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TGF-Beta-1 Regulation of Chemokine Receptors in Rat Microglia and Human Macrophages

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TGF-Beta-1 Regulation of Chemokine Receptors in Rat Microglia and Human Macrophages
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CHEN, SHUZHEN ( Author, Primary )
Copyright Date:
2008

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Chemokine receptors ( jstor )
Chemokines ( jstor )
HIV 1 ( jstor )
Infections ( jstor )
Macrophages ( jstor )
Messenger RNA ( jstor )
Microglia ( jstor )
Phosphorylation ( jstor )
Rats ( jstor )
Receptors ( jstor )

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University of Florida
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University of Florida
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Copyright Shuzhen Chen. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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4/30/2006
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72823302 ( OCLC )

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TGFREGULATION OF CHEMOKINE RECEPTORS IN RAT MICROGLIA AND HUMAN MACROPHAGES By SHUZHEN CHEN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2004

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This dissertation is dedicated to my mom Baozhu, my dad Wenshi and my aunt Qiaoling.

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ACKNOWLEDGMENTS First, I want to thank my mentor Dr. Jeffrey K. Harrison. He has been a wonderful mentor ever since I came to rotate in this lab. In research, he constantly guided me in science and gave me moral support. He gave me lots of ideas, some of which worked out to be great and some didn’t; some I agreed with and some I didn’t; but they were sure exciting. He taught me how to write and present my ideas and data, which helped me to be an independent researcher. Jeff also showed me how to be critical of the data. Beside work, Jeff has been a professor to me on English, which is my second language; he introduced me to American culture through those interesting chats. At the same time, I would like to thank Dr. Wolfgang J. Streit, who is an expert of neuroimmunology. Dr. Streit introduced me to microglia and let me use his lab freely. He gave me lots of advice and support during the past four years. Without his lab and his help, I would not have been able to finish my graduate studies. I would also like to thank my two other committee members, Dr. Paul S. Oh and Dr. Peter P. Sayeski, for their wonderful advice, critical comments and support. They are always there to help me when I need it. Second of all, I want to thank all of the labs that I collaborated with. Dr. Maureen M. Goodenow gave us full support and critical insights on the HIV-1 project. Dr. Daniel L. Tuttle and Joseph Oshier helped me in the lab and had lots of interesting discussions with me. We collaborated with Dr. Henry Baker’s lab with microarray analysis. I would like to thank Dr. Henry Baker and Maria C. Lopez for their help. I want to express me iii

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gratitude toward Dr. Harm J. Knot for his great comments and the confocal microscopy technical support. Thirdly, I want to thank all the former and present members in Jeff’s lab that I worked with. Dr. Violetta Zujovic has been helping me daily from ideas to bench work. She is one of my greatest friends and makes my life more interesting and fun. Defang Luo, our lab manager, always tried to get lab supplies whenever I needed them. Being the expert on many techniques, she has taught me and helped me a great deal. Christopher Davis, my fellow graduate student in the lab, helped in trouble-shooting lots of experiment, which made my projects move faster. We interact daily about our work and helped each other a lot; I also appreciate him very much for helping me with all the computer work. Peter Swain taught me lots of techniques and also about American culture. I thank him dearly. Last but least, I want to thank my parents, for all the sacrifice they made; for their constant love, support and encouragement. They are the greatest motivation for all my hard work. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iii LIST OF TABLES...........................................................................................................viii LIST OF FIGURES...........................................................................................................ix ABSTRACT.........................................................................................................................x CHAPTER 1 INTRODUCTION........................................................................................................1 Chemokines and Chemokine Receptors.......................................................................1 Fractalkine and CX3CR1......................................................................................3 SDF-1 and CXCR4................................................................................................4 Chemokines and Chemokine Receptors in the Central Nervous System.....................4 Chemokines and Chemokine Receptors in CNS Development and Physiology...5 Chemokines and Chemokine Receptors in CNS Pathology and Inflammation....7 Fractalkine and CX3CR1 in CNS Diseases...................................................9 Microglia: the Immune Cells Resident in the CNS.............................................10 Chemokines and Chemokine Receptors in the Peripheral Immune System...............11 Fractalkine and CX3CR1 in Immune Response and Diseases............................11 SDF-1 and CXCR4..............................................................................................13 Chemokine Receptors and HIV-1.......................................................................13 Macrophage and HIV-1.......................................................................................16 TGF-: a Multifunctional Growth Factor.................................................................16 Regulation of G-protein Coupled Receptor Signal Transduction by RGS Proteins...20 2 TGF-UPREGULATES CX3CR1 EXPRESSION AND INHIBITES FRACTALKINE-STIMULATED SIGNALING IN RAT MICROGLIA.................24 Introduction.................................................................................................................24 Materials and Methods...............................................................................................27 Primary Microglial Cell Cultures........................................................................27 Northern Blot Analysis........................................................................................28 Whole Cell Radioligand Binding........................................................................28 v

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Analysis of ERK1/2 and p38 MAPK Phosphorylation by Western Immunoblotting................................................................................................29 Isolation of DNA Sequences of the Rat CX3CR1 Promoter...............................30 Results.........................................................................................................................30 TGF-1 Enhances Microglial CX3CR1 mRNA in a Time and Concentration-dependent Manner............................................................................................30 TGF-1 Induces CX3CR1 Protein: Time and TGF-1 Concentration Dependence......................................................................................................31 Fractalkine-induced ERK1/2 Phosphorylation is Inhibited by Pretreatment with TGF-1............................................................................................................32 TGF-1 Does Not Affect CX3CR1 mRNA Stability..........................................33 Discussion...................................................................................................................34 3 TGF-1-INDUCIBLE RGS2 AND RGS10 REGULATE CX3CR1 FUNCTION IN RAT MICROGLIA...............................................................................................48 Introduction.................................................................................................................48 Materials and Methods...............................................................................................50 Cell Culture.........................................................................................................50 Microarray Genechip Hybridization and Data Analysis.....................................51 Northern Blot.......................................................................................................52 Transfection of HEK 293-T cells........................................................................52 Analysis of ERK1,2 Phosphorylation by Western Immunoblotting...................53 Immunostaining of RGS10..................................................................................53 Results.........................................................................................................................53 TGF-1 Regulation of Gene Expression Profile in Rat Microglia......................53 RGS2 and RGS10 Attenuated CX3CR1-mediated ERK1,2 Stimulation............54 Kinetics of RGS10 Inhibition of CX3CR1 Activation........................................55 Effect of CX3CR1 on Cellular Localization of RGS10......................................55 Discussion...................................................................................................................55 4 TGF-1 INCREASES CXCR4 EXPRESSION, SDF-1-STIMULATED SIGNALING AND HIV-1 ENTRY IN HUMAN MONOCYTE-DERIVED MACROPHAGES......................................................................................................64 Introduction.................................................................................................................64 Materials and Methods...............................................................................................66 Primary Cultures of Human MDMs and Rat Microglia......................................66 Northern Blot Analysis........................................................................................67 Flow Cytometry and Immunofluorescence Microscopy.....................................67 SDF-1 Stimulation of ERK1,2 Phosphorylation..............................................68 HIV-1 Entry.........................................................................................................69 Statistic Analysis.................................................................................................70 Results.........................................................................................................................70 TGF-1 Increased CXCR4 mRNA in MDMs.....................................................70 vi

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TGF-1 Increased MDM Cell Surface CXCR4 and Altered its Cellular Localization......................................................................................................71 SDF-1-stimulated ERK1,2 Phosphorylation was Enhanced by TGF-1 Pretreatment.....................................................................................................72 TGF-Increased CXCR4 and Enhanced SDF-1-stimulated ERK1,2 Phosphorylation in Rat Microglia....................................................................72 TGF-1 Increased DX4 HIV-1 Infection of MDMs...........................................73 TGF-1 Did not Alter CCR5 mRNA or R5 Viral Entry of MDMs....................73 Discussion...................................................................................................................74 5 GENERAL DISCUSSION.........................................................................................85 LIST OF REFERENCES...................................................................................................90 BIOGRAPHICAL SKETCH...........................................................................................108 vii

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LIST OF TABLES Table page 1-1 Chemokine receptors and their ligands....................................................................23 2-1 Kd and Bmax values of 125I-fractalkine binding to CX3CR1 on TGF-1-treated and untreated microglial cells.........................................................................................41 3-1 Genes regulated by TGF-in rat microglia...........................................................59 viii

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LIST OF FIGURES Figure page 2-1 Effects of TGF-1 on induction of CX3CR1 mRNA expression in rat microglial cells...........................................................................................................................42 2-2 TGF-1 affects the expression of CX3CR1 in microglia but not astrocytes...........43 2-3 TGF-1 increases CX3CR1 protein expression on rat microglial cells...................44 2-4 Stimulation of intracellular signaling by fractalkine in TGF-1-treated and control microglial cells.........................................................................................................45 2-5 The half-life of CX3CR1 mRNA is unaltered in TGF-1-treated microglia...........46 2-6 Schematic structure of the rat CX3CR1 gene..........................................................47 3-1 Genes regulated by TGFin rat microglia...........................................................60 3-2 RGS10 and RGS2 attenuated fractalkine-stimulated ERK1,2 phosphorylation......61 3-3 Kinetics of RGS10 inhibition of CX3CR1-mediated ERK1,2 phosphorylation..62 3-4 Effect of CX3CR1 on the localization of RGS10....................................................63 4-1 TGF1 increased CXCR4 mRNA expression in human MDMs...........................79 4-2 TGF-1 increased cell surface expression of CXCR4 on MDMs and changed its cellular localization..................................................................................................80 4-3 SDF-1-stimulated ERK1,2 phosphorylation in control and TGF1 treated cells..81 4-4 TGF1 increased CXCR4 mRNA and enhanced SDF-1-stimulated ERK1,2 phosphorylation in primary cultures of rat microglia...............................................82 4-5 TGF1 increased the susceptibility of MDMs to D-X4 HIV-1 entry....................83 4-6 TGF1 had no effect on human MDM CCR5 mRNA or R5 HIV-1 entry into MDMs and decreased CCR5 mRNA in rat microglia..............................................84 5-1 Model of TGF-regulation of chemokine receptors.............................................89 ix

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy TGF-REGULATION OF CHEMOKINE RECEPTORS IN RAT MICROGLIA AND HUMAN MACROPHAGES By Shuzhen Chen May, 2004 Chair: Jeffrey K. Harrison Major Department: Pharmacology and Therapeutics Deregulation of cytokine and chemokine networks is a common theme in a variety of diseases. Chemokines are small cytokines that mainly mediate leukocyte migration and activation. In addition, they also play important roles in embryonic development, hematopoiesis, lymphopoiesis, angiogenesis, tumor biology and viral pathogenesis. Chemokines exert these functions through receptors which belong to the seven-transmembrane, G-protein coupled receptor superfamily. The expression and function of chemokine receptors are under extensive regulation in developmental, physiological and pathological conditions. However, the mechanism of their regulation is not well characterized. My dissertation is focused on the regulation of expression and function of chemokine receptors by the cytokine TGF-1. Following rat peripheral nerve transection, both CX3CR1 and TGF-1 are increased in a time-dependent manner within the injured facial motor nucleus, leading us to examine the relationship between TGF-1 and CX3CR1 in the CNS. TGF-1 increased CX3CR1 mRNA, cell surface protein expression but blunted fractalkine-stimulated ERK1,2 phosphorylation in rat microglia. To explore the possible mechanism x

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by which TGF-inhibited CX3CR1-mediated signaling in spite of the increased receptor expression, gene profiling with microarray analysis was performed on TGF--treated and -untreated rat microglia. Among other genes, RGS2 and RGS10 were increased by TGF-Co-expression of RGS2 or RGS10 with CX3CR1 attenuated CX3CR1-mediated ERK1,2 phosphorylation, with RGS10 having a greater effect. CXCR4 is one of major co-receptors for HIV-1 expressed on macrophages. In HIV-1 infected patients, blood and tissue levels of TGF-1 are increased. We examined the effects of TGF-1 on CXCR4 expression in human MDM and rat microglia. TGF-1 increased CXCR4 expression and SDF-1-stimulated ERK1,2 phosphorylation in both cell types. The increased CXCR4 expression in human MDMs resulted in increased susceptibility of the cells to entry by dual-tropic CXCR4-using HIV-1. In contrast, TGF-1 failed to increase CCR5 expression or infection by CCR5-using virus in MDMs. These results suggest increased expression of CXCR4 on macrophages may contribute to the emergence of dual-tropic X4 viral variants at later stages of HIV-1 infection. In summary, my study demonstrates that TGF-1 can directly and indirectly regulate the function of chemokine receptors in peripheral immune and central nervous systems. xi

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CHAPTER 1 INTRODUCTION Chemokines (Chemoattractant cytokines) are a group of cytokines that were first described to mediate leukocyte migration and activation. Recent studies show that they also play important roles in cell adhesion, cytokine secretion, angiogenesis, embryonic development, cancer and viral pathogenesis. Chemokines exert their functions through receptors that are members of the seven-transmembrane, G-protein coupled receptor (GPCR) superfamily. Once the ligands bind to the receptors, multiple intracellular signaling pathways can be activated. The expression and function of chemokines and their receptors are under extensive regulation in both physiological and pathological conditions. In the past decade, great achievements in identifying chemokines and their functions have been made. However, much of the data is still quite descriptive and the mechanism of their regulation and function in both physiological and pathological conditions still remains largely unknown. This dissertation addressed the regulation of expression and function of chemokine receptors in the peripheral immune and central nervous systems by the multifunctional cytokine TGF-Transforming growth factor-1). Chemokines and Chemokine Receptors Chemokines are structurally related small cytokines. More than 40 chemokines have been discovered and most of them are small secreted proteins of about 8-14 kDa, except fractalkine and CXCL16, which are tethered to cell surface via a mucin-like stalk. Based on the number of amino acids in between the first two conserved cysteines, 1

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2 chemokines can be classified into four subfamilies: C, CC, CXC and CX3C. There is only a single member of the CX3C subfamily, fractalkine (CX3CL1). The CXC chemokine subfamily includes such members as SDF-1 (Stromal derived factor-1) (CXCL12), IL-8 (Interleukin-8) and IP-10 (Interferon-inducible protein 10). MCP-1 (Monocyte chemotactic protein-1), MIP-1 (Macrophage inflammatory protein-1) and RANTES (Regulated on activation normal T-cell expressed and secreted) are examples of the chemokines that belong to the CC subfamily. Lymphotactin-and -comprise the C chemokine family. Chemokines exert their biological function through cell surface receptors. Chemokine receptors are members of the seven-transmembrane, G-protein coupled receptor superfamily. They are named according to the subfamilies of their ligands. Eighteen chemokine receptors have been identified by molecular cloning methods so far, including ten CC, six CXC, one C and one CX3C receptors (Table 1-1). Some chemokines and their receptors are promiscuous, which means one chemokine can bind to several receptors with high affinity, and vice versa. For example, CCR1 can bind MIP-1, RANTES, and MCP-2 among other chemokines with high affinity; MIP-1 can bind to CCR1 and CCR5. On the other hand, some ligands and receptors are very exclusive for each other. For example, fractalkine only binds to CX3CR1 with high affinity; CXCR4 has one known agonist, SDF-1. Once a chemokine binds to its receptor, multiple intracellular signal transduction pathways are activated. The patterns of signaling pathways activated by these receptors are quite common irrespective of the kind of chemokines or the type of cells. Most, but not exclusively, responses are mediated by pertussis toxin-sensitive Gi subfamily of G

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3 proteins. The downstream signaling includes such events as Ca2+ mobilization, activation of PI3 kinase and MAPK pathways and cytoskeleton polymerization. These cascades culminate in biological functions of receptor-expressing cells including migration, activation and phagocytosis. Fractalkine and CX3CR1 Fractalkine is the only known member in the CX3C subfamily. Two different groups first identified it in 1997 (Bazan et al., 1997; Pan et al., 1997). It has 373 amino acids, consisting of a signal peptide, a chemokine module, a mucin-like stalk with multiple glycosylation sites, a single transmembrane domain and a short intracellular region. This membrane-tethered form can be cleaved by TNF--converting enzyme (TACE [ADAM17]) and ADAM10 into soluble fractalkine (Garton et al., 2001; Tsou et al., 2001). It is widely expressed in various organs including brain, lung, heart, kidney and adrenal gland (Bazan et al., 1997; Harrison et al., 1998). Neurons and endothelial cells are the main sources of fractalkine. CX3CR1 is the exclusive receptor for fractalkine. It is also called V28 for human CX3CR1 and RBS11 for rat CX3CR1. The gene is located on human chromosome 3p21 where many CC chemokine receptor genes cluster (Maho et al., 1999). It is expressed on monocytes, T cells, NK (natural killer) cells, microglia and astrocytes (Harrison et al., 1998; Imai et al., 1997). Fractalkine is unique among all the chemokines in that it can exist as soluble and membrane attached forms. Soluble fractalkine mainly induces the migration of CX3CR1-expressing cells, while the membrane-tethered form functions as an adhesion molecule (Imai et al., 1997). Indeed, fractalkine-CX3CR1 mediates leukocyte capture and firm

