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Expression of chemokine receptors in rat central nervous system

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Expression of chemokine receptors in rat central nervous system
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Jiang, Yan, 1964-
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CC chemokines ( jstor )
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Chemokine receptors ( jstor )
Chemokines ( jstor )
Glioma ( jstor )
HIV 1 ( jstor )
Messenger RNA ( jstor )
Microglia ( jstor )
Rats ( jstor )
Receptors ( jstor )

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EXPRESSION OF CHEMOKIh4E RECEPTORS IN RAT CENTRAL
NERVOUS SYSTEM
















By

YAN JIANG














A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1998




























THIS DISSERTATION IS DEDICATED TO ALL THAT I LOVE













ACKNOWLEDGMENTS

I would like to thank my mentor, Dr. Jeffrey K. Harrison, who was courageous enough to let me (someone who didn't have much experience in research) to work in his lab to build up my career in medical research science. During the past four years, he patiently guided me in every step along the way of my research from pipetting to scientific thinking, writing and presentation. His constant challenge prompted me to try harder and be better which made my years in graduate school very rewarding. I would also like to express my appreciation to present and former members of the laboratory for providing an enjoyable working environment. A special thank goes to Mina Salafranca for teaching me many of the techniques necessary to complete this dissertation.

I would also like to thank every member of my committee, Drs. Wolfgang J.

Streit, Thomas C. Rowe, John M. Petitto, and Joel Schiffenbauer. I am grateful for their understanding and support. Special thanks to Dr. Streit and members of his laboratory for generously giving me unlimited access to their knowledge, facilities and friendship.

I want to express my gratitude to all of the faculty members, secretary staffs and fellow graduate students of the Department of Pharmacology and Therapeutics for making my years in graduate school a truly pleasant experience. I would like to show my appreciation to Lynn Raynor for her friendship, encouragement and enlightening conversations, especially during difficult days of my life.





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Finally, I would like to thank my family, in particular, my husband, who has always been there for me throughout our ten years of marriage, and my mother, who taught me a principle that carries my life to a truly blessed one-a principle that says it is fine if you are not the best but it is detrimental if you don't try your best.












































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TABLE OF CONTENTS
page


ACKNOWLEDGMENTS..................................................................... i

LIST OF TABLES ............................................................................. viii

LIST OF FIGURES .............................................................................. ix

ABSTRACT....................................................................................xi

CHAPTERS

1. INTRODUCTION.............................................................................. I

Chemokine: Identification, Expression, and Function..............................1.
Identification of Chemokine Peptides .............................................. 2
Production, Expression, and Functions of Chemokines ......................... 4
Molecular Cloning and Expression of Chemokine Receptors....................... 7
Molecular Cloning of CXC Chemokine Receptors ............................... 7
Molecular Cloning of CC Chemokine Receptors ................................. 9
Cloning of CX3CRI, XCRl .............I.......................................... 12
Signal Transduction Properties of Chemokine Receptors.......................... 13
Expression of Chemokines and Chemokine Receptors in the Central
Nervous System ................................................................ 15
Chemokines and Receptors Expression in EAE and Other Models for
MS................................................................................ 15
The Expression of Chemokines and Their Receptors in MS Patients......... 19 Chemokines and Receptors in Other Neuropathologies........................ 20
Microglia-the Brain Immune Responsive Cells ................................ 23
Chemokines, Chemokine Receptors and HIV........................................ 24
Some Chemokine Receptors are HIV Entry Cofactors ......................... 24
Inhibition of HIV Entry by Chemokine Ligands ................................. 26
Chemokine Receptors and W-V in the CNS ...................................... 27
Specific Aims............................................................................ 28

2. MATERIALS AND METHODS............................................................ 32

Materials................................................................................. 32
Methods ................................................................................. 33


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Polymerase Chain Reaction (PCR) Amplification of Novel DNA
Sequences ............................................................................................. 33
Isolation of Rat Genomic Clones ............................................................... 34
Southern Blot Analysis of Rat Genomic DNA ........................................... 34
Transfection of HEK293 Cells .................................................................... 34
Measurement of Intracellular Calcium Levels ........................................... 35
Expression of Rat CCR5 in Xenopus Laevis Oocytes ................................ 36
RNA Isolation, RNase Protection Assay, and Northern Analysis .............. 36
Preparation of Primary Cultures of Rat Microglia ....................................... 37
Induction of Rat EAE .................................................................................. 38
Molecular Cloning and Sequencing of Rat Fractalkine cDNA ................... 39
Expression and Functional Characterization of Rat CX3CR1 in
Transfected Cells ................................................................................. 39
In situ Hybridization Analysis .................................................................... 40
Lectin Staining and Immunohistochemical Analysis .................................. 42
Facial Nerve Transections ........................................................................... 43
Tumor Inoculation ....................................................................................... 44
Terminal Transferase-Mediated UTP Nick End Labeling (TUNELLabeling) ............................................................................................... 44

3. CHEMOKINE RECEPTOR EXPRESSION IN CULTURED GLIA AND RAT
EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS ................................. 46

Introduction ...................................................................................................... 46
R esults ................................................................................................................... 4 8
Isolation of Rat Genomic DNA Containing Sequences Encoding Novel
G-Protein Coupled Receptors ............................................................... 48
Signal Transduction Properties of Rat CCR2 and CCR5 .......................... 49
G enom ic A nalysis ...................................................................................... 50
Tissue Specific Expression of Rat CCR2 and CCR5 ................................. 50
Expression and Regulation of the Rat CCR2 and CCR5 in Cultured
M icroglia ............................................................................................... 51
Modulation in Rat EAE ............................................................................. 51
D iscussion ........................................................................................................ 52

4. ROLE FOR NEURONALLY-DERIVED FRACTALKINE IN MEDIATING
INTERACTIONS BETWEEN NEURONS AND CX3CR1-EXPRESSING
MICROGLIA-AN IN VIVO STUDY IN THE FACIAL MOTOR NEURON
REGENERATION PARADIGM ........................................................................... 65

Introduction ...................................................................................................... 65
Results68
Molecular Cloning of Rat Fractalkine ........................................................ 68
Tissue Distribution of Rat Fractalkine ......................................................... 68
Distribution of Rat Fractalkine in the Brain ................................................ 68
Identification of RBS 11 as the Receptor for Rat Fractalkine .................... 69


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Expression and Regulation of Rat CX3CR1 mRNA in Cultured
M icroglia ................................................................................................ 69
Expression and Regulation of rat CX3CR1 In Vivo in the Facial Motor
Nucleus After Facial Nerve Transection ................................................ 70
Expression of Rat Fractalkine in FMN After Facial Nerve Transection ......... 71
D iscussion ........................................................................................................ 71

5. DIFFERENTIAL EXPRESSION OF CX3CR1 IN TWO RAT GLIOMA
MODELS-ROLE FOR TGF-[ AND CX3CRI IN MICROGLIAL CELL
DEATH WITHIN RAT BRAIN TUMORS ........................................................... 84

Introduction ...................................................................................................... 84
R esults ................................................................................................................... 87
Expression of Rat CX3CR1 in C6 Glioma .................................................. 87
Rat CX3CR1 mRNA Expression by Microglia in C6 Gliomas .................. 87
Different Microglial-Infiltrating Patterns Between C6 and RG2
G liom as .................................................................................................. 88
Microglial Apoptosis Within C6 and RG2 Gliomas .................................... 88
D iscussion ........................................................................................................ 89

6. CONCLUSIONS AND FUTURE DIRECTIONS .................................................... 97

LIST O F REFEREN CES ................................................................................................. 101

BIOGRAPHICAL SKETCH ........................................................................................... 126

























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LIST OF TABLES

Table page

1-1. Ligand Specificity of Chemokine Receptors .................................................. 30

3-1. Amino Acid Identities of the Rat, Murine and Human Chemokine Receptors,
C C R2 and C C R 5 .................................................................................................... 57





































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LIST OF FIGURES

Figure page

1-1. Amino Acid Alignment of Representative Members of the Human Chemokine
Superfam ily ................................................................................................................ 29

1-2. Lectin (GSA I-B4) Staining of Facial Motor Nucleus Four Days After Facial
N erve Transection .................................................................................................. 31

3-1. Sequence Comparison of Rat and Murine C-C Chemokine Receptors ............ 56

3-2. Calcium Mobilization Response to MCP-1 By HEK293 Cells Expressing Rat
C C R 2 ..................................................................................................................... 58

3-3. Functional Responses of Cells Expressing Rat CCR5 ...................................... 59

3-4. Southern Blot Analysis of Restriction Endonuclease-Digested Rat Genomic
D N A ....................................................................................................................... 60

3-5. Rat CCR2 RNA Tissue Distribution in Rat Solid Organs and Cells ................ 61

3-6. Rat CCR5 mRNA Tissue Distribution in Rat Solid Organs and Cells ............. 62

3-7: Dose-Dependent Increase in Rat CCR5/2 mRNA by Interferon-y (IFNy) Treated
M icroglia .................................................................................................................... 63

3-8. Expression of CCR2, CCR5, CXCR4, and RBSI1 mRNAs in Lumbar Spinal
Cords of EAE Rats as Detected by RNase Protection Assay or Northern Blot
A nalysis ..................................................................................................................... 64

4-1.Amino Acid Sequence of Rat Fractalkine (Genbank accession number R75309)..75 4-2. Nase Protection Analysis of Fractalkine mRNA in Various Tissues of Adult Rat 76 4-3. Ddistribution of Rat Fractalkine mRNA in Adult Rat Brain ............................ 77

4-4. Whole Cell Radioligand Binding Analysis of CHO-CX3CR1-Expressing Cells..78




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4-5. Calcium Mobilization Response to Different Forms of Fractalkine by CHO Cells Expressing Rat CX 3CR1 ..................................................................................... 79

4-6. Dose-Dependent Increases in Rat CX3CR1 mRNA by Transforming Growth Factor-P (TGF-P) Treated M icroglia .................................................................... 80

4-7. In Situ Hybridization Analysis of CX3CR1 Expression in the Rat Facial Nucleus After M otor Neuron Axotomy ............................................................................. 81

4-8. Colocalization of Rat CX3CR1 mRNA With Microglial Cells ....................... 82

4-9: Fractalkine Expression in the Rat Facial Nucleus After Motor Neuron Axotomy 83 5-1. Expression of Rat CX3CR1 in C6 Glioma-Northem Blot Analysis .............. 92

5-2. Expression of Rat CX3CR1 in C6 Glioma-In Situ Hybridization Analysis ........ 93

5-3. Expression of Rat CX3CR1 in Infiltrating Microglia in C6 Gliomas .............. 94

5-4. Lectin (GSA I-B4) Staining of RG2 (A, B) and C6 (C, D) Sections ................ 95

5-5. TUNEL (Terminal Transferase dUTP Nick End-Labeling) Analysis in RG2 and C 6 G liom as ................................................................................................................ 96


























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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


EXPRESSION OF CHEMOKINE RECEPTORS IN RAT CENTRAL NERVOUS SYSTEM

By

Yan Jiang

December, 1998
Chairman: Jeffrey K. Harrison
Major Department: Pharmacology and Therapeutics

Chemokines (Chemoattractive cytokines) are a fast growing family of more than 40 structurally related proteins that are responsible for leukocyte chemoattraction and activation during inflammatory processes. Receptors for the chemokine peptides are members of the G-protein coupled receptor superfamily.

While the expression and functional roles of chemokine and chemokine receptors in peripheral systems have been well characterized, related information regarding their role in the central nervous system (CNS) is very limited. A number of chemokine peptides and receptors have been found to be expressed or up regulated in neuropathological circumstances such as EAE (Experimental Allergic Encephalomyelitis), an animal model of human multiple sclerosis.

This study examined expression characteristics of chemokine receptors in the rat CNS. Two genes of the rat orthologs of human CC chemokine receptors, CCR2 and



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CCR5, have been isolated. While both are expressed in a number of peripheral tissues of adult rat, CCR5 mRNA is also found in rat brain, spinal cord, and cultured microglia. IFN-y treatment of cultured microglial upregulates CCR5 expression in a dose-dependent manner. While CCR5 mRNA expression was elevated in EAE rats, CCR2 mRNA becomes detectable in the spinal cord of these diseased rats.

We also studied the expression of RBS 11, a chemokine receptor-like gene, which has recently been identified as receptor for a newly characterized chemokine termed fractalkine. Both fractalkine and RBS 11 (CX3CRI) are constitutively expressed in a wide variety of regions of the brain. In situ hybridization analysis revealed that rat CX3CRI is expressed primarily in microglia whereas its ligand, fractalkine, is expressed in neurons. Interactions between this ligand and receptor may provide a mechanism by which neurons communicate with microglia.

Lastly, we examined the expression of fractalkine and CX3CRI in two rat glioma models: C6 and RG2 gliomas. Although rat fractalkine was not detectable in either of the gliomas, rat CX3CR1 mRNA was detected in C6 but not RG2 gliomas. The differential expression of CX3CR1 in these gliomas suggests that CX3CRlexpression may be one of the markers of the functional state of microglia in the CNS.















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CHAPTER 1
INTRODUCTION

Chemokines (Chemoattractive cytokines) are a fast growing family of more than 40 structurally related proteins that are responsible for leukocyte chemoattraction and activation during inflammatory processes (Luster 1998, Baggiolini et al., 1994, Baggiolini et al., 1997). These proteins can be divided into at least four subfamilies based primarily on the relative position of four highly conserved cysteine residues in their amino acid sequences. Receptors for some of the chemokine peptides have been identified by molecular cloning techniques. They are members of the G-protein coupled receptor superfamily. Chemokine receptors can induce a number of cellular signaling molecules after being stimulated by their ligands. While the expression and function of chemokines and their receptors have been widely explored in peripheral systems, there is very limited information regarding the expression and function of these molecules in the central nervous system (CNS). Studies carried out in this dissertation have focused on characterizing the expression and regulation of chemokine receptors in the rat central nervous system (CNS).

Chemokine: Identification, Expression, and Function


Chemokines are a group of proteins that are involved in leukocyte recruitment

during inflammation. To date, this family consists of more than 40 members that can be divided into four subfamilies according to the arrangement of highly conserved cysteine



I





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residues in their amino acid sequences (Figure 1-1). By far, CC and CXC chemokine subfamilies are the largest among these four subfamilies. Members of both subfamilies have four highly conserved cysteine residues. The first cysteine forms a disulfide bond with cysteine 3 and the second cysteine is linked to cysteine 4 by a disulfide bridge. In the CXC chemokine subfamily, the first two cysteines are separated by one amino acid whereas in CC chemokines, the first two cysteines are adjacent to each other. Many CXC chemokines are chemotactic for neutrophils while most CC chemokines have chemotactic activity toward monocytes, macrophages, lymphocytes, eosinophils and basophils.

Representative members of CXC chemokine subfamily are IL-8 (interleukin 8), PF-4 (platelet factor 4), GRO (growth-regulated oncogene) -c, -3, -y, IP-10 (interferoninducible protein 10) and SDF-1 (stromal cell-derived factor 1). Representative members of the CC chemokine subfamily include the MCPs (monocyte chemoattractant protein-1,

-2, -3, -4, -5), the MIPs (macrophage inflammatory protein-I, 13, and -3), RANTES (regulated upon activation normal T cell expressed and secreted) and 1-309.

Lymphotactin is the only chemokine, identified to date, that has only two

cysteines in its amino acid sequence. It is chemotactic for lymphocytes and is designated as the first member of C chemokine subfamily (Kelner et al., 1994). Recently, a novel chemokine peptide, termed fractalkine, has been characterized that has three amino acids between the first two cysteine residues. It is designated as the first member of CX3C chemokine subfamily (Bazan et al., 1997, Pan et al., 1997). Identification of Chemokine Peptides

The first identified chemokine peptide was PF-4 (Deuel et al., 1977, Hermodson et al., 1977, Walz et al., 1977). Because of its high affinity for heparin, this peptide was








isolated using heparin-sepharose chromatography. Its amino acid sequence was determined by treatment of this peptide with different peptidases and ultimately analyzed by protein sequencing. PF-4 is a 70-residue peptide with a highly negatively charged Nterminal region, characteristic of most of the chemokine peptides.

Ten years after the discovery of PF4, human IL-8 was identified by three

independent groups. Walz et al. (1987) and Yoshimura et al. (1987) purified this peptide from conditioned media of human blood monocyte cultures treated with E.coli lipopolysaccharide (LPS). Simultaneously, Schmid and Weissman (1987) isolated an IL8 cDNA clone from a cDNA library of SEA (staphylococcal enterotoxin A)-treated human peripheral blood leukocytes by the technique of differential colony hybridization (St. John and Davis 1979). IL-8 was found to be 35% identical to PF-4 in their amino acid sequences.

Among the CC chemokine subfamily, MIP-lca (Obaru et al., 1986) and MIP-1 13 (Lipes et al., 1988) were the first to be identified. A cDNA encoding human MIP-l a was isolated from a cDNA library constructed from the mRNA of TPA (12-o-tetradecanoyl phorbol-13-acetate) or PHA (phytoemagglutinin)-stimulated human tonsillar lymphocytes by differential colony hybridization. MIP- 113P was isolated in a similar manner from an activated T-cell library. MCP-1 is the most characterized CC chemokine. It was isolated by several groups using either protein purification and sequencing or sequence homology screening techniques (Yoshimura et al., 1989a-c, Matsushima et al., 1989, Furutani et al., 1989, Rollins et al., 1989). In the past ten years, more than 40 CXC and CC chemokine peptides have been identified by techniques including protein purification and sequencing, sequence homology screening, or signal sequence trapping (Yoshie et al.,





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1997). These peptides are generally 90-99 amino acids long with a 20-25 amino-acid signal sequence.

The only member of the C chemokine subfamily, lymphotactin, was isolated from a complementary DNA library generated from activated mouse pro-T cells (Kelner et al., 1994). Its gene is located on human chromosome one. While Kelner reported its chemotactic activity for lymphocytes, Yoshida et al, (1998) experienced difficulties in reproducing these results. The functional activity of this peptide has yet to be determined.

Fractalkine, a newly identified chemokine, is the first member of the CX3C chemokine subfamily (Bazan et al., 1997, Pan et al., 1997, Rossi et al., 1998) and has some very unique properties. The full-length amino acid sequence is comprised of five regions: a signal peptide, a chemokine domain, a highly glycosylated mucin-like stalk which tethers the chemokine domain with a transmembrane domain and a short intracellular region. There are three amino acids in between the first two of the four highly conserved cysteine residues in its chemokine domain. This peptide can exist as both secreted soluble and membrane anchored forms. The soluble form is chemotactic for monocytes and lymphocytes while the membrane anchored form can promote a strong adhesion of these leukocytes to endothelial cells. Unlike most chemokine peptides, fractalkine is expressed in non-hematopoietic tissues including brain, kidney, lung, heart and adrenal gland.

Production, Expression, and Functions of Chemokines

A wide variety of cells can produce chemokine peptides including

monocytes/macrophages, T cells, B cells, fibroblasts, endothelial cells, epithelial cells, keratinocytes, platelets, neutrophils, smooth muscle cells and a number of tumor cell





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lines. In the CNS, microglia, astrocytes as well as neurons have been shown to synthesize chemokine peptides including 1L-8, MCP- 1, MIP- I a, MIP- 113, TCA3 (T-cell activation3), and IP-10; expression has been demonstrated from both in vitro and in vivo studies (Van Meir et al., 1992, Ransohoff et al., 1993, Vanguri and Farber, 1994, Huurwitz et al., 1995, Hayashi et al., 1995, Glabinski et al., 1997, Xia et al., 1998). A number of brain tumor tissues can also make chemokine peptides (Desbaillets et al., 1994, Takeshima et al., 1994).

Chemokines act on different types of cells such as monocytes/macrophages, T cells, basophils, fibroblasts, endothelial cells, keratinocytes, melanoma cells, and neutrophils to elicit their functions. The biological functions of chemokines include chemotaxis (Colditz et al., 1989, Rampart et al., 1989); changing shape of the target cells that is important for cell motion (Omann et al., 1987); release of histamine, elastase, 3glucuronidase, myeloperoxidase, and other enzymes (Kuna et al., 1992, Walz et al., 1987, Peveri et al., 1988); promotion of adherence (Detmers et al., 1991); induction of the respiratory burst (Rollins et al., 1991); and increased cell growth (Bordoni et al., 1990). In addition, chemokines play some roles in "normal" cellular physiology as well. They have been shown to be involved in such diverse processes as development, hematopoiesis (Nagasawa et al., 1998), angiogenesis (Strieter et al., 1995) and apoptosis (Hesselgesser et al., 1998). In the past few years, numerous evidence has indicated that a number of chemokines including MIP-lac, MIP- 113, RANTES (Cocchi et al., 1995), SDF-a (Oberlin et al., 1996, Bleul et al., 1996), MCP-2 (Gong et al., 1998), MDC (macrophage-derived chemokine) (Pal et al., 1997) and eotaxin (Choe et al., 1996) can inhibit HIV entry and/or viral replication within host cells.





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The expression or induction of expression of chemokines is found in a variety of tissues during inflammation (Luster 1998). For instance, the concentration of IL-8 and ENA-78 were increased in bronchoalveolar during bacterial pneumonia (Chollet-Martin et al., 1993, Huffnagle, 1996); MCP-1, RANTES, IP-10 mRNA were found during glomerulonephritis (Tang, et al., 1995); MCP-1 I and MCP-4 were seen in the plaque of atherosclerosis (Nelken et al., 1991, Berkhout et al., 1997); MCP-1, MIP-l ct, eotaxin, IP10, IL-8 were found to be markedly increased in inflammatory bowel disease (GarciaZepeda et al., 1996b, Grimm and Doe, 1996, Reinecker et al., 1995); RANTES, MIP-lp, IL-8, ENA-78 (epithelial derived neutrophil attractant) were detected in rheumatoid arthritis (Robinson et al., 1995, Koch et al., 1994). The appearance of different groups of chemokines in different inflammatory diseases where there is a subset of leukocyte infiltration suggests that in vivo, chemokines have the capacity to control precisely the type of leukocyte infiltration.

