R & D Systems. Values represent mean release (pg/ml) ± S E M (n = 3). ANOVA p < 0.001; * p < 0.05 as compared to unstimulated HBMEC. Values shown are the results of one of two independent, representative experiments. 108 Figure 10: R N A and intracellular protein expression of the former C C R 1 1 . RNA expression (panel A) was determined using semi-quantitative R T - P C R . After P C R amplification with gene-specific primers, samples were run on a 6% polyacrylamide gel and stained with ethidium bromide for visualization. Control samples (U) and cytokine-treated (S, 100 U/ml T N F a + 500 U/ml IFNy for 24 hours) showed similar levels of R N A expression. The data shown are representative of one of two experiments. Immunoperoxidase staining using a rabbit polyclonal anti-human CCR11 antibody showed strongly positive cytoplasmic perinuclear staining of the majority of HBMEC for CCR11 (B). (C) Significant downregulation of intracellular protein expression occurred following cytokine stimulation (100 109 Figure 11: C C L 2 and C C L 3 expression in acute MS lesions. Frozen sections from a patient diagnosed with acute MS were incubated with either ant i -CCL2 (Chemicon, 25 ug/ml, panel A) or ant i-CCL3 (Peprotech, 25 ug/ml, panel B). Both sections show strong staining for the respective chemokines in and around E C (indicated with arrows), perivascular cells, astrocytes (indicated with arrowheads) and macrophages. In control sections incubated with secondary antibody only, there was no staining observed (panel C). 110 CNS Inflammation f GlyCAM-l, CD-34. MAdCAM-1 Figure 12: Proposed hypothesis for the role of chemokines in C N S inflammation. 111 


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