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4 adhesion in both static and physiological flow conditions (Fong et al., 1998; Goda et al., 2000; Imai et al., 1997). The adhesion function of fractalkine is independent of G-protein activation and signal transduction (Haskell et al., 1999). SDF-1 and CXCR4 SDF-1, a member of the CXC subfamily, has 89 amino acids with 21 of them as signal peptide. It has at least another isoform, SDF-1and more recently a third isoform, SDF-1 was cloned in rat (Gleichmann et al., 2000). The gene is located on human chromosome 10q and on murine chromosome 6F1. The sequence is highly conserved through the evolution; human and murine SDF-1 differs only by one amino acid. CXCR4 is the only known receptor for SDF-1 and also very conserved in different species. The gene is located on human chromosome 2q21. It is ubiquitously expressed on a wide variety of cell types including most hematopoietic cell types, vascular endothelial cells, neurons, microglia and astrocytes (Lazarini et al., 2003; Nagasawa et al., 1998). Chemokines and Chemokine Receptors in the Central Nervous System It has been well documented that chemokines and their receptors are expressed in the CNS, mainly in neuropathological situations where the levels of chemokines and their receptors change dynamically. Chemokines identified to be present in CNS tissues and cells include MCP-1, MIP-1 and , RANTES, IL-8, Gro(Growth-regulated oncogene), IP-10, SDF-1 as well as fractalkine. Chemokine receptors that are expressed in the CNS include CXCR2, CXCR3, CXCR4, CXCR5, CCR1-3, CCR5, DARC (Duffy-antigen-related chemokine) and CX3CR1. The discovery of constitutive expression of SDF-1-CXCR4, fractalkine-CX3CR1 and gene deletion studies of the former ligand

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5 receptor, has demonstrated that chemokines are crucial in CNS development and homeostasis. Chemokines and Chemokine Receptors in CNS Development and Physiology The importance of chemokines and chemokine receptors in CNS development has been highlighted by analysis of either SDF-1 or CXCR4 gene deleted mice (Ma et al., 1998; Zou et al., 1998). In these mice, the development of the cerebellum was disrupted and upon further examination, revealed the premature movement and proliferation of external granule layer (EGL) cells into the cerebellum. In wild type mice, granule cells proliferate in the EGL zone in the first postnatal week and migrate radially through the Purkinje cell layer to form the internal granule cell layer (IGL). During this time, there are high levels of CXCR4 and SDF-1 mRNA in the granule cells and in the pia mater overlaying the cerebellum, respectively. Therefore, it is likely that granule cells are retained in the EGL by SDF-1. The development of the hippocampal dentate gyrus is also defective in CXCR4 knockout mice, suggesting it may also be involved in hippocampal granule neuron organization and dentate gyrus neurogenesis (Bagri et al., 2002; Lu et al., 2002). Fractalkine and CX3CR1 are constitutively expressed in the CNS, generally at higher levels than the peripheral tissues. Fractalkine is mainly expressed by neurons and CX3CR1 is primarily expressed by microglia, although there are differences in the reports about their localization (Harrison et al., 1998; Nishiyori et al., 1998; Pan et al., 1997). This expression pattern suggested that fractalkine and CX3CR1 plays important roles in mediating communication between neurons and microglia. In vitro studies on microglia show that fractalkine can induce calcium mobilization, activate PI3 kinase and MAPK pathways and promote chemotaxis in these cells (Harrison et al., 1998;

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6 Maciejewski-Lenoir et al., 1999), suggesting that fractalkine is important in microglial survival, activation and migration. Indeed, fractalkine inhibited Fas-mediated microglial apoptosis (Boehme et al., 2000). Fractalkine also has anti-inflammatory effects on microglia. It inhibited TNFrelease from LPS-induced microglia, resulting in less neuronal apoptosis in a neuron-microglia coculture paradigm (Zujovic et al., 2000). These data suggest that fractalkine is important in maintaining the CNS homeostatic state and immune responses of the brain. In addition to their roles in development, chemokines play important roles in neuronal physiology. SDF-1, Gro-, Groand IL-8 induced spontaneous postsynaptic currents and enhance neurotransmitter release in Purkenje cells in cerebellar slices or cell cultures, suggesting that these chemokines act as neuromodulators (Giovannelli et al., 1998; Limatola et al., 2000; Ragozzino et al., 1998). In hippocampal neurons, MDC (Monocyte-derived chemokine) and fractalkine could inhibit spontaneous glutamatergic excitatory postsynaptic currents and the latter chemokine also inhibited voltage-dependent Ca2+ current (Meucci et al., 1998). Chemokines have also been shown to have neurotrophic and neuroprotective functions. IL-8 promoted hippocampal neuron survival compared to control neurons in cell culture (Araujo and Cotman, 1993). MDC, RANTES, SDF-1 and fractalkine inhibited apoptosis of hippocampal neurons on removal of their glial cell feeder layer (Meucci et al., 1998). These chemokines reduced neuronal apoptosis induced by HIV-1 envelope protein gp120. Further experiments on fractalkine proved that PI3 kinase pathway activation was involved in these neuroprotective effects (Meucci et al., 2000).

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7 In summary, chemokines are important in embryonic development and normal physiology. However, most of the data was established using in vitro cell culture systems and thus, their roles in vivo await further investigation. More recently, studies of fractalkine or CX3CR1 gene deleted mice indicated no abnormalities in development or response to peripheral nerve injury (Cook et al., 2001; Jung et al., 2000). Nonetheless it is hard to imagine constitutive expression of fractalkine and CX3CR1 without significant importance. Therefore, more investigation needs to be carried out to characterize the functions of fractalkine in the CNS. Chemokines and Chemokine Receptors in CNS Pathology and Inflammation The expression of chemokines and their receptors in the CNS have been mostly characterized in neuropathological situations. Increased expression of chemokines and their receptors has been detected in such challenges as brain ischemia (Tarozzo et al., 2002), bacterial infection (Zwijnenburg et al., 2001), stab wound (Ghirnikar et al., 1996), brain tumors (Barbero et al., 2002), multiple sclerosis (Babcock and Owens, 2003) as well as HIV-1 encephalitis and AIDS dementia (Martin-Garcia et al., 2002). Neurons, microglia, astrocytes and infiltrating leukocytes have been identified as the sources of chemokines and chemokine receptors. Although most investigations suggest the central roles of chemokines in the CNS are as chemotactic factors, their functions are not clearly determined. Leukocyte infiltration into the CNS is a central feature in the pathogenesis of a variety of inflammatory brain diseases. Chemokines may promote leukocyte migration into the brain and also amplify local inflammatory networks. There are few chemokines constitutively expressed in the CNS, however, in response of inflammatory cytokines, such as TNFor challenges including brain trauma and bacterial infection, expression of

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8 chemokines can be induced from cells intrinsic to the CNS or infiltrating leukocytes. For example, in multiple sclerosis patients expression of MCP-1, MCP-2 and MCP-3 has been detected in lesions (McManus et al., 1998) and the levels of MIP-1, IP-10 and RANTES in the cerebrospinal fluid is increased (Sorensen et al., 1999; Miyagishi et al., 1995). The lesions are surrounded by T cells and macrophages infiltrated into the CNS. In animal models of multiple sclerosis, EAE (experimental autoimmune encephalomyelitis), anti-MIP-1treatment prevented the development of acute but not relapsing EAE while anti-MCP-1 antibodies reduced the severity of relapsing EAE, which suggests they are involved in different stages of the diseases (Karpus and Kennedy, 1997). The involvement of chemokines and their receptors in the CNS extends to neurodegenerative diseases such as Alzheimer’s disease and AIDS dementia. A is able to stimulate the production of IL-8, MCP-1, MIP-1 and MIP-1 from human monocytes (Fiala et al., 1998). MCP-1 has also been detected in brains from AD patients and localized to mature senile plaques and reactive microglia (Ishizuka et al., 1997). The chemokine receptor, CXCR2 is significantly increased in brain tissue of AD patients and it was localized to dystrophic neurites of senile plaques (Xia et al., 1997). In patients suffering from AIDS dementia, levels of MIP-and -are increased compared to AIDS patients without dementia (Schmidtmayerova et al., 1996). Our complete understanding of the roles of chemokines in these diseases is still at a very early stage and the data is very preliminary. More extensive studies will be necessary to reveal the functions of chemokines in these diseases.

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9 Fractalkine and CX3CR1 in CNS Diseases Studies of the involvement of fractalkine/CX3CR1 in CNS pathologies are less extensive and the existing data are quite fragmentary and mostly descriptive. The best studied examples of fractalkine functions in CNS diseases are EAE, ischemia, systematic injection of inflammatory reagents and rat motor facial nerve axotomy. In animal models of stroke, cerebral ischemia is usually induced by two methods: transient or permanent unilateral middle cerebral artery occlusion, and temporary occlusion of the carotid arteries to create transient forebrain global ischemia. Ischemic events are associated with activation and migration of inflammatory cells such as leukocytes and microglia. After ischemia-reperfusion injury to the rat brain, fractalkine expression is increased in neurons of ischemic penumbra and endothelial cells of the infarcted area; CX3CR1 is upregulated in macrophages/microglia inside the infarcted tissues (Tarozzo et al., 2002). In fractalkine deficient mice, a reduction in both infarction size and mortality rate has been noted (Soriano et al., 2002). These data suggest that fractalkine and CX3CR1 may participate in the activation and migration of microglia into the infarcted tissue and thus the inflammation process after brain ischemia. In EAE and a prion model of neurodegeneration, CX3CR1 expression is increased in microglia (Jiang et al., 1998; Hughes et al., 2002). LPS injection into the CNS increased expression of fractalkine but decreased that of CX3CR1 (Boddeke et al., 1999; Pan et al., 1997). Overall, it is reasonable to conclude that changes of fractalkine and CX3CR1 expression are associated with CNS pathologies. These likely include the migration and activation of infiltrating leukocytes and microglia; as well as contributing to the immune response intrinsic to the injured CNS.

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10 Microglia: the Immune Cells Resident in the CNS Microglia are the immunocompetent cells resident in the CNS. Their nature and origin have long been debated, nonetheless, it is now generally accepted that ontogenetically they are related to mononuclear phagocytic cells (Hickey et al., 1992; Ling and Wong, 1993). However, it has been postulated that microglia belong to true glia of neuroectodermal lineage. They are often the first cell type in the CNS to respond to injuries where they go through rapid transformation from a resting to an activated state. Microglia are characteristic of typical immune cells expressing such molecules as MHC antigens, complement receptors, CD4 co-receptor, cytokine and chemokine receptors. CX3CR1, CXCR4 and CCR5 have been shown to be expressed on microglia constitutively (Jiang et al., 1998). In response to certain stimuli, microglia become activated to perform the functions of macrophages including antigen presentation, free radical and NO secretion, synthesis of cytokines, chemotaxis and phagocytosis. In a variety of CNS diseases such as multiple sclerosis (Li et al., 1993; Prineas and Wright, 1978), EAE (Bauer et al., 1994), Alzheimer’s disease (Meda et al., 1995) and AIDS dementia (Price et al., 1988), microglia become activated and release substance toxic to neurons and oligodendrocytes. In contrast to their cytotoxic activities, microglia can also be protective to other cells in the CNS. An invaluable animal model to study the activation stages and functions of microglia is the facial motor nerve axotomy that leaves the blood-brain barrier relatively unimpaired with little to no infiltration of peripheral leukocytes. Within hours after transection of facial motor nerves, microglia within the facial nucleus become activated and proliferate surrounding the injured neurons (Graeber et al., 1988). They do not become phagocytic and eventually, the motor neurons regenerate. In this scenario,

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11 microglia produce lots of TGF-1 that could promote tissue repair (Kiefer et al., 1993). Previous data in our lab showed that CX3CR1 mRNA was increased in the facial nucleus in a time-dependent manner similar to TGF-, which leads us to speculate that TGF-and fractalkine-CX3CR1 systems interact. A major goal in this dissertation was to characterize this interaction in order to help us understand the function of both TGF-and fractalkine in the CNS. Chemokines and Chemokine Receptors in the Peripheral Immune System Chemokines can mediate leukocyte trafficking and activation, angiogenesis, hematopoiesis and tumor growth. Collective data have shown that they are involved in both innate and acquired phases of a normal immune response to host insult (Luster, 2002; Matsukawa et al., 2000). However, excessive activities from chemokines are also implicated in such diseases as asthma (Lukacs et al., 2003), rheumatoid arthritis (Szekanecz et al., 2003), allograft rejection (el Sawy et al., 2002) and atherosclerosis (Bursill et al., 2004). Fractalkine and CX3CR1 in Immune Response and Diseases In the peripheral immune system, fractalkine is a chemoattractant for monocytes, natural killer cells and T cells (Bazan et al., 1997; Imai et al., 1997). Since soluble and membrane attached forms have chemoattractant and cell adhesion functions respectively, fractalkine can mediate leukocyte extravasation through the vascular wall and into surrounding tissues in a selectinand integrin-independent manner. Fractalkine also plays roles in cytotoxicity. Almost all CD16+ NK cells express CX3CR1 and soluble fractalkine can induce the transmigration of these cells (Bazan et al., 1997; Imai et al., 1997) in association with enhanced cytolytic function against NK sensitive target cells

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12 (Yoneda et al., 2000). CX3CR1-expressing lymphocytes, including NK cells, T cells and CD8+ cells, but not those without CX3CR1, show terminally differentiated cytotoxic phenotypes such as intracellular perforin and granzyme B (Nishimura et al., 2002). These CX3CR1 positive cells have higher cytotoxic activity than CX3CR1 negative cells. Furthermore, interaction of these cells with membrane-bound fractalkine promotes their migration ability towards a second chemokine including IL-8 and MIP-1(Nishimura et al., 2002). Therefore, fractalkine may mediate cytotoxic activity of cells and induce their migration in response to other chemokines, resulting in coordinated immune defense against infection. In a variety of pathological conditions including atherosclerosis, allograft rejection and renal diseases, excessive immune responses can result in tissue damage and diseases. Acute allograft rejection is characterized by the infiltration of circulating leukocytes into the inflamed graft. Fractalkine expression is significantly enhanced in this condition and is particularly prominent on vascular tissues and endothelium (Robinson et al., 2000). Treatment with anti-CX3CR1 blocking antibodies significantly prolonged cardiac allograft survival in mice (Robinson et al., 2000). The survival time of cardiac allograft was also increased in CX3CR1 knock out mice with a reduction in the infiltration of macrophages, NK cells and other leukocytes (Haskell et al., 2001). The data demonstrate that fractalkine is important in orchestrating the response of specific set of leukocytes during inflammation. Thus, it is tempting to address fractalkine and CX3CR1 as targets for developing new therapeutic drugs to treat these clinical diseases.

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13 SDF-1 and CXCR4 SDF-1 and CXCR4 are generally expressed in most organs, including brain, thymus, heart, lung, liver, kidney, spleen, intestine and bone marrow. SDF-1or CXCR4-deficient mice die at a late embryonic stage or within a few hours after birth, demonstrating a crucial role in embryonic development (Ma et al., 1998; Nagasawa et al., 1996; Zou et al., 1998). These mice have severely reduced B-lymphopoiesis, reduced fetal liver myelopoiesis and absence of bone marrow myelopoiesis (Ma et al., 1998; Nagasawa et al., 1996; Zou et al., 1998). In addition, they also have ventricular septum defects (Nagasawa et al., 1996). Collectively, these data demonstrate that SDF-1 and CXCR4 are essential for hematopoiesis, cardiogenesis, immune system and CNS development. Besides their functions in embryonic development, SDF-1 and CXCR4 are also involved in the pathogenesis of infectious and inflammatory process of several diseases such as rheumatoid arthritis (Nanki et al., 2000), atherosclerosis (Abi-Younes et al., 2000) and allergic airway disease (Gonzalo et al., 2000). Rheumatoid synovial T cells express high levels of CXCR4 while synovial endothelial cells express SDF-1, which together contribute to T cell accumulation within rheumatoid synovium (Nanki et al., 2000). SDF-1 can induce aggregation of CXCR4-expressing platelets and is highly expressed in smooth muscle cells, endothelial cells, and macrophages in human atherosclerotic plaques but not in normal vessels (Abi-Younes et al., 2000). Chemokine Receptors and HIV-1 Another important consequence of chemokine receptor expression is to mediate HIV-1 infection. CCR5 and CXCR4 are the two major co-receptors for HIV-1. The envelope glycoprotein gp120 binds to CD4, which triggers a structural change and

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14 exposes the binding site for the co-receptor. Further structural changes happen after the co-receptor is bound and finally viral and cellular membranes fuse together. In vivo, HIV-1 mainly infects cells that express CD4 and CCR5 or CXCR4 including CD4+ T lymphocytes, macrophages and dendritic cells. Macrophage (M)-tropic viruses primarily use CCR5 to infect cells of myeloid lineage and CD4+ primary T cells. T-tropic viruses mainly use CXCR4 to enter CD4+ T cells. However this distinction is blurred since primary isolated X4 variants of HIV-1 have been reported to infect macrophages productively (Simmons et al., 1998; Verani et al., 1998; Yi et al., 1998; Yi et al., 1999). During viral transmission, cells of myeloid lineage are the first to be infected, preferentially by R5 viruses and contribute to the viral pathogenesis throughout the whole course of the disease (Spira et al., 1996; van't Wout et al., 1994; Zhu et al., 1993). The role of CCR5 in HIV transmission is illustrated in a 32 CCR5 polymorphism where a 32 base pair deletion in the coding region results in failure of the receptor to reach the cell membrane (Benkirane et al., 1997). Homozygous individuals are healthy, CCR5 negative, and resistant to HIV infection via sex, blood contact or mother-to-child transmission (Philpott et al., 1999; Wilkinson et al., 1998). At later stages of HIV-1 infection, T-tropic viruses emerge when AIDS-defining symptoms are first observed. T-tropic viruses are more cytopathic and infected CD4+ T-lymphocytes have a short half life of 1-1.5 days. Several mechanisms have been put forward to explain the tropism evolution in the progress of AIDS, including cytokine upregulation of CXCR4 and downregulation of CCR5 on CD4+ T-lymphocytes (Valentin et al., 1998), increased expression of R5 variants blocking chemokines at the sites of HIV-1 infection (Tedla et al., 1996; Trumpfheller et al., 1998) and increased expression