There is a group of newly identified CC chemokines, including ELC (EBI I -ligand chemokine, Yoshida et al., 1997), SLC (secondary lymphoid-tissue chemokine, Nagira et al., 1997), TARC (thymus and activation-regulated chemokine, Imai et al., 1996), TECK (thymus-expressed chemokine, Vicari et al., 1997) and DC-CK 1 (dendritic cell-derived chemokinel, Adema et al., 1997), that are expressed constitutively in lymphoid tissues. All of them are found to be chemotactic for lymphocytes, including T cells, B cells or naive lymphocytes, but not for monocytes or neutrophils. The unique properties of these peptides suggest that they may constitute a new group of CC chemokines that are responsible for trafficking and homing of lymphocytes to particular lymphoid tissues, as part of their immune surveillance function.





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Molecular Cloning and Expression of Chemokine Receptors


Many of the receptors for these chemokine peptides have been characterized by molecular cloning techniques (Table 1-1). They are members of the rhodopsin family of the G-protein coupled receptor superfamily, which are characterized by their putative seven hydrophobic transmembrane helices in their amino acid sequences. Molecular Cloning of CXC Chemokine Receptors

To date, five human CXC chemokine receptors have been identified. Two

receptors for IL-8 were the first chemokine receptors to be identified. In 1991, William Woods' group characterized a receptor by expression cloning of a human neutrophil cDNA library that was transfected into COS-7 cells as pools and examined for L-8 binding activity (Holmes et al., 1991). The pure clone obtained encodes a 350-aminoacid protein that not only binds to, but also responds to IL-8, to form a transient intracellular calcium increase in transfected cells. Its amino acid sequence was found to be most closely related to two other G-protein coupled receptors, the fMLP and C5a receptors. On the other hand, Philip Murphy's group (Murphy and Tiffany, 1991) identified another IL-8 receptor gene in an attempt to isolate the human ortholog of a rabbit N-formyl peptide receptor (F3R). They designed an oligonucleotide probe corresponding to nucleotides of the cDNA sequence of F3R to screen the cDNA library of a leukemia cell line (HL-60). The isolated gene contained a 1065-bp open reading frame (orf), encoding a 355-amino-acid protein. It is 77% identical to the other IL-8 receptor. It can bind to IL-8 and several other CXC chemokines including MGSA (melanoma growth stimulatory activity)/GRO and NAP (neutrophil activation protein)-2. Application of





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these peptides to Xenopus oocytes that express this receptor triggered an intracellular Ca2+ mobilization (Murphy et al., 1991). These two IL-8 receptors were later designated as CXCR1 and CXCR2. Both are expressed on neutrophils. CXCR2 is also found in myelomonocitic cell lines and its expression can be stimulated by G-CSF (granulocyte colony stimulating factor) and TNF (tumor necrosis factor)-ct (Sprenger et al., 1994).

RT-PCR technique was used to isolate human CXCR3. Degenerate

oligonucleotide primers were designed based on the sequence of transmembrane domain2 (TM2) and TM7 of other CXC and CC chemokine receptors (Loetscher et al., 1996). This receptor gene encodes a protein of 368 amino acids with putative seven transmembrane domains. The CXC chemokines Mig (monokine induced by interferon-y) and IP-10 were identified as ligands for this receptor because of their ability to stimulate Ca2+ mobilization and chemotaxis of receptor transfected cells. Northern blot analysis revealed that this receptor was expressed in T cells stimulated with 1L-2. Recently, another CXC chemokine I-TAC (Interferon-inducible T cell Alpha Chemoattractant, Cole et al., 1998) was found to be the ligand of human CXCR3 and a CC chemokine peptide 6Ckine (Soto et al., 1998) was shown to be the ligand for murine CXCR3.

In 1994, Loetscher et al. identified an "orphan" seven transmembrane domain

receptor that was expressed in neutrophils, activated T cells and some leukemia cell lines. This receptor was later found to be a co-receptor for T-cell line tropic HIV entry (Feng et al., 1996). Two years later, a CXC lymphocyte chemoattractant, SDF-1 I (stromal cellderived factor 1), was proved to be the ligand of this receptor (Bleul et al., 1996 and Oberlin et al., 1996). According to the accepted nomenclature of chemokine system, it was designated as human CXCR4. Two CXCR4 "knock out" studies reported most








recently (Zou et al., 1998, Tachibana et al., 1998) suggest that CXCR4 plays a very important role in embryonic development of the cerebellum as well as vascularization of the gastrointestinal tract.

Human CXCR5 was also isolated as an orphan receptor that was expressed in B cells and Burkitt's lymphoma tissue (Dobner et al., 1992). Forster et al. (1996) provided evidence indicating the involvement of this receptor in B cell migration and localization by using CXCR5 "knock out" mice. Recently, Gunn et al. (1998) and Legler et al. (1998) characterized a CXC chemokine BLC (B-lymphocyte chemoattractant)/BCA-1 I (B cellattracting chemokine 1) that can bind and stimulate Ca2 mobilization and chemotaxis of cells transfected with the CXCR5 gene.

Molecular Cloning of CC Chemokine Receptors

To date, ten human CC chemokine receptors have been characterized by different groups. Neote et al. (1993) and Gao et al. (1993) discovered human CCRI1 gene using sequence homology cloning techniques in their attempt to isolate chemokine receptors that are closely related to IL-8 receptors and the fMLP receptor. Specific binding and Ca 2 mobilization analysis revealed that RANTES, MIP-lat are ligands of this receptor. CCR1 mRNA was detected in B-lymphocytes and some human myeloid precursor cell lines e.g. HL-60, U937 and THP-1. Additional chemokine peptides have been characterized in recent years; MCP-2 (Gong et al., 1997), MCP-3 (Combadiere et al., 1995b), Leukotactin- 1 (Youn et al., 1997), and Hemofiltrate C-C chemokine (HCC)-1 (Tsou et al., 1998) have since been identified as ligands of CCR1.

Human CCR2a and CCR2b are MCP-1 I receptors with alternatively spliced

carboxyl tails (Charo et al., 1994). RT-PCR technique was performed using mRNA from





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a monocytic cell line as the template to isolate these receptor genes. Degenerate primers were designed based on conserved sequences of CCR1, CXCR1 and CXCR2. Northern blot analysis indicated that both CCR2 receptors are expressed in monocytes and THP-1 and MonoMac 6 cell lines. While MCP-1 is the only known ligand for CCR2a, MCP-2, MCP-3 and MCP-4 are found to be additional ligands for CCR2b (Gong et al., 1997, Combadiere et al., 1995b, Godiska et al., 1997).

Similarly, CCR3 was cloned by several different groups (Combadiere et al.,

1995a, Ponath et al., 1996, Daugherty et al., 1996, Kitaura et al., 1996). This receptor is expressed in eosinophils and basophils (Uguccioni et al., 1997) and weakly in monocytes and neutrophils. It is the only receptor, to date, for eotaxin-1 and eotaxin-2 (White et al., 1997). However, RANTES, MCP-1 (Daugherty et al., 1996), MCP-2 (Heath et al., 1997), MCP-3 (Ponath et al., 1996), MCP-4 (Garcia-Zepeda et al., 1996a) Leukotactin-1 I (Youn et al., 1997) are also ligands for CCR3.

Human CCR4 was isolated from a basophil cell line, whose amino acid sequence shares only 49% identity to CCR1 (Power et al., 1995). It is expressed at high levels in thymus, T cells, B cells, monocytes, platelets, and to a lower level in spleen. The identification of its ligand(s) has been controversial. While Power et al. demonstrated detectable Ca2+ dependent Cl currents in CCR4 expressing oocytes by MIP-l ca, MCP-1 and RANTES application, Imai et al. (1997a) failed to stimulate Ca 2 mobilization in CCR4 expressing 293/EBNA cells with these ligands. However, they successfully identified TARC (thymus and activation-regulated chemokine) as a specific ligand for CCR4. Recently, Imai et al. (1998) demonstrated that MDC (macrophage-derived chemokine) is also a ligand for CCR4.





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A number of different groups (Samson et al., 1996a, Raport et al., 1996,

Combadiere et al., 1996) isolated CCR5 by RT-PCR technique. These investigators were able to identify MIP-l c, MIP- 113P, and RANTES as its ligands. Gong et al. (1998) provided evidence demonstrating that MCP-2 is also a ligand for CCR5. This receptor was found to play an important role in macrophage-tropic HIV-1 infection as a fusion cofactor (Deng et al., 1996, Dragic et al., 1996, Alkhatib et al., 1996).

Human CCR6 (Zaballos et al., 1996, Liao, et al., 1997a), CCR7 (Schweickart et al., 1994), and CCR8 (Zaballos et al., 1996, Napolitano et al., 1996, Samson et al., 1996b) were isolated via RT-PCR and initially reported as orphan receptors. LARC (liver and activation-regulated chemokine)/MIP-3a/Exodus- 1 was found to be the ligand of CCR6 (Baba et al., 1997, Power et al., 1997, Greaves et al., 1997, Liao et al., 1997b). This receptor is expressed in spleen, lymph node, gut, pancreas and peripheral blood lymphocytes, monocytes/macrophages. Two CC chemokine peptides, MIP-3p/CKb1 I/ELC (EBIl-ligand chemokine) and SLC (secondary lymphoid-tissue chemokine), can specifically bind to CCR7 expressing cells and stimulate a transient increase of the intracellular Ca2+ level (Yoshida et al., 1997, Willimann et al., 1998). A number of lymphoid organs such as tonsil, spleen, and lymph node constitutively express CCR7. This receptor was also found in several B- and T-cell lines but not in the myeloid cell lines. Northern blot analysis was carried out on different tissue samples to determine the expression of CCR8. CCR8 mRNA was detectable in spleen, lymph nodes and very weakly in appendix and lymphocytes. To date, 1-309 is the only ligand found to be able to interact with CCR8 expressing cells and stimulate Ca2+ mobilization (Roos et al., 1997, Goya et al., 1998).





12


Choe et al. (1998) have presented data demonstrating that human CCR9 can serve as a coreceptor for HIV-1 and SIV entry. However, they did not present in detail how this receptor was cloned and its amino acid sequence, neither did they discuss the tissue distribution pattern of this receptor. Human CCR10 was identified by screening a human lung cDNA library with a 32p labeled probe containing known EST (Expressed Sequence Tag) # 149079 sequence (Bonini et al., 1997). The resulting cDNA contains a coding region of 1,1 52bp for a 384-amino-acid protein. Its amino acid sequence is around 30% identical to the other CC chemokine receptors. MCP-1, MCP-3, MCP-4 and RANTES can bind to CCRlO expressing COS-7 cells in nanomolar concentration level. However, binding of MCP-3 to the receptor did not stimulate a transient increase of the intracellular Ca21 levels. Thus, the real ligand(s) for CCR1O remains to be determined. Cloning of CX3CRI, XCRI

The receptor for fractalkine, designated as CX3CR1, was identified recently (Imai et al., 1997b). Previously, it was considered to be an orphan receptor (Harrison et al., 1994, Raport et al., 1995, Combadiere et al., 1995c). It was found in many lymphoid cell lines and is broadly expressed in a variety of tissues including many discrete regions of the brain.

Most recently, the receptor for lymphotactin, designated as XCRI, has been

identified by Yoshida et al. (1998). It is expressed in placenta and weakly in spleen and thymus among various human tissues. Lymphotactin binds to XCR1 and triggers an intracellular Ca increase and stimulates chemotaxis of XCR1 expressing cells. The functional target of lymphotactin remains to be determined because of the controversial results between two groups (Kelner et al., 1994, Yoshida et al., 1998).





13


Finally, there is an interesting chemokine receptor termed Duffy antigen receptor for chemokines (DARC) which interacts with most of the CC and CXC chemokines but does not signal (Chaudhuri et al., 1993, Horuk et al., 1993). This gene encodes a 338amino-acid protein that also contains seven hydrophobic transmembrane regions conserved in all G-protein coupled receptors. Its function is likely to serve as reservoir of chemokines in the body.

The murine and rat orthologs of many of these human chemokine receptors have also been identified (Bozic et al., 1994, Cerretti et al., 1993, Harada et al., 1994, Heinrich and Brano, 1995, Lee et al., 1995, Dunstan et al., 1996, Nagasawa et al., 1996, Heesen et al., 1996, Gao and Murphy, 1996, Hoogeworf et al., 1996, Post et al., 1995, Boring et al., 1996, Kurihara and Brano, 1996, Gao and Murphy, 1995, Meyer et al., 1996, Schweickart et al., 1994, Zingoni et al., 1998), (Table 1). These murine and rat orthologs will be useful in studying the expression, regulation and functional role of chemokines and chemokine receptors in vivo under physiological and pathological conditions using a number of murine or rat animal models of human diseases.

Signal Transduction Properties of Chemokine Receptors


Chemokines act on their receptors to stimulate a number of signal transduction pathways to elicit their functions. Almost all receptors stimulate a transient intracellular calcium increase upon activation. In most cases, except for MCP-2, this response can be abolished by pertussis toxin pretreatment (Baggiolini et al., 1997, Sozzani et al., 1994) suggesting a Gi protein involvement in their signal transduction pathways. Complementation experiments in cotransfected COS-7 cells indicated that CXCR1 and





14


CXCR2 can couple to many G-protein subtypes including Gi2, Gi3, G14, G15, and G16 (Wu et al., 1993). Kuang et al. also examined the selective G protein coupling by CC chemokine receptors in cotransfected COS-7 cells (1995). Their data revealed that while both CCR1 and CCR2a couple to G14, only CCR2a couples to G16. Neither CCR1, CCR2a nor CCR2b couple to Gq/11. A similar study using both COS-7 and HEK-293 cell lines suggests that receptor-G-protein interactions are highly cell type specific (Arai and Charo, 1996). The GI3y subunits of Gi proteins have been shown to activate specific isoforms of PLCP (Wu et al., 1993, Kuang et al. 1995) and mediate chemotaxis (Neptune and Bourne, 1997 and Arai et al., 1997). When human neutrophils are activated by 1L-8, but not GRO-a. or NAP-2, there is a stimulation in phospholipase D activity in these cells which is accompanied by the increase of respiratory burst (L'Heureux et al., 1995, Jones et al., 1996). RANTES has also been shown to be able to stimulate phospholipase D activity in Jurkat cells (Bacon et al., 1998). The inhibition of cAMP (Shyamala et al., 1997), induction of arachidonic release (Locati et al., 1994) and stimulation of inositol phosphate formation (Arai et al., 1996) have been well documented in chemokine receptor transfected cell systems when exposed to the chemokine peptides. Bacon et al. have demonstrated that the induction of mobilization of Ca2+ in T cells by RANTES is mediated by both a G protein coupled pathway and activation of protein tyrosine kinases (1995). Other studies have also provided evidence of the activation of a number of different tyrosine kinases when the cells are treated with chemokine peptides (Schraw and Richmond, 1995, Bacon et al., 1996, Ganju et al., 1998). Studies from two groups revealed that chemokines can stimulate the activation of MAP-kinases (Jones et al., 1995, Knall et al., 1996), phosphoinositide 3'-kinases (Knall et al., 1997, Turner et al., 1995),





15

and protein kinase B (Tilton et al., 1997) in the chemokine receptor expressing cells. Recently, Wong et al. reported the activation of STATs (Signal Transducers and Activators of Transcription) in T cells by RANTES and MIP-la (Wong et al., 1998).

In conclusion, different chemokine peptides use different G proteins in different cells to stimulate different signal transduction pathways for different cellular responses. However, more studies are needed in order to understand, more completely, the signal transduction pathways mediated by chemokines and chemokine receptors.

Expression of Chemokines and Chemokine Receptors in the Central Nervous System


While the expression and functional roles of chemokines and their receptors have been widely explored in the periphery, there is very limited information available regarding the expression and function(s) of chemokines and chemokine receptors in the CNS. Most of this information has been collected from studies on experimental allergic encephalomyelitis (EAE), the best known laboratory animal model of human multiple sclerosis (MS).

Chemokines and Receptors Expression in EAE and Other Models for MS

EAE is an inflammatory demyelinating disease of the central nervous system, which has many clinical and pathological features in common with multiple sclerosis

(MS). In 1933, Rivers and his coworkers discovered that immunization of susceptible animals with brain tissue could induce an acute or chronic encephalomyelitis. This model has evolved considerably over the last few decades. EAE can be produced in monkeys, mice, rats, guinea pigs and rabbits using a variety of antigen preparations, including whole spinal cord, MBP (myelin basic protein) (Kies et al., 1960), PLP (proteolipid





16


protein) (Madrid et al., 1982), and peptides of these proteins (Pettinelli et al., 1982, Touhy, 1994). EAE can also be transferred via intravenous injection of lymphocytes derived from sensitized donors (Paterson, 1960) or MBP-reactive cells (Ben Nun et al., 1981).

Pathological findings of EAE animals include intense inflammation and some demyelination of brain and spinal cord. Clinical signs occur within 9-15 days after injection, which consist of weight loss, lethargy, loss of tail tonicity, different degrees of paraparesis, and incontinence.

Because of the extensive inflammation involved in EAE, it has become a very useful animal model for studying the expression and function of cytokines, including chemokines, in the CNS. In 1993, Ransohoff et al. demonstrated expression of the chemokine peptides MCP-1 and IP-10 in the spinal cords of EAE mice using RT/PCR and in situ hybridization analysis. Their data suggested that astrocytes were the source of MCP-1 and IP-10 mRNA. At the same time, Berman's group demonstrated expression of MCP-1 mRNA and protein in spinal cords of EAE rats. MCP-l mRNA levels were elevated immediately before the onset of clinical signs, peaked with the height of clinical disease, and declined with resolution of the disease (Hulkower et al., 1993). Later, they defined the localization of MCP- 1 by immunohistochemistry and in situ hybridization (Berman et al., 1996). Their data indicated that at the onset of inflammation, prior to clinical signs, MCP-1 was detectable in lymphocytes and endothelial cells in subarachnoid locations whereas after the onset of the clinical signs, MCP-1 was widely distributed in the spinal cord in lymphocytes, macrophages, astrocytes and endothelial cells.





17

Godiska and colleagues examined the expression of a number of chemokines in EAE mice using RT/PCR and Northern blot analysis and found that mRNAs encoding RANTES, MIP-la, MIP-1B, TCA3, IP-10, MCP-1 KC/GRO and MCP-3 were expressed in the spinal cords of EAE mice. Also, SDF was expressed in normal as well as diseased spinal cords and no expression of MIP-2 was detected in either normal or diseased tissue (1995). Using in situ hybridization analysis combined with immunohistochemistry, Miyagishi et al. identified the cellular localization of RANTES and MIP-113 in rat EAE. Their evidence indicated that mRNAs for RANTES and MIP-1B were mostly expressed by CD3-positive T-cells located around blood vessels. These mRNAs were also colocalized with GFAP-positive astrocytes as well as OX42-positive macrophages/microglia (1997). They were able to detect MIP-l a expression in the spinal cord by day 10 post injection, but they failed to identify its cellular colocalization due to the fact that the combination of immunohistochemistry and in situ hybridization was much less sensitive using MIP- la probes.

Glabinski and colleagues examined the time course of the expression of five

chemokines (MCP- 1, MIP-I a, KC/GRO-a, IP- 10, and RANTES), during spontaneous relapses of chronic EAE mice (1997), using RT-PCR, ELISA, and in situ hybridization analysis. They found that all five chemokines were simultaneously up regulated during relapses in the brain and spinal cord, in addition, expression was confined to the CNS. In situ hybridization revealed that IP-10 and MCP-1 are expressed by GFAP-positive astrocytes. The nuclear morphology and location of KC-expressing cells suggested astrocytes as the cellular expressions of this chemokine. MIP-I oa and RANTES are produced exclusively by mononuclear leukocytes in perivascular cuffs. Interestingly,





18

while other groups failed to demonstrate any expression of MIP-2, a CXC chemokine peptide, Adamus et al. (1997) were able to detect MIP-2 mRNA expression in Lewis rats at day 10 after MBP injection, the time of disease onset. Their in situ hybridization data suggested that MCP- 1 was expressed in astrocytes and infiltrating cells in the white matter.

The involvement of chemokines in EAE development became evident when

Karpus et al. (1995) demonstrated that injection (i.p.) of an antibody towards MILP-la to SJL/J mice passively transferred with EAE can prevent the development of clinical disease as well as infiltration of mononuclear cells into the CNS. Antibody therapy could also be used to ameliorate the severity of ongoing clinical disease of these EAE mice. However, in their report, they didn't see the production of MCP-1 and MIP-2 peptides by ELISA in diseased animal spinal cord, anti-MCP- 1 failed to prevent the disease development. In another study with the attempt to intervene the development of EAE, Wojcik et al. (1996) revealed that intrathecal administration of antisense oligonucleotides to MBP-injected Lewis rats could significantly reduce EAE scores at the height of clinical disease.

Recently, Lane et al. (1998) examined the expression of a number of chemokines using another model, virus-induced encephalomyelitis. Transcripts of CRG-2 (cytokineresponse gene-2)/IP- 10, RANTES, MCP- 1, MCP-3, MP- 1 3, and MIP-2 were seen in the brain of mouse hepatitis virus-infected mice 3 days postinfection. These transcripts were increased markedly in brains and spinal cords at day 7, which coincides with the occurrence of acute viral encephalomyelitis. Their in situ hybridization data confirmed that CGR-2/IP-10 was expressed by astrocytes.





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The Expression of Chemokines and Their Receptors in MS Patients

A number of studies have focused on examining the expression of chemokines in CNS tissue and cerebrospinal fluids (CSF) of MS patients or cell lines derived from MS patients. Miyagishi et al. (1995) observed an increase of MIP-la concentration in CSF of MS patients by ELISA. Based on this evidence, Bennetts et al. (1997) looked at the potential protection effect of CCR5 (one of the receptors for MIP- lx) deletion mutation against MS. Two CCR5 deletion mutation individuals have been diagnosed with MS. This suggested that the absence of CCR5 is not protective against MS. It is likely that other MIP- 1 x receptors are responsible for MIP- 1 x related pathogenesis in MS patients.

Since the demyelination process that occurs in MS patient is, in part, due to an

inflammatory response in which CD4+ and CD8+ T cells and macrophage infiltrate white matter, Biddison et al. (1997, 1998) did a series of in vitro studies demonstrating that CD8+ T cell lines derived from MS patients are capable of secreting MIP-Icc, MIPl I3, and IP-10. Soluble products of these CD8+ T cells are chemotactic for CD4+ T cells. Antisera treatment revealed that the majority of this chemotactic activity is mediated by IP- 10. Another study using post-mortem tissue samples of MS patients revealed that the expression of RANTES mRNA level in MS brain is higher than control brain. In situ hybridization analysis indicated that RANTES mRNA localization was associated with perivascular T cells (Hvas et al., 1997). These data further suggested that chemokines play a proinflammatory role in the pathogenesis of MS.





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Chemokines and Receptors in Other Neuropathologies

The expression of chemokines was also characterized in a number of other

neuropathological conditions. A traumatic injury to adult mammalian central nervous system results in reactive astrogliosis and the migration of hematogenous cells into the damaged neural tissue. Several groups looked at expression of chemokines in models of mechanical injury of the brain based on the hypothesis that chemokines might play an important role in this cellular accumulation after the injury. Glabinski et al. (1996) identified the expression of MCP-1 I mRNA in mouse brain after penetrating mechanical injury within 3 hours postinjury of the brain. MCP-1 mRNA levels stayed high 12 hours after injury and came back to base line around 24 hours postinjury. Using ELISA, they revealed that MCP- I protein level was elevated in the homogenates of the lesioned tissue by 12 hours postinjury and approached baseline by 48 hours. Their in situ hybridization analysis again showed that MCP-1 was expressed by GFAP (glial fibrillary acidic protein) expressing astrocytes. In another study, Berman et al. (1996) also found the upregulation of MCP-1 mRNA and protein level in the rats subjected to stab wounds of the cortex. However their immunohistochemistry data indicated that MCP-1 was expressed by macrophages and endothelial cells from morphological assessment. Ghimikar et al. (1996) found the expression of two other chemokines; namely RANTES and MIP- 1 3, in the rat stab wound brain by immunohistochemical staining. Doublelabeling studies showed that MIP- 1 P, but not RANTES, was expressed by astrocytes and macrophages near the lesion site.

Hausmann et al. (1998) compared the type of chemokine expression between

cortical lesions under sterile conditions or with LPS (lipopolysaccharide) injection. Only





21'

MCP- 1 expression was detectable in the sterile lesion. However, in the lesioned tissues of LPS injection, they found expression of mRNAs of all chemokines examined, including MCP-1, MIP-Io, MIP-l 13, RANTES, IEP-10 and KC. They also examined the leukocytes and macrophage densities after the lesion and revealed that macrophage/microglia accumulation occurred more rapidly; activated microglia further from the lesion border; and more cells accumulated in LPS injected injury than sterile injury. This indicated that chemokines are instrumental in the initiation of repair processes following brain injury. Infusion of MCP-1 antisense oligonucleotides to the stab wound injured rats not only reduced MCP- 1 protein expression but also decreased macrophage accumulation in the lesioned area (Ghimikar, et al., 1998). This data more directly suggested a functional role of MCP- I in macrophage chemoattraction after mechanical brain injury.

Focal cerebral ischemia of rat induced by middle cerebral artery occlusion

(MCAO) is a frequently used animal model for ischemic stroke. Ischemic brain injury secondary to an arterial occlusion is characterized by acute local inflammation. Using this model, Liu et al. (1993) detected CINC/GRO/KC mRNA expression in the ischemic tissue. Gourmala et al. (1997) identified MCP- I mRNA expression in the ischemic tissue of the rat after MCAO. Recently, Kostulas et al. (1998) examined expression of 1-8 in the peripheral blood of healthy individuals or patients with ischemic stroke. They found that plasma 11-8 concentration was increased in patients that suffered a stroke. This correlated positively to E1-8 mRNA expression in mononuclear cells of examined patients.





22

Another useful model for stroke is reperfusion after a transient brain ischemia because reperfusion after ischemia is a frequently encountered clinical condition that often causes greater tissue damage than persistent ischemia itself. Using a rabbit reperfusion model, Matsumoto et al. (1997) revealed that anti-IL-8 antibody significantly reduced brain edema and infarct size caused by reperfusion after ischemia in comparison to rabbits receiving a control antibody.

In patients suffering from pyogenic meningitis, there is a significant elevation of levels for IL-8, GRO-a, MCP-1, and MIP-1 3 in the CSF in comparison with noninflammatory CSF of control individuals (Spanaus et al., 1997). The CSF of these patients was chemotactic for neutrophils and monocytes. A number of chemokine genes including CRG-2/IP- 10, RANTES, MCP- 1, MIP- I 3, MCP-3, lymphotactin, C 10, MIP-2 and MIP- I c were found to be induced in the brains of mice with lymphocytic choriomeningitis (Asensio and Campbell, 1997). In a study using an osmotic stimulation model, Koike et al. (1997) found CINC gene expression in the rat hypothalamus. In an excitotoxic injury model, MCP-1 was found to be expressed in the brain of intrahippocampal NMDA injected postnatal rats (Szaflarski et al., 1998).

Expression of chemokine receptors in the CNS have been reported. Horuk et al. (1997) examined the protein expression of DARC, CXCRI and CXCR2 in various regions of the brain by immunohistochemical staining. Their data revealed that DARC was expressed exclusively by Pukinje cells in the cerebellum. While CXCR1 was not expressed in the CNS, CXCR2 was expressed at high levels by subsets of projection neurons in diverse regions of the brain and spinal cord. CXCR2 was also found in the neuritic portion of plaques surrounding deposits of amyloid of patients with Alzheimer's





23


disease (AD, Horuk et al., 1997). Immunohistochemical analysis has revealed that CCR3 and CCR5 are expressed on microglia in normal and AD brains (Xia et al., 1998). However, not all ligands of these two receptors are found in the brain. MIP- 1 P was found predominantly in a subpopulation of active astrocytes, which were more widespread in AD than control brains. MIP- 1a appeared to be constitutively expressed at low levels by neurons and microglia in both AD and control brain. Eotaxin, RANTES and MCP-3 immunoreactivity were not detected. Recently, Spleiss et al. (1998) found induction of CCR5 mRNA in the lesioned region of rats that underwent brain ischemia or LPS injection.

Microglia-the Brain Immune Responsive Cells

Microglia are the immune responsive cells in the central nervous system. Thus, it is conceivable that function of chemokines and receptors in the CNS could relate to their expression in microglia. There is evidence demonstrating the expression of some chemokines and chemokine receptors in microglia. These molecules include EL-8 (Lipovski et al., 1998), IP-10, MCP-1 (Sun et al., 1997), MIP-la, MIPI-p, RANTES (Janabi et al., 1998), SDF-3 (Ohtani et al., 1998), CCR3, CCR5, and CXCR4 (Shieh et al., 1998). To study microglia activation in vivo, the facial nerve regeneration model is a very useful animal model and has been well characterized. A transection of facial nerve in the periphery stimulates a response in the facial motor nucleus. This response involves microglia proliferation and activation. Facial motoneuron cell bodies are ensheathed by these activating microglia (Figure 1-2). The advantage of this model is that one can generate a microglia response without breaking the blood brain barrier so that only the





24

endogenous cells are responsible for the change. This model is very useful in our studies examining the expression of chemokines and chemokine receptors in the CNS.

In conclusion, by far, most studies in regard to chemokines and chemokine

receptors in the CNS still remain at a descriptive level. However, these data implicate important role(s) of chemokines and their receptors in a broad variety of neuropathological conditions.

Chemokines, Chemokine Receptors and HIV


In the past two years, chemokines and chemokine receptors have been brought to the spotlight largely because of the discovery of the functional role chemokines and chemokine receptors play in HIV (Human immunodeficiency virus) infection. Some Chemokine Receptors Are HIV Entry Cofactors

The primary interaction site between HIV-1 and infectable cells is the CD4

molecule. This interaction is mediated by the viral surface glycoprotein gpl20. In 1995, Cocchi et al. demonstrated that chemokine peptides, MIP-i t, MIP- I 1, RANTES, were the major HIV suppressive factors produced by CD8+ T cells. One year later, a G-protein coupled receptor, termed fusin, was characterized as an HIV-1 entry cofactor based on the fact that CD4+ cells made to express this receptor became susceptible to T cell-tropic HIV-1 infection (Feng et al., 1996). Fusin was identified later as the receptor for a unique CXC chemokine, SDF-1.

Five independent groups published their work identifying a CC chemokine

receptor, CCR5, as the co-receptor for Macrophage-tropic HIV-1 I entry into CD4+ cells (Deng et al., 1996, Dragic et al., 1996, Alkhatib et al., 1996, Doranz et al. 1996, Choe et





25

al., 1996). Evidence from Choe et al. (1996) demonstrated that 1) CCR5, when expressed along with CD4, allowed cell lines resistant to most primary HIV-1 isolates to be infected, 2) CCR3 facilitated infection by a more restricted subset of primary viras, 3) eotaxin, one of the ligands for CCR3, inhibited infection by these isolates, and 4) the sequence of the third variable region of the HIV gpl20 exterior envelope glycoprotein is the important region for HIV-chemokine receptor interaction. Doranz et al. (1996) demonstrated that besides CCR5 and CXCR4, the receptors CCR3 and CCR2b are entry cofactors for a dual-tropic HIV-1 isolate (89.6). Later, V28 (human CX3CR1), and CCR8 were identified as coreceptors by certain strains of HIV- 1 (Rucker et al., 1997, Combadiere et al., 1998, Jinno et al., 1998).

The role of CCR5 as a cofactor for HIV-I entry became unequivocal when Liu et al. (1996) identified two individuals that had been multiply exposed to IVf-I but remained uninfected, had inherited a homozygous defective CCR5. The encoded defective CCR5 cannot be detected at the cell surface. Thus, These cells are resistant to HIV infection.

In order to determine the important regions for the fusion property of CCR5, a

number of studies using chimeras have been carried out. Rucker et al. (1996) determined regions in CCR5 and CCR2b that set HIV-1 tropic specificity by making CCR5/CCR2b chimeras. They found that residues 2-5 in the N-terminus of CCR5 are important in Mtropic virus fusion whereas residues 6-9 are important for dual-tropic (89.6) virus fusion. Atchison and colleagues (1996) demonstrated that various segments of extracellular regions of human CCR5 are sufficient to reconstitute fusion in the human CCR5/mouse





26

CCR5 chimera. In addition, data from studies of CCR5/CCR2 chimeras suggests that either the N-terminus or the rest of CCR5 are sufficient for M-tropic HIV fusion. Inhibition of HIV Entry by Chemokine Ligands:

Since Cocchi et al. first reported the inhibitory effect of MIP- 1 a, MIP- 3, RANTES on HIV infection, there have been several studies examining the role of chemokine ligands in suppressing HIV infection. With the identification of MCP-2 as a ligand for CCR5, Gong et al. (1998) demonstrated that this peptide blocks CD4/CCR5mediated HIV-1 entry/replication. Evidence from other groups proved the ability of inhibitors for either CCR5 or CXCR4 in blocking HIV-1 entry of the respective strain (Simmons et al., 1997. Howard et al., 1998). While the mechanisms by which chemokines and coreceptor inhibitors block HIV-1 infection are not fully elucidated, studies from Amara et al. (1997) and Howard et al. (1998) indicated that these molecules induced coreceptor internalization.

Interactions between HIV and coreceptors have been shown to inhibit the binding between chemokine peptides and their receptors. Studies from Wu et al. (1996) and Trkola et al. (1996) suggested that CD4 could induce the interaction between gpl20 glycoprotein of HIV-1 and CCR5 resulting in inhibition of the binding of MIP- I a and MIP- IP to CCR5. On the other hand, studies carried out by Dolei et al. (1998) suggested that ligands for CCR5, MIP- la, MIP-I 3, and RANTES, increased the replication of some T-cell-tropic HIV-1 strains and induced expression of the CXCR4 coreceptor.

To further explore the effect of HIV envelope protein on target cells after binding to these chemokine receptors, Davis and colleague's (1997) work indicated that HIV-1 envelope glycoproteins from both T-tropic and M-tropic strains activated a signal





27

tansduction pathway that induced tyrosine phosphorylation of the protein tyrosine kinase Pyk2 of CXCR4+ or CCR5+ target cells. These effects of HIV-1 I on target cells through chemokine receptors are helpful for better understanding the pathogenesis of HIV-1. Chemokine Receptors and HIV in the CNS

In the CNS, microglia are the principal target cells for HIV infection. Several

groups examined the coreceptors that were responsible for HIV- 1 infection and got some contradictory data. He, et al. (1997) reported the expression of CCR3 and CCR5 in microglia of primary human brain cultures. Their data suggested that anti-CCR3 and anti-CCR5 antibody can both block M-tropic and brain-tropic HIV-1 isolates infection to these microglia, indicating that CCR3 and CCR5 are cofactors for some strains of HIV-1 infection in microglia. Another study carried out by Shieh et al. (1998) suggested that predominantly CCR5, and to a much less extent CCR3 or CXCR4 are coreceptors for some of the HIV-1 isolates in cultured human microglia infection. However, while Ghorpade et al. confirmed the expression of CCR3 and CCR5 in cultured human microglia, their usage of antibodies to CCR3 or CCR5 failed to demonstrate similar levels of blocking of HIV-1 I infection or replication. They concluded that there might be other chemokine receptors in microglia that are responsible for HIV-1 I infection.

Elucidation of the roles of chemokine receptors in HIV infection will be very useful in developing therapeutic regimes in AIDS treatment.





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Specific Aims


There is great interest in characterizing the expression, regulation, and functional roles of chemokine receptors in the CNS. Thus, the specific aims of this dissertation addressed three major areas.

1. The molecular cloning and functional characterization of rat CCR2, CCR5, and

examination of the expression of these receptors in the CNS both in vitro (cultured

microglia) and in vivo (EAE).

2. Characterization of the expression of a chemokine:receptor pair, rat

fractalkine:CX3CR1, in the CNS. Determination of expression site(s) of these

molecules in the facial motor neuron regeneration model.

3. Examination of expression of rat fractalkine and CX3CR1 in rat glioma models (RG2

and C6 gliomas).












CC chemokine family

12 3 4
K[P-IS APNGSDP"k CCrMARn PRNrVVDTY. nSSICWA VVFQTXRS3CO VCWVSZSWV WVYDZSIX ......
Ktg-la .... ADTPTA CC"TTSAOI WWXAD7F. ZTSS=KM VllrLMRQ VCADPSZEWV QKTVSDixLs A .....
RAMM SVYSSDT.TP CCFAXI"PL PPAHXKZYF. YTSGKCSNPA VVFWPXMtQ VCAWZKKWV RZTINSLZHS ......
WN-1 OVDAIUMVT CCYNVINRKX SVORLKSTRR 1283KCIKZA VXIrK IVAJM ICADPKQKWV QDSHDHZDKQ TQTPKT 1-309 KSKVQPVSR CCFSFAZQZX PLRAILCYR NTBSICSNZG LUKLXRGRE ACAWTVGWV QRHRKMLRHC PSKM.



CXC chemokine family

1 2 3 4
&TO GKZZSLDSDL YAWJRCM= TTO.GIMPKN ZQSZ.ZVXGKO TUWQVXVXA TL.XD=ZIC LDRDAPRIKE ZVOMAME $AD.
GRO-0 ....... ASV ATXLROOCLQ TLO.GIRM XQSVNVKSPO PWAQTZVXA TLANGWW LNPASPIV= Xlzm4LNSDK SN..
"4 ...... zAzz DaDzacLcvz 2Tx.ovRPjtH XTSZXV PM:PTAQLXA TL.ZN=KZC LDLOAPLYKK ZIKMJEAS .. ....
XIo-I ......... 3 AKUJK*= TYSIKPFIUW XKELRVXESG PSCAUTZIrv KL.SD=ELC ZA)PKENNVQR VVEJMIUUtAz US..
XV-10 ....... VPL SRTVRCTCXS ISNQPVNPRS LZKLRIXPAS OFCPRVZIXA TMKXKGEKRC LNPZSKAIRN LLKAVSKEMS KRSP



C chemokine family

2
Un EGVGTZVL ZZSSCVNLQT QRLVVQKXKT YIIWXMM VMrVTIMGLK ICADPEAKW WUI"VDGR ASTRMOMT VIGTGAQRST STAITLTG



CX3C chemokine family

1 2 3 4
TzaCt&Mdn6 QHKGVTXWI TCSKKTSKXV VALLIHYQQN QASCGXMX LETRQHRLFC ADPKZQWVKD AMRWROAA ALTRN( =Z KOIGZVKPRT TPAAGOWSS WLZPZATGE SSSLZPTPSS QZAQRAL(;TS PZLPTGMS SGTRLPPTPK AWWPVGTE LrRVPPVSTA ATHOSSAPHO PGISLKUM TSZAPSTQDP STOASTASSP APEENAPSEG ORVWGWOSP RPENSLZPZE NGPVPAHTDA TONGPGSM HVSVVPVSSE
GTPSRZPVAS GSWTPKABEP IHATMPOP.L MITPVIDA OS LQGCPRKMAG F&MGLRYIP
RSC(;SNSYVL VPV





Figure I 1: Amino Acid Alignment of Representative Members of the Human Chemokine Family

The line indicates the location of predicted membrane spanning segment of fractalkine. Arabic numbers above the sequence refers to the four highly conserved cysteine residues.









Table 1-1. Ligand Specificity of Chemokine Receptors




Human Genes I.IggandS Human Genes Ligands
CCRs CXCRs
CCRI MIP-1a, RATES, MCP-2, CXCR1 IL-A GCP-2
MCP-3, Lkn-1, HCC-1
CXCR2 IL-k GRO (c4 6, y),
CCR2A MCP-1 NAP-2, GCP-2, ENA-78

CCR2B MCP-1, -2,-3,4 CXCR3 IP-10, MIG, I-TAC
CCR3 FA)taxin-1, Eotaxin-2, RANTES, CXCR4 SDF-I
MCP-2,.3,4
CXCRS BLC
CCR4 TARC, MDC
CCRS MIP-1a, -1% RATES, MCP-2
CX3CR
CCR6 Exodus- I/LARCIMIP-3c& CX3CR1 Fradalkine

CCR7 MIP-38/ELC,
SLC/Exodus-2/6Ckine ICA
XCRI Lymphotactin
CCRS 1-309






















Control '
-CT


F 4b1'A .- t, ,
.,,::..




Axotomized














Figure 1-2. Lectin (GSA I-B4) Staining of Facial Motor Nucleus Four Days After Facial Nerve Transection Microglia are stained by lectin from griffonia simplicifolia. Upper panel shows facial motor nucleus of the unoperated side and lower panel shows facial motor nucleus four days after facial nerve transection. Figure was kindly provided by Dr. Wolfgang J. Streit.













CHAPTER 2
MATERIALS AND METHODS




Materials


DNA modifying enzymes, RNA polymerases and the random primer labeling kit were purchased from Promega (Madison, WI) with the exception of DNA polymerase (pfu) which was purchased from Stratagene (La Jolla, CA). Reverse transcriptase, TRIzol Reagent, DMEM, trypsin, penicillin/streptomycin, LipofectAMINE and rat recombinant interferon-y were from Life Technologies, Inc. (Grand Island, NY). In vitro transcription kit was purchased from Ambion (Austin, TX). [Alpha-32P]dCTP (3000 Ci/mmol) and [alpha- 3P]dATP (2000 Ci/mmol) were from DuPont/NEN (Wilmington, DE). [Alpha33P]rUTP (2500 Ci/mmol) was from Amersham (Arlington Heights. IL). DNA sequencing kit (Sequenase, v. 2.0) was from U.S. Biochemical Corp. (Cleveland, OH). HEK293 cells were from ATCC (Rockville, MD). FURA2-A/M was from Molecular Probes (Eugene, OR). Xenopus laevis were purchased from Nasco (Fort Atkinson, WI). The mammalian expression vectors, pcDNA3 and pCLDN-10 Ob, were from Invitrogen (San Diego, CA) and SmithKline Beecham (King of Prussia, PA), respectively. Guinea pig myelin basic protein, complete Freund's adjuvant (CFA), lectin (GSA I-B4), and monoclonal-GFAP antibody were from Sigma (St. Louis, MO). Biotinylated anti-mouse IgG and horseradish peroxidase-avidin D were from Vector (Burlingame, CA).


32





33

Mycobacterium tuberculosis (H37Ra) was from Difco (Detroit, MI). Male Lewis rats (200-250 g) and male Sprague Dawley rats (170-200) were obtained from Harlan (Indianapolis, IN). Murine MCP-1, murine MIP-lot, murine MIP-113P, human RANTES, rat fractalkine, human TGF-P were from R&D system (Minneapolis, MN). NTB2 autoradiographic emulsion D 19 developer and fixer were from Kodak (Rochester, NY). TdT transferase and biotin-16-dUTP were from Boehringer Mannheim (Indianapolis, IN). An 829-base pair cDNA containing sequences encoding rat cyclophilin was kindly provided by Dr. A. John Maclennan (University of Florida, Gainesville, FL). Rat genomic DNA libraries, HaelI partial digest (lambda Charon 4A) was a kind gift of Dr. Kevin R. Lynch (University of Virginia, Charlottesville, VA).