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15 of CC chemokines by X4 virus infection (Margolis et al., 1998). The phenomenon is still perplexing and the answers are not yet satisfying. The principal pathway for HIV-1 to enter the CNS is through infected monocytes. Chemokines secreted by microglia and astrocytes, such as MCP-1, seem to regulate monocyte migration through the blood brain barrier (Weiss et al., 1998). In the CNS, HIV-1 mainly infects microglia, although astrocytes and neurons also express CCR5 and CXCR4 (Watkins et al., 1990; Wiley et al., 1986). Microglia express CCR5, CXCR4 and other potential HIV-1 co-receptors such as CCR3 (He et al., 1997; Albright et al., 1999). Among these receptors, CCR5 is the main co-receptor to mediate HIV-1 infection (Albright et al., 1999). CXCR4 and CCR3 have also been reported to mediate HIV-1 infection, but at much lower efficiency (Gabuzda and Wang, 1999). Infection of the brain by HIV-1 can cause reactive astrocytosis, myelin pallor and infiltration of monocytoid cells, culminating in HIV encephalitis. Chemokine receptor expression, such as CXCR4, is increased in HIV encephalitis and localized on microglia, astrocytes, neurons and infiltrating leukocytes (Sanders et al., 1998; Westmoreland et al., 2002; McManus et al., 2000). When HIV infection progress towards AIDS, infected macrophages and microglia are activated and release inflammatory cytokines and neurotoxic substance, causing neuron loss and HIV-associated dementia (HAD) (Kolson et al., 1998). In addition to the major co-receptors, CCR5 and CXCR4, other chemokine receptors, CCR2 (Doranz et al., 1996), CCR3 (He et al., 1997; Doranz et al., 1996), CCR8 (Horuk et al., 1998; Jinno et al., 1998; Rucker et al., 1997), CCR9 (Choe et al., 1998) and CX3CR1 (Combadiere et al., 1998; Rucker et al., 1997) have been demonstrated to mediate HIV-1 infection in primary or transfected cells in vitro. For

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16 example, CX3CR1 expressed on NIH3T3 cells can function as a co-receptor in an HIV-1 envelope-mediated cell-cell fusion assay, and fractalkine specifically inhibits this fusion (Combadiere et al., 1998). A recently identified CX3CR1 haplotype which affects two amino acids, I249 and M280, is associated with accelerated AIDS progression in a French Caucasian cohorts (Faure et al., 2000). This study suggested a possible role of CX3CR1 in mediating HIV-1 infection and/or rapid progression to the AIDS phenotype. Macrophage and HIV-1 Macrophages are one of the predominant cell types infected with HIV-1 in many tissues, including brain, lung and lymph nodes (Koenig et al., 1986; Pantaleo et al., 1991; Salahuddin et al., 1986). Tissue macrophages including alveolar macrophages, peritoneal macrophages, placental macrophages and microglia are major targets for infection. Unlike infected CD4+ T-lymphocytes that have a short half life of 1-1.5 days, macrophages are quite resistant to the cytopathic effects of the virus and thus, may provide a reservoir for persistent infection and virus dissemination. The two major co-receptors of HIV-1, CXCR4 and CCR5 are both expressed on macrophages. Macrophages are primarily infected by CCR5 utilizing viruses (R5) in early stages of infection but at later stages of the disease they are often infected by dual tropic CXCR4 using variants (X4), which are associated with disease progression. TGF-: a Multifunctional Growth Factor TGF-1 was discovered in 1981 by its capacity to induce anchorage-independent growth of normal rat kidney and fibroblast cell lines, i.e., to induce transformation (Moses et al., 1981; Roberts et al., 1981). Three isoforms of TGFhave been identified, TGF-1, -2 and -3, in mammals. They are 64-82% identical in amino acid sequence

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17 and largely interchangeable in biological functions. However, each is encoded by a separate gene and unique promoter and displays distinct spatial and temporal patterns of expression (Roberts and Sporn, 1992; Sporn and Roberts, 1990). TGFis synthesized as a preproprotein, consisting of a signal peptide, prodomain and mature protein from Nto C-terminal. Cleavage of the signal peptide and pro-domain releases the mature protein of 110-140 amino acids and homoor hetero-dimerization of these proteins generates the biologically active molecules. Newly secreted TGFis inactive due to its association with latency-associated peptide and the latent TGFbinding protein. Low pH, irradiation, binding to thrombospondin and proteolysis break the association and activate the protein. Platelets are an abundant source of TGF-1 (Assoian et al., 1983). In addition, many other cell types express TGF-1, including monocytes, macrophages, astrocytes and lots of other cell types. TGF-1 is a multifunctional growth factor and plays crucial roles in embryonic development, hematopoiesis, angiogenesis, cell proliferation and apoptosis, immune functions, wound healing and tumor biology. TGF-1 elicits responses through activation of two types of serine/threonine kinase receptors: type I (TRI) and type II (TRII) receptors. TGF-1 initially binds to TRII and subsequently recruits and activates TRI. The activated receptors then phosphorylate and activate receptor-regulated Smads (R-Smads), which include Smad-2 and -3 for TGFactivation. R-Smads bind to common mediator Smads (Co-Smads) forming heteromeric complexes that translocate into the nucleus to activate gene transcription. Co-factors and coactivators are often required to bind to DNA together with Smads for activation. The third class of Smads, inhibitory

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18 Smads, counteract the activity of R-Smad/Co-Smad complexes by competing for the binding sites on activated receptors (Massague and Wotton, 2000). TGF-1 has complicated functions in immune response. This is illustrated in TGF-1 null mice (Kulkarni et al., 1993). These mice develop a rapid waste syndrome after 2 weeks of normal growth and die at 3-4 weeks old. Furthermore, excessive inflammatory responses with multiorgan leukocyte infiltration are evident in these animals. Accumulative studies show that TGF-1 bidirectionally regulates of macrophages and microglia (Ashcroft, 1999). At early stages of inflammation, TGF-1 induces potent migration of these cells and mediates their infiltration through the endothelial wall into sites of inflammation. TGF-1 also stimulates the production of cytokines, chemokines and chemokine receptors from these cells. In addition, TGF-1 has anti-inflammatory effects on macrophages and microglia. Microglia synthesize TGF-1 in response to pro-inflammatory cytokines such as IL-1, IFNand TNF-(da Cunha et al., 1993; Chao et al., 1995; Hu et al., 1995). In turn, TGFcan inhibit TNF--induced superoxide production (Hu et al., 1995), IFNinduced cytokine (IL-1, IL-6 and TNF-) synthesis and GM-CSF induced proliferation in microglia (Suzumura et al., 1993). In macrophages TGF-1 suppresses proinflammatory cytokine production, including IL-8, GM-CSF and TNF-(Fadok et al., 1998). In the CNS, TGF-1 has profound functions on neurons, astrocytes, microglia and oligodendrocytes. Knock-out of TGF-1 resulted in widespread increase of neuronal degeneration and animal susceptibility to neuronal injury (Brionne et al., 2003). GFAP-driven overexpression of TGF-1 in astrocytes spared neurons from acute and chronic

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19 injury (Brionne et al., 2003), suggesting neuroprotective functions for TGF-. Intracerebroventricular administration of TGF-induced astrocyte hypertrophy and upregulation of GFAP, which is similar to the astrocyte reaction seen after experimental injury or in Alzheimer’s diseases (Laping et al., 1994). In diseases such as AIDS, Alzheimer’s disease and multiple sclerosis, TGF-1 expression is altered, implicating this cytokine in the pathogenesis of these diseases. An inverse relationship between the levels of TGF-1 and severity of multiple sclerosis is noted (Link, 1994; Link et al., 1994). TGF-1 can reduce the incidence and severity of paralytic episodes in antigen stimulated or adoptive transfer-mediated models of EAE (Johns et al., 1991; Kuruvilla et al., 1991; Racke et al., 1991; Santambrogio et al., 1993). In TGFoverexpressing, aged transgenic mice, an increase in A clearance and decrease of plaque burden was seen (Wyss-Coray et al., 2001). In individuals infected with HIV-1, blood and tissue levels of TGF-1 are increased and it is induced from monocytes, macrophages and astrocytes (Hu et al., 1996; Kekow et al., 1990; Navikas et al., 1994; Wahl et al., 1991). Although the data are limited, TGF-1 regulation of chemokine receptors is implicated in immune system development, immune response, tumor and viral pathogenesis. Treatment of immature dendritic cells with TGF-1 resulted in increased CCR1, CCR3, CCR5, CCR6, CXCR4 and an enhanced migration towards the correspondent ligands (Sato et al., 2000). On the other hand, TGF-1 enhanced TNF--induced downregulation of these chemokine receptors and inhibited their migration towards the ligands (Sato et al., 2000). CXCR3 and CXCR4 expression is increased by TGF-1 on NK cells and so are their migration tendency towards IP-10 and SDF-1

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20 (Inngjerdingen et al., 2001). Homing of nave T-cells in mice was enhanced due to upregulation of CXCR4 and subsequent chemotaxis towards SDF-1 by TGF-1 (Franitza et al., 2002). TGF-1 regulation of chemokine receptors also are involved in AIDS. It increases CXCR4 expression on dendritic cells which results in their susceptibility of HIV-1 infection (Zoeteweij et al., 1998). In the CNS, TGF-1 upregulated CCR1 expression on murine astrocytes and in turn their migration capacity towards MIP-1(Han et al., 2000). Regulation of G-protein Coupled Receptor Signal Transduction by RGS Proteins Chemokine receptors are members of the seven transmembrane, G-protein coupled receptor superfamily. They are coupled to heterotrimeric G proteins, which consist of , and subunits. There are four subfamilies of G subunits based on their function and amino acid sequence homology: Gs, Gi, Gq and G12. Most G subunits that mediate chemokine receptor function are Gi. The binding of chemokines to receptors induces conformational change of the receptor and enhances guanine-nucleotide-exchanging activity of the receptor, resulting in the GDP exchange for GTP on G subunit. This leads to the dissociation of and subunits. Freed and subunits then transduce signals including inhibition of adenyly cyclase, activation of phospholipase C and stimulation of MAPK pathway. The intrinsic GTPase activity of G hydrolyzes GTP into GDP and G is deactivated. In this model, the lifetime of GTP loaded G subunit determines the duration of the activated signaling. There are positive regulators that accelerate the GTP-GDP exchange on G and negative regulators that increase the GTPase activity. RGS (Regulators of G-protein signaling) proteins are GAPs (GTPase-activating proteins) for heterotrimeric G

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21 proteins that accelerate the hydrolysis of GTP into GDP by G subunit, ultimately attenuating GPCR signaling. RGS proteins are highly diversified and multifunctional signaling proteins. They share a conserved signature 120 amino acid RGS domain that binds directly to the G subunit to regulate GPCR signaling. So far, more that 30 mammalian RGS proteins have been identified and classified into 7 subfamilies according to amino acid identities within the RGS domain. Investigations of RGS regulation of chemokine receptor function are limited. In 1998, Bowman et al. demonstrated that RGS1, RGS3 and RGS4 inhibited chemotaxis of L1/2 cells to MCP-1 and IL-8 (Bowman et al., 1998). Subsequently, several groups have reported interaction between RGS and chemokine receptors, most of which were focused on lymphocyte activation and migration. In antigen activated B cells, RGS1 and RGS2 were upregulated and RGS1 could inhibit the migration of a mature B cell line to SDF-1, BLC (B-lymphocyte chemoattractant) and ELC (EBV-induced molecule 1 ligand chemokine). These changes contribute to the regulation of B cell migration within lymphoid tissues (Reif and Cyster, 2000). In transgenic mice that express RGS16 in CD4+ and CD8+ cells, T lymphocyte migration toward SDF-1 was dramatically inhibited. Migration of CD4+ lymphocytes that express CCR3, CCR5 and CXCR4 into the lung after acute inhalation challenge with allergen was also reduced. These data showed that RGS16 could attenuate the responsiveness of CCR3, CCR5 and CXCR4 towards inflammatory stimuli (Lippert et al., 2003)The profound effects of RGS proteins on chemokine receptors in normal immune response and in disease states make them a group of tantalizing drug targets with therapeutic potentials. However, there are

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22 still gaps of knowledge needed to be filled. For example, i) the specificity of RGS proteins on chemokine receptors is not well characterized; ii) we are still not clear how RGS proteins interact with G-proteins and chemokine receptors dynamically; and iii) a more complete understanding of regulation and tissue expression of RGS proteins is still required to understand their roles in physiology and pathological conditions. Specific Aims: Based on the background information, we proposed that TGF-1 could regulate the expression and function of chemokine receptors in the CNS and peripheral immune systems. Three specific aims evolved from this line of investigation. They are: I). to characterize the effects of TGF-on microglial expression and function of CX3CR1. II). to identify a potential mechanism to account for the TGF-effects on fractalkine-stimulated activation of ERK1,2. III). to characterize the effects of TGF-1 on human macrophage expression and function of CXCR4.

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23 Table 1-1. Chemokine receptors and their ligands. Ligands Chemokine Receptor Acronyms Systematic Name CC Chemokine Receptors CCR1 MIP-11RANTES MCP-1, 2, 3, 4 HCC-1,4 MPIF-1 CCL3, CCL4 CCL5 CCL2, 8, 7, 13 CCL14, 16 CCL23 CCR2 MCP-1, 2, 3, 4, 5 CCL2, 8, 7, 13 CCR3 Eotaxin, Eotaxin-2,3 RANTES MIP-1 MCP-2,3 CCL11, CCL24, 26 CCL5 CCL15 CCL8,7,13 CCR4 RANTES MDC TARC CCL5 CCL22 CCL17 CCR5 MIP-1RANTES CCL3, 4 CCL5 CCR6 MIP-3CCL20 CCR7 MIP-3SLC CCL19 CCL21 CCR8 I-309 vMIP-I CCL1 CCR9 TECK CCL25 CCR10 Eskine CCL27 CCR11 MCP-1, 2, 4 CCL2, 8, 13 CXC Chemokine Receptors CXCR1 IL-8 GCP2 CXCL8 CXCL6 CXCR2 IL-8 GCP2 ENA-78 NAP-2 GroCXCL8 CXCL6 CXCL5 CXCL7 CXCL1, 2, 3 CXCR3 IP-10 MIG I-TAC CXCL10 CXCL9 CXCL11 CXCR4 SDF-1CXCL12 CXCR5 BCA-1 CXCL13 CX3C Chemokine Receptor CX3CR1 Fractalkine CX3CL1 C Chemokine Receptor XCR1 Lymphotactin-XCL1, 2

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CHAPTER 2 TGF-UPREGULATES CX3CR1 EXPRESSION AND INHIBITES FRACTALKINE-STIMULATED SIGNALING IN RAT MICROGLIA Introduction It is now well established that fractalkine (CX3CL1) and its receptor, CX3CR1, are constitutively expressed in the CNS although the expression of these genes are regulated in a variety of CNS diseases and pathological insults. Fractalkine, the unique member of CX3C chemokine family, is primarily expressed on neurons while the majority of CX3CR1 is associated with microglial cells (Harrison et al., 1998; Nishiyori et al., 1998). This pattern of expression suggests a crosstalk between neurons and microglial cells through this ligand–receptor pair. Additional roles for fractalkine in the CNS could involve recruitment of CX3CR1-expressing cells into the CNS subsequent to inflammatory insults to the brain or spinal cord. At a cellular level, recent studies have established that fractalkine can induce, via activation of CX3CR1, a cascade of signal transduction events in microglial cells, including stimulation of intracellular Ca2+ mobilization (Harrison et al., 1998; Boddeke et al., 1999), as well as ERK1/2 and Akt/PKB phosphorylation (Maciejewski-Lenoir et al., 1999). Fractalkine also has been shown to inhibit LPS-induced TNFrelease from microglia, implicating an anti-inflammatory function of this chemokine (Zujovic et al., 2000). Collectively, these data suggest that fractalkine modulates microglial migration, activation and survival. However, studies aimed at establishing roles for fractalkine and CX3CR1 in CNS pathology are still deplete, with most data derived from studies of either cell culture 24

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25 systems or expression analysis of fractalkine and CX3CR1 in normal or pathologic tissue. Results from the characterization of the expression of fractalkine and CX3CR1 in the CNS suggest that changes in fractalkine expression are directed toward attracting or activating CX3CR1-expressing cells within sites of injury. However, CNS phenotypes associated with either fractalkine or CX3CR1 gene disrupted mice have not provided evidence for these or any such roles (Jung et al., 2000; Cook et al., 2001). A recent report suggests that fractalkine may participate in post ischemic brain injury; a slight reduction in infarct size subsequent to transient focal cerebral ischemia was apparent in fractalkine gene depleted mice (Soriano et al., 2002). Microglial cells serve as resident immune cells in the brain. In response to certain stimuli, they become activated to perform the function of macrophages including antigen presentation, free radical and NO secretion, synthesis of a variety of cytokines, chemotaxis and phagocytosis. In a variety of CNS diseases such as multiple sclerosis (Li et al., 1993; Prineas and Wright, 1978), and its animal model EAE (Bauer et al., 1994), Alzheimer’s disease (Meda et al., 1995) and AIDS dementia (Price et al., 1988), microglial cells become activated and release substances toxic to neurons and oligodendrocytes. In these various scenarios, the expression levels of chemokines and their receptors have been shown to change, implying some involvement in the disease process (Asensio and Campbell, 1999). A few chemokine receptors such as CCR5, CXCR4 and CX3CR1 have been localized to microglial cells and changes in their expression may be critical in regulating chemokine-dependent functions in the CNS (Bacon and Harrison, 2000).