Methods


Polymerase Chain Reaction (PCR) Amplification of Novel DNA Sequences

Two degenerate oligonucleotides 5'-TAC CTS CTS AAC CTG GCC HTL JSY GA-3' and 5'-ATD AQR GGP TTP ALP CAG CHG TG-3' (where Y=T or C, S=G or C, P=A or G, R=T or G, H=A/T, L=C/T/G, Q=T/C/A, J=A/T/G and D=A/G/C) were synthesized and used as primers for the amplification of DNA fragments (using rat genomic DNA as the template) encoding novel rat GCRs. The amino acid sequences encoded by these oligonucleotides correspond to highly conserved regions of G-protein coupled receptors (and specifically chemokine receptors). The conditions for the amplification of DNA in the polymerase chain reaction (PCR) were: 94 oC (1 minute), 450C (2 minutes), and 720C (3 minutes), 30 cycles. PCR products were subcloned to the





34

SmaI site of pGEM7Zf(+) (Promega) and subjected to DNA sequence analysis (Sanger dideoxy chain termination, Sequenase kit).

Isolation of Rat Genomic Clones

Approximately 1 x 106 independent recombinants of a Haelln partially digested Sprague Dawley rat genomic DNA library (lambda Charon 4A) was screened with the 32P-radiolabeled 0.7-kbp (pCrec 13) PCR products. Fifteen pCrec 13 hybridizing positive signals were plaque purified and phage DNAs purified by standard methods (Sambrook et al., 1989). Restriction-digested recombinant rat genomic DNAs hybridizing to the pCrec 13 DNA were subcloned to pGEM7Zf(+) and subjected to partial DNA sequence analysis (Sanger dideoxy chain termination, Sequenase kit). Southern Blot Analysis of Rat Genomic DNA

Rat genomic DNA (15 ptg, from normal adult Wistar rat liver) was restriction

endonuclease-digested and subsequently electrophoresed through 0.85% agarose gel and subjected to Southern blot analysis (Sambrook et al., 1989). The transferred DNAs were hybridized to the 32P-radiolabeled 1.0 kbp ClaI/BamHI fragment of the rat CCR5 gene using the method of Church and Gilbert (1984). Final wash temperatures were 500C and 650C.

Transfection of HEK293 Cells:

The open reading frame (orf) of rat CCR2 was amplified using the PCR and the cloned genomic DNA as the template. Two oligonucleotides were synthesized and used in the PCR (30 cycled of 94oC for 1 minute, 550C for 2 minutes, and 720C for 3 minutes). The oligonucleotides used were 5'-TTC TCT GTC CAC AGA ATC and 5'-CTA CTT





35

ACT TTA CAA CCC. The PCR product was reacted with the Klenow fragment of DNA poll in the presence of dNTPs and cloned subsequently to the EcoRV site of pcDNA3. The resultant expression plasmid construct was subject to DNA sequence analysis to verify the fidelity of the Pfu DNA polymerase used in the PCR. The DraJ/BamHI fragment containing the rat CCR5 orf was cloned to the HindI and BamHI sites of pCLDN-10b; the HindHIf site was made blunt by the action of the Klenow fragment of DNA poll in the presence of dNTPs. A KpnI/BamHI fragment of this pCLDN-10b construct was subcloned subsequently to the same sites of pcDNA3. HEK293 cells were grown in DMEM supplemented with 10% heat inactivated fetal bovine serum (FBS) and penicillin/streptomycin (complete media). HEK293 cells (2 x 105 cells) were transfected with 10 ig of plasmid DNA using LipofectAMINE reagent according to the manufacturer's recommended procedure. Approximately 72 hours post transfection, cells were replaced in complete media containing the neomycin analog G418 (0.8 mg/ml). Clonal cells resistant to G418 were isolated and propagated for further analysis. Cells expressing (c)RNA hybridizing to G418 were isolated and propagated for further analysis. Cells expressing (c)RNA hybridizing to the ClalBamHI DNA fragment of rat CCR5 were characterized further.

Measurement of Intracellular Calcium Levels

HEK293 cells (-5x 106) which stably express rat CCR2 were dissociated from the culture dish and diluted subsequently into 10 ml MEM (serum- and antibiotic-free). The suspension was made in 5 mM of FURA-2/AM and was incubated for 30 minutes in the dark at 37C. The cells were then washed twice with PBS buffer and subsequently resuspended in 2 ml HBSS buffer. The cell suspension was transferred to cuvettes and





36


placed in a SLM 8000 model spectrofluorimeter (driven by an IBM PC). FURA-2 fluorescence was monitored at 510 nm, with automatic slewing between excitation wavelengths at 340nm and 380nm. Ratios at these two excitations were automatically calculated.

Expression of Rat CCR5 in Xenopus Laevis Oocytes

Plasmid (pcDNA3) constructs containing the open reading frames of rat CCR2 and rat CCR5 were linearized, and complementary RNAs were synthesized in an in vitro transcription reaction using T7 RNA polymerase. The transcription products were confirmed by Northern blot analysis. Xenopus laevis oocytes were injected with 50 ng of RNA in a total volume of 25 nl per oocyte. Electrophysiological recordings were made 34 days after injection in oocytes superfused with Oocytes Ringer's solution at room temperature under voltage-clamped conditions using two microelectrodes (both filled with 3 M KCI), with the membrane potential routinely clamped at -50 mV using a TEV200 voltage clamp instrument. Test chemokines were dissolved in distilled water and diluted to a final concentration of 250 nM in Oocytes Ringer's solution. Thirty pl of each chemokine was applied directly onto voltage-clamped oocytes for 120s, and the current induced was monitored on a Tektronix 5113 dual beam storage oscilloscope linked to an IBM-PC.

RNA Isolation, RNase Protection Assay, and Northern Analysis

Total RNA was isolated using TRIzol Reagent according to the manufacturer's recommended procedure. The RNase protection assay was performed as previously described (Feng et al., 1994). The antisense riboprobe for rat CCR2 was generated from an in vitro transcription reaction using a plasmid construct containing the Dral/EcoRI





37

fragment (365-base pair) of rat CCR2. The antisense probe for rat CCR5 was made in a similar manner using an EcoRV/BgllI restriction fragment (310-base pair) of rat CCR5. The antisense probe for rat fractalkine and RBS 11 were made using total RNA (five tg) and 32P-UTP-labeled riboprobes generated from in vitro transcription reactions utilizing plasmid construct templates containing either a 0.4 kbp NcoI fragment of rat fractalkine cDNA or a 0.45 kbp SmaI/KpnI fragment ofRBSl I cDNA.

Total RNA (10 pg/lane) extracted from various cell and tissue preparations was electrophoresed through denaturing 1.2% agarose and subjected to Northern blot analysis (Church and Gilbert, 1984). For hybridization probes, the following DNAs were used: the 1.5-kbp Clal/BamHI fragment of the rat CCR5 gene, a 1.1-kbp RBS 11 protein coding DNA sequence, a 700 base pair cDNA fragment for rat fractalkine, and an 829-base pair cDNA encoding rat cyclophilin, all of which were 32P-radiolabeled by the random primer method (1-2 x 109 dpm/[tg).

Preparation of Primary Cultures of Rat Microglia

Microglial cultures were prepared as previously described, with minor

modifications (Giulian and Baker, 1986). In brief, brains were removed from neonatal rats and placed in an isotonic salt solution containing 100 U of penicillin G, 100 jg of streptomycin and 0.25 Vg of amphotericin B (Fungizone) per ml (pH 7.4). All blood vessels and pia mater were removed, and the diencephalon were dissected out and chopped into approximately 2 to 3 mm chunks. The brain pieces were then dissociated by trituration at 370C in 20 ml of isotonic saline solution (pH 7.4) containing 0.25% trypsin (wt/vol) and DNase-I (8 ptg/ml). The dissociated cell suspension was added to an equal volume of DMEM containing 10% FBS; the suspension was then centrifuged at





38

1000 x g for 10 min and the resulting pellet washed with 50 ml DMEM containing 10% FBS. Cells were resuspended in DMEM containing 10% FBS to a density of 1 x 105 cells/ml and 10 ml of this suspension were added to 100 mm poly-L-lysine precoated culture dishes and incubated at 37C in a humidified incubator with 5% C02/95% air. On day 3, the culture medium was replaced with fresh DMEM containing 10% FBS. After 11 aditional days of incubation of the mixed primary astroglial/microglial cultures, the flasks were rotary-shaken for one hrs (100 rpm, 37C) and the culture media, which contained dislodged free-floating microglia, was removed and centrifuged at 1000 x g for 15 min. The resulting microglial pellet was resuspended in DMEM and plated onto 100 mm dishes at a density of 5 x 106 cells per dish. Microglia were cultured for approximately 24 48 hours prior to treatment. Induction of Rat EAE:

Male Lewis rats (-200 250 g) were injected in the hind foot pad, under

methoxyflurane anesthesia, with an emulsion (0.1 ml) of 125 ptg purified guinea pig myelin basic protein (MBP) in Complete Freund's Adjuvant (CFA, Difco) supplemented with 50 tg Mycobacterium tuberculosis (H37Ra). Clinical disease, i.e. various degrees of paraparesis, was typically evident between days 10 and 12 post injection. Animals (EAE and control) were screened for neurological signs on a daily basis according to the following scoring system: 0: no signs; 1: flaccid tail, general depression of activity; 2: mild paraparesis, unsteady gait; 3: moderate paraparesis, legs splayed apart but voluntary movements of affected extremities possible; 4: complete paraparesis, extremities without voluntary movements; 5: death due to EAE. If the clinical sign remained in one stage for 2-3 days or reached stage 4, the rat was perfused intracardially with PBS and the lumbar





39


enlargements of the spinal cords removed. Total RNA was isolated and subsequently subjected to either RNase protection assay or Northern blot analysis as described above. Molecular Cloning and Sequencing of Rat Fractalkine cDNA

A rat brain cDNA library (Strategene, CA) was screened by hybridization using a rat factalkine cDNA probe. The hybridization probe was generated by PCR using primers derived from a mouse cDNA (Genbank accession number R75309). The rat fractalkine cDNA was sequenced by standard methods (Sanger dideoxy chain termination, Sequenase kit).

An EcoRI/SalI DNA fragment containing sequences encoding full length rat fractalkine protein was subcloned to the mammalian cell expression vector, pCDM8 (Invitrogen). DNA containing human fractalkine protein coding sequence was generated by PCR and subcloned into the same vector. To generate peptidase-resistant membraneanchored fractalkine, both human and rat fractalkine mutant forms were also prepared by PCR, in which the putative dipeptidase site Arg-Arg, adjacent to the transmembrane spanning domain, was mutated into Ser-Ala. Expression and Functional Characterization of Rat CX3CRI in Transfected Cells

A cDNA containing the open reading frame of RBS 11 was cloned to the EcoRV site of pcLDN- 10Ob. CHO cells (grown in Ham's F 12/10% heat inactivated fetal bovine serum (FBS)/penicillin/streptomycin) were transfected with 10 tg of plasmid DNA using LipofectAMINE reagent (Life Technologies, Inc.). G418-resistant clonal cells expressing

(c)RNA hybridizing to RBS 11 cDNA were characterized further in binding and functional assays. HEK293T cells (grown in DMEM/10% FBS/pen/strep) were





40

transfected using LipofectAMINE and 10 gg of rat or human fractalkine encoded DNA/100 mm plate and used three days post-transfection.

Synthetic chemokines were generated by native chemical ligation of peptides synthesized by solid-phase methods (Applied Biosystems 430A Peptide Synthesizer), purified by reverse-phase high-performance liquid chromatography, and characterized by electrospray mass spectrometry (Dawson et al., 1994). Purified synthetic chemokines were reconstituted in phosphate buffered saline (PBS) and stored frozen, until use, at minus 700C.

Whole cell binding analysis was performed on three million PBS-rinsed cells per 35 mm well. Cells were incubated (1 hour at room temperature) in 1 ml HBSS/0.1% BSA, 125I-labeled human fractalkine/chemokine domain (100 Ci/mmol) and various concentrations of cold competing chemokine ligands. At the end of the incubation, the cells were rinsed 3 x 2 ml with ice-cold HBSS/0.1% BSA. Total radioactivity retained in the wells was removed with 0.2 N NaOH and quantitated by gamma spectroscopy. In Situ Hybridization Analysis

In situ hybridization was carried out according to previously published procedures (McNamara and Routtenberg, 1995). Radiolabled [33P] (c)RNA probes were prepared by in vitro transcription from T7 or Sp6 RNA polymerase promoters in the pGEM7Zf(+) plasmid system. For RBS11 probes, a full length cDNA clone in pGEM7Zf(+) was digested with KpnI and a resultant 450 bp fragment was isolated and subcloned to the SmaI site of pGEM7Zf(+). The antisense probe for RBS 11 was prepared by linearizing the pGEM7Zf(+) plasmid with HindUl. For preparation of the sense strand probe for RBS11 the plasmid was digested with EcoRI. For rat fractalkine probes, a cDNA clone





41

encoding chemokine domain of rat fractalkine was digested with NcoI. An approximately 400 bp fragment was purified and subcloned to the Sinai site of pGEM7Zf(+). Antisense probe was generated by digesting this construct with EcoRI and sense strand probe was obtained with BamHI digestion. In vitro transcription and labeling of RNA probes was performed as described (McNamara and Routtenberg, 1995) with mild modifications. Briefly, 100I Ci of [alpha-33p]-rUTP was added in a total 10 pl reaction [40 mM Tris; 6 mM MgCl2, 2 mM spermidine; 10 mM NaCl; 4 mM dithiothreitol; 0.8 U/ml RNase inhibitor; 0.5 mM each ATP, CTP, GTP; linearized template equivalent to 0.5 pg of whole plasmid; and 10 U of either T7 or Sp6 RNA polymerase]. The reaction was allowed to proceed for 60 minutes at 370C. The DNA template was removed by RNase-free DNase (10 U) for 15 minutes at 370C. Proteins and free ribonucleotides were removed by NH4Ac and ethanol precipitation. Brain tissues were embedded in O.C.T. compound (Miles Inc., Elkhart, IN) and 12-20 pm thick frozen sections were made and placed on Superfrost/Plus microscope slides (Fisher Scientific, Pittsburgh, PA). After fixed in 4% paraformaldehyde buffered PBS, pH 7.4. for 10 min in 40C, tissue sections were washed in DEPC treated water and acetylated in a freshly prepared solution of 0.1 M triethanolamine with acetic anhydride, pH 8.0, for 10 minutes. After washing in 2x SSC, sections were dehydrated through graded ethanols. Sixty jil of the hybridization mixture (50% formamide, 0.3 M NaCI, 20 mM Tris, pH 8.0, 5 mM EDTA, 1 x Denhardt's, 10% Dextran Sulfate, 10 mM DTT) containing 1.5x 106 cpm counts of radiolabeled RNA probe was applied to each slid and covered with a siliconized coverslip. Hybridization was carried out for 16-18 hours at 60-650C in a humidified chamber. The excess radioactivity was removed by washing the slides with two 20-min





42

rinses of standard saline citrate (SSC) buffer (0.3 M NaCI, 30 mM sodium citrate), and a 30-min digestion in RNAse A at 370C in buffer containing 500 mM NaCI, 10 mM Tris, pH 8.0, and 1 mM EDTA. After two more rinses with SSC, sections were stringently washed in 0.1X SSC at 600C for two hours, followed by two 10-min rinses in 0.5X SSC at room temperature and dehydrated in graded ethanols containing 300 mM ammonium acetate. Sections hybridized with "sense" radiolabelled riboprobe served as a control. Lectin Staining and Immunohistochemical Analysis

Following in situ hybridization and prior to emulsion autoradiography, some sections were stained with either isolectin from Griffonia simplicifolia (GSA I-B4, to identify microglia) according to previously published procedures (Streit, 1990) or with anti-GFAP (glial fibrillary acidic protein, to identify astrocytes) in order to determine cellular localization of RBS11 or fractalkine.

For lectin staining, sections were soaked in PBSC (PBS containing 0.3% Triton X-100 and 0.1 mM CaCl2, MgC12, and MnCl2 ) at 4oC for 1 hour before being incubated with GS I-B4-HRP (10 ptg/ml) in PBSC overnight at 4C. The sections were briefly rinsed with PBS and the lectin binding sites were visualized using 3,3' diaminobenzidine-H202.

For GFAP staining, sections were rehydrated in PBS for 10 minutes and blocked with PBS/10% goat sera for 30minutes before incubating overnight in 40C with antiGFAP antibody diluted 1:1000 in PBS/10% goat sera. After washing in PBS 3x 10 minutes, sections were then incubated with biotinylated anti-mouse-IgG (from goat) for 45 minutes in room temperature. With another extensive wash using PBS, 3x 10





43

minutes, sections were treated with horseradish peroxidase avidin D for 45-60 minutes and rinsed with PBS before exposed to 3,3' -diaminobenzidine-H202.

Sections were soaked in PBS for 5 minutes and allowed to air dry. The slides were then exposed to films for 24 hrs and developed in D 19 developer for 5 min, rinsed in water 5min, and fixed for 8 min. If the films showed that the hybridization was successful, the sections were exposed to Kodak NTB2 autoradiographic emulsion at 40C for a period of time depending on the intensity of the signals on the films generally between 2-6 weeks and then developed in D 19 developer for 2 minutes, rinsed in tap water for 5 minutes, and fixed for 5 minutes in fixer. After washing in tap water, the slides were counterstained in cresyl violet or hematoxylin, dehydrated in ascending ethanols and coverslipped with Permount.

Facial Nerve Transections

Facial motor nerve transections on methoxyflurane-anesthetized male SpragueDawley rats (175 200 g) were carried out according to previously published procedures (Streit et al., 1988). In brief, Male Sprague-Dawley rats (175-200 g) were anesthetized with methoxyflurane. The right facial nerve is exposed and transected at the level of the stylomastoid foramen. The unoperated contralateral side serves as the control. Following 1, 4, 7, 14, and 21 days after axotomy, rats (n = 3 per time point) were sacrificed with an overdose of sodium pentobarbital (75 mg/kg) and perfused transcardially with lx PBS (pH 7.0) followed by 4% paraformaldehyde/0.1 M PO4 buffer (pH 7.4). Brains were saturated in 30% sucrose/0.1 M PO4 buffer (pH 7.2) and frozen in 2-methylbutane (chilled to -80oC before use), and then stored at -800C. At a later time, the brain slices





44

were prepared for in situ hybridization and immunohistochemistry analysis as described above.

Quantitation of areal grain densities of bilateral facial motor nuclei was performed on emulsion-dipped slides under dark field conditions using a video imaging system (MicroComputer Imaging Device) and image analysis program (Imaging Research Inc.) interfaced via a video camera (Sony XC77) connected to a Zeiss microscope (Axioscop 20). Statistical comparisons (one-way analysis of variance) were made between ipsilateral and contralateral values within each post-lesion time point. Tumor Inoculation

The RG-2 or C6 glioma cell lines were maintained in a monolayer culture in

DMEM with 10% FBS. Female Fisher rats were anesthetized with sodium pentobarbital. Tumor cells (5x 105/5 d) were injected stereotactically into the right cerebral hemisphere through a burr hole. Five gl of saline solution was injected into a group of rats that served as controls.

Terminal Transferase-Mediated UTP Nick End Labeling (TUNEL-Labeling)

TUNEL-labeling was performed following the protocols by Gavrieli and

colleagues (1992) with some adjustments. RG2 or C6 cell inoculated Fisher rats were sacrificed, fixed and cryoprotected as described for in situ hybridization analysis. After rinse in water 2x5 minutes, endogenous peroxidase was inactivated by incubating the slides in 3% water for 10 minutes at room temperature. Tissues were then exposed to 0.2% triton X-100/PBS for 5 minutes in room temperature. Slides were incubated in water at 600C for one hour and cooled down in room temprature before TdT treatment. After pre-incubating in TdT buffer (30 mM Tris-HCl, pH 7.2, 140 mM sodium





45

cacodylate, 1 mM cobalt chloride) for 5 minutes, the tissue sections were incubated in TdT buffer containing 2.5 U/pl terminal transferase and 10 [mol biotinylated 16-dUTP in a humid atmosphere for 1 hour at 370C. The reaction was stopped by transferring the slides into 300 mM sodium chloride, 30 mM sodium citrate for 20 minutes in room temperature. After blocking the coverslips for 30 minutes in 2% bovine serum albumin (BSA), they were incubated with the ABC kit for 30 minutes at room temperature and developed with 3,3'-diaminobenzidine as substrate. Negative controls were carried out by omission of terminal transferase and biotinylated UTP.













CHAPTER 3
CHEMOKINE RECEPTOR EXPRESSION IN CULTURED GLIA AND RAT
EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS Introduction


Chemokines are a growing family of peptides that are involved in leukocyte recruitment and activation. These peptides can be divided into four subfamilies (CC, CXC, C, and CX3C subfamilies) based on the relative positions of highly conserved cysteine residues in their amino acid sequences.

Some of the receptors for chemokines have been identified by molecular cloning techniques and they are members of the rhodopsin superfamily of G-protein coupled receptors (GCRs). These receptors are characterized by their seven transmembrane spanning helices in their amino acid sequences. Although most chemokine receptors interact with more than one chemokine peptides, in general, they can only be stimulated by chemokines from the same subfamily. To date, the primary structures of at least sixteen functional human chemokine receptors have been determined. Five of these receptors bind, and are activated by, C-X-C chemokines nine receptors are activated by C-C chemokine peptides, one receptor binds the C chemokine lymphotactin and one receptor is bound and activated by the CX3C chemokine fractalkine. Genes encoding chemokine receptors have also been identified in the mouse and rat. It has been demonstrated that human CXCR4, CCR5, CCR2, CCR3, CCR8, and CX3CR1 act as cofactors for entry into cells of primary isolates of HIV. While the expression and 46





47

functions of chemokines and chemokine receptors have been extensively studied in the periphery, less information, regarding these genes, is available in the central nervous system.

Microglia, as the immune responsive cells in the CNS, comprise 5-20% of the total glial cell population (Graeber et al., 1990, Kreutzberg 1996). These cells react to even minor pathological changes in the CNS and serve as a key factor in the defense of the neural parenchyma against infectious agents, inflammation, trauma, ischemia, brain tumors and neurodegeneration. Several chemokines and chemokine receptors have been shown to be expressed in microglia including MCP-l, MIP-lca, MIP-1 3 (McManus et al., 1998), RANTES (Miyagishi et al., 1997), SDF-3 (Ohtani et al., 1998), IP-10 (Ren et al., 1998), CXCR4 (Lavi et al., 1997), CCR3, and CCR5 (Xia et al., 1998).

Microglia activation and proliferation occurs in experimental allergic

encephalomyelitis (EAE). EAE is a cell-mediated autoimmune disease of the central nervous system induced by a number of different antigen preparations including myelin basic protein. Its histopathology is characterized by perivascular mononuclear cell infiltration and microglia and astrocyte activation. Clinical signs of EAE consist of hindlimb and tail paresis as well as paralysis. Since it has many clinical and pathological features in common with multiple sclerosis, it is considered to be an animal model of this disease. Several groups have demonstrated that a number of chemokine peptide were expressed or up regulated in the spinal cords of EAE animals. These chemokines include RANTES, MIP-la, MIP-1B, TCA3, IP-10, MCP-1, KC/GRO, MCP-3, SDF, and MIP-2 (see chapter one).





48

Towards our goal of defining chemokine receptor expression in the CNS, I have isolated, by molecular cloning techniques, rat genes encoding the chemokine receptors CCR2 and CCR5. I describe expression patterns of these genes in cells and tissues of the CNS and report regulation of chemokine receptor expression in vitro in cultured rat microglia and demonstrate in vivo modulation of chemokine receptor expression in EAE rats.

Results


Isolation of Rat Genomic DNA Containing Sequences Encoding Novel G-Protein Coupled Receptors

Degenerate oligonucleotide primers were designed based on the highly conserved regions (TM2, TM3, and TM7) of the known human chemokine receptors (hulL-8RA, hulL-8RB, huCCR1, and huCCR2 receptors) and the orphan rat chemokine receptor-like gene, RBS11 (Holmes et al., 1991; Murphy et al., 1991; Neote et al., 1993; Gao et al., 1993; Charo et al., 1994; Harrison et al., 1994). DNA fragments encoding novel rat chemokine receptors were generated by PCR reactions using these oligonucleotides and rat genomic DNA as the template. Consequently, these PCR products were utilized for further screening of a Sprague Dawley rat genomic library under high stringency conditions in order to isolate the full length DNA sequenses. Genomic library screening using one of the fragments, pCrec 13, resulted in 15 plaque pure bacteriophage clones. Restriction endonuclease digestion and DNA sequence analysis of each of these clones revealed two distinct sets of bacteriophage containing open reading frames (orfs) encoding novel G-protein coupled receptors with high sequence identity to other known chemokine receptors. Hereafter, we designate the orfs containing the pCrec 13 sequences





49

as rat CCR2 and rat CCR5. Figure 3-1 depicts a multiple alignment of rat, human and murine CCR2 and CCR5. Rat CCR2 encodes a protein of 373 amino acids with a calculated molecular mass of 42,763 Da, while rat CCR5 is a protein of 354 amino acids and a calculated molecular mass of 41,017 Da. Both receptors contain structural characteristics of CC chemokine receptors. There is a preponderance of acidic amino acid residues in their amino acid sequences of the respective N-termini. Putative Nlinked glycosylation sites (N-X-S/T) can also be identified. The C-termini of each of these rat receptors are serine rich, suggesting possible receptor regulation by phosphorylation.

A comparison of amino acid identities of the rodent, murine and human receptors is shown in Table 3-1. Rat CCR2 is 79% and 93% identical to human and marine CCR2 respectively while rat CCR5 is 83% and 92% identical to human and murine CCR5, respectively. The most variable regions between rat and human orthologs were found in putative extracellular portions (N-termini and loops). The high amino acid identity between rat and human or murine CC chemokine receptors suggested the identity of these novel rat chemokine receptors and allowed us to designate these rat receptors according to nomenclature standardized for the chemokine receptor family. Signal Transduction Properties of Rat CCR2 and CCR5

In order to confirm the identities of rat CCR2 and CCR5, functional assays were carried out on cells expressing each of these receptor genes. Application of murine MCP1 stimulated a transient elevation in intracellular calcium ([Ca2+]j) levels in rat CCR2 expressing HEK293 cells (Fig. 3-2) in a dose dose-dependent manner. The EC50 value for this effect is approximately 0.4 rM. However, rat CCR5 expressing HEK293 cells





50

did not show a detectable change in [Ca2 ]i in the presence of either murine MCP-l or MIP- la. Electrophysiological experiments were carried out to examine the calcium mobilization of CCR5 expressing Xenopus laevis oocytes in response to ligand application. Figure 3-3 demonstrates traces of Ca2+-activated Cl" current recorded from Xenopus laevis oocytes microinjected with CCR5 (c)RNA and exposed to MIP-la or MIP-1B. No response can be detected when the cells were exposed to the buffer only (data not shown).

Genomic Analysis

Southern blot analysis of restriction-digested rat genomic DNA probed with

radiolabeled DNA encoding rat CCR5 showed consistent hybridization to two distinct sets of genomic fragments (Fig. 3-4). These hybridizing restriction fragments were of identical size to the hybridizing restriction fragments from the isolated rat genomic bacteriophage clones. These two sets of the hybridizing signals can be seen even under more stringent wash conditions as shown in the left panel of Figure 3-4 where the blot was washed at 650C as compared to the right panel with the wash temperature at 50C. Tissue Specific Expression of Rat CCR2 and CCR5

The tissue and cell distribution of rat CCR2 and CCR5 mRNAs were determined by RNase protection assay. Analysis of total RNA from solid rat organs and cells probed with an antisense riboprobe of rat CCR2 revealed that adult rat lung, spleen, kidney, thymus, and macrophages are sources of CCR2 (Fig. 3-5). CCR2 mRNA was not detected in rat liver or rat brain (data not shown). Similar to that of rat CCR2, rat CCR5 mRNA was also found to be expressed in rat lung, spleen, kidney, thymus, and





51

macrophages (Fig. 3-6). However, in addition, CCR5 mRNA was detected in extracts of whole rat brain.

Expression and Regulation of the Rat CCR2 and CCR5 in Cultured Microglia

The expression and regulation of the CCR2 and CCR5 gene in the cultured rat microglia was investigated by Northern blot analysis shown in Figure 3-7. RNA from microglial cells treated with various concentrations of IFN-y was isolated and a Northern blot was generated as described in chapter 2. This blot was subsequently probed with a 1.5 kbp ClaI/BamHI fragment of rat CCR5. Steady state levels of CCR5 mRNA present in the cultured microglia were dose-dependently increased by IFN-y treatment. CCR2 mRNA was not detected in primary cultures of rat microglia (either control or IFN-ytreated, Figure 3-5).

Modulation in Rat EAE

A number of cytokines, including [FN-y and chemokine peptides, are known to be elevated in spinal cords from EAE animals (Merrill et al., 1992; Weinberg et al., 1993; Hulkower et al., 1993; Ransohoff et al., 1993; Godiska et al., 1995; Miyagashi et al., 1995; Berman et al., 1996; Glabinski et al., 1997). In order to examine the expression of the receptors for chemokine peptides in vivo, in collaboration with Dr. Wolfgang J. Streit, we examined for the expression of chemokine receptor genes in rat EAE. EAE was induced in Lewis rats by hind footpad injection of myelin basic protein. Total RNA from the lumbar enlargements of the spinal cords from animals at various stages of the disease were examined for the presence of chemokine receptor mRNAs. Figure 3-8 depicts an analysis of multiple chemokine receptor (CCR2, CCR5, CXCR4, and RBS 11) mRNA





52

levels in the lumbar enlargement of the EAE rats, as assessed by either RNase protection analysis or Northern blot analysis. For each receptor, increases in the levels of mRNA were observed in animals displaying clinical symptoms of the disease. In animals that did not display symptoms, either no increases in mRNA (as for CCR2, CCR5, and CXCR4) or minimal increases (as for RBS 11) were seen. A correlation between the increase in receptor RNA and the degree of clinical symptoms was observed for CCR2 and CCR5, i.e. a greater increase in mRNA for these receptors was evident in the animal displaying complete hind limb paralysis.

Discussion


In the past few years, many research groups have carried out studies to determine the expression of chemokine peptides and chemokine receptors in the CNS. This project launched our series of studies in an ongoing effort to characterize the expression, regulation and functional roles of chemokine peptides and receptors in the central nervous system. Our results demonstrate that the rat genome contains orthologous genes encoding the chemokine receptors CCR2 and CCR5. Unlike rat CCR2, which is not constitutively expressed in the brain or cultured microglia, rat CCR5 mRNA is constitutively expressed in these tissues. In addition, steady state levels of chemokine receptor mRNA(s) are regulated both in vitro in the cultured microglia and in vivo in rat EAE.

Two very closely related CC chemokine receptor genes, rat CCR2 and CCR5 have been identified via sequence homology techniques. The deduced amino acid sequences contain putative seven hydrophobic transmembrane alpha helices, which confirmed that they are members of the G-protein coupled receptor superfamily. Similar to many other





53

chemokine receptors (Ahuja and Murphy, 1993), there is a preponderance of acidic amino acid residues in the conceptualized amino acid sequences of the respective N-termini, a region that is likely to participate in binding with the positively charged N-termini of chemokine peptides. A serine rich region is found in the carboxy-terminal domain of both receptors, suggestive of potential phosphorylation regulation of these proteins. The amino acid sequences also carry potential N-linked glycosylation sites (N-X-S/T) for both receptors. The overall amino acid sequence identity between human and rat CCR2 and CCR5 are 79% and 83% respectively. The high amino acid sequence identity prompt us to hypothesize that these genes encode rat orthologs of the two human chemokine receptors.

Functional analysis of these rat chemokine receptors in receptor-expressing-cell systems finalized their identity as rat CCR2 (MCP-1 receptor) and CCR5 (receptor for MIP-la, MIP- 1 P and RANTES), respectively. Similar to its human ortholog, rat CCR2 can be stimulated by MCP-1 in low nanomolar concentrations. Although we failed to demonstrate a transient intracellular Ca2+ increase in rat CCR5 expressing HEK cells, we were able to detect a Ca2+ activated Cl" current in rat CCR5 expressing oocytes in response to murine MIP-la, MIP-Ip and human RANTES. The identical ligand specificity between rat and human orthologs indicates that the rat can serve as a good model in studying the functional roles of human CCR2 and CCR5 under physiological and pathological circumstances.

Rat CCR2 and CCR5 are found in a number of cells and tissues that can either

produce or respond to chemokine peptides. In addition to the peripheral cells and tissues that express both rat CCR2 and CCR5, rat CCR5 is also constitutively expressed in the





54

brain as well as cultured microglia. This observation is in agreement with the finding that the human gene is expressed in human microglia present in primary brain cultures (He et al., 1997). In vivo studies will be necessary to prove unequivocally that rat CCR5 is expressed in microglial cells in the CNS.

My data revealed that steady state levels of rat chemokine receptor mRNA were regulated in cultured microglia in vitro as well as in an EAE animal model in vivo. After IFN-y treatment, the expression of rat CCR5 mRNA is up-regulated in cultured microglia. On the other hand, the lesion of EAE involves in infiltration of the IFN-y producing T lymphocytes due to the break down of the blood brain barrier (Merrill et al., 1992, Weinberg et al., 1993). Taken together, it is conceivable that in normal rat spinal cord, the CCR5 hybridizing signal was contributed by endogenous microglia. The microglial expression of CCR5 is promoted by these IFN-y producing T lymphocytes during EAE. This hypothesis is supported by my data demonstrating the increased expression of CCR5 mRNA in the spinal cords of EAE rats. While constitutive expression of CCR2 is absent in rat spinal cord, its expression is detectable in EAE rats. It is known that infiltrating macrophages are also found in the spinal cords of EAE rats (Huitinga et al., 1995). Cultured macrophages are capable of expressing CCR2, CCR5 and another chemokine receptor gene, RBS 11, in vitro (Combadiere et al., 1995b; Raport et al., 1995). Thus, macrophages are likely to be the sources of CCR2 hybridizing signal and part of the increased CCR5 and RBS 11 hybridizing signals in the spinal cords of diseased rats. Studies combining in situ hybridization and histochemical analysis should clarify the cell type expression of each of these receptors in the EAE model.





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The expression of chemokine receptor mRNA in the brain and spinal cord as well as cultured microglia indicates that these molecules may play some functional roles in the CNS under neurophathological and neurophysiological conditions. It is known that a number of chemokine peptide genes are induced in EAE, including MCP-1, MCP-3, MIP-lcta, MIP-1B, RANTES, TCA3, KC/CINC, and IP-10 (Hulkower et al., 1993; Ransohoffet al., 1993; Godiska et al., 1995; Miyagashi et al., 1995; Berman et al., 1996; Glabinski et al., 1997). For some of these peptides, astrocytes have been identified as the cellular source (Ransohoff et al., 1993; Berman et al., 1996; Glabinski et al., 1997), while for others, infiltrating leukocytes were determined to be the source (Glabinski et al., 1997). Our data demonstrated the presence of receptors of some of these chemokine peptides in the CNS, particularly in spinal cord of EAE rats. These data suggest a potential interaction between these chemokine peptides and their receptors in this neuropathological model of human multiple sclerosis which may play some functional roles during the disease process. Our identification of the rat genes encoding chemokine receptors CCR2 and CCR5 should enhance fundamental studies of these receptors in normal and pathological states of the CNS.






56


60
ratCCR5 MFQGSI PTYIYDMYS KSMCOKM K0UQLLPP
surOCR5 *HDrQG$V PTTSYDIDYG NBAPCQKINV KOZAAGLWP
h'ICXM *MDYQVSS P.. IYDnM TSZPCOKDfV KQIAARLLPP
ratCCR2 NEDSNNLPQF IRGILSTSRS I"RSIQZLD WvATTPYDYD DMPCIMTSV KOIGMILPP murCCR2 NZDWGCLPQr IRGIL3TSRS UrTRSIQZLD 3GATTPIDHD Dawamsv KQIGMILPP huCCR2b .......... ... WATERS RrXRMMSG ZZVTTrl'DYD YGNPCXKrDV NOXGAQLLF.P

61 120
rateCR5 LYSLVrlrW VGNHKVrLIL ISCKXLKSHT DXYLTNLAIS DLLrLLTLPT WARYAANZWV murccr5 LYSLVFrIW VGNHKWLIL ISCXKLKSVT DXYLLNLAIS DLLTLLTLPIP WARYAMM
huCCR5 LYSLVIPXFW VGWMVILIL niCRRLXSMT DXYLIA"IS DLrrLLTwr wwrmhM ratCCR2 LYSLVFXFW VGWMVIIIL ISCX3aJMNT DXYLTNLAIS DLLIrLLTLPr WARYAAMM murCCR2 LYSLVIrUW VGWMVXIIL IOC30MKSKT DXYLLWTAIS DLLrLLTLIr WARYAMMW
huCCR2b INClUa.KCLT DT
z zz
121 ISO
ratCCR5 M XMCXLFT GLYRIGYrGG zrrxlLLTID RYL&rVHAVr ArKARTVTFG VITSVVTMVV nurccrS TWINCKM GLYHIGYTGG IrTlILLTID RYLUVRAW ALXVRTVNFG VIT3VVTWAV
huCCRS rONTHCOLLT GLYrIGrrSG IPTIILLTID RYLAVVRAVr ALKRRTVTFG VVTSVXTWVV r&tCCR2 r=IMCKLIrT GLYHIGYrGG INTZILLTID RYLhIVRAVr A27.ARTVTFG VITSVVTWVV murCCR2 rGWIHCKVrT GLYRIGYrGG irrxlLLTID RYLhXMVtP ALKhRrVTTG VXTSVVTWVV huCCR2b RYLhIVKAVr ALKRRTVTrG VVTSVYTNLV
zzz
181 240
ratoCR5 AVrVSLPZII rNMKZGSR YTCSPKIPLHI QYPYWKWIQT LKKVILSLIL PLLVNVICYS mur=5 AVrASLPZII rTRSQXBM YTCSPHrPHT QYHrMUWW LKKVILSLIL SLLVNVICYS
huCM5 AvrASLPGir FMOKZGLH YTC3SRrPYS QyQrWXMPQT LxrVIIaLVL PLLVWICYS r&tCCR2 AVt'ASLPGIX rTRSZMDQ WMGPYrP.. ..TIMOUVT IMMILSLIL PLLVKVICYS murCCR2 AVFGSLPGri rTKSXOWKH YTOOPYrT.. ..OLMOMOT DWXILSLIL PLLVKVICYS
huCCR2b 3V YVCGPYFP..
zV V
241 300
ratCCR5 GILNTUrRCR NZKXRHRAVR LIrAIMXVYIP TA'NTPYNIVL LLTTMZYrO laNC333NRL oir=5 GILHTUMM HElOaUUtAVR LIrAn41VYr LIATPYNIVL LLTTTQZrlrG IjQCSSSNRL
huCCR5 GILICTIL= XZX)UUMAVR LIrTIKIVY1r LrK"YNIVL LLNTTQZFIrG JARICSSSMM r&tCCR2 GIUITLFRCR NZKKRKRhVR LXFAIMXM UP WT TNM nTTrQZFW MSNCVVDNHL
mur=2 GILRTUrRCR NZKKRWtAVR LIIPAIMIGV LVWPYSM nTTFMW MSNCVMKKL
huCCR2b GILKTLLPCR NEIUUUMAVR trWTPVWrVT LUITrQEM LSN=STSQL
Vz
301 360
ratCCR5 DOAMQVTZTL GKTRCCLNPV IYArVWJWR HYLSVrrRKH IVKRFCM= IFQC)%WDRV
nur=5 D0MQATZTL GKTHMLNPV IYArVGZXrR SYLSVrrRXR NVXRrC1M-S IrQQDMPDRA
huCCR5 DQAMOVTZTL GMMCCXWI IYArVGZKrR HYLLVrTWA IAXRTCMCCS ITQQZRPZRA rst=2 DQAMOVTETL GKTRCCVNPI IYAFVGWR RYLSIFTRKH IAKULCY= VrYRETADRV
nurWR2 DOAMOVTZTL GKTR=rNPV IYARVOCKM RYLSIrn= IAKRLCKOCP VrYRZTADRV
huCCR2b RYLSVMUal ITXRrCXOCP VrYRZ2VDGV
Vzx
361 377
ratCCR5 SSVYTRSTGE QZVSTGL murccr5 SSVYTRSTGZ HZVSTGL huCCR5 SSVrZRST= QZISVGL r&tCCR2 SSTMSGZ Q8VSVGL murC=2 SS2rTPST= QIVVSVGL hu= b TSTPMSTW QZVSWL



Figure 3-1. Sequence Comparison of Rat and Murine C-C Chemokine Receptors Multiple alignment of two rat CC chemokine receptor amino acid sequences (ratCCR2
and ratCCR5) with the two functional human CC chemokine receptor (huCCR2 and
huCCR5) and the two marine CC chemokine receptor (murCCR2 and murCCR5).
Alignment was achieved using the PILEUP algorithm of the GCG package (39). Lines
denote putative transmembrane regions. Genbank Accession Numbers:
(huCCR2b:U03905, huCCR5:X91492, murCCR2:U47035, murCCR5:U47036, ratCCR2:U77349, ratCCR5:U77350).





57








Table 3-1. Amino Acid Identities of the Rat, Murine and Human Chemokine Receptors, CCR2 and CCR5

Murine ortholog Human ortholog
Rat CCR2 93% 79%
Rat CCR5 92% 83%





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0.45

AF

0.35 -2 min




1.2

1.0

0.8

0.6

;-o 0.6 f. 0.4


0.2
0 2 4 6 8 10
murMCP-1 (nM)


Figure 3-2. Calcium Mobilization Response to MCP- 1 By HEK293 Cells Expressing Rat CCR2. HEK293 cells stably expressing rat CCR2 were prepared as outlined in chapter two. Transfected cells were loaded with FURA-2A/M. FURA-2 fluorescence was monitored before and after addition of MCP- 1. Panel A shows a representative trace. Panel B depicts the dose-dependent stimulation of intracellular calcium mobilization by MCP- 1 in cells expressing rat CCR2.





59

















murMIP-la murMIP-13
(500 nM) (500 nM)



9s






9 sec 9 sec












Figure 3-3. Functional Responses of Cells Expressing Rat CCR5 Xenopus laevis oocytes expressing rat CCR5 were prepared as described in chapter two. These oocytes were exposed to 500nM murine MIP-1 a (A) or 500 nM murine MIP-110
(B). The current induced was monitored on a dual beam storage oscilloscope linked to an IBM-PC.





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A B




12 -12

--"j-7



3.2 .3.23- 2.9 -3
2.7- 2.71.1



65 OC 50 OC







Figure 3-4. Southern Blot Analysis of Restriction Endonuclease-Digested Rat Genomic DNA.
Wistar rat genomic DNA (15 jg) was completely digested with the indicated restriction endonucleases and then subjected to Southern blot analysis using the 1.5 kbp [32p]radiolabeled Clal/BamHI fragment of rat CCR5 and the method of Church and Gilbert. Final wash temperatures were 650C (A) and 500C (B). The blots were exposed to Kodak XAR film with an intensifying screen at -80 'C for 1-3 days. The sizes of the hybridizing bands are indicated (in kbp) and were estimated by comparison to a GIBCO/BRL 1-kbp ladder.