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26 Cytokines such as IFNand TGF-, together with LPS and other factors, play major roles in regulating the expression of chemokine receptors. TGFconsists of three closely related isoforms (-1, -2 and -3), displaying broad functional diversity including inhibition and stimulation of cell proliferation, immune suppression, neuroprotection and modulation of cytokine production. TGFis nearly absent in normal brain while increased expression by astrocytes and microglia is evident in the injured or diseased CNS (O’Brien et al., 1994; Kiefer et al., 1995; Pratt and McPherson, 1997). TGFmay function as a potent suppressor of microglial cell activation and play an important role in neurodegenerative diseases and CNS trauma (Suzumura et al., 1993; Semple-Rowland et al., 1995; Lodge and Sriram, 1996). It has been shown to modulate chemokine and chemokine receptor expression both in peripheral systems and within the CNS. Previous data showed that TGF-1 upregulated CXCR3 and CXCR4 expression on human NK cells (Inngjerdingen et al., 2001) and CCR1 expression on murine astrocytes (Han et al., 2000). TGFexerts its functions through a group of signaling proteins called Smads. TGFactivates cytoplasmic, receptor-regulated Smads (Smad2, Smad3), which in turn associate with common mediator Smads (Smad4). These complexes translocate into the nucleus, bind DNA, and activate gene transcription (Massague and Wotton, 2000). Co-factors and co-activators such as AP1 and SP1 are often required with Smads to bind to DNA and promote transcriptional activation (Wrana, 2000). Following facial motor nerve axotomy in the adult rat, microglial cells are activated and recruited rapidly to the injured motor neuron pool. In this scenario, microglial cells do not become phagocytic and the injured facial motor nerves regenerate. Following peripheral facial motor nerve injury, both microglial TGF-1 and CX3CR1 are

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27 upregulated in a comparable time-dependent manner within the facial nucleus suggesting that these two cytokine systems interact (Kiefer et al., 1993; Harrison et al., 1998). To explore the relationship between TGF-1 and fractalkine in the CNS, we examined the effects of TGF-1 on CX3CR1 mRNA, protein and fractalkine-dependent stimulation of signal transduction cascades in primary cultures of rat microglia. In this report, we show that TGF-1 can increase CX3CR1 expression on rat microglial cells both at the mRNA and protein levels without changing its affinity for fractalkine. Although CX3CR1 is increased on microglial cells, stimulation of ERK1/2 by fractalkine is diminished by TGF-1. Furthermore, the mechanism by which TGF-1 increases CX3CR1 in microglia is likely due to enhanced transcription of the CX3CR1 gene. Materials and Methods Primary Microglial Cell Cultures Cerebra were dissected from newborn Sprague–Dawley rats, stripped of meninges and mechanically minced in solution D (137 mM NaCl, 5.4 mM KCl, 0.2 mM NaH2PO4, 0.2 mM KH2PO4, 1 g/l glucose, 0.25 g/ml fungizone, 106 U/l penicillin/streptomycin, pH 7.4). The tissues were digested with 0.25% trypsin at 37C on a bidirectional rotator for 30 min. An equal volume of DMEM (Life Technologies, Rockyville, MD) containing 10% FBS (Life Technologies) was added to stop the trypsin and the mixture was passed through 130-M Nitex filter (Tetko, Brairdiff Manor, NY). The mixture was centrifuged at 400 g for 10 min. The pellet was resuspended, filtered with 40-M Nitex filter and plated into 0.01 g/l poly-L-lysine(Sigma, St. Louis, MO) coated T175 flasks (Sarstedt, Newton, NC) at a density of 1.5 brains per flask in DMEM containing 10% FBS. The cultures were incubated at 37 C with 8% CO2. After 3 days, the medium was changed

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28 and left for another 4 days to favor the proliferation of microglia. To harvest microglia, the flasks were shaken at 100 rpm for 1 h. The medium was centrifuged and the cells were replated in DMEM 10% FBS at the indicated cell densities for the various experiments described below. After 1 h, the medium was aspirated to remove any nonadherent cells and replaced with fresh medium. The adherent cells contain more than 95% microglia. Two days after plating, the cells were used for experiments. Northern Blot Analysis Microglial cells were seeded at a density of 4 million cells/100 mm dish. Different concentrations of human TGF-1 (R&D Systems, Minneapolis, MN) were applied to the cells for various times as indicated. Total RNA was extracted with TRIZOL reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. Equal amounts of RNA were electrophoresed through agarose, transferred to nylon membranes, and subjected to Northern blot analysis (Church and Gilbert, 1984). 32P-labeled cDNAs encoding rat CX3CR1 and rat cyclophilin were generated using random prime labeling of the isolated inserts and were labeled to a specific activity of 1-2 9 dpm/g DNA. The washed blots were exposed to X-ray films and bands were visualized by autoradiography. Whole Cell Radioligand Binding Microglial cells (500,000/well) were seeded into 12-well plates. The cells were stabilized for 1 days prior to the binding assay. Subsequent to the TGF-1 treatment, cells were then washed once with room temperature PBS and incubated in binding buffer (HBSS, 0.1% BSA, pH 7.4) with various concentrations of 125I-labeled fractalkine at room temperature for 1 h. Nonspecific binding was determined in parallel reactions using

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29 30 nM of fractalkine chemokine domain (R&D Systems, Minneapolis, MN). In some instances, 0.01% sodium azide was included in the binding reaction. Binding reactions were terminated by washing the cells with ice-cold binding buffer three times. Bound radioactivity was determined by lysing the cells with 0.2 M NaOH and counting in a gamma counter. Data were analyzed using GraphPad Prism. Analysis of ERK1/2 and p38 MAPK Phosphorylation by Western Immunoblotting Microglia (26 cells) were seeded into the wells of a six-well plate. Two days later, the cells were treated with serum-free DMEM with or without 2 ng/ml TGF-1 for 16 h. Cells were rinsed with serum-free DMEM and further incubated in serum-free DMEM for 2 h. Various concentrations of fractalkine were then added to the cells and incubated for times indicated below. To terminate the reactions, cells were treated with lysis buffer (PBS, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, pH 7.4), sonicated and centrifuged at 14,000 rpm for 10 min. SDS-PAGE sample buffer was added to aliquots of supernatant and then boiled for 5 min. The proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane (Osmonics, Westerbrough, MA). The membranes were blocked in 5% nonfat milk TBS-t (20 mM Tris, 150 mM NaCl, 0.1% Tween, pH 7.4) for 30 min and then incubated in primary antibody overnight at 4 C. The membranes were washed three times (10 min per wash) with TBS-t and subsequently incubated in secondary antibody for 1 h at room temperature. The membranes were washed and then developed for 5 min with Pierce chemiluminescent kit (Pierce, Rockford, IL). The membrane was finally exposed to ECL film (Amersham, Buckinghamshire, UK). To detect the amount of total ERK1/2 and p38 MAPK, membranes were stripped with 0.2 N NaOH for 2 min at room temperature, washed twice with TBS-t and re-blocked with 5%

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30 nonfat milk TBS-t. The following primary antibodies (all obtained from Cell Signaling Technology, Beverly, MA) were used in the Western blot analysis: anti-phosphorylated ERK1/2 monoclonal antibody; anti-phosphorylated p38 MAPK polyclonal antibody; anti-ERK1/2 polyclonal antibody; anti-p38 MAPK polyclonal antibody. The secondary antibodies used were anti-mouse IgG antibody (Sigma) and anti-rabbit IgG antibody (Cell Signaling Technology). Isolation of DNA Sequences of the Rat CX3CR1 Promoter A rat genomic DNA library (Lambda Fix II, Stratagene) was screened (1.6 million pfu) with a pair of complimentary and overlapping oligonucleotides. The specific oligonucleotides were 5V-TGG CAC TTC CTG CAG-3V and 5’-GAG CAG CTG GGG ACG GGG ACT TCT GCA GGA-3’. The oligonucleotide sequences correspond to DNA sequences in the most 5’ region of the rat CX3CR1 cDNA and lie in an exon distinct from the exon that contains the protein coding sequences. The oligonucleotides were annealed at room temperature in the presence of labeled 32P-dCTP, dATP, dGTP, dTTP and the Klenow fragment of DNA polymerase I. Five bacteriophage hybridizing to the radiolabeled oligonucleotide pair were plaque purified and two independent bacteriophage clones were subjected to restriction endonuclease and DNA sequence analysis. Results TGF-1 Enhances Microglial CX3CR1 mRNA in a Time and Concentration-dependent Manner Previous studies have shown that mRNA levels of both TGF-1 and CX3CR1 were upregulated, in a comparable time-dependent manner, within the rat facial motor nucleus after peripheral nerve axotomy (Kiefer et al., 1993; Harrison et al., 1998). To investigate

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31 the relationship between TGF-1 and expression of CX3CR1, primary cultured rat microglial cells were treated with various concentrations of TGF-1 (0.001 ng/ml) for 16 h and total RNA was extracted. Northern blot analysis of stimulated microglial cells showed a TGF-1 concentration-dependent effect on CX3CR1 mRNA accumulation, reaching a maximal increase of 7– 8-fold after treatment with 1 ng/ml TGF-1 (Fig. 2-1A). In subsequent experiments, a TGF-1 concentration of 2 ng/ml was chosen in order to characterize the time dependence of TGF-1-induced CX3CR1 mRNA expression. Fig. 2-1B shows that as early as 4 h after TGF-1 treatment CX3CR1 mRNA was increased and reached a maximal level after 16 h of treatment. It has been shown that primary cultures of both microglial cells and astrocytes express CX3CR1 (Jiang et al., 1998; Maciejewski-Lenoir et al., 1999). To examine the effect of TGF-1 on the expression of CX3CR1 in both cell types, primary cultures of rat astrocytes and microglial cells were individually treated with 2 ng/ml TGF-1 for 16 h and Northern blot analysis was carried out. Fig. 2-2 shows that CX3CR1 was expressed in astrocytes but not increased with TGF-1 treatment. The data indicated that the effect of TGF-1 on CX3CR1 expression is cell-type specific. TGF-1 Induces CX3CR1 Protein: Time and TGF-1 Concentration Dependence Microglial cells were treated with different concentrations of TGF-1 for various times. 125I-fractalkine binding analysis was carried out on TGF-1-treated and untreated microglial cells in order to determine the effect of TGF-1 on CX3CR1 protein expression. Whole cell binding analysis demonstrated that TGF-1 stimulated CX3CR1 protein expression by microglial cells in a timeand -concentration-dependent manner. Maximal levels of CX3CR1 protein were seen after treatment of microglial cells with 1–

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32 10 ng/ml TGF-1 for 16 h (Fig. 2-3A and B). Inclusion of sodium azide in the binding reactions (at 0.01%) had no effect on the binding of 125I-fractalkine to either the TGF-1-treated or control treated cells (Fig. 2-3C), indicating that the increase in 125I-fractalkine associated with the TGF-1-treated cells is due to increased cell surface binding as opposed to an increase in the amount of receptor-dependent internalization of the radioligand. To confirm this, a more comprehensive binding analysis was undertaken. Scatchard analysis indicated that the affinity of CX3CR1 for fractalkine in the TGF-1-treated cells was similar to that of the control treated microglial cells. Fig. 2-3D depicts a representative Scatchard analysis of 125I-fractalkine binding to control and TGF-1-treated microglia while Table 2-1 summarizes the results from five independent 125Ifractalkine binding experiments. The data indicate that TGF-1 upregulated CX3CR1 expression on microglial cells 34-fold without changing its affinity for fractalkine. Fractalkine-induced ERK1/2 Phosphorylation is Inhibited by Pretreatment with TGF-1 Previous data showed that fractalkine can induce ERK1/2 phosphorylation in microglial cells (Maciejewski-Lenoir et al., 1999). Since CX3CR1 was upregulated by TGF-1, we hypothesized that the fractalkine-induced ERK1/2 stimulation would be enhanced in the TGF-1-treated cells, due to either an increase in the efficacy of fractalkine in stimulating phosphorylation of ERK1/2 and/or an increase in the potency for the stimulation. We treated microglial cells with 2 ng/ml TGF-1 or serum-free medium for 16 h, changed into fresh medium for 2 h and then treated with fractalkine for 10 min. Microglial cells treated with medium alone were stimulated significantly by fractalkine in a concentration dependent way, showing maximal activation at 10-30 nM

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33 fractalkine (Fig. 2-4). However, little, if any, fractalkine-stimulated phosphorylation of ERK1/2 was seen in TGF-1treated cells. The basal levels of phosphorylated ERK1/2 were also diminished by TGF-1. We suspected that fractalkine stimulation could be shifted to other MAPK pathways, so we investigated fractalkine activation of p38 MAPK signaling. No obvious increase in p38 MAPK phosphorylation after 30 min of fractalkine treatment was seen in either control or TGF-1-treated microglial cells. However, the basal level of p38 MAPK phosphorylation was increased by TGF-1. We have previously reported that cultured microglia do not respond to the chemokine domain of fractalkine in terms of increasing intracellular calcium levels (Harrison et al., 1998). Both TGF-1-treated and control microglia were examined for fractalkine-dependent stimulation of intracellular calcium mobilization with the anticipation that the increased receptor density on the TGF-1-treated cells could potentially make them responsive to fractalkine in this assay system. However, similar to control treated microglia, TGF-1-treated microglia did not show elevations of intracellular calcium upon stimulation with fractalkine chemokine domain (data not shown). TGF-1 Does Not Affect CX3CR1 mRNA Stability To begin to address the mechanism by which TGF-1 upregulates CX3CR1 mRNA expression, experiments were performed to determine the effect of TGF-1 on the stability of CX3CR1 mRNA. Microglial cultures were treated with medium alone or 2 ng/ml TGF-1 for 16 h and then Actinomycin D was added to inhibit transcription. Total RNA was extracted at various time points and examined for the level of CX3CR1 mRNA by Northern blot analysis. The half-lives of CX3CR1 mRNA in TGF-1-treated or untreated microglia were found to be similar, both about 3 h (Fig. 2-5). This indicates that

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34 TGF-1 did not change the stability of CX3CR1 mRNA in microglial cells, further suggesting that the increase in steady state levels of CX3CR1 mRNA is due to regulation of the CX3CR1 gene at the transcriptional level. In support of this, we have determined that the rat CX3CR1 gene contains at least two potential Smad binding elements (SBEs) in the putative promoter sequence. Our previous report indicated the rat CX3CR1 gene contains at least two exons (Harrison et al., 1994). We screened a rat genomic DNA library with radiolabeled pair of overlapping oligonucleotides whose sequences correspond to the most 5’ sequences of the known cDNA for rat CX3CR1. Two independent bacteriophage clones were isolated that each contained 3.3 kbp BamHI, 7 kbp EcoRI and 8 kbp HindIII fragments that hybridized to this probe. The BamHI, EcoRI and HindIII fragments are identical in size to the minor rat CX3CR1 cDNA hybridizing bands previously reported. Fig. 2-6 depicts a schematic of the rat CX3CR1 gene. The gene is comprised of two exons and one intron; the second exon contains all of the protein coding sequences. DNA sequence analysis of the 3.3 kbp BamHI fragment, upstream of the first exon sequences (~2.4 kbp), revealed several putative SP1 and AP1 transcription factor binding sites as well as motifs for NF-B binding. Two potential SBEs, one at position -1268 to -1261 (Smad 3), and another at -1154 to -1147 (Smad 4) of the sequence, were also identified. These latter elements likely represent targets for the TGF-1 regulation of the CX3CR1 gene. Discussion The principal goal of this study was to determine the effect of TGF-1 on CX3CR1 expression and function in primary rat microglial cells. The major findings of this work are three fold. Firstly, TGF-1 upregulated CX3CR1 expression, in terms of both steady

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35 state mRNA and cell surface protein levels. Secondly, despite the significant TGF-1dependent increase in CX3CR1, the function of the receptor was inhibited in the TGF-1-pretreated cells, as fractalkine was unable to stimulate any known signaling pathways normally associated with CX3CR1 activation. Thirdly, the TGF-1-dependent increase in CX3CR1 mRNA was not due to a change in message stability, and isolation of DNA sequences of the putative promoter region of the rat CX3CR1 gene revealed sequence motifs of Smad binding elements. These latter data indicate that the regulation of CX3CR1 in microglia by TGF-1 is due to enhanced transcription of the gene. A number of reports have shown that fractalkine is abundant in neurons while CX3CR1 is expressed on microglia in the CNS. The cellular expression patterns of these genes indicate an important contribution of fractalkine-CX3CR1 signaling involved in communication networks between neurons and microglia. More recent studies suggest that fractalkine is important in microglial migration, activation, survival and proliferation. Despite the preponderance of data from in vitro experimentation, no precise in vivo role for this chemokine in the CNS has emerged. No obvious CNS developmental abnormality in either fractalkine or CX3CR1 knockout mice has been revealed from characterization of the respective gene disrupted animals (Jung et al., 2000; Cook et al., 2001). Thus, the function of this chemokine system in the CNS requires more extensive characterization. Increased TGFexpression in the CNS has been reported for a number of diseases and following injury. TGFupregulation has been seen in brain ischemic injury, EAE and AIDS. In the normal adult brain, there is little or no TGFexpression in glial cells. However, following injury, astrocytic and microglial expression of TGFis increased. Pro-inflammatory cytokines such as TNFand IL-1 can also increase microglial

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36 expression of TGF-1 (da Cunha et al., 1993; Chao et al., 1995). Numerous studies show that TGF-1 suppresses the activation of microglia under inflammatory conditions. In the animal model of rat facial motor nerve axotomy, increased mRNA of TGF-1 was localized to microglia in the facial nucleus (Kiefer et al., 1993). Interestingly, the mRNA levels of both TGF-1 and CX3CR1 dynamically change in a correspondent time dependent manner, implicating some relationship between these two cytokine systems. Northern blot and radioligand binding analysis showed that both CX3CR1 mRNA and protein expression in the cultured microglia were upregulated by TGF-1 treatment in a timeand TGF-1 concentration-dependent manner. The regulation of steady state levels of CX3CR1 mRNA by TGF-1, in terms of this time and concentration dependence, is similar to other genes known to be regulated by this pleiotropic growth factor. In experiments designed to determine the stability of CX3CR1 mRNA in microglial cells treated with and without TGF-1, it was found that the half-life of the CX3CR1 mRNA was essentially identical in the control and TGF-1-treated cells. This result indicates that the observed increase in steady state levels of CX3CR1 mRNA was not due to a TGF-1-dependent increase in the stability of this transcript. Thus, the most likely mechanism for the increased mRNA is due to enhanced transcription of the CX3CR1 gene. In support of this, two potential Smad binding elements in the putative promoter region of the rat CX3CR1 gene were identified. The actual sequences of these two SBEs are similar to known consensus sequences for SBEs and represent binding motifs for both Smad-3 and Smad-4. The specific role and/or contributions of each of these SBEs to the TGF-1-dependent increase in CX3CR1 mRNA remains to be determined and will require promoter deletion analysis.