61



















CCR2







L32


L32












Figure 3-5. Rat CCR2 RNA Tissue Distribution in Rat Solid Organs and Cells Two gg of total RNA from rat tissues or cells were subjected to RNase protection assay (see chapter two). The RNAs were hybridized simultaneously with 32P-radiolabeled 365base pair rat CCR2 and 93-base pair ribosomal RNA (L32) riboprobes. The dried gel was exposed to Kodak X-RP film at -80 oC for 72 hours.





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CCR5
CCR5CCR5



GAPDH
GAPDH















Figure 3-6. Rat CCR5 mRNA Tissue Distribution in Rat Solid Organs and Cells Two gg of total RNA from rat tissues or cells were subjected to RNase protection assay (see chapter two). The RNAs were hybridized simultaneously with [32P]-radiolabeled 230 bp rat CCR5 and 114 bp GAPDH riboprobes. The dried gel was exposed to X-ray film at
-80 oC for 10 hours.





63





IFN-y









28S




18S








cyclophilin



Figure 3-7: Dose-Dependent Increase in Rat CCR5/2 mRNA by Interferon-y (IFNy) Treated Microglia
Northern analysis of rat CCR5/2 mRNA in primary cultures of rat microglia treated with IFNy. Cultured microglia were incubated for six hours with DMEM (control) or DMEM containing the specified concentration of IFNy. At the end of the incubation, total RNA was isolated from the culture dishes using the Trizol Reagent (Gibco/BRL). Ten tg of total RNA from each group was subject to Northern analysis. Immobilized RNA was hybridized with 32P-labelled CCR5 (ClaI/BamHI fragment) by the method of Church and Gilbert. Final wash temperature was 650C. The positions of migration of 18 S and 28 S ribosomal RNA are shown. Exposure to Kodak X-AR film was for 3 days (-800C). The lower panel depicts a subsequent hybridization of the blot with 32P-labelled DNA encoding rat cyclophilin and was used to control for lane loading. Exposure time for the cyclophilin hybridized membrane was 18 hours.





64






.0 0 0 00.P








L32 i




CCR2 CCR5





18 S CXCR4


28 S RBS11


18S


"yclophilin

Figure 3-8. Expression of CCR2, CCR5, CXCR4, and RBS1 1 mRNAs in Lumbar Spinal Cords of EAE Rats as Detected by RNase Protection Assay or Northern Blot Analysis EAE was induced in Lewis rats and animals were scored for clinical symptoms as described in chapter two. Total RNAs from control and EAE rat spinal cords (5 gg for RNase protection assay and 10 gg per lane for Northern blot analysis) were subjected to RNase protection assay (upper panel) and Northern blot analysis (lower panels) as described in chapter 2. Exposure time for the RNase protection assay gel was 18 hours (at -80 'C). Exposure times for the Northern blots were 18 hours (at -80 'C) for the RBS 11 and cyclophilin hybridized membranes, and 7 days (at -80 'C) for the CXCR4 hybridized membrane.













CHAPTER 4
ROLE FOR NEURONALLY-DERIVED FRACTALKINE IN MEDIATING INTERACTIONS BETWEEN NEURONS AND CX3CR1-EXPRESSING
MICROGLIA-AN IN VIVO STUDY IN THE FACIAL MOTOR NEURON REGENERATION PARADIGM


Introduction


Among the four subfamilies of chemokine peptides, the newly identified

chemokine termed fractalkine/neurotactin (Bazan et al., 1997 and Pan et al., 1997) has some very unique properties. Its full-length amino acid sequence is comprised of five regions: a signal peptide, a chemokine domain, a highly glycosylated mucin-like stalk which tethers the chemokine domain with a transmembrane domain and a short intracellular region. There are three amino acids in between the first two of the four highly conserved cysteine residues in its chemokine domain which makes it the first member of the CX3C chemokine subfamily. This peptide can exist as both secreted soluble and membrane anchored forms. The soluble form is chemotactic for monocytes and lymphocytes while the membrane anchored form can promote a strong adhesion of these leukocytes to endothelial cells. Unlike most chemokine peptides, fractalkine is expressed in non-hematopoietic tissues including brain, kidney, lung, heart and adrenal gland.

An "orphan" rat G-protein coupled receptor termed RBS 11 has a tissue

distribution pattern that is very similar to fractalkine (Harrison et al., 1994). It is widely expressed in the adult rat. Although its amino acid sequence is most similar to other 65





66

chemokine receptors, functional screening assays using a variety of CC and CXC chemokine peptides failed to identify its ligand. The similar tissue distribution pattern of fractalkine and RBS 11 prompted us to hypothesize that RBS 11 is the receptor for rat fractalkine. The human ortholog of rat RBSI11, termed V28, was also identified (Raport et al., 1995 and Combadiere et al., 1995). Recently, studies identifying V28 to be a receptor for human fractalkine have been reported (Imai et al., 1997, Combadiere et al., 1998). V28 is designated human CX3CR1.

In our continuous effort to characterize the role(s) of chemokines and chemokine receptors in the CNS, we employed an animal model-the facial motor neuron regeneration model-to explore the expression site(s) of the chemokines and chemokine receptors in the CNS. A transection of the facial nerve in the periphery can trigger a dramatic response in the facial motor nucleus (FMN) of the CNS. This response includes microglia activation and proliferation as well as astrocyte activation. Ultimately, the damaged motoneurons are regenerated. The advantage of this model is that one can challenge the brain tissue without breaking the blood brain barrier. Therefore, only endogenous cells are responsible for any reactions in the facial motor nucleus in the CNS. The other phenomenon that is associated with this model is so called "synaptic stripping" where activated microglia occupy perineuronal positions, forming a sheath around the motoneurons and displacing synaptic terminals from the surface of regenerating motoneurons (Blinzinger and Kreutzberg., 1968, Graeber and Streit, 1990a, Graeber et al., 1990b).

Microglia activation in FMN after facial nerve transection was determined not

only by morphological but also immunohistochemical studies. After axotomy, microglia





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antigenic profiles have changed dramatically indicative of an activated state of these cells. Increase of CR3 is found as early as 24 hours after axotomy (Graeber et al., 1988a). De novo synthesis of MHC (major histocompatibility complex) class I occurrs around 3-4 days after the lesion followed by the expression of MHC class II and CD4 antigens (Streit et al, 1988, Streit et al., 1989,). These findings demonstrate that microglia can express molecules involved in communication with other immune responsive cells. However, since there is no inflammation and leukocyte infiltration in this paradigm, the relevance of these molecules in the regeneration process is not apparent.

After facial nerve transection, the production of a number of cytokines,

neurotrophic factors and cell matrix proteins by microglia in the lesioned FMN is elevated including interleukin IL-6 (Kiefer et al., 1993), TGF-p l (Kiefer et al., 1993), vimentin (Graeber et al., 1988b), and a polymeric ECM protein TSP (thrombospondin, Moller et al., 1996). TSP has been shown to promote neuritic outgrowth from neurons in the in vitro system (Lawer et al., 1986). The up-regulation of these molecules may play an important role for microglia to elicit their function as neuroprotective cells in the motoneuron regeneration paradigm.

Since microglia are immunoresponsive cells in the CNS and chemokine peptides and receptors are immunologically relevant molecules, we hypothesized that chemokines and/or their receptors are expressed in microglia and play a role in microglia function. In this study, we examined the expression of rat fractalkine and its receptor CX3CR1 in the FMN after facial nerve transection.





68

Results


Molecular Cloning of Rat Fractalkine

A rat brain cDNA library (Strategene, CA) was screened by hybridization using a rat factalkine cDNA probe. The hybridization probe was generated by PCR using primers derived from a mouse cDNA (Genbank accession number R75309). The rat fractalkine cDNA was sequenced by standard methods and conceptualized protein sequence is shown in Figure 4-1. The conceptualized amino acid sequence derived from the cDNA predicts a 392 amino acid protein with a molecular mass of 42,161 Da. The amino acid sequence identities of rat fractalkine with human and murine forms are 64% and 81%, respectively. Analogous to these human and murine proteins, rat fractalkine contains a signal peptide, chemokine module, mucin-like stalk, transmembrane spanning region, and a short intracellular C-terminus.

Tissue Distribution of Rat Fractalkine

In collaboration with Dr. Lili Feng (The Scripps Research Institute), we carried

out RNase protection assays in order to determine the tissue distribution of rat fractalkine. Analogous to its human and murine orthologs, rat fractalkine is expressed in a number of non-hematopoietic tissues including kidney, lung, heart, and adrenal gland. Among the tissues that we've examined, brain was identified as the most abundant source of rat fractalkine (Figure 4-2).

Distribution of Rat Fractalkine in the Brain

In situ hybridization analysis revealed that rat fractalkine is highly expressed in discrete regions of rat brain including cortex, hippocampus, caudate putamen, thalamus,





69

and olfactory bulb (Figure 4-3) and was significantly lower or absent in cerebellum and white matter regions including corpus collosum and fimbria/fornix. Identification of RBS 11 As the Receptor for Rat Fractalkine

Figure 4-4 demonstrates the ability of radiolabeled chemokine domain of

fractalkine bind to RBSI 1-transfected CHO cells. Wild type CHO cells did not display any significant binding. Inhibition binding constants (IC50s) of 2 and 3 nM for the chemokine domains of rat and human fractalkine, respectively, were determined. Other CC or CXC chemokine peptides that we tested, including MIP-lc a, RANTES, MCP-1 MIP-2, and SDF-1, did not displace the binding of labeled fractalkine to RBS 11 expressing CHO cells. This evidence further corroborated that the binding between fractalkine and RBS 1I transfected cells was specific. Figure 4-5 depicts the functional properties ofRBS 11 in the transfected CHO cells. Application of either soluble or membrane-tethered forms of fractalkine stimulated a transient elevation of intracellular calcium levels in the RBS 11 expressing CHO cells. These data, collected by Dr. Jeffrey Harrison, unequivocally identified that RBS 11 is a functional receptor for rat fractalkine. Expression and Regulation of Rat CX3CR1 mRNA in Cultured Microglia

To identify the expression site(s) of rat CX3CR1, Northern blot analysis was

carried out. Primary cultures of rat microglia were incubated for eight hours with DMEM (control) or DMEM containing increasing concentrations of TGF-P3. At the end of the incubation, total RNA was isolated from these microglia and subjected to Northern blot analysis. Figure 4-6 depicts constitutive expression of rat CX3CRI mRNA in cultured microglia. Steady state levels of CX3CRl mRNA present in the cultured microglia were dose-dependently increased by TGF-P treatment.





70

Expression and Regulation of Rat CX3CR1 in vivo in the Facial Motor Nucleus After Facial Nerve Transection

The facial motor neuron regeneration model was used to determine the

localization and regulation of expression of CX3CR1 in rat CNS. In situ hybridization analysis was used to assess the expression of CX3CR1 in the facial motor nucleus over a 21 day period during which the facial motor neurons undergo regeneration. At time points of 4, 7, and 14 days post-axotomy, the levels of CX3CRI hybridizing mRNA were significantly elevated in the injured facial motor nucleus (Figure 4-7), as compared to the contralateral nucleus. Highest levels of CX3CR1 mRNA were days 4 and 7 postaxotomy. At the 21 day time point CX3CR1 mRNA had returned to a level comparable to that detected in the contralateral nucleus. The graph depicts quantitative analysis of rat CX3CR1 mRNA hybridizing signal in the FMN of control and lesioned side.

The widespread distribution of rat CX3CR1 in normal rat brain (Harrison et al., 1994) and its presence on cultured microglia indicate that the CX3CR1 gene might also be expressed on these cells in vivo. To determine the expression site(s) of rat CX3CR1 in the CNS, in situ hybridization analysis was combined with histochemical staining using sections of brain stem containing FMN four days after facial nerve transection. Lectin (from Griffonia simplicifolia, GSA-I-B4) staining was used to identify microglia cells whereas anti-GFAP (glial fibrillary acidic protein) antibody was applied to identify astrocytes. Figure 4-8 demonstrates the colocalization of rat CX3CR1 mRNA hybridizing signals (silver grains) with lectin staining positive microglia but not antiGFAP positive astrocytes (data not shown) in the FMN before and after axotomy. This data confirmed that rat CX3CR1 is expressed principally by microglia in vivo.





71

Quantitative analysis indicated that, in the control FMN, only 30% of the lectin staining microglia were CX3CR1 positive (a cluster of>10 silver grains over a single cell) whereas in the lesioned FMN, there is approximately 60% of the lectin staining microglia expressing CX3CR1. In addition, the intensity of CX3CR1 mRNA signal, represented by numbers of silver grains found over the microglia, appear to be comprable between the control and lesioned FMN. This suggests that CX3CR1 mRNA expressed by each single microglia did not change after facial nerve axotomy. Expression of Rat Fractalkine in FMN After Facial Nerve Transection

The expression of fractalkine mRNA in the facial motor nucleus following axotomy was also examined. In situ hybridization analysis demonstrated that the motoneurons were the primary source of fractalkine mRNA (Figure 4-9 A-D). Fractalkine positive cells were not colocalized with either lectin positive microglia or anti-GFAP positive astrocytes. A decrease in the hybridization signal in motoneurons four days after axotomy was also evident. Western blot analysis done by Elizabeth Wees in our laboratory demonstrated that lower molecular mass forms of rat fractalkine protein levels were upregulated in the lesioned FMN.

Discussion


The expression of some of the chemokine and chemokine receptors in the CNS has been reported by a number of different groups. In this study, we isolated the rat ortholog of human and murine fractalkine by homology screening technique and examined the expression and regulation of rat fractalkine and its receptor rat CX3CR 1, in the facial motor neuron regeneration paradigm. Consistent with its human and murine





72

orthologs, rat fractalkine gene encodes a protein that has a chemokine domain followed by a highly glycosylated mucin-like stalk, a transmembrane domain and a short intracellular domain. Its mRNA can be found in some non-hematopoietic tissues including lung, heart, kidney, and adrenal gland. However, its expression is most abundant in the brain where it is expressed principally by neurons.

By in situ hybridization analysis, we demonstrated that the expression of CX3CR 1 mRNA was transiently up regulated in the facial motor nucleus (FMN) after axotomy. The time course of this up-regulation parallels the transient increase in microglial cell numbers after axotomy. It is also known that, at this time point, the majority of axotomized motoneurons are tightly ensheathed by the perineuronal microglial cells (Kreutzberg 1996, Streit 1996). In an in vivo study, Kiefer et al. (1993) demonstrated that TGF-P mRNA level was up regulated transiently in the lesioned FMN. It peaked between days 2-7 and gradually comes down after that and returned to normal by three weeks post-lesion. Our in vitro study indicated that cultured microglia constitutively express rat CX3CR1. The expression is up regulated in a dose dependent manner after TGF-P3 treatment. Collectively, it can be speculated that TGF-P may play an important role in regulation of microglial expression of CX3CR1 in vivo as well.

Compared to the other CNS inflammatory models such as EAE, the motor neuron regeneration model provides the advantage of confined cell population within the CNS because of the intact blood brain barrier. This brings a great advantage in our effort to identify the cellular localization of the genes that we are interested in. Our data unequivocally demonstrated that in the FMIN, microglia are the principal source of CX3CR1 whereas fractalkine is primarily expressed by motor neurons. Our collaborator





73


Dr. Lili Feng's group collected similar data demonstrating that neurons are the source of fractalkine in cortex, hippocampus and thalamus of adult rat (data not shown). The interaction between fractalkine and its receptor CX3CR1 may be an important mechanism by which neurons communicate with microglia and elicit their functional roles during facial motor neuron regeneration.

The present study did not define the specific form(s) of rat fractalkine that was expressed in FMN. Our functional analysis shown in figure 4-2 indicated that the response of CX3CRI expressing cells to fractalkine various based upon the different forms of the peptide (membrane vs. soluble). Membrane-anchored forms of fractalkine activated a greater calcium mobilizing response compare to soluble forms. Previous studies (Imai et al., 1997) demonstrated that a soluble form of fractalkine stimulates chemotaxis while a membrane anchored form promotes adhesion. Thus, it is conceivable that the larger calcium response triggered by membrane anchored form is related to cell adhesion whereas the minor response stimulated by soluble form results in chemotaxis. Alternatively, these forms of fractalkine may serve other functions for the CX3CRl expressing cells than chemotaxis or adhesion. Analogous to the receptor-transfected CHO cell model, studies carried out by our collaborator Dr. Kevin Bacon's group demonstrated that differential stimulation of microglia intracellular calcium mobilization by fractalkine was dependent on the form present (data not shown). Membrane-anchored forms of fractalkine stimulated robust changes in intracellular calcium levels while a soluble form of fractalkine had no effect. Our speculation based upon these data is that potentially, fractalkine-expressing neurons may function in regulating CX3CR1 expressing microglial activity by providing different forms of fractalkine to the





74

microenvironment. However, the physiological relevance of these relative intracellular calcium levels in both transfected cells and microglia still remains to be determined. We hypothesize that the soluble form of fractalkine stimulates an unknown signaling pathway which results in microglial chemotaxis towards facial motor neurons whereas the membrane anchored form elevates intracellular calcium levels which functions to promote the adhesion of microglia to these neurons and causes the ensheathement of neurons by microglia, known as "synaptic stripping".

In contrast to the up regulation of CX3CR1 mRNA in FMN after nerve

transection, the mRNA level of fractalkine is down-regulated four days after the lesion. However, our Western blot analysis indicates the up-regulation of some lower molecular mass forms of fractalkine protein in the lesioned FMN (data not shown). This discrepancy between mRNA and protein levels of rat fractalkine in FMN after nerve transection is suggestive of the dynamic regulation of fractalkine expression in the CNS.

The constitutive expression of fractalkine and CX3CRI in discrete regions of the CNS indicates that they are involved in fundamental processes of communication between neurons and microglia under both pathological as well as physiological circumstances. A better understanding of the functional roles of these molecules will eventually lead to the development of therapeutic reagents that can interrupt disease processes that these molecules are involved in.





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Rat Fractalkine (CX3C)

MA P S Q LA W L L R LA A F F H LC T L L AG
Q I LGMT K CN ITCH KMTS P I PVT
L I H Y Q LN Q S C G K R A I I L T R
Q H R I F CAD P K E KW V Q DA MN K B LD
EQ TAAL T R N G
G K F E K R V D N VT P R I T S A T R G L S PTAL Q S LS Q P Q W K T L LW N R LL F P R S P KA M GT SQ E P P A AV T G S S P S T S K A Q DA G L A K P Q S T G I S E V
A AV S T I W P S S A V Y Q S G S S L WA
E E K A T E S P P T I A L S T Q A S T T S S P K Q NV G S E G Q P PW V QE Q D S T P E K S PG P E E T N P V T DI F QD R G P G
S TV V P S VA P T S S E K T P S P E L V A
S G S Q A P K V E E P I H A T A D P Q K L S V F IT PV P D S Q AA T R R Q AV G L LA FL G L L F C L G VA MN F
A Y Q S L Q G C P R K MA G E M V E G L R Y V P R S C G S N S Y VL V P V








Figure 4-1.Amino Acid Sequence of Rat Fractalkine (Genbank accession number R75309)
A rat brain cDNA library (Strategene, CA) was screened by hybridization using a rat factalkine cDNA probe. The hybridization probe was generated by PCR using primers derived from a mouse cDNA (Genbank accession number R75309). The rat fractalkine cDNA was sequenced by standard methods (Sanger dideoxy chain termination, Sequenase kit).





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b






c-Frac



2-L32
II




















Figure 4-2. RNase Protection Analysis of Fractalkine mRNA in Various Tissues of Adult Rat
Five pg of total RNA from rat tissues or cells were subjected to RNase protection assay (see Materials and Methods). The RNAs were hybridized simultaneously with 32pradiolabeled 400-base pair NcoI fragment of rat fractalkine cDNA and 93-base pair ribosomal RNA (L32) riboprobes. The dried gel was exposed to Kodak X-RP film at -80 oC for 72 hours.





77









































Figure 4-3. Distribution of Rat Fractalkine mRNA in Adult Rat Brain Hybridization analysis using "anti-sense" (A) and "sense" (B) [35S]-UTP-labeled riboprobes generated from rat fractalkine cDNA. Hybridized horizontal sections were exposed to film for 18 hours (abbreviations used: Olf: olfactory bulb; ctx: cortex; cc: corpus collosum; CPu: caudate putamen; Th: thalamus; ca3: ca3 region of the hippocampus; gc: granule cell layer of the hippocampus; CG: central gray; cbm: cerebellum).





78














100



0
IMI

20

0"7

-10 J -9 -8 -f
[Inhibitor], log M




Figure 4-4. Whole Cell Radioligand Binding Analysis of CHO-CX3CRI-Expressing Cells
Competition of 1 nM labeled peptide with the chemokine domain of rat fractalkine (0), the chemokine domain of human fractalkine (@, rat MCP-I (@, rat MIP-lIa (), rat RANTES (* ), rat SDF-I (v), and murine MIP-2 (a). Wild type CHO cells did not display any significant specific binding of 125I-fractalkine (V). Data were calculated as the fraction of specific binding in the presence of the specified concentration of unlabeled chemokine peptides [CPMs bound per 3 million cells were: Total: 7945 + 428; Non-specific: 2078 + 231, N = 7]. The results (from triplicate determinations) are presented as the mean + S.E.M. of 3 experiments (rat fractalkine), 4 experiments (human fractalkine) or the mean of two experiments (all other chemokines). The calculated IC50s for inhibition of labeled peptide binding by the chemokine domains of either rat or human fractalkine are 2.2 nM (95% confidence interval: 0.9 5.3 nM) and 2.9 nM (95% confidence interval: 1.9 4.5 nM), respectively.