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37 In a similar fashion, TGF-1 increased CX3CR1 protein expression in the cultured microglial cells. 125I-fractalkine binding analysis indicated that TGF-1 increased CX3CR1 protein in a similar timeand concentration-dependent manner. Further analysis using sodium azide, a known inhibitor of receptor internalization, revealed that the apparent increase in 125I-fractalkine binding to the TGF-1treated microglial cells was not due to enhanced receptor internalization. Scatchard analysis showed that although CX3CR1 was upregulated on microglial cells, its affinity for fractalkine was not changed significantly. There was a trend toward reduced affinity of the CX3CR1 for fractalkine in the TGF-1-treated cells. This may be due to a relative increase in the ratio of receptors for G-proteins (most likely a Gi member). A common feature of agonist binding to GPCRs is a reduced affinity with increasing receptor density. The biological significance of this slight reduction in the affinity of CX3CR1 for fractalkine remains to be determined as the levels of fractalkine in the CNS, under either normal or pathological states are not formally known. Since CX3CR1 was upregulated by TGF-1, we anticipated that fractalkine would be either more potent and/or more efficacious in stimulating downstream signal transduction events in microglial cells. Fractalkine stimulation of ERK1/2 phosphorylation in microglial cells has been previously demonstrated and regulation of this intracellular signaling pathway may be involved in microglia migration, proliferation and synthesis of secretable cytokines (MaciejewskiLenoir et al., 1999). We have also determined that fractalkine can stimulate ERK1/2 phosphorylation in a fractalkine concentration-dependent manner. However, and to our surprise, fractalkine-induced ERK1/2 phosphorylation was inhibited by pretreatment with TGF-1. Microglia

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38 associated ERK1/2 phosphorylation has also been shown to be involved in the synthesis of pro-inflammatory factors such as TNFand nitric oxide (Bhat et al., 1998). Tan et al. (1999) showed that inhibition of ERK1/2 phosphorylation is the mechanism of the TGF-1 inhibition of CD40-induced microglial TNFsynthesis. Thus, inhibition of ERK1/2 phosphorylation may be a means by which TGF-1 could inhibit fractalkine-induced activation of microglia. A potential lack of potentiated ERK1/2 phosphorylation also prompted us to examine other signaling pathways with the hypothesis that a shift towards activation of these other intracellular signaling cascades underlay mechanisms of TGF-1 regulation of CX3CR1 signaling. Activation by fractalkine of p38 MAPK in MonMac6 cells has been previously reported (Cambien et al., 2001). However, we did not identify augmented signaling in the TGF-1-pretreated cells with respect to activation of p38 MAPK or stimulation of intracellular calcium mobilization. Despite our inability to identify enhanced signaling in the TGF-1treated cells, it is still possible that other unknown fractalkinestimulated signal transduction pathways may be augmented by the TGF-1 increase in CX3CR1 expression. Alternatively, TGF-1 may convert CX3CR1 from a signaling to a non-signaling receptor. Several non-signaling chemokine binding proteins have been identified, including Duffy and D6 (Murphy et al., 2000). Relevant to our findings, expression levels of CCR1, CCR2 and CCR5 on dendritic cells remain elevated after exposure of the cells to LPS and IL-10. Nonetheless, these receptors were unable to respond to the appropriate chemokine ligands in terms of promoting migration or stimulating intracellular signaling events (D’Amico et al., 2000). The physiological significance of an upregulated and uncoupled receptor is perplexing, although it could include a mechanism to scavenge chemokine ligand.

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39 We have previously reported increased levels of low molecular weight forms of fractalkine in the injured facial motor nucleus (Harrison et al., 1998). While the final destination of this injury-dependent increased fractalkine was not determined, it is intriguing to consider a role for TGF-1 in converting CX3CR1 from a signaling molecule to a scavenging receptor. Nonetheless, our results of fractalkine binding in the presence of sodium azide indicate that the binding of fractalkine to microglia is predominately a cell surface association, and the increased level of fractalkine binding was due to enhanced cell surface accumulation and not increased receptor internalization. While receptor internalization provides an efficient means to clear ligand from the cellular environment, it is still possible that the threeto fourfold increase in cell surface expression of CX3CR1 is sufficient to provide a molecular sink for increased extracellular fractalkine. Fractalkine differs from nearly all other chemokine family members in that it can be expressed as either a membrane-tethered or soluble form. While soluble fractalkine can induce microglia migration and stimulate a variety of intracellular signaling cascades, membrane-bound fractalkine is more likely playing a role in mediating cell adhesion phenomena between neurons and microglia. Fractalkinedependent cell adhesion has been determined to be independent of G-protein activation (Fong et al., 1998; Haskell et al., 1999). Although TGF-1 upregulated CX3CR1 expression on microglia, it simultaneously uncoupled the receptor from stimulating downstream signaling cascades. Therefore, it is attractive to speculate that TGFupregulated CX3CR1 expression could promote or increase adhesion of microglia to neurons expressing fractalkine. In this scenario, the role for the increased expression f CX3CR1 is to favor interactions between

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40 neurons and microglia such that other cell–cell signaling mechanisms are utilized. For instance, an enhanced microglial cell adherence to injured neurons may be a prerequisite for microglial cells to release factors that are critical for neuronal recovery and survival. Finally, it is worth noting that other chemokine receptor systems in the CNS have been shown to be modulated by TGF-1. CCR1 expression on astrocytes is upregulated by TGF-1 (Han et al., 2000), and unlike our observations with CX3CR1, has been shown to enhance astrocyte chemotaxis to the CCR1 ligand MIP-1. While astrocytes express CX3CR1, the receptor is not regulated by TGF-1 in this cell type. It is possible that Smad signaling in astrocytes and microglia differ or that different co-activators as well as other signal transduction events control the effects of TGF1 on CX3CR1 expression in astrocytes. Thus, the level of control by TGF-1 on chemokines in the CNS likely depends on the specific chemokine system as well as the specific cell types in which the interplay between these two cytokine systems is operating.

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41 Table 2-1. Kd and Bmax values of 125I-fractalkine binding to CX3CR1 on TGF-1-treated and untreated microglial cells. Shown are mean (S.E.M.) Kd and Bmax values of 125I-fractalkine binding to TGF-1-treated and -untreated microglial cells from five independent experiments. *, P<0.05 significantly different from control. *P<0.0553,440 12,900*4063521007974524472363365TGF-116,870 2,3471850410058231661952813078ControlMean S.E.M54321ExperimentBmax (sites/cell)0.93 0.340.800.302.240.540.78TGF-1 0.45 0.110.550.240.840.320.29ControlMean S.E.M54321ExperimentKd (nM) *P<0.0553,440 12,900*4063521007974524472363365TGF-116,870 2,3471850410058231661952813078ControlMean S.E.M54321ExperimentBmax (sites/cell)0.93 0.340.800.302.240.540.78TGF-1 0.45 0.110.550.240.840.320.29ControlMean S.E.M54321ExperimentKd (nM)

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42 CX3CR1CyclophilinAB Time:04816 24(hrs) TGF-1:--+ -+ -+ -+(2 ng/mL)CX3CR1Cyclophilin00.010.10.001101TGF-1:(ng/mL) Con-3-2-101 0 1 2 3 4 5 6 7 Log [TGF-1] (ng/mL) CX3CR1 mRNA Con481624 0 1 2 3 4 5 6 7Control TGF-1 Time (hr)CX3CR1 mRNA Fig. 2-1. Effects of TGF-1 on induction of CX3CR1 mRNA expression in rat microglial cells. (A) Concentration-dependent effect of TGF-1 on microglial CX3CR1 mRNA accumulation. Microglial cells were treated with medium alone or various concentrations of TGF-1 (0.001 ng/ml) for 16 h. Total RNA was isolated and subjected to Northern blot analysis (9 g/lane) as described in Materials and methods . The intensity of the CX3CR1 signal was quantified by densitometry and normalized to that of cyclophilin mRNA. The bar graphs depict the data normalized to expression in the control treated cells. The results are representative of two experiments. (B) Time dependence of TGF-1 effect on microglial CX3CR1 mRNA accumulation. Microglial cells were treated with medium alone or 2 ng/ml TGF-1 for the indicated times. The extracted RNA was subjected to Northern blot analysis (5 g/lane) as described in Materials and methods . The bar graphs depict the data normalized to expression in the control treated cells. The results are representative of two experiments.

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43 CX3CR1CyclophilinAstrocytes Microglia Con TGF-1 Con TGF-1 Fig. 2-2. TGF-1 affects the expression of CX3CR1 in microglia but not astrocytes. Rat microglial cells and astrocytes were each treated with and without 2 ng/ml TGF-1 for 16 h. Total RNA was extracted and subjected to Northern blot analysis (10 g/lane astrocyte RNA; 5 g/lane microglia RNA) as described in materials and methods. The results are representative of two experiments.

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44 0 10 20 30 0 1 2 3 4 5Control TGF-1 Time (hr)Relative BindingABC 00.01% 0 2 4 6 8Control TGF-1 [Sodium Azide]Relative Binding 0 1 2 3 4 5 6 7 0.00 0.05 0.10 0.15Control TGF-1 Bound ( 104sites/cell)Bound/Free (sites/cell/nM)D 0.001 0.01 0.1 1 10 0 0 1 2 3 4TGF-1 (ng/mL)Relative Binding Fig. 2-3. TGF-1 increases CX3CR1 protein expression on rat microglial cells. (A) Time dependence of TGF-1 effects on microglial CX3CR1 upregulation. Microglial cells were treated with medium alone or 2 ng/ml TGF-1 for various times (8 h), and then levels of 125I-fractalkine binding to the cells were determined in the whole cell binding assay. The concentration of 125I-fractalkine used in the assay was 0.8 nM; nonspecific binding was determined in the presence of 50 nM unlabeled fractalkine chemokine domain. The results represent the mean S.E.M. of three experiments. (B) Concentration dependence of TGF-1 effects on microglial CX3CR1 upregulation. Microglial cells were treated with different concentrations of TGF-1 (0.001–10 ng/ml) for 16 h and subjected to whole cell binding analysis as described above. The results represent the mean S.E.M. of four experiments. (C) Effect of sodium azide (0.01%) on 125I-fractalkine binding to TGF-1-treated and untreated microglial cells. Microglial cells were treated with medium alone or TGF-1 and then levels of 125I-fractalkine binding to the cells were determined in the whole cell binding assay. The results are expressed as the mean S.E.M. from five experiments. (D) Scatchard analysis of 125I-fractalkine binding to TGF-1-treated and untreated microglial cells. Whole cell binding analysis was conducted using increasing concentrations of 125I-fractalkine. Nonspecific binding was determined in the presence of 30 nM unlabeled fractalkine chemokine domain. The data represent one of five experiments.

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45 FKN Control TGF-10 1 3 10 30 0 1 3 10 30 nM Phospho-ERK1Phospho-ERK2 ERK2 ERK1 0131030 0.0 0.5 1.0 1.5 2.0 2.5Control TGF-1 ****FKN (nM)Phospho-ERK 1,2 FKNControl TGF-10 1 3 10 30 0 1 3 10 30 nMAB Phospho-p38 MAPKP38 MAPK Fig. 2-4. Stimulation of intracellular signaling by fractalkine in TGF-1-treated and control microglial cells. Microglial cells were treated with 2 ng/ml TGF-1 or medium alone for 16 h and changed into fresh serum free medium for 2 h. (A) Cells were then treated with increasing concentration of fractalkine for 10 min and the level of phosphorylated ERK1/2 was determined by Western blot analysis according to procedures outlined in materials and methods. The results represent the mean S.E.M. of four experiments. *P<0.05, significantly stimulated by fractalkine; **P<0.01, significantly stimulated by fractalkine. (B) Cells were treated with various concentrations of fractalkine for 30 min and phosphorylated p38 MAPK was examined as described in Materials and methods. The results are representative of four experiments.

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46 CX3CR1 CX3CR1Control:TGF-1 treated:Time: 0 0.5 1 2 4 6 (h) 0 1 2 3 4 5 6 10 100 Control TGF-1 Time (h)Percentage of CX3CR1mRNA Remaining Fig. 2-5. The half-life of CX3CR1 mRNA is unaltered in TGF-1-treated microglia. Microglial cells were treated with medium alone or 2 ng/ml TGF-1 for 16 h, before the addition of 4 g/ml Actinomycin D. Total RNA was extracted at the indicated time points and subjected to Northern blot analysis (8 g/lane control treated microglia RNA; 5 g/lane TGF-1-treated microglia RNA). The CX3CR1 mRNA levels before the addition of Actinomycin D (0 h time point) were plotted as 100%. The results are representative of two experiments.

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47 AAATAAA AACCAGACGTCTCGAC5’CX3CR1-1268 SBE-1154 -2367 SBE //// -52 Fig. 2-6. Schematic structure of the rat CX3CR1 gene. The positions (relative to the putative start of transcription) and sequences of the two potential Smad Binding Elements (SBE) are shown. The filled black box represents the protein coding sequence of rat CX3CR1. The open boxes represent 5'and 3'-untranslated regions. DNA sequences have been deposited in the Genbank database (accession no. AF547167).

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CHAPTER 3 TGF-1-INDUCIBLE RGS2 AND RGS10 REGULATE CX3CR1 FUNCTION IN RAT MICROGLIA Introduction Microglia serve as immune cells in the central nervous system. They are the first group of cells to respond to challenges to the brain and perform functions of macrophages, such as antigen presentation, synthesis of cytokines and chemokines, free radical and NO production, migration and phagocytosis. These functions have been described in the normal brain immune response; however, excessive brain inflammation is also involved in multiple sclerosis (Li et al., 1993; Prineas and Wright, 1978), Alzheimer’s diseases (Meda et al., 1995) and AIDS dementia (Price et al., 1988), where they become toxic to neurons and other cells in the brain. So the survival, proliferation and activation of microglia are important in physiological and pathological conditions in the CNS and under strict regulation. In the CNS, TGF-1 is a potent inhibitor of microglia activation. It also modulates chemokine receptor expression on different kind of cells. CCR1 expression on murine astrocytes was increased by TGF-1 (Han et al., 2000), so was CX3CR1 expression on rat microglia (Chen et al., 2002). Chemokines were first described to mediate migration and activation of leukocytes, including microglia; in addition, later studies show that they are also important in embryonic development, cell proliferation and apoptosis, cytokine production, tumor and viral pathogenesis. Chemokine receptors that have been shown to be constitutively expressed on microglia include CX3CR1, CXCR4 and CCR5 (Jiang et al., 1998). CCR148

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49 3, CXCR2 and CXCR3, among other chemokine receptors, have also been described on microglia in pathological situations (He et al., 1997; Boddeke et al., 1999; Filipovic et al., 2003; Biber et al., 2002). CX3CR1 is the receptor for fractalkine, which is the only member of CX3C chemokine subfamily. Fractalkine is mainly expressed on neurons and the receptor is primarily expressed on microglia (Harrison et al., 1998), suggesting that they mediate the communication between neurons and microglia. Fractalkine can induce microglia migration, activation and survival. It can also inhibit LPS-induced TNFrelease (Zujovic et al., 2000) and Fas-induced apoptosis in microglia (Boehme et al., 2000). These data demonstrate that fractalkine contributes to maintaining the homeostasis of microglia. CX3CR1 is a member of the seven-transmembrane, G-protein coupled receptor superfamily. Once ligands binds to G-protein coupled receptors, GTP replaces GDP on G subunit of the G-protein and this leads to the dissociation of G from GThe activated G-protein then starts a cascade of down-stream signaling, including Ca2+ mobilization and activation PI3 kinase and MAPK pathways (Maciejewski-Lenoir et al., 1999). In this scenario, there are positive regulators that accelerate the exchange of GDP for GTP and negative regulators that enhance the hydrolysis of GTP into GDP. RGS (regulator of G-protein signaling) proteins are one type of negative regulating GAPs (GTPase-activating proteins). They are a group of proteins with structural variation yet all containing a highly homologous region of about 120aa (Hollinger and Hepler, 2002). In cells, RGS proteins have been shown to localize to the plasma membrane, cytoplasm, nucleus and Golgi. Different mechanisms were proposed for them to access to the plasma membrane in order to function. G protein was shown to be sufficient to transfer RGS2

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50 and RGS4 from cytoplasm to plasma membrane; however, they also show that receptors alone can achieve these effects (Roy et al., 2003). The mechanism needs to be better characterized and probably for different receptors, the way that RGS proteins function is different. Previously we reported that TGF-1 upregulated CX3CR1 expression, yet however, inhibited fractalkine-stimulated ERK1,2 phosphorylation in rat microglia (Chapter 2). To investigate this seemingly counterintuitive data, we profiled gene expression in TGF-1-treated and -untreated microglia with microarray analysis. Among more than 200 genes that changed significantly by TGF-1, we show for the first time that RGS2 and RGS10 were upregulated by TGF-1, which was confirmed by Northern blot analysis. In cotransfected cells, RGS2 and RGS10 attenuated fractalkine-stimulated ERK1,2 phosphorylation. Attenuation by RGS10 was sustained throughout the time course examined. Furthermore, co-expression of CX3CR1 altered the cellular localization of RGS10. These data identified a potential mechanism by which the multifunctional growth factor TGF-1 could regulate the chemokine receptor activation of MAPK pathway. Materials and Methods Cell Culture Primary culture of rat microglia was prepared as describe before. Briefly, cerebra were isolated from new born Sprague-Dawley rats, stripped of meninges and minced in Solution D (137 mM NaCl, 5.4mM KCl, 0.2mM NaH2PO4, 0.2 mM KH2PO4, 1 g/L Glucose, 20 g/L Sucrose, 0.25 g/L Fungizone, 106 U/L penicillin/streptomycin, pH 7.4). DMEM with 10% FBS and 106 U/L penicillin/streptomycin was added to stop digestion

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51 by 0.25% Trypsin in 37 C and the mixture was passed through 130 M filter (Tetko, Brairdiff Manor, NY). The filtered mixture was then spun down, resuspended and passed through a 40 M filter (Tetko, Brairdiff Manor, NY). It was seeded into Poly-L-Lysin (Sigma, St. Louis, MO) coated T-175 cm2 flasks (Sarstedt, Newton, NC) in DMEM with 10% FBS and incubated in 37 C, 8% CO2. Three-four days after, the medium was changed. After another 3-7 days, microglia were harvested by shaking the flasks and collecting the medium. The cells were spun down and replated into dishes. One hour later, the medium was changed to remove any non-adherent cells. One day later, the cultures containing >95% microglia were used for experiments. HEK 293-T cells were maintained in DMEM with 10% FBS and 106 U/L penicillin/streptomycin in 37 C, 5% CO2 incubator. Microarray Genechip Hybridization and Data Analysis Microglia seeded at the density of 4 million/60mm dish were treated with or without 2 ng/mL human TGF-1 (R&D Systems, Minneapolis, MN) for 16 h in serum free medium. TriZol reagent was used to extract total RNA according to manufacturer’s instruction (Invitrogen, Carlsbad, CA). Five g total RNA was used for double strand cDNA synthesis (Affymetrix Inc., Santa Clara, CA) and Biotin labeled cRNA was prepared by in vitro transcription. Twenty-four g cRNA was fragmented and hybridized to rat gene chip 230A (Affymetrix Inc., Santa Clara, CA) for 16 h. Chips were washed, stained and scanned with an Affymetrix scanner. Scanned data were analyzed with Affymetrix Microarray Suite Version 5. The hip-to-chip normalization was accomplished by setting mean signal intensity of all probes on each chip at 500. Probe sets whose signal intensities were not detected above the

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52 background noise were removed from the data set. The signal intensities of the left probe sets were then ranked according to the coefficient of variation and the top 50% of the data set were normalized to a mean of 0 and a standard deviation of 1. Hierachical analysis was performed and displayed with the software package Cluster and TreeView developed by Eisen et al (Eisen et al., 1998). The data were also analyzed with BRB Array Tools 3.01 (developed by Richard Simon and Amy Peng) to identify genes that were different among the groups: TGF--treated and -untreated (p< 0.001). “Leave-one-out” cross-validation with each of the four methods was used to predict the likelihood of a gene would be changed by TGFtreatment: nearest-neighbor prediction, three-nearest-neighbors prediction, linear discrimination analysis and nearest-centroid analysis. Northern Blot Human RGS2, RGS10, HA-tagged RGS2 and HA-tagged RGS10 were purchased from Guthrie cDNA Resource Center (Sayre, PA). Six g rat microglia total RNA was electrophoresed through agarose gel, transferred to nylon membrane and subjected to Northern blot analysis. 32P-labeled human RGS2, RGS10 and rat cyclophilin cDNA were used to probe the membrane. The temperature for hybridization and wash was lowered from standard 65 C to 60 C to decrease the stringency when human cDNAs were used. The blots were exposed to X-ray films and signals were visualized by autoradiography. Transfection of HEK 293-T cells Two days after they were plated, cells were transfected with human CX3CR1 alone or together with RGS plasmids, as indicated in Results, using Lipofectamine reagent according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA). Two days later, the

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53 cultures were assayed for fractalkine-stimulated ERK1,2 phosphorylation or immunocytochemistry. Analysis of ERK1,2 Phosphorylation by Western Immunoblotting Transfected HEK 293-T cells were washed once with serum free medium and incubated in serum free medium for 2 h. Cells were then stimulated with or without 20 nM human fractalkine (R&D Systems, Minneapolis, MN) in serum free medium for various times as indicated in Results. They were washed with ice-cold PBS and lyzed with lysis buffer (PBS, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 100 g/mL PMSF, 20 g/mL Aprotinin, 1 mM Na orthovanadate, 1 mM DTT, pH 7.4). The Western immunoblotting was carried out as described previously (Chen et al., 2002). Immunostaining of RGS10 Transfected 293-T cells seeded on cover slips were washed once with PBS, fixed in 4% paraformaldehyde for 10 min and permeabilized with 0.5% Triton-X in PBS for 2 min. After washing twice with PBS, the cells were incubated in 1:1000 mouse anti-HA antibody (Sigma, St. Louis, MO) in 1% BSA PBS at room temperature for 1h. The cells were then incubated in FITC-conjugated 1:1000 sheep anti-mouse secondary antibody (Sigma, St. Louis, MO) in 1% BSA PBS after washed three times. The immunofluorescence was examined by confocal microscopy. Results TGF-1 Regulation of Gene Expression Profile in Rat Microglia Among ~10,000 genes that were examined, 232 genes were changed significantly (P<0.001), 89 of which were downregulated by more than 40% and 134 of which were increased by more than 1.5 fold (Fig. 3-1A). Pro-inflammatory cytokines and chemokines including IL-1, MIP-1MIP-2were dramatically decreased by TGF-1 treatment,

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54 which was consistent with previous data demonstrating that TGF-1 is a very potent inhibitor of microglia activation (Suzumura et al., 1993; Maltman et al., 1996). MMP9 (Matrix metalloproteinase 9), a proteolyitc enzyme that is secreted by activated microglia (Gottschall et al., 1995), was inhibited about 10 fold by TGFtreatment. On the other hand, genes related to cell-cell connection and extracellular matrix were upregulated, including tight junction protein 1 and fibronectin 1. RI has been reported to be upregulated by TGF-in microglia (Morgan et al., 2000), which was also confirmed in our microarray analysis. Consistent with our previous data, chemokine receptors CX3CR1 and CXCR4 were increased by TGF(Chapter 2 and 4) (Table 3-1). More interestingly, we identified two RGS genes, RGS2 and RGS10, to be upregulated in TGF--treated cells (Table 3-1). Since RGS proteins can attenuate G-protein coupled receptor signaling, this could explain the phenomenon that TGF-1 upregulated CX3CR1 while inhibiting its activation of ERK1,2 phosphorylation in rat microglia. Thus Northern blot analysis was used to confirm the data from microarray analysis. RGS2 and RGS10 were upregulated by TGFabout 2-fold in Northern blot analysis, which is consistent with the microarray analysis (Fig. 3-1B). RGS2 and RGS10 Attenuated CX3CR1-mediated ERK1,2 Stimulation To examine if RGS10 could inhibit CX3CR1-mediated downstream signaling, HEK 293-T cells co-transfected with human CX3CR1 and different amounts of DNA encoding human RGS10 were stimulated with fractalkine and ERK1,2 phosphorylation was evaluated. Fractalkine markedly increased ERK1,2 phosphorylation in CX3CR1-expressing cells, in the absence of RGS10. However, fractalkine-stimulated ERK1,2 phosphorylation was attenuated by co-expression of RGS10. The inhibition was

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55 enhanced when the amount of RGS10 DNA was increased (Fig. 3-2A). RGS2 could also inhibit CX3CR1-mediated ERK1,2 activation, but with a lower effect (Fig. 3-2B). The inhibition was not due to a reduction in CX3CR1 expression since there was no change in 125I-fractalkine binding to cells co-expressing RGS proteins (data not shown). Kinetics of RGS10 Inhibition of CX3CR1 Activation To determine if RGS10 altered the kinetics of fractalkine activation of ERK1,2, HEK 293-T cells transfected with CX3CR1 alone or CX3CR1 and RGS10 were stimulated with 20 nM fractalkine for 2, 7 or 15 min. Fractalkine stimulated ERK1,2 phosphorylation by 3.9 fold at 2 min and the stimulation reached a peak level, about 4.5 fold, at 7 min in CX3CR1 alone transfected cells. At the15 min time point, fractalkine stimulation was minimal. Throughout the time course we examined, RGS10 attenuated CX3CR1-mediated ERK1,2 phosphorylation (Fig. 3-3). Effect of CX3CR1 on Cellular Localization of RGS10 RGS10 is required to interact with G-proteins in order for them to exert their GAP function. To examine the localization of RGS10, HA-tagged RGS10 was expressed in HEK 293-T cells with or without CX3CR1. In the absence of CX3CR1, HA-RGS10 was localized mainly in the cytoplasm. However, in the presence of CX3CR1, the cellular localization of HA-RGS10 was shifted to regions proximal to the plasma membrane (Fig. 3-4). Discussion Previously we reported that TGF-1 upregulated CX3CR1 expression on rat microglia but fractalkine-stimulated ERK1,2 phosphorylation was inhibited (Chapter 2). To investigate the possible mechanism underlying the seemingly contradictory phenomenon, we performed microarray analysis on TGF-1-treated and -untreated rat

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56 microglia. Gene profiling found two RGS proteins, RGS2 and RGS10, to be upregulated by TGF-1. In vitro cotransfection studies demonstrated that RGS2 and RGS10 could attenuate CX3CR1-mediated ERK1,2 phosphorylation, with RGS2 having a lower effect than RGS10. RGS10 attenuation was seen at all time points we examined, suggesting that RGS10 interacted with the receptor signaling complex prior to fractalkine stimulation. This was supported by immunocytochemical analysis of RGS10 localization in cells co-expressing CX3CR1. TGF-is a potent inhibitor of microglia activation. Our microarray analysis showed that inflammatory genes such as IL-1MIP-1and MIP-2were downregulated by TGFtreatment. MMP9 is a protelytic enzyme that is capable of degrading the extracellular matrix. It has been detected in multiple sclerosis lesions (Cuzner and Opdenakker, 1999) and microglia have been shown to express MMP9 (Gottschall et al., 1995). MMPs can disrupt the blood brain barrier, mediate influx of leukocyte into the CNS and degrade myelin (Chandler et al., 1995; Gijbels et al., 1993), suggesting an involvement in the pathogenesis of CNS diseases such as multiple sclerosis. Our data showed that TGF-1 inhibited MMP9 production dramatically in cultured microglia, suggesting a possible mechanism by which TGF-1 has a protective role in the context of multiple sclerosis. Among the genes that are significantly upregulated by TGF-RGS2 and RGS10 attracted our attention. RGS2 and RGS10 have been shown to be expressed in the CNS, and here we demonstrate that they are expressed by cultured microglia. More importantly, they are upregulated by TGF-treatment, which proves that TGF-and RGS proteins interact and provides another means for TGFto regulate the function of

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57 G-protein coupled receptors. Co-transfection experiments showed that RGS2 and RGS10 attenuated CX3CR1-mediated ERK1,2 stimulation. However, RGS2 was less inhibitory than RGS10 when equal amounts of DNA were transfected. This could be due to two possible reasons: i). RGS2 has lower expression level than RGS10, which was difficult to prove because there are no good antibodies against RGS proteins; and ii) RGS2 has lower affinity towards the G-protein activated by CX3CR1 than RGS10, which would be consistent with RGS10 selectively regulating this chemokine receptor. The microarray analysis showed that RGS10 is more abundant in microglia than RGS2, indicating that the former RGS protein is more likely to regulate CX3CR1 signaling to MAPK. We examined the kinetics of RGS10 inhibition of CX3CR1 activation. Since there was no apparent lag in the RGS10 inhibition of CX3CR1 activation, it is reasonable to speculate that RGS10 interacts with CX3CR1 or G-protein in the absence of fractalkine stimulation. Our immunocytochemistry data showed that expression CX3CR1, without any activation, changed the localization of RGS10 from the cytoplasm probably to the plasma membrane. TGFinhibits fractalkine-stimulated ERK1,2 phosphorylation despite the increased CX3CR1 expression on the cell surface. The data presented here provide a possible mechanism for this observation. TGFregulates the expression of chemokine receptors and RGS proteins may add another layer of functional regulation. When fractalkine binds to CX3CR1 on microglia, it activates Ca2+ mobilization, PI3 kinase and MAPK pathways as well as actin reorganization. RGS inhibition of MAPK activation may shift CX3CR1 function towards other known or unknown signaling pathways. Fractalkine can exist in soluble and membrane-bound forms, with the former one mainly

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58 responsible for cell migration and activation and the latter one mediating cell adhesion of CX3CR1-expressing cells. Fractalkine-mediated cell adhesion is independent of downstream signaling, thus it is possible that while TGF-increased CX3CR1 expression on rat microglia, RGS10 and/or RGS2 converts the receptor from a signaling to a purely cell adhesion molecule. In summary, our studies show that RGS2 and RGS10 are expressed in rat microglia and are upregulated by TGF-RGS2 and RGS10 can change the responsivesness of CX3CR1-expressing cells to chemokines, contributing to the complex regulation of their functions in the immune and central nervous systems.

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59 Table 3-1. Genes regulated by TGF-in rat microglia. Gene Ratio (TGF1/CON) MMP9 0.104 IL-1 0.157 MIP-1 0.187 MIP-2 0.217 Genes Downregulated by TGF-1 Gene Ratio (TGF-1/CON) Smad7 14.492 CX3CR1 4.926 TRI 4.651 CXCR4 3.663 Fibronectin 1 3.165 Tight Junction Protein 1 2.833 RGS2 2.577 IGF1 2.532 RGS10 1.894 Genes Upregulated by TGF

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60 Fig. 3-1. Genes regulated by TGFin rat microglia. (A). Microarray analysis of gene expression profile in TGF-1-treated and -untreated rat microglia. Total RNA was extracted from rat microglia treated with or without 2 ng/mL TGF-1 for 16 h and subjected to microarray analysis. The graph summarizes the expression profile of 232 genes from four independent experiments. (B). Northern blot analysis of RGS2 and RGS10. Total RNA from rat microglia treated with or without 2 ng/mL TGF-1 for 16 h was subjected to Northern blot analysis and normalized to that of cyclophilin. The data is representative of two independent experiments.

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61 Fig. 3-2. RGS10 and RGS2 attenuated fractalkine-stimulated ERK1,2 phosphorylation. (A). HEK 293-T cells were transfected with 0.25 g/well CX3CR1 alone or with different amount of RGS10 and stimulated with 20 nM fractalkine for 7 min. The levels of ERK1,2 phosphorylation were examined by Western blot. (B). HEK-293 T cells were transfected with CX3CR1 alone, or with RGS10 or RGS2. The shown is the representative of three independent experiments that were summarized in the graph.

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62 Fig. 3-3. Kinetics of RGS10 inhibition of CX3CR1-mediated ERK1,2 phosphorylation. Cells were transfected with 0.25 g/well CX3CR1 and 0.75 g/well RGS10 and stimulated with 20 nM fractalkine for various time. The experiments were done in duplicates and the graph summarizes the data from three independent experiments.

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63 Fig. 3-4. Effect of CX3CR1 on the localization of RGS10. HEK 293T cells were transfected with HA-RGS10 alone or together with CX3CR1. The cells were then immunostained with anti-HA antibody and visualized with confocal microscopy.

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CHAPTER 4 TGF-1 INCREASES CXCR4 EXPRESSION, SDF-1-STIMULATED SIGNALING AND HIV-1 ENTRY IN HUMAN MONOCYTE-DERIVED MACROPHAGES Introduction Macrophages are one of the predominant cell types infected with HIV-1 in many tissues, including brain, lung and lymph nodes (Koenig et al., 1986; Pantaleo et al., 1991; Salahuddin et al., 1986). Unlike infected CD4+ T-lymphocytes which have a short half life of 1-1.5 days, macrophages are quite resistant to the cytopathic effects of the virus and thus, may provide a reservoir for persistent infection and virus dissemination. The two major co-receptors of HIV-1, CXCR4 and CCR5, are both expressed on macrophages. Macrophages are primarily infected by CCR5 utilizing viruses (R5) in early stages of infection (Zhu et al., 1993) but at later stages of the disease they are often infected by dual-tropic CXCR4 using variants (D-X4) (Singh and Collman, 2000), which are associated with disease progression (Connor et al., 1993). The susceptibility of macrophages to HIV-1 infection is dependent on multiple factors, including the stage of differentiation of the cells as well as a variety of host factors (Kedzierska et al., 2003). Previous studies showed that R5 HIV-1 inefficiently infects monocytes. However, as monocytes differentiate into macrophages, mRNA and cell surface expression of CCR5 increases while CXCR4 decreases. Concurrently, the infection level of the cells by R5 viruses also increases (Tuttle et al., 1998; Naif et al., 1998). Host factors, including cytokines and growth factors, play important roles in regulating pathogenesis of HIV infection. TGF-1 is a multifunctional growth factor that 64

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65 is important in development, immune responses, tumor biology and angiogenesis (Letterio and Roberts, 1998; Massague and Chen, 2000). In patients infected with HIV-1, blood levels of TGF-1 are increased (Navikas et al., 1994). TGF-1 production can be induced from macrophages, peripheral blood mononuclear cells (PBMCs) and astrocytes infected with HIV-1 (Kekow et al., 1990; Hu et al., 1996; Wahl et al., 1991). TGF-1 increases CXCR4 expression, X4-envelope-mediated syncytium and X4-infection levels on human mature dendritic cells (Zoeteweij et al., 1998). MDM expression of CXCR4 has also been shown to be regulated by TGF-1(Wang et al., 2001a). In addition, TGF-1 can overcome the replicative restriction of T-tropic isolates of HIV-1 in macrophages (Lazdins et al., 1992). However, the mechanism of how TGF-1 regulates macrophage susceptibility to X4 HIV-1 infection is not well characterized. CXCR4 is the only known receptor for the CXC subfamily chemokine SDF-1. SDF-1 and CXCR4 are essential in CNS development, angiogenesis, lymphopoiesis and tumorigenesis (Lazarini et al., 2003; Nagasawa et al., 1998). SDF-1 activates Ca2+ mobilization, PI3 kinase and MAPK pathways and chemotaxis in CXCR4-expressing cells (Bleul et al., 1996; Ganju et al., 1998). CXCR4 expression is under extensive regulation in both physiological and pathological states. For example, a previous study showed that the interaction with thymic epithelial cells (TEC) induced an upregulation of CXCR4 expression on CD4+ mature thymocytes (Schmitt et al., 2003). In the CNS, CXCR4 is expressed on astrocytes, microglia and neurons. CXCR4 on microglia can mediate HIV-1 entry into the CNS, although the efficiency of the viral-cell interaction is higher with CCR5 (Albright et al., 1999; Shieh et al., 1998).