79









0.2
AFL
iv 1 min.
1zfr v vi

VIIvill IX


10




C 40
0
1


J0


10 2b
[Fractalkine], nM

Figure 4-5. Calcium Mobilization Response to Different Forms of Fractalkine by CHO Cells Expressing Rat CX3CR1 Representative traces of CHO-CX3CR1 expressing cells in response to application of either 293T cells transiently expressing rat (i, ii) or human fractalkine (iv, v), or the chemokine domain of rat (iii) or human (vi) fractalkine. Wild-type fractalkines (i, iv) or mutant fractalkines (ii, v) in which arginine-arginine residues adjacent to the TM domain were changed to serine-alanine, were expressed transiently in 293T cells. Lower traces depict application of mock transfected 293T cells (vii) to CHO-CX3CRl-expressing cells or addition of the chemokine domain of rat fractalkine (viii), or the wild-type membranetethered form of rat fractalkine (ix) to wild-type CHO cells. The graph depicts concentration-dependent effect of the chemokine domain of human fractalkine on intracellular calcium levels in CHO-CX3CRl-expressing cells. Data are plotted as the fraction of the maximal response seen in the presence of a high dose of fractalkine. Values are the mean of at least two determinations at each concentration. The calculated EC50 from these experiments is 3 nM.





80


















-28S





-18S





cyclophilin




Figure 4-6. Dose-Dependent Increases in Rat CX3CR1 mRNA by Transforming Growth Factor-3 (TGF-P3) Treated Microglia
Northern analysis of rat CX3CRI mRNA in primary cultures of rat microglia treated with TGF-P. Cultured microglia were incubated for eight hours with DMEM (control) or DMEM containing the specified concentration of TGF-P3. At the end of the incubation, total RNA was isolated from the culture dishes using the Trizol Reagent (GibcoBRL). Ten ptg of total RNA from each group was subject to Northern analysis. Immobilized RNA was hybridized with 32P-labelled 1.1 kbp CX3CRI cDNA fragment by the method of Church and Gilbert. Final wash temperature was 650C. The positions of migration of 18 S and 28 S ribosomal RNA are shown. Exposure to Kodak X-AR film was for 4 days (-800C). The lower panel depicts a subsequent hybridization of the blot with 32P-labelled DNA encoding rat cyclophilin and was used to control for lane loading. Exposure time for the cyclophilin hybridized membrane was 18 hours.





81

F

1.0
0.8

0.6
B 0.4

00
0 0.2

0 5 10 15 20 25 Days Post-Lesion




G




















Figure 4-7. In Situ Hybridization Analysis of CX3CR1 Expression in the Rat Facial Nucleus After Motor Neuron Axotomy In situ hybridization was performed on rat brainstem sections from animals sacrificed at 1
(A), 4 (B), 7 (C), 14 (D), and 21 (E) days post nerve transection using [35S]-UTP-labeled riboprobe. Panel F depicts a quantitative analysis of the hybridization signal on the operated (o) and contralateral (o) sides. Results are presented as the mean + S.E.M. of data derived from at least three sections from three different animals. The hybridization signal was specific for CX3CR1 as no increase in signal was observed using a "sense" riboprobe (H) when compared to a corresponding "anti-sense" riboprobe hybridizedsection (G).





82





A:w

N








*1 9
B






















Figure 4-8. Colocalization of Rat CX3CRI mRNA With Microglial Cells Rat CX3CR1 mRNA hybridization signals (silver grains) from "anti-sense" [33P]-UTPlabeled riboprobe hybridized-sections are found principally over lectin (GSA-I-B4) staining microglia in the neuropil of the unoperated facial motor nucleus (B). Several CX3CR1 mRNA containing microglial cells are found perineuronally [arrows in (A)] surrounding axotomized motorneurons (N: motor neuron cell body). (C) "Sense" [33P]UTP-labeled riboprobe-hybridized section showing only a few scattered silver grains.





83



























W, 1W VV
A 8k



~ J
















Figure 4-9: Fractalkine Expression in the Rat Facial Nucleus After Motor Neuron Axotomy
In situ hybridization was performed on rat brainstem sections from animals sacrificed 4 days post nerve transection. Hybridization analysis of the contralateral (A and B) and ipsilateral (C and D) facial motor nuclei using "anti-sense" (A and C) and "sense" (B and D) [33P]-UTP-labeled riboprobes generated from rat fractalkine cDNA. Hybridization signals are found principally over the motor neurons.













CHAPTER 5
DIFFERENTIAL EXPRESSION OF CX3CR1 IN TWO RAT GLIOMA MODELSROLE FOR TGF-3 AND CX3CR1 IN MICROGLIAL CELL DEATH WITHIN RAT BRAIN TUMORS


Introduction


It has been known for sometime that tumor growth in vivo can create an immunosuppressive environment (Sawamura and De Tribolet, 1990). What role chemokines and their receptors play in this environment is a question that needs to be addressed.

Some tumor cell lines and tumors have been found to express chemokine

peptides. For instance, IL-8 was found to be expressed in lung carcinoma, osteosarcoma, melanoma, renal carcinoma, fibrosarcoma, astrocytoma and glioblastoma cell lines; Gro peptides were found in melanoma, renal carcinoma and bladder carcinoma cell lines; MCP-1 expression was seen in fibrosarcoma, glioma, histocytoma, melanoma, osteosarcoma and sarcoma cell lines. In vivo studies revealed that MCP-1 is expressed in ovarian cancer (Negus et al., 1995), prostate adenocarcinoma (Mazzucchelli et al., 1996), and human invasive ductal breast cancer (Valkovic et al., 1998). In these studies, ovarian cancer cells and breast cancer cells were found to be the sources of MCP-1 I whereas prostate adenocarcinoma cells were not. The expression and function of CXC chemokines in tumor development have been studied rather extensively mainly based on their angiogenic or antiangiogenic functions. 1L-8 and IP 10 have been found in human



84





85

non-small cell lung cancer tissues. Immunohistochemical studies revealed that neoplastic cells are the principal sources of these peptides (Yatsunami et al., 1998, and Arenberg et al., 1996). Multiple studies carried out by a number of groups demonstrated that IL-8 promotes tumorigenesis because of its angiogenic function whereas IP10 has tumor growth inhibitory function based primarily on its angiostatic activity (reviewed by Arenberg et al., 1997). There is evidence to support the speculation that the ELR motif in the N-terminus of some of the CXC chemokine peptides plays an important role in the angiogenic activity of these peptides and a lack of a ELR motif in other CXC chemokine peptides confers their angiostatic activity. The mechanism of these phenomena remains to be elucidated.

There is very limited information regarding the expression of chemokines and their receptors in brain tumor tissue. Studies from several different groups have shown that several astrocytoma derived cell lines express MCP- 1 either constitutively or after induction with IL-I beta and TNF alpha (Kasahara et al., 1991). MCP-1 mRNA and protein expression has also been demonstrated in vivo from human glioma tumor cells, endothelial cells and possibly infiltrating macrophages (Takeshima et al., 1994). The evidence that MCP- 1 expression correlated with the degree of macrophage infiltration into the tumor (Takeshima et al., 1994) and that MCP-l containing cyst fluids derived from brain tumors exhibited chemotactic activity for macrophages in vitro (Desbaillets et al., 1994) suggests that MCP-1 is biologically active in the tumors in vivo. Another study carried out by Barnes et al. (1996) demonstrated that astrocytoma cells produced RANTES when stimulated with TNF-c or IL- 1 P.





86

The experimental rat glioma models are useful in studying chemokine and

chemokine receptor expression and function in brain tumors. RG2 and C6 gliomas are two very widely used rat models in experimental neuro-oncology (Barth et al., 1998). Both gliomas are chemically induced with different immunogenic responses. RG2 glioma appears to be either weakly or non-immunogenic. This tumor is refractory to a variety of therapeutic modalities and has an invasive growth pattern as well as uniform lethality. Compared to RG2 glioma, C6 glioma has a potential to evoke an alloimmune response partly because it is not syngeneic to any inbred strain. Morioka et al. (1992) has reported that in RG2 gliomas, increased numbers of microglia were formed around and infiltrated into the tumor. This indicated that tumor growth in the brain causes microglial activation and proliferation. However, the question of whether or not these microglial cells are functional as tumor surveillance cells remains unanswered. The microglial response in C6 glioma has not been carefully studied.

Our data from the previous chapter demonstrated that CX3CRI mRNA is

expressed in microglia in vivo. We then set out to determine if this is also true in tumor tissues. The specific aim of this study was to determine the expression and to specify the cellular localization of fractalkine and CX3CR1 in experimental rat glioma tissues (RG2 and C6 gliomas). Results generated by this study may provide more information in understanding brain tumor biology as well as some aspects of the function of fractalkine and CX3CR1 in the CNS.





87

Results


Expression of Rat CX3CR1 in C6 Glioma

To examine the expression of rat fractalkine as well as CX3CR1 in RG2 and C6 gliomas, Northern blot and in situ hybridization analysis were carried out. After tumor cell inoculation, female Fisher rats were sacrificed at the point in time when the first C6 or RG2 inoculated animal developed the first signs of neurological deficit. It normally takes 2-3 weeks for RG2 glioma and 4-6 weeks for C6 glioma inoculated rats to develop some symptoms. For Northern blot analysis, tumor tissues were dissected out on an ice cold surface and surrounding normal tissues were removed. RNA isolation was carried out using Trizol reagent. For in situ hybridization, rats were sacrificed as described in chapter two and frozen sections were made subsequently. Both Northern blot and in situ hybridization analysis demonstrated that these gliomas express little if any fractalkine mRNA. C6 tumors, but not RG2 tumors, expresses CX3CRI mRNA (Figure 5-1, Figure 5-2). In collaboration with Sharon Walters and Dr. Wolfgang Streit, we also examined the ability of these gliomas to express TGF-3 mRNA. Figure 5-2 (E-F, K-L) demonstrats that RG2 tumors express much higher levels of TGF-P mRNA than C6 tumors do. Emulsion dipped slides confirmed that tumor cells rather than microglia were the sources of TGF-P3 mRNA (data not shown).

Rat CX3CR1 mRNA Expression by Microglia in C6 Gliomas

To identify the cellular localization of CX3CR1 in C6 gliomas, lectin (GSA I-B4) staining was carried out in combination with in situ hybridization as described in chapter two. Slides were then exposed to Kodak NTB2 autoradiographic emulsion at 4'C for





88

approximately 3-4 weeks before being developed. Our data indicated that rat CX3CRl mRNA is expressed in microglia within C6 gliomas as clustered silver grains were evident over lectin binding positive cells (Figure5-3A). In RG2 gliomas rat CX3CR1 mRNA is expressed by microglia on the border of the tumor but not lectin positive cells within the tumor (data not shown).

Different Microglial-Infiltrating Patterns Between C6 and RG2 Gliomas

Lectin (GSA I-B4) staining of both C6 and RG2 gliomas were carried out to characterize microglial-infiltrating patterns of these two gliomas. Figure 5-4 demonstrates that within RG2 tumors, lectin positive microglia tend to cluster together to form isolated islands (A); many cells have pyknotic nuclei-one of the characteristics of apoptotic cells (B); many areas within RG2 tumors have none or very little microglial infiltration (data not shown). On the other hand, inside C6 gliomas, infiltrating microglia are evenly distributed throughout the tumor (C); these cells conserve the morphology of normal microglial cells (D).

Microglial Apoptosis Within C6 and RG2 Gliomas

We further examined apoptosis within C6 and RG2 gliomas by TUNEL staining (Terminal Transferase dUTP Nick End-Labelling) as described in chapter two. TUNEL is a staining assay that labels cells with fragmented DNA ends, characteristic of apoptotic cells. Figure 5-5 demonstrates that within RG2 tumors, TUNEL staining positive cells are clustered to form isolated islands similar to what we see in RG2 tumors with lectin staining (A). However, in the C6 gliomas that I tested, there are only a few TUNEL positive cells (data not shown) and the major areas of the tumor mass were TUNEL negative (B).




Full Text
87
Results
Expression of Rat CX3CR1 in C6 Glioma
To examine the expression of rat fractalkine as well as CX3CR1 in RG2 and C6
gliomas, Northern blot and in situ hybridization analysis were carried out. After tumor
cell inoculation, female Fisher rats were sacrificed at the point in time when the first C6
or RG2 inoculated animal developed the first signs of neurological deficit. It normally
takes 2-3 weeks for RG2 glioma and 4-6 weeks for C6 glioma inoculated rats to develop
some symptoms. For Northern blot analysis, tumor tissues were dissected out on an ice
cold surface and surrounding normal tissues were removed. RNA isolation was carried
out using Trizol reagent. For in situ hybridization, rats were sacrificed as described in
chapter two and frozen sections were made subsequently. Both Northern blot and in situ
hybridization analysis demonstrated that these gliomas express little if any fractalkine
mRNA. C6 tumors, but not RG2 tumors, expresses CX3CR1 mRNA (Figure 5-1, Figure
5-2). In collaboration with Sharon Walters and Dr. Wolfgang Streit, we also examined
the ability of these gliomas to express TGF-P mRNA. Figure 5-2 (E-F, K-L) demonstris
that RG2 tumors express much higher levels of TGF-P mRNA than C6 tumors do.
Emulsion dipped slides confirmed that tumor cells rather than microglia were the sources
of TGF-P mRNA (data not shown).
Rat CX3CR1 mRNA Expression by Microglia in C6 Gliomas
To identify the cellular localization of CX3CR1 in C6 gliomas, lectin (GSA I-B4)
staining was carried out in combination with in situ hybridization as described in chapter
two. Slides were then exposed to Kodak NTB2 autoradiographic emulsion at 4C for


Expression and Regulation of Rat CX3CR1 mRNA in Cultured
Microglia 69
Expression and Regulation of rat CX3CR1 In Vivo in the Facial Motor
Nucleus After Facial Nerve Transection 70
Expression of Rat Fractalkine in FMN After Facial Nerve Transection 71
Discussion 71
5. DIFFERENTIAL EXPRESSION OF CX3CR1 IN TWO RAT GLIOMA
MODELSROLE FOR TGF-p AND CX3CR1 IN MICROGLIAL CELL
DEATH WITHIN RAT BRAIN TUMORS 84
Introduction 84
Results 87
Expression of Rat CX3CR1 in C6 Glioma 87
Rat CX3CR1 mRNA Expression by Microglia in C6 Gliomas 87
Different Microglial-Infiltrating Patterns Between C6 and RG2
Gliomas 88
Microglial Apoptosis Within C6 and RG2 Gliomas 88
Discussion 89
6. CONCLUSIONS AND FUTURE DIRECTIONS 97
LIST OF REFERENCES 101
BIOGRAPHICAL SKETCH 126
Vll


104
Boring, L., Gosling, J., Monteclaro, F.S., Lusis, A J., Tsou, C.-L., and Charo, I.F.
(1996) Molecular cloning and functional expression of murine JE (monocyte
chemoattractant protein 1) and murine macrophage inflammatory protein la receptors. J.
Biol. Chem. 271:7551-7558.
Bozic, C.R., Gerard, N.P., von Uexkull-Guldenband, C., Kolakowski, Jr., L.F.,
Conklyn, M.J., Breslow, R., Showell, H.J., and Gerard, C., (1994) The murine interleukin
8 type B receptor homologue and its ligands; expression and biological characterization.
J. Biol. Chem. 269:29355-29358.
Cerretti, D.P., Nelson, N., Kozlosky, C.J., Morrissey, P.J., Copeland, N.G.,
Gilbert, D.J., Jenkins, N.A., Dosik, J.K., and Mock, B.A. (1993) The murine homologue
of the human interleukin-8 receptor type B maps near the Ity-Lsh-Bcg disease resistance
locus. Genomics 18:410-413.
Charo, I.F., Myers, S.J. Herman, A., Franci, C., Connolly, A.J., and Coughlin,
S.R. (1994) Molecular cloning and functional expression of two monocyte
chemoattractant protein 1 receptors reveals alternate splicing of the carboxyl-terminal
tails. Proc. Natl. Acad. Sci. U.S.A.99\:2152-2756.
Chaudhuri, A., Polyakova, J., Zbrzezna, V., Williams, K., Gulati, S., and Pogo,
A.O. (1993) Cloning of glycoprotein D cDNAA, which encodes the major subunit of the
Duffy blood group system and the receptor for the Plasmodium vivax malaria parasite.
Proc. Natl. Acad., Sci. USA 90:10793-10797.
Choe, H., Farzan, M., Konkel, M., March, K., Sun, Y., Marcon, L., Cayabyab, M.,
Berman, M., Dorf, M.E., Gerard, N., Gerard, C., and Sodroski, J. (1998) The orphan
seven-transmembrane receptor Apj supports the entry of primary T-cell-line-topic and
dualtropic human immunodeficiency virus type 1. J. Virol. 72:6113-6118.
Choe, H., Farzan, M., Sun, Y., Sullivan, N., Rollins, B., Ponath, P.D., Wu, L.,
Mackay, C.R., LaRosa, G., Newman, W., Gerard, N., Gerard, C., and Sodroski, J. (1996)
The p-chemokine receptors CCR3 and CCR5 facilitate infection by primary HTV-1
isolates. Cell 85:1135-1 148.
Chollet-Martin, S., Montravers, P., Gibert, C., Elbim, C., Desmonts, J.M., Fagon,
J.Y., Gougerot-Pocidalo, M.A. (1993) High levels of interleukin-8 in the blood and
alveolar spaces of patients with pneumonia and adult respiratory distress syndrome.
Infect. Immun. 61:4553-4559.
Church, G.M., and Gilbert, W. (1984) Genomic sequencing. Proc. Natl. Acad. Sci.
USA. 81:1991-1995.


8
these peptides to Xenopus oocytes that express this receptor triggered an intracellular
Ca2~ mobilization (Murphy et al., 1991). These two EL-8 receptors were later designated
as CXCR1 and CXCR2. Both are expressed on neutrophils. CXCR2 is also found in
myelomonocitic cell lines and its expression can be stimulated by G-CSF (granulocyte
colony stimulating factor) and TNF (tumor necrosis factor)-a (Sprenger et al., 1994).
RT-PCR technique was used to isolate human CXCR3. Degenerate
oligonucleotide primers were designed based on the sequence of transmembrane domain-
2 (TM2) and TM7 of other CXC and CC chemokine receptors (Loetscher et al., 1996).
This receptor gene encodes a protein of 368 amino acids with putative seven
transmembrane domains. The CXC chemokines Mig (monokine induced by interferon-y)
and IP-10 were identified as ligands for this receptor because of their ability to stimulate
Ca2r mobilization and chemotaxis of receptor transfected cells. Northern blot analysis
revealed that this receptor was expressed in T cells stimulated with IL-2. Recently,
another CXC chemokine I-TAC (Interferon-inducible T cell Alpha Chemoattractant,
Cole et al., 1998) was found to be the ligand of human CXCR3 and a CC chemokine
peptide 6Ckine (Soto et al., 1998) was shown to be the ligand for murine CXCR3.
In 1994, Loetscher et al. identified an orphan seven transmembrane domain
receptor that was expressed in neutrophils, activated T cells and some leukemia cell lines.
This receptor was later found to be a co-receptor for T-cell line tropic HIV entry (Feng et
al., 1996). Two years later, a CXC lymphocyte chemoattractant, SDF-1 (stromal cell-
derived factor 1), was proved to be the ligand of this receptor (BleuI et al., 1996 and
Oberlin et al., 1996). According to the accepted nomenclature of chemokine system, it
was designated as human CXCR4. Two CXCR4 knock out studies reported most


78
[Inhibitor], log M
Figure 4-4. Whole Cell Radioligand Binding Analysis of CHO-CX3CR1-Expressing
Cells
Competition of 1 nM labeled peptide with the chemokine domain of rat fractalkine (), the
chemokine domain of human fractalkine (A), rat MCP-1 (), rat MIP-la (4), rat RANTES (
), rat SDF-1 (?), and murine Mff-2 (a). Wild type CHO cells did not display any
significant specific binding of I25I-fractalkine (?). Data were calculated as the fraction of
specific binding in the presence of the specified concentration of unlabeled chemokine
peptides [CPMs bound per 3 million cells were: Total: 7945 + 428; Non-specific: 2078 +
231, N = 7], The results (from triplicate determinations) are presented as the mean +
S.E.M. of 3 experiments (rat fractalkine), 4 experiments (human fractalkine) or the mean
of two experiments (all other chemokines). The calculated IC50s for inhibition of labeled
peptide binding by the chemokine domains of either rat or human fractalkine are 2.2 nM
(95% confidence interval: 0.9 5.3 nM) and 2.9 nM (95% confidence interval: 1.9 4.5
nM), respectively.