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66 In this study, we examined the effects of TGF-1 on CXCR4 expression, CXCR4-mediated signaling, and HIV-1 infection in human MDMs. We found that TGF-1 increased CXCR4 expression at both the mRNA and cell surface protein levels. Distribution of CXCR4 in the cells was also changed by TGF-1 treatment. The increase in CXCR4 expression resulted in enhanced SDF-1 stimulated ERK1,2. In the CNS, TGF-1 also upregulated CXCR4 mRNA expression and potentiated SDF-1-stimulated signaling in primary cultures of rat microglia. More importantly, CXCR4-mediated D-X4 HIV-1 virus entry into human MDMs was enhanced by TGF-1. In contrast, neither CCR5 expression nor R5 virus entry into MDM was changed by TGF-1 treatment. Our studies implicate TGF-1-regulated CXCR4 on macrophages as one possible mechanism responsible for the emergence of X4 HIV-1 variants during later stages of AIDS. Materials and Methods Primary Cultures of Human MDMs and Rat Microglia Two types of media were used in the preparation of human MDM culture. Serum free medium: RPMI1640 medium (Life Technologies, Rockyville, MD), 100,000 U/L penicillin, 100 mg/L Streptomycin, pH 7.4. Complete medium: RPMI1640 medium, 15% human serum (Sigma, St. Louis, MO), 100,000 U/L penicillin, 100 mg/L Streptomycin, pH 7.4. Uninfected PBMCs from fresh HIV-1-, CMV-, and HBV-negative leukopheresis residues were isolated from adult donors by Ficoll gradient centrifugation. Briefly, Leukopheresis residues were washed once with wash buffer (HBSS, 2.5mM EDTA, 2% FBS [Life Technologies, Rockyville, MD], 100,000 U/L penicillin, 100 mg/L Streptomycin, pH7.4) and then incubated with RosetteSep Human Monocyte Enrichment Cocktail (StemCell Technologies, Vancouver, BC, Canada) for 20 min at room

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67 temperature before Ficoll gradient centrifugation. The enriched monocytes were washed twice with wash buffer and plated with complete medium into 75 cm2 flasks (Sarstedt, Newton, NC). After 1-2 h incubation at room temperature, cultures were rinsed twice with serum free medium. Monocytes were incubated in complete medium supplemented with 100 ng/mL GM-CSF (Sigma, St. Louis, MO) at 37 C, 5% CO2 for a week. After cultures matured into macrophages, the medium and unattached cells were aspirated off. Cell monolayers were washed twice with PBS, scraped gently off the flasks, and replated for experiments. One day after plating, the cells were used for experiments. The cultures contain >95% macrophages. Primary cultures of rat microglia were prepared from neonatal rat brain as described previously (Chen et al., 2002). Northern Blot Analysis MDMs were plated at a density of 1-1.5 million/well into 6-well plates (Corning Incorporated, Corning, NY). Microglia were seeded at a density of 4 million cells per 100 mm dish. Serum free medium containing various concentrations of human TGF-1 (R&D Systems, Minneapolis, MN) was applied to the cells. At various times after addition of TGF-1, as indicated in Results, total RNA was extracted with TriZol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions and subjected to Northern blot analysis (Church and Gilbert, 1984). [32P]-labeled cDNAs encoding human CXCR4, CCR5 and GAPDH, or rat CXCR4 and cyclophilin were used as hybridization probes. The washed membranes were exposed to film and detected by autoradiography. Flow Cytometry and Immunofluorescence Microscopy For flow cytometry analysis, MDMs seeded into 6-well plates at a density of 1-1.5 million/well were treated with 20 ng/mL TGF-1 for 48 h. Cells were collected by gentle

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68 scraping, washed once with fluorescence-activated cell sorter (FACS) washing buffer (PBS, 0.02% Sodium Azide, 10% FBS and 2% human serum) and incubated subsequently with either allophycocyanin (APC)-conjugated anti-human CXCR4 antibody (mAb 12G5, BD Biosciences Pharmingen, San Diego, CA) or APC-conjugated anti-human IgG2 antibody (BD Biosciences Pharmingen, San Diego, CA) for 15 min at 4 C. Cells were washed twice with FACS washing buffer, then fixed with fixing buffer (PBS, 1% paraformaldehyde, 0.02% sodium azide) and analyzed with FACScan machine (Becton-Dickinson). For immunofluorescence microscopy, 200,000-300,000 cells were seeded onto cover slips in 24 well plates. After 48 h treatment with TGF-1, cells were fixed in 4% paraformaldehyde for 30 min at room temperature, permeabilized with PBS-1% Tween for 15 min and then blocked with blocking buffer (PBS, 1% BSA) for 1 h. The cells were incubated in 1:100 goat anti-human CXCR4 (C-20) antibody (Santa Cruz Biotechnology, Santa Cruz, CA) in blocking buffer at 4 C overnight and further with fluorescein isothiocyanate (FITC)-conjugated bovine anti-goat secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature for 1 h. The cells were analyzed using regular immunofluorescent microscopy (Zeiss Axoskop2 Plus with SPOT II camera) and confocal microscopy (Solamere Technology Group, Salt Lake City, UT) with a Stanford Photonics XR-Mega 10 camera (Palo Alto, CA). SDF-1 Stimulation of ERK1,2 Phosphorylation MDMs (500,000/well) in 12-well plates were treated for 48 h with serum free medium containing TGF-1, incubated in serum free medium for an additional 2 h, and stimulated subsequently with 50 nM human SDF-1(R&D Systems, Minneapolis, MN)for 2 or 5 min. The cells were collected in lysis buffer with proteinase inhibitor

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69 cocktail (PBS, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 100 g/mL PMSF, 20 g/mL Aprotinin, 1 mM Na orthovanadate, 1 mM DTT, pH 7.4) and Western blot analysis was carried out as described previously (Chen et al., 2002). Mouse anti-phospho-ERK1,2 antibody (Cell Signaling Technology, Beverly, MA), rabbit anti-ERK1,2 antibody (Cell Signaling Technology, Beverly, MA), HRP-conjugated sheep anti-mouse secondary antibody (Sigma, St. Louis, MO), and HRP-conjugated goat anti-rabbit secondary antibody (Cell Signaling Technology, Beverly, MA) were used. HIV-1 Entry Single cycle recombinant viruses were produced by cotransfection of a luciferase-tagged HIV-1 backbone vector and an envelope expression vector containing env sequences, as described previously (Tuttle et al., 2002). The vector pNL4-3.Luc.Eencoded the HIV-1NL4-3 provirus with a frameshift in env that prevent expression of the envelope glycoprotein and was tagged with firefly luciferase in the nef reading frame. The envelope expression vector was prepared by inserting the entire env reading frame from either HIV-1JR-FL or HIV-1D-X4 into the cytomegalovirus promoter-based expression vector, pcDNA3.1+ (Stratagene, La Jolla, CA). The two vectors were cotransfected into 293 cells with Superfect transfection reagent (Qiagen, Chatsworth, CA). Viral supernants were harvested two days later, pooled, cleared of cell debris by 0.45-m pore size filtration, assayed for p24 antigen content and stored in aliquots at -80C. MDMs were plated into wells of 48-well plates at a density of 250,000 cells/well. After 48 h treatment with 20 ng/mL TGF-1, cells were washed once with PBS and fed with complete medium. Cells were inoculated with 60 ng of viral p24 and cell lysates were prepared with cell culture lysis reagent (Promega, Madison, WI) 4 days after

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70 infection. The amount of luciferase activity in each lysate was determined by a standard luminometer assay (Monolight 2010; Analytical Luminescence Laboratories, San Diego, CA), which reported results in relative light units (RLU). Statistic Analysis Student’s t-test and two-way analysis of variance were done in GraphPad Prism 3.1 (GraphPad Software, San Diego, CA) as indicated in the results. The significance level was set at p<0.05. Results TGF-1 Increased CXCR4 mRNA in MDMs Blood levels of TGF-are increased in individuals infected with HIV-1 and it can be induced from macrophages, PBMCs and astrocytes (Hu et al., 1996; Kekow et al., 1990; Navikas et al., 1994; Wahl et al., 1991). To examine the effects of TGF-on CXCR4 expression in macrophages, human monocytes were differentiated for 1 week in the presence of GM-CSF into mature macrophages. Macrophages were treated with different concentrations (0-20 ng/mL) of TGF-in serum free medium for 48 h, total RNA was extracted and subjected to Northern blot analysis. The levels of CXCR4 were quantified and normalized to GAPDH hybridization signals. Figure 4-1A shows that TGF-upregulated CXCR4 mRNA in MDMs in a concentration-dependent manner. Maximal induction of CXCR4 mRNA was 2-fold and occurred at TGFconcentrations of 10 and 20 ng/mL. The kinetics of the TGFdependent increase in CXCR4 mRNA were also characterized. At each of the time points examined, a 2-fold increase in CXCR4 mRNA was evident (Figure 4-1B). In cells treated with TGF-in the presence of human serum, there was no increase of CXCR4 mRNA (data not shown).

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71 TGF-1 Increased MDM Cell Surface CXCR4 and Altered its Cellular Localization A direct relationship between levels of mRNA and surface expression of CXCR4 and CCR5 has been established (Tuttle et al., 1998). Since CXCR4 mRNA was upregulated by TGF-it was probable that CXCR4 on the cell surface of MDMs would be upregulated also. MDMs were treated with 20 ng/mL of TGF-1 for 48 h and subjected to flow cytometry analysis in order to evaluate cell surface expression of CXCR4. TGF-1 treatment increased mAB 12G5 immunoreactivity indicating that CXCR4 expression on the cell surface was elevated; isotype control staining of control and TGF-1 treated cells were less than staining by 12G5 (Figure 4-2A). A summary of 8 independent donors indicated that TGF-1 increased cell surface CXCR4 protein expression by 50 16% (Figure 4-2A). The localization of CXCR4 in TGF-1-treated and untreated MDMs was determined by staining permeabilized cells with an anti-CXCR4 antibody directed against the C-terminus of human CXCR4. Specificity of the immunoreactivity was established by two methods. First, no staining was observed when the primary antibody was omitted and second, HEK293T cells engineered to over-express human CXCR4 displayed higher immunoreactivity, as compared to mock-transfected cells (data not shown). Consistent with the flow cytometry analysis, the fluorescence intensity of the cells and particularly the cell surface of MDMs was increased by TGF-1 (Figure 4-2B and 4-2C). Furthermore, CXCR4 immunoreactivity appeared in a punctate pattern in control treated cells, a cellular distribution similar to previous reports (Wang et al., 2001a; Volin et al., 1998). On the other hand, CXCR4 was more evenly distributed throughout the TGF-1-treated MDMs, although more intense perinuclear immunoreactivity was observed (Figure 4-2C).

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72 SDF-1-stimulated ERK1,2 Phosphorylation was Enhanced by TGF-1 Pretreatment Binding of SDF-1 to CXCR4-expressing cells stimulates intracellular Ca2+ mobilization, as well as PI3 kinase and MAPK pathways. We examined if the increased CXCR4 on MDMs enhanced SDF-1 stimulation of ERK1,2. MDMs were treated with TGF-1 for 48 h, changed into serum free medium for 2 h and then stimulated with 50 nM SDF-1 for 2 and 5 min. At both time points, TGF-1 pretreatment enhanced SDF-1-stimulated ERK1,2 phosphorylation (Figure 4-3). At 2 and 5 min time points, SDF-1 increased pERK1,2 levels 3.5and 2.7fold respectively in TGF-1-treated cells. In control cells, SDF-1only increased pERK1,2 2.3and 1.5fold (2 and 5 min, respectively). TGF-Increased CXCR4 and Enhanced SDF-1-stimulated ERK1,2 Phosphorylation in Rat Microglia Microglia are immune cells resident in the CNS and can perform such functions of macrophages as antigen presentation, cytokine synthesis and phagocytosis (Kreutzberg, 1996). These cells also constitutively express CXCR4, which can be activated by SDF-1 and mediate HIV-1 infection within the CNS. We investigated if TGF-1 could alter the expression of CXCR4 on rat microglia. Primary cultured neonatal rat microglia were treated with 2 ng/mL TGF-1 for 8, 16, and 24 h and total RNA was extracted and subjected to Northern blot analysis. As shown in Figure 4-4A, as early as 8 h after TGF-1 treatment, a dramatic increase (about 4-fold) of CXCR4 mRNA was evident; the increase was maintained through 24 h of TGF-1 treatment. Steady state levels of CXCR4 mRNA in primary cultures of rat astrocytes were unaltered by TGF-1 (Figure 4-4B). TGF-1-treated and untreated microglia were also subjected to analysis of

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73 CXCR4 function. As observed with the human MDM, TGF-1 significantly enhanced the SDF-1-stimulated ERK1,2 phosphorylation (Figure 4-4C). A decreased basal level of ERK1,2 phosphorylation in TGF--treated rat microglia was also observed, consistent with our previously published data (Chen et al, 2002). TGF-1 Increased DX4 HIV-1 Infection of MDMs Although macrophages are primarily infected with R5 viruses, variants of HIV-1 can also use CXCR4 to gain entry into these cells (Crowe, 1995; Simmons et al., 1998; Verani et al., 1998; Yi et al., 1998; Yi et al., 1999). We evaluated the susceptibility of TGF-1-treated MDMs to X4 HIV-1 virus infection. To this end we used a dual-tropic (D-X4) virus that can use CXCR4 to infect both macrophages and CD4+ T-lymphocytes. MDMs treated with TGF-1 for 48 h were infected with recombinant virus and four days later, entry levels were measured by a luciferase activity assay. TGF-1 increased viral entry amongst 7 of 8 donors that were examined, although levels of virus entry for the various donors were quite variable (Figure 4-5). Statistical analysis of all 8 donors demonstrated a significant 2-fold enhancement by TGF-1. While previous data showed that TGF-1 could overcome the replicative restriction of T-tropic HIV-1 virus in MDMs (Lazdins et al., 1992), in our system, HIV-1LAI virus was unable to infect either TGF-1or control-treated MDMs (data not shown). TGF-1 Did not Alter CCR5 mRNA or R5 Viral Entry of MDMs CCR5 is the other major co-receptor expressed on MDMs. Macrophages are primarily infected with R5 viruses following transmission and throughout the course of the disease (Zhu et al., 1993; Crowe, 1995). To examine if TGF-1 had any effects on CCR5 mRNA expression, total RNA of controland TGF-1-treated MDMs were

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74 subjected to Northern blot analysis. Figure 4-6A shows the results of a representative experiment demonstrating that steady state levels of CCR5 mRNA were unaltered after 48 h of 20 ng/mL TGF-1 treatment. The effect of TGF-1 on the infection capacity of R5 variants on MDMs with JR-FL recombinant virus was also assessed. Among the three donors that were examined, MDMs were easily infected with the R5 virus indicated by the high levels of luciferase activity. However, there was no difference in infection levels between control and TGF-treated cells (Figure 4-6A, right panel). In microglia, TGF-1 reduced steady state levels of CCR5 mRNA (Figure 4-6B). TGF-1 did not affect CCR5 mRNA in cultured astrocytes (Figure 4-6B). Discussion Macrophages express both major HIV-1 co-receptors, CXCR4 and CCR5. In this report, we demonstrated that the pleiotropic growth factor/cytokine, TGF-1, increased CXCR4 expression in human MDM and rat microglia which resulted in two functional consequences. These included increased activation of ERK1,2 by SDF-1 and, in the case of human MDM, an enhanced susceptibility to D-X4 HIV-1 infection. This latter change was very likely due to the increase in surface expression of CXCR4 because the expression of the other major HIV-1 co-receptor, CCR5, was unaltered by TGF-1 in MDMs and so was the susceptibility of the cells to a R5 HIV-1 variant. CXCR4 expression and function are under extensive regulation in both physiological and pathological conditions. TGF-1 can increase CXCR4 expression on nave T cells, which results in increased homing of these cells to the spleen (Franitza et al., 2002). In the CNS, CXCR4 expression is upregulated in HIV encephalitis, experimental allergic encephalomyelitis and brain tumors. In these disease scenarios, the

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75 increased expression was localized primarily to microglia, astrocytes and infiltrating leukocytes (Jiang et al., 1998; McManus et al., 2000; Sanders et al., 1998; Sehgal et al., 1998). In our study, Northern blot, flow cytometry and immunostaining demonstrated that TGF-1 could increase CXCR4 expression at both mRNA and protein levels in human macrophages and rat microglia. In addition to increasing steady state levels of CXCR4 mRNA and cell surface protein, TGF-1 also changed the localization of CXCR4 in human MDMs. CXCR4 immunostaining and confocal microscopy showed that CXCR4 on the plasma membrane was increased by TGF-1, which was consistent with data from the flow cytometry analysis. More surprisingly, in control cells, CXCR4 immunoreactivity appeared in a punctate pattern in the cytoplasm. However, in TGF-1-treated cells, a more even cellular distribution was observed, although more intensive staining was seen around the nucleus. CXCR4 and CCR5 have been shown by immunoelectron microscopy to form homogenous clusters in human macrophages, mostly on microvilli (Singer et al., 2001). CCR5 microclusters were also found in secretory vesicles of the Golgi apparatus and they were speculated to be transported to the cell membrane (Singer et al., 2001). The CXCR4 punctate pattern is consistent with this earlier report. TGF-1 may be increasing the rate of transportation of CXCR4 from these vesicles to the plasma membranes. Concurrently, a TGF--dependent signal to increase de novo synthesis of CXCR4 could explain the enhanced immunofluorescence intensity around the nucleus. Nonetheless, further investigation will be required to verify this hypothesis. SDF-1-mediated ERK1,2 phosphorylation was enhanced in TGF-1 pretreated cells indicating that the increased CXCR4 expression functionally enhanced activation of

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76 this MAPK signaling pathway. ERK1,2 phosphorylation is part of an important set of intracellular signaling pathways that are involved in cell survival, proliferation and activation (Bajetto et al., 2001; Ganju et al., 1998; Kayali et al., 2003). ERK1,2 activation has also been shown to be involved in regulating HIV-1 infectivity and production. Exposure of HIV-1 infected cells to SDF-1 enhanced the production of X4 HIV-1 that was dependent on SDF-1-stimulated ERK1,2 activation (Montes et al., 2000). ERK1,2 activated by serum or constitutively active Ras, Raf or MEK also enhanced the infectivity of HIV-1 virions (Yang et al., 1999; Yang and Gabuzda, 1999). Thus, TGF--enhanced SDF-1-stimulated ERK1,2 activation may result in greater activation of macrophages in immune responses and increased HIV-1 infectivity. TGF-1 also increased CXCR4 expression and SDF-1-stimulated intracellular signaling in the central nervous system macrophage, i.e. microglia. We previously reported that fractalkine-stimulated ERK1,2 phosphorylation in microglia was diminished by TGF-1 pretreatment although it upregulated CX3CR1 expression (Chen et al., 2002). These contrasting effects of TGF-1 on CX3CR1 and CXCR4 functions suggest that TGF-1 differentially and specifically regulates the responsiveness of mononuclear cells to the chemokine ligands fractalkine and SDF-1. Blood levels of TGF-1 have been shown to be elevated in individuals infected with HIV-1 (Navikas et al., 1994). Increased production of TGF-1 has been demonstrated from HIV-1 infected monocytes, macrophages and astrocytes (Hu et al., 1996; Kekow et al., 1990; Wahl et al., 1991). When HIV-infected individuals progress towards AIDS, variants using CXCR4 emerge compared to predominantly R5 variants in early infection. Our results suggest that one possible mechanism to achieve this change in

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77 viral tropism is to increase CXCR4 expression on macrophages while keeping CCR5 expression unchanged. The entry of DX4 into human MDMs was upregulated 2-fold in TGF-1 pretreated cells. Our experiments used single cycle viruses that were replication incompetent. Thus it is reasonable to predict the increase of viral entry in TGF--treated cells could result in exponential amplification of virus production in subsequent replication cycles. Further amplification of DX4 virus infection of macrophages, in vivo, could result from increased TGF-1 from HIV-1 infected macrophages, as well as enhanced number of these cells as a consequence of the potent monocyte chemoattractant property of TGF-1(Wahl et al., 1987; Wiseman et al., 1988). TGF-1 also increases CXCR4 expression in primary CD4+ T cells(Wang et al., 2001b). Since D-X4 variants can use CXCR4 to get into both macrophages and CD4+ T cells, the increased production of these viruses by macrophages exposed to TGF-1 could provide a mechanism to promote further infection of CD4+ T cells. Other cytokines can also contribute to increasing HIV-1 infection of macrophages. IFN-and IL-6 increased the capacity of MDMs to favor CXCR4 using HIV-1 infection while decreasing infection by R5 variants (Zaitseva et al., 2000). Cytokine regulation of cell susceptibility to HIV-1 infection by changing co-receptor expression levels has also been seen in other cells. IL-2, IL-4, IL-7 and IL-15 induced functional cell surface expression of CXCR4 on CD4+CCR7+ human memory T cells, and increased the infection by X4 strains of HIV-1 in these cells (Hu et al., 1996; Kekow et al., 1990; Wahl et al., 1991). IL-7 has been reported to increase CXCR4 expression on CD4+ mature thymocytes and X4 virus replication in these cells (Schmitt et al., 2003). Our results, together with others, demonstrate that the cytokine

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78 milieu contributes to the complex regulation of an individual’s susceptibility to HIV-1 infection and viral tropism change (Kedzierska et al., 2003). A previous report showed that TGF-1 could overcome replicative restriction of X4 HIV-1 (LAI) in macrophages (Lazdins et al., 1992). However, in our experiments, MDM cultures derived from multiple donors were resistant to infection by LAI with or without TGF-1 pretreatment (data not shown). While the different donor populations could account for this discrepancy, it may reflect the nature of the experimental paradigm. Our experiments were specifically designed to evaluate the effects of TGF-1 on viral entry, as the virus-reporter system we utilized was not replication competent. Thus, the difference between the two sets of results suggests that there could be multiple TGF-1-regulatable steps during the process of viral infection. Nonetheless, a clear correlation between CXCR4 surface expression and viral entry is evident from our study. In summary, we report that TGF-1 increased CXCR4 expression and SDF-1-stimulated intracellular signaling in macrophages in both the peripheral immune and the central nervous systems. Furthermore, the increased CXCR4 expression results in enhanced susceptibility of the cells to entry of dual-tropic X4 HIV-1 variants. Our data demonstrate that TGF-1 regulates the expression of chemokine receptor on human macrophages and rat microglia with clear functional consequences on viral pathogenesis as well as potential alterations in mechanisms whereby chemokines contribute to host immune responses.

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79 Fig. 4-1. TGF1 increased CXCR4 mRNA expression in human MDMs. (A) Concentration-dependence of TGF1 upregulation of CXCR4 mRNA in MDMs. Human MDMs were treated with serum free medium alone or in the presence of the indicated concentrations of TGF1 for 48 h and total RNA was extracted and subjected to Northern blot analysis. The intensity of CXCR4 mRNA was quantified by densitometry and normalized to that of GAPDH mRNA. (B) Time dependent effects of TGF1 on CXCR4 mRNA. Human MDMs were treated with serum free medium with (filled bars) or without (open bars) 20 ng/mL TGF1. Total RNA was extracted at various time points indicated and subjected to Northern blot analysis. The results are representative of two independent experiments.

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80 Fig. 4-2. TGF-1 increased cell surface expression of CXCR4 on MDMs and changed its cellular localization. (A) FACS analysis of CXCR4 expression in control and TGF-1-treated cells. MDMs were treated with medium with or without 20 ng/mL TGF-1 for 48 h and then examined for cell surface CXCR4 expression with anti-CXCR4 or isotype control antibody staining and flow cytometry. The lower bar graph summarizes results of experiments on 8 different donors. Solid line, isotype control staining of control cells; dotted line, isotype control staining of TGF-1 treated cells; dash-dotted line, mAb 12G5 staining of control cells; bold line, mAb 12G5 staining of TGF-1 treated cells. The data was analyzed by student's t-test, *, p<0.05. (B) Immunofluorescence microscopy analysis of CXCR4 expression in control and TGF-1-treated cells. Human MDMs treated with medium with or without TGF-1 were immunostained with anti-CXCR4 antibody and visualized by immunofluorescence microscopy. The data are representative of 5 independent experiments. The microscope magnification used was 40. (C) Localization of CXCR4 in control and TGF-1-treated MDMs. The same slides in Figure 4-2B were visualized by confocal microscopy. The microscope magnification used was 40.

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81 Fig. 4-3. SDF-1-stimulated ERK1,2 phosphorylation in control and TGF1 treated cells. Human MDMs were treated with or without 20 ng/mL TGF1 for 48 h, washed twice and incubated with serum free medium for an additional 2 h. Cells stimulated with medium alone (open bars) or medium containing 50 nM SDF-1(filled bars)for 2 min and 5 min were then lysed and subjected to Western blot analysis. The levels of Phospho-ERK1,2 were normalized to those of total ERK1,2. The data was analyzed by two-way ANOVA. *, p<0.05, data represent means SEM of four experiments on four different donors.

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82 Fig. 4-4. TGF1 increased CXCR4 mRNA and enhanced SDF-1-stimulated ERK1,2 phosphorylation in primary cultures of rat microglia. (A) Rat microglia were treated with (filled bars) or without (open bars) 2 ng/mL TGF1 and total RNA was collected at the indicated time points. The levels of CXCR4 mRNA were analyzed by Northern blot analysis and normalized to the cyclophilin hybridization signal. The results are representative of multiple experiments. (B) Rat microglia and astrocytes were treated with (filled bars) or without (open bars) 2 ng/mL TGF1 for 16 h. The levels of CXCR4 mRNA were analyzed by Northern blot analysis and normalized to the cyclophilin hybridization signal. The results are representative of multiple experiments. (C) Rat microglia were treated with or without 2 ng/mL TGF1 for 16 h, washed twice and incubated with serum free medium for an additional 2 h. Cells stimulated with medium alone (open bars) or medium containing 50 nM SDF-1(filled bars) for 2 min were then lysed and subjected to Western blot analysis. The data represent means SEM of three independent experiments and analyzed with Two-way ANOVA. *, p<0.05

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83 Fig. 4-5. TGF1 increased the susceptibility of MDMs to D-X4 HIV-1 entry. Cells treated with medium with or without 20 ng/mL TGF1 for 48 h were washed once with PBS, incubated with complete medium, and subsequently inoculated with recombinant D-X4 HIV-1. Four days lter, cells were lysed and infection levels examined by determining the luciferase activity. The table lists the raw RLUs derived from experiments on 8 different donors. The bar graph summarizes the mean SEM. The data was analyzed by student t-test. *, p<0.05.

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84 Fig. 4-6. TGF1 had no effect on human MDM CCR5 mRNA or R5 HIV-1 entry into MDMs and decreased CCR5 mRNA in rat microglia. (A) Total RNA was isolated from MDMs treated with serum free medium with or without 20 ng/mL TGF1 for 48 h and subjected to Northern blot analysis. The data are representative of 3 independent experiments. Human MDMs were treated with serum free medium with or without 20 ng/mL TGF1 and then infected with JR-FL recombinant virus. The table summarizes results from three independent experiments. (B) Total RNA was isolated from rat primary microglia or astrocyte cultures treated with serum free medium with or without 2 ng/mL TGF1 for 16 h and subjected to Northern blot analysis. The data are representative of 3 independent experiments.

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CHAPTER 5 GENERAL DISCUSSION It is now well established that cytokine and chemokine networks are important in regulating immune responses. They play roles in orchestrating the development and function of a variety of immunecompetent cells. In addition to their roles in immune responses, cytokines and chemokines are also important in embryonic development, hematopoiesis, angiogenesis, cancer biology and viral pathogenesis. Their expression changes dynamically, which are necessary to regulate their functions. On the other hand, dysregulation of cytokine and chemokine network is a common theme in a variety of diseases. In the past decades, lots of data have been accumulated to describe the alteration of chemokines and chemokine receptors in immune responses and diseases; however, the mechanism and the biological consequences are not well characterized. My dissertation focused on how TGFregulated the expression and function of chemokine receptors in the CNS and immune systems. My data demonstrate that TGF-upregulated CX3CR1 and CXCR4 expression in rat microglia and human macrophages. CX3CR1 upregulation has been seen in such situations as brain ischemia and EAE (Jiang et al., 1998; Tarozzo et al., 2002). CXCR4 upregulation occurred in HIV encephalitis and AIDS dementia (Martin-Garcia et al., 2002), EAE (Jiang et al., 1998) and brain tumors (Barbero et al., 2002). However, the direct factors that cause these changes are not known. We identified one possible candidate, TGF-. TGF-expression also changed dynamically in diseases including AIDS (Hu et al., 1996; Kekow et al., 1990; Navikas et al., 1994; Pratt and McPherson, 85

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86 1997; Wahl et al., 1991). Thus we linked the multifunctional cytokine TGF-1 with chemokine receptors and proposed one possible mechanism to explain how these two specific chemokine receptors were upregulated in vivo. Furthermore, the biological consequences of upregulated CX3CR1 and CXCR4 expression were characterized. TGF-1 had opposite effects on downstream signaling mediated by these two different receptors (Figure 5-1). TGF-1 inhibited fractalkine-mediated ERK1,2 phosphorylation while it enhanced SDF-1 activation of ERK1,2. ERK1,2 phosphorylation is implicated in cell survival, activation and synthesis of different proteins such as cytokines. In the case of CX3CR1, TGF-1 inhibition of microglial activation may override the activating function of fractalkine. It is also possible that TGF-1 shifted CX3CR1 from activating downstream signaling pathways towards mainly mediating cell adhesion. A shift in chemokine receptor function as a consequence of cytokine regulation has been observed in other systems. Expression of CCR1, CCR2 and CCR5 were upregulated by LPS and IL-10, but the receptors lost their ability to promote cell migration or stimulate intracellular signaling. Instead, these receptors were converted into scavenger receptors (D'Amico et al., 2000). In the case of CXCR4, TGF-1 augmented its function: it upregulated CXCR4 expression and concurrently, enhanced CXCR4-mediated ERK1,2 phosphorylation. The increased CXCR4 expression on human macrophages has another biological meaning since it enhanced the entry of D-X4 HIV-1 variants into these cells, which may contribute to the emergence of X4 HIV-1 at later stage of AIDS. Another major finding of my study was that TGF-1 could regulate the expression of RGS proteins in microglia (Figure 5-1). TGF-1-inducible RGS2 and RGS10 could

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87 attenuate fractalkine-stimulated ERK1,2 phosphorylation, which may explain how TGF-1 inhibited fractalkine-stimulated ERK1,2 phosphorylation in spite of upregulated CX3CR1 expression. TGF-1 upregulation of RGS proteins has general biological significance because we showed for the first time that the cytokine TGF-1 and RGS protein systems interact. In addition to directly regulating the expression and function of G-protein coupled receptors, TGF-1 change of RGS protein expression adds another layer of regulation, which could be tuned to each specific ligand-receptor system. In summary, my data demonstrated that TGF-1 could upregulate the expression of chemokine receptors, namely CX3CR1 and CXCR4 in human macrophage and rat microglia. Furthermore it specifically regulated their functions directly or indirectly, possibly through TGF-1-inducible RGS proteins, in immune responses and pathogenesis of diseases such as AIDS. However, there are still questions unanswered. TGF-1 increased CX3CR1 expression in rat microglia, but the biological consequences remain unknown. One possible function could be to increase chemotactic tendency towards the ligand. However, our data (not shown) demonstrated that TGF-1 alone was a very potent chemoattractant for microglia, which obscured their migratory response to fractalkine. As I discussed earlier, membrane bound fractalkine can mediate cell adhesion and it is independent of downstream signaling. Therefore, it will be very interesting to determine if increased CX3CR1 expression can enhance the adhesion between neurons and microglia, thereby favoring direct cell-cell communication. This will be very meaningful since microglia then can release some growth factors and cytokines to help injured neurons recover specifically.

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88 To explore the mechanism of how TGF-1 inhibited fractalkine-mediated ERK1,2 phosphorylation in spite of increased CX3CR1 expression, we proposed that TGF-1-inducible RGS proteins could attenuate this activation. However, this is only indirect evidence. To prove if this is truly what happened in the primary cells, we should introduce RGS proteins into microglia. This raises another problem since microglia are notorious for transfection. Actually this was one of my major difficulties during the study. Therefore, RGS proteins need to be expressed in some kind of virus that can transduce microglia, such as AAV2. My data was derived from primary cultured cells which has advantages such as ease of investigation of specific mechanisms. Nonetheless, the results will be more significant if we translated the study into in vivo systems. For instance, we should examine if TGF-1 could regulate the expression of these chemokine receptors and RGS proteins in TGF-1 overexpressing animals. Overall, my studies have contributed to understanding the roles of TGF-1 and chemokine receptors in physiological and pathological conditions.

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89 Fig. 5-1. Model of TGF-regulation of chemokine receptors. TGFinduced CX3CR1 and CXCR4 expression in microglia and macrophages. However, it had opposite effects on intracellular signaling mediated by these two receptors. TGF-1 enhanced CXCR4-mediated ERK1,2 phosphorylation but inhibited CX3CR1-activated ERK1,2 phosphorylation. TGF--induced RGS proteins may add another layer of regulation and shift CX3CR1 from inducing downstream signal transduction to mainly mediating cell adhesion between neurons and microglia.

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BIOGRAPHICAL SKETCH In January 1978, Shuzhen was born in a village in Yiwu, Zhejiang Province, of southeast China. She spent most of her childhood in the beautiful fields full of animals and flowers when she was not in school. From then, she developed the sense of how to appreciate the great nature and how to work with her hands. At age of 14, she left her family to go to high school in the city of Yiwu. In 1995, she was admitted into the Department of Cell Biology and Genetics, College of Life Sciences, in Beijing University. In this university full of free spirits, she took lots of classes and read lots of books about science, philosophy, history and literature. Also she spent a great amount of time in the lab learning the basic technique of physics, chemistry and biology. She met lots of interesting and inspiring schoolmates and professors, some of whom went on to be long term friends. In 1999, Shuzhen joined the Interdisciplinary Program in Biomedical Sciences at University of Florida in Gainesville, FL. After three rotations, she chose Dr. Jeffrey K. Harrison’s lab in the Department of Pharmacology and Therapeutics to study the regulation and function of chemokine receptors. She had all the ups and downs of science experience, yet it was a rewarding five year experience for her, so she is ready to move on to bigger challenges in the field of biomedical sciences. 108