22
Another useful model for stroke is reperfusion after a transient brain ischemia
because reperfusion after ischemia is a frequently encountered clinical condition that
often causes greater tissue damage than persistent ischemia itself. Using a rabbit
reperfusion model, Matsumoto et al. (1997) revealed that anti-IL-8 antibody significantly
reduced brain edema and infarct size caused by reperfusion after ischemia in comparison
to rabbits receiving a control antibody.
In patients suffering from pyogenic meningitis, there is a significant elevation of
levels for IL-8, GRO-a, MCP-1, and MIP-ip in the CSF in comparison with
noninflammatory CSF of control individuals (Spanaus et al., 1997). The CSF of these
patients was chemotactic for neutrophils and monocytes. A number of chemokine genes
including CRG-2/IP-10, RANTES, MCP-1, MIP-lp, MCP-3, lymphotactin, CIO, MIP-2
and MIP-1 a were found to be induced in the brains of mice with lymphocytic
choriomeningitis (Asensio and Campbell, 1997). In a study using an osmotic stimulation
model, Koike et al. (1997) found CINC gene expression in the rat hypothalamus. In an
excitotoxic injury model, MCP-1 was found to be expressed in the brain of intra-
hippocampal NMDA injected postnatal rats (Szaflarski et al., 1998).
Expression of chemokine receptors in the CNS have been reported. Horuk et al.
(1997) examined the protein expression of DARC, CXCR1 and CXCR2 in various
regions of the brain by immunohistochemical staining. Their data revealed that DARC
was expressed exclusively by Pukinje cells in the cerebellum. While CXCR1 was not
expressed in the CNS, CXCR2 was expressed at high levels by subsets of projection
neurons in diverse regions of the brain and spinal cord. CXCR2 was also found in the
neuritic portion of plaques surrounding deposits of amyloid of patients with Alzheimers


CHAPTER 6
CONCLUSIONS AND FUTURE
DIRECTIONS
Since the discovery of chemokine receptors functioning as HIV entry cofactors,
there has been growing interest in studies of the expression and function of chemokine
peptides and receptors. Chemokine peptides are primarily found to be involved in
leukocyte recruitment and activation during inflammatory processes. Recently, these
molecules have been shown to participate in such diverse processes as development,
hematopoiesis, angiogenesis, lymphocyte homing and trafficking, and apoptosis.
Studies carried out in this dissertation were aimed at characterizing the expression
of chemokine receptors in the central nervous system (CNS) with the goal to begin to
understand the functional role(s) these molecules perform during neurophathological as
well as neurophysiological conditions.
In the first part of this study, we isolated two closely related rat CC chemokine
receptor genes, CCR2 and CCR5, using sequence homology screening techniques. The
identities of these two receptors were determined based on their high amino acid
sequence identity to the human orthologs and the functional responses of receptor
expressing cells to their specific ligands. While both CCR2 and CCR5 are expressed in a
number of tissues in the periphery, CCR5 is expressed constitutively in the brain and
cultured microglia. The steady state expression of CCR5 is up regulated after the
treatment of the cultured microglial cells with IFN-y. An animal model, Experimental
97


73
Dr. Lili Fengs group collected similar data demonstrating that neurons are the source of
fractalkine in cortex, hippocampus and thalamus of adult rat (data not shown). The
interaction between fractalkine and its receptor CX3CR1 may be an important mechanism
by which neurons communicate with microglia and elicit their functional roles during
facial motor neuron regeneration.
The present study did not define the specific form(s) of rat fractalkine that was
expressed in FMN. Our functional analysis shown in figure 4-2 indicated that the
response of CX3CR1 expressing cells to fractalkine various based upon the different
forms of the peptide (membrane vs. soluble). Membrane-anchored forms of fractalkine
activated a greater calcium mobilizing response compare to soluble forms. Previous
studies (Imai et al., 1997) demonstrated that a soluble form of fractalkine stimulates
chemotaxis while a membrane anchored form promotes adhesion. Thus, it is conceivable
that the larger calcium response triggered by membrane anchored form is related to cell
adhesion whereas the minor response stimulated by soluble form results in chemotaxis.
Alternatively, these forms of fractalkine may serve other functions for the CX3CR1
expressing cells than chemotaxis or adhesion. Analogous to the receptor-transfected
CHO cell model, studies carried out by our collaborator Dr. Kevin Bacons group
demonstrated that differential stimulation of microglia intracellular calcium mobilization
by fractalkine was dependent on the form present (data not shown). Membrane-anchored
forms of fractalkine stimulated robust changes in intracellular calcium levels while a
soluble form of fractalkine had no effect. Our speculation based upon these data is that
potentially, fractalkine-expressing neurons may function in regulating CX3CR1
expressing microglial activity by providing different forms of fractalkine to the


89
Discussion
Towards our goal for determining expression of fractalkine and CX3CR1 in the
rat glioma models, my data demonstrated that neither C6 nor RG2 gliomas express rat
fractalkine mRNA in vivo. While C6 gliomas express relatively high levels of rat
CX3CR1 mRNA, there is no detectable expression of rat CX3CR1 in RG2 gliomas by
both Northern blot and in situ hybridization analysis. Infiltrating microglia are the
primary sources of rat CX3CR1 mRNA in C6 gliomas.
In another study collaborating with Sharon Walters from Dr. Wolfgang J. Streits
laboratory, we found that RG2 gliomas, as compared to C6 gliomas, express very high
levels of TGF-P mRNA. However, our in vitro study shown in chapter four (Figure 4-6)
demonstrated that the expression of rat CX3CR1 mRNA in cultured microglia is up-
regulated in a dose dependent manner by TGF-P treatment. The paradox became
apparent: since microglia are the primary sources of CX3CR1 and RG2 gliomas express
very high levels of TGF-P, one would predict that RG2 gliomas are abundant sources of
ratCX3CRl mRNA. Hoever, our data indicate the opposite. Besides the argument that
in vitro systems dont necessarily always represent what happens in vivo, we also looked
into the alternative explanations. Combination of lectin (GSA I-B4) staining with
TUNEL staining using RG2 as well as G6 glioma tissues allows us to examine the
survival state of the infiltrating microglia in these two gliomas. In RG2 gliomas, lectin
staining and TUNEL staining positive cells form a very similar patternclustered
together to form isolated islands, many of which have comparable shapes; cell
morphology of lectin positive cells resembles TUNEL positive cells with many pyknotic


94
Figure 5-3. Expression of Rat CX3CR1 in Infiltrating Microglia in C6 Gliomas
After exposure to emulsion film for 3-4 weeks, sections containing RG2 or C6 gliomas
were developed and counter stained with cresyl violet. C6 glioma sections were probed
with antisense (A) or sense (B) CX3CR1 riboprobe.


38
1000 x g for 10 min and the resulting pellet washed with 50 ml DMEM containing 10%
FBS. Cells were resuspended in DMEM containing 10% FBS to a density of 1 x 103
cells/ml and 10 ml of this suspension were added to 100 mm poly-L-lysine precoated
culture dishes and incubated at 37C in a humidified incubator with 5% C02/95% air.
On day 3, the culture medium was replaced with fresh DMEM containing 10% FBS.
After 11 aditional days of incubation of the mixed primary astroglial/microglial cultures,
the flasks were rotary-shaken for one hrs (100 rpm, 37C) and the culture media, which
contained dislodged free-floating microglia, was removed and centrifuged at 1000 x g for
15 min. The resulting microglial pellet was resuspended in DMEM and plated onto 100
mm dishes at a density of 5 x 106 cells per dish. Microglia were cultured for
approximately 24 48 hours prior to treatment.
Induction of Rat EAE:
Male Lewis rats (-200 250 g) were injected in the hind foot pad, under
methoxyflurane anesthesia, with an emulsion (0.1 ml) of 125 pg purified guinea pig
myelin basic protein (MBP) in Complete Freunds Adjuvant (CFA. Difco) supplemented
with 50 pg Mycobacterium tuberculosis (H37Ra). Clinical disease, i.e. various degrees
of paraparesis, was typically evident between days 10 and 12 post injection. Animals
(EAE and control) were screened for neurological signs on a daily basis according to the
following scoring system: 0: no signs; 1: flaccid tail, general depression of activity; 2:
mild paraparesis, unsteady gait; 3: moderate paraparesis, legs splayed apart but voluntary
movements of affected extremities possible; 4: complete paraparesis, extremities without
voluntary movements; 5: death due to EAE. If the clinical sign remained in one stage for
2-3 days or reached stage 4, the rat was perfused intracardially with PBS and the lumbar


47
functions of chemokines and chemokine receptors have been extensively studied in the
periphery, less information, regarding these genes, is available in the central nervous
system.
Microglia, as the immune responsive cells in the CNS, comprise 5-20% of the
total glial cell population (Graeber et al., 1990, Kreutzberg 1996). These cells react to
even minor pathological changes in the CNS and serve as a key factor in the defense of
the neural parenchyma against infectious agents, inflammation, trauma, ischemia, brain
tumors and neurodegeneration. Several chemokines and chemokine receptors have been
shown to be expressed in microglia including MCP-1, MOM a, MIP-lp (McManus et al.,
1998), RANTES (Miyagishi et al., 1997), SDF-P (Ohtani et al., 1998), IP-10 (Ren et al.,
1998), CXCR4 (Lavi et al., 1997), CCR3, and CCR5 (Xia et al., 1998).
Microglia activation and proliferation occurs in experimental allergic
encephalomyelitis (EAE). EAE is a cell-mediated autoimmune disease of the central
nervous system induced by a number of different antigen preparations including myelin
basic protein. Its histopathology is characterized by perivascular mononuclear cell
infiltration and microglia and astrocyte activation. Clinical signs of EAE consist of
hindlimb and tail paresis as well as paralysis. Since it has many clinical and pathological
features in common with multiple sclerosis, it is considered to be an animal model of this
disease. Several groups have demonstrated that a number of chemokine peptide were
expressed or up regulated in the spinal cords of EAE animals. These chemokines include
RANTES, MIP-la, MIP-113, TCA3, IP-10, MCP-1, KC/GRO, MCP-3, SDF, and MIP-2
(see chapter one).


103
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This dissertation was submitted to the Graduate Faculty of the College of
Medicine and to the Graduate School and was accepted as partial fulfillment of the
requirements for the degree of Doctor of Philosophy.
December 1998
Dean, College of Medicine


70
Expression and Regulation of Rat CX3CR1 in vivo in the Facial Motor Nucleus After
Facial Nerve Transection
The facial motor neuron regeneration model was used to determine the
localization and regulation of expression of CX3CR1 in rat CNS. In situ hybridization
analysis was used to assess the expression of CX3CR1 in the facial motor nucleus over a
21 day period during which the facial motor neurons undergo regeneration. At time
points of 4, 7, and 14 days post-axotomy, the levels of CX3CR1 hybridizing mRNA were
significantly elevated in the injured facial motor nucleus (Figure 4-7), as compared to the
contralateral nucleus. Highest levels of CX3CR1 mRNA were days 4 and 7 post-
axotomy. At the 21 day time point CX3CR1 mRNA had returned to a level comparable
to that detected in the contralateral nucleus. The graph depicts quantitative analysis of rat
CX3CR1 mRNA hybridizing signal in the FMN of control and lesioned side.
The widespread distribution of rat CX3CR1 in normal rat brain (Harrison et al.,
1994) and its presence on cultured microglia indicate that the CX3CR1 gene might also
be expressed on these cells in vivo. To determine the expression site(s) of rat CX3CR1 in
the CNS, in situ hybridization analysis was combined with histochemical staining using
sections of brain stem containing FMN four days after facial nerve transection. Lectin
(from Griffonia simplicifolia, GSA-I-B4) staining was used to identify microglia cells
whereas anti-GFAP (glial fibrillary acidic protein) antibody was applied to identify
astrocytes. Figure 4-8 demonstrates the colocalization of rat CX3CR1 mRNA
hybridizing signals (silver grains) with lectin staining positive microglia but not anti-
GFAP positive astrocytes (data not shown) in the FMN before and after axotomy. This
data confirmed that rat CX3CR1 is expressed principally by microglia in vivo.


124
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108
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43
minutes, sections were treated with horseradish peroxidase avidin D for 45-60 minutes
and rinsed with PBS before exposed to 3,3 -diaminobenzidine-fBCb.
Sections were soaked in PBS for 5 minutes and allowed to air dry. The slides
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successful, the sections were exposed to Kodak NTB2 autoradiographic emulsion at 4C
for a period of time depending on the intensity of the signals on the films generally
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water for 5 minutes, and fixed for 5 minutes in fixer. After washing in tap water, the
slides were counterstained in cresyl violet or hematoxylin, dehydrated in ascending
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Facial Nerve Transections
Facial motor nerve transections on methoxyflurane-anesthetized male Sprague-
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(Streit et al., 1988). In brief, Male Sprague-Dawley rats (175-200 g) were anesthetized
with methoxyflurane. The right facial nerve is exposed and transected at the level of the
stylomastoid foramen. The unoperated contralateral side serves as the control. Following
1, 4, 7, 14, and 21 days after axotomy, rats (n = 3 per time point) were sacrificed with an
overdose of sodium pentobarbital (75 mg/kg) and perfused transcardially with lx PBS
(pH 7.0) followed by 4% paraformaldehyde/0.1 M P04 buffer (pH 7.4). Brains were
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(chilled to -80oC before use), and then stored at -80C. At a later time, the brain slices


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55
The expression of chemokine receptor mRNA in the brain and spinal cord as well
as cultured microglia indicates that these molecules may play some functional roles in the
CNS under neurophathological and neurophysiological conditions. It is known that a
number of chemokine peptide genes are induced in EAE, including MCP-1, MCP-3,
MIP-la, MIP-1B, RANTES, TCA3, KC/CINC, and IP-10 (Hulkower et al., 1993;
Ransohoff et al., 1993; Godiska et al., 1995; Miyagashi et al., 1995; Berman et al., 1996;
Glabinski et al., 1997). For some of these peptides, astrocytes have been identified as the
cellular source (Ransohoff et al., 1993; Berman et al., 1996; Glabinski et al., 1997), while
for others, infiltrating leukocytes were determined to be the source (Glabinski et al.,
1997). Our data demonstrated the presence of receptors of some of these chemokine
peptides in the CNS, particularly in spinal cord of EAE rats. These data suggest a
potential interaction between these chemokine peptides and their receptors in this
neuropathological model of human multiple sclerosis which may play some functional
roles during the disease process. Our identification of the rat genes encoding chemokine
receptors CCR2 and CCR5 should enhance fundamental studies of these receptors in
normal and pathological states of the CNS.


79
rrM)wU*#'
|W
Vlll
III
1 min.
Vl
¡mM{**(*
Figure 4-5. Calcium Mobilization Response to Different Forms of Fractalkine by CHO
Cells Expressing Rat CX3CR1
Representative traces of CHO-CX3CR1 expressing cells in response to application of
either 293T cells transiently expressing rat (i, ii) or human fractalkine (iv, v), or the
chemokine domain of rat (iii) or human (vi) fractalkine. Wild-type fractalkines (i, iv) or
mutant fractalkines (ii, v) in which arginine-arginine residues adjacent to the TM domain
were changed to serine-alanine, were expressed transiently in 293T cells. Lower traces
depict application of mock transfected 293T cells (vii) to CHO-CX3CR1-expressing cells
or addition of the chemokine domain of rat fractalkine (viii), or the wild-type membrane-
tethered form of rat fractalkine (ix) to wild-type CHO cells. The graph depicts
concentration-dependent effect of the chemokine domain of human fractalkine on
intracellular calcium levels in CHO-CX3CR1-expressing cells. Data are plotted as the
fraction of the maximal response seen in the presence of a high dose of fractalkine.
Values are the mean of at least two determinations at each concentration. The calculated
EC50 from these experiments is 3 nM.


CHAPTER 1
INTRODUCTION
Chemokines (Chemoattractive cytokines) are a fast growing family of more than
40 structurally related proteins that are responsible for leukocyte chemoattraction and
activation during inflammatory processes (Luster 1998, Baggiolini et al., 1994, Baggiolini
et al., 1997). These proteins can be divided into at least four subfamilies based primarily
on the relative position of four highly conserved cysteine residues in their amino acid
sequences. Receptors for some of the chemokine peptides have been identified by
molecular cloning techniques. They are members of the G-protein coupled receptor
superfamily. Chemokine receptors can induce a number of cellular signaling molecules
after being stimulated by their ligands. While the expression and function of chemokines
and their receptors have been widely explored in peripheral systems, there is very limited
information regarding the expression and function of these molecules in the central
nervous system (CNS). Studies carried out in this dissertation have focused on
characterizing the expression and regulation of chemokine receptors in the rat central
nervous system (CNS).
Chemokine: Identification, Expression, and Function
Chemokines are a group of proteins that are involved in leukocyte recruitment
during inflammation. To date, this family consists of more than 40 members that can be
divided into four subfamilies according to the arrangement of highly conserved cysteine
1


61
Figure 3-5. Rat CCR2 RNA Tissue Distribution in Rat Solid Organs and Cells
Two (ig of total RNA from rat tissues or cells were subjected to RNase protection assay
(see chapter two). The RNAs were hybridized simultaneously with 32P-radiolabeled 365-
base pair rat CCR2 and 93-base pair ribosomal RNA (L32) riboprobes. The dried gel was
exposed to Kodak X-RP film at -80 C for 72 hours.


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Identification, cloning and characterization of an immune activation gene. Proc. Natl.
Acad. Sci. USA 85:9704-9708.
Lipovsky, M.M., Gekker, G., Hu, S., Ehrlich, L.C., Hoepelman, A.I., and
Peterson, P.K. (1998) Cryptococcal glucuronoxylomannan induces interleukin (IL)-8
production by human microglia but inhibits neutrophil migration toward IL-8. J. Infect.
Dis. 177:260-263.


6
The expression or induction of expression of chemokines is found in a variety of
tissues during inflammation (Luster 1998). For instance, the concentration of H-8 and
ENA-78 were increased in bronchoalveolar during bacterial pneumonia (Chollet-Martin
et al., 1993, Huffnagle, 1996); MCP-1, RANTES, IP-10 mRNA were found during
glomerulonephritis (Tang, et al., 1995); MCP-1 and MCP-4 were seen in the plaque of
atherosclerosis (Nelken et al., 1991. Berkhout et al., 1997); MCP-1. MIP-la, eotaxin, IP-
10, LL-8 were found to be markedly increased in inflammatory bowel disease (Garcia-
Zepeda et al., 1996b, Grimm and Doe, 1996, Reinecker et al., 1995); RANTES, MIP-ip,
IL-8, ENA-78 (epithelial derived neutrophil attractant) were detected in rheumatoid
arthritis (Robinson et al., 1995, Koch et al., 1994). The appearance of different groups of
chemokines in different inflammatory diseases where there is a subset of leukocyte
infiltration suggests that in vivo, chemokines have the capacity to control precisely the
type of leukocyte infiltration.
There is a group of newly identified CC chemokines, including ELC (EBI1-ligand
chemokine, Yoshida et al., 1997), SLC (secondary lymphoid-tissue chemokine, Nagira et
al., 1997), TARC (thymus and activation-regulated chemokine, Imai et al., 1996), TECK
(thymus-expressed chemokine, Vicari et al., 1997) and DC-CK1 (dendritic cell-derived
chemokine 1, Adema et al., 1997), that are expressed constitutively in lymphoid tissues.
All of them are found to be chemotactic for lymphocytes, including T cells, B cells or
naive lymphocytes, but not for monocytes or neutrophils. The unique properties of these
peptides suggest that they may constitute a new group of CC chemokines that are
responsible for trafficking and homing of lymphocytes to particular lymphoid tissues, as
part of their immune surveillance function.



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109
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58
Figure 3-2. Calcium Mobilization Response to MCP-1 By HEK293 Cells Expressing Rat
CCR2.
HEK293 cells stably expressing rat CCR2 were prepared as outlined in chapter two.
Transfected cells were loaded with FURA-2A/M. FURA-2 fluorescence was monitored
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depicts the dose-dependent stimulation of intracellular calcium mobilization by MCP-1 in
cells expressing rat CCR2.


85
non-small cell lung cancer tissues. Immunohistochemical studies revealed that neoplastic
cells are the principal sources of these peptides (Yatsunami et al., 1998. and Arenberg et
al., 1996). Multiple studies carried out by a number of groups demonstrated that IL-8
promotes tumorigenesis because of its angiogenic function whereas IP 10 has tumor
growth inhibitory function based primarily on its angiostatic activity (reviewed by
Arenberg et al., 1997). There is evidence to support the speculation that the ELR motif in
the N-terminus of some of the CXC chemokine peptides plays an important role in the
angiogenic activity of these peptides and a lack of a ELR motif in other CXC chemokine
peptides confers their angiostatic activity. The mechanism of these phenomena remains
to be elucidated.
There is very limited information regarding the expression of chemokines and
their receptors in brain tumor tissue. Studies from several different groups have shown
that several astrocytoma derived cell lines express MCP-1 either constitutively or after
induction with EL-1 beta and TNF alpha (Kasahara et al., 1991). MCP-1 mRNA and
protein expression has also been demonstrated in vivo from human glioma tumor cells,
endothelial cells and possibly infiltrating macrophages (Takeshima et al., 1994). The
evidence that MCP-1 expression correlated with the degree of macrophage infiltration
into the tumor (Takeshima et al., 1994) and that MCP-1 containing cyst fluids derived
from brain tumors exhibited chemotactic activity for macrophages in vitro (Desbaillets et
al., 1994) suggests that MCP-1 is biologically active in the tumors in vivo. Another study
carried out by Barnes et al. (1996) demonstrated that astrocytoma cells produced
RANTES when stimulated with TNF-a or IL-ip.


LIST OF TABLES
Table page
1-1. Ligand Specificity of Chemokine Receptors 30
3-1. Amino Acid Identities of the Rat, Murine and Human Chemokine Receptors,
CCR2 and CCR5 57
vm


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90
nuclei. These data support my hypothesis that in RG2 tumors, most infiltrating microglia
are going through apoptosis. On the other hand, in C6 gliomas, microglial cells are
evenly distributed through out the tumors that have very little apoptotic cells. Taking
together, these evidences suggest that infiltrating microglia are stimulated to go through
apoptosis in RG2 gliomas; these apoptotic cells no longer capable of expressing
CX3CR1. However, infiltrating microglia in C6 gliomas do not go through apoptosis and
thus, are capable of expressing CX3CR1.
Based on the results of our in situ hybridization analysis that TGF-P mRNA
expression in RG2 tumors is substantially higher than C6 tumors, we hypothesize that
TGF-P, may play a very important role in mediating microglial apoptosis in RG2
gliomas. However, previous studies have given us controversial results. Data from Xiao
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compared to the control group. A similar study performed by Jochen Gehrmann (1995)
indicated that TGF-P did not have such an effect on cultured microglia. Some
preliminary results from my collaborator Sharon Walters did not show any significant
increase of microglial cell death after TGF-P treatment. Further studies remain to be
done to clarify this issue. On the other hand, since CX3CR1 expression of cultured
microglia is elevated as a result of TGF-P treatment, fractalkine/CX3CRl could
potentially also play a role in mediating microglial apoptosis.
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of neutrophil chemoattractant including IL-8 and CINC (Yamanaka et al., 1994,


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64
L32
cu
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o
A#
^ // .b ^ ^ ^
>> V
O b b-
£ 00V V V < 5- £ O 0 V V V *
w c
CCR2 CCR5
Figure 3-8. Expression of CCR2, CCR5, CXCR4, and RBS11 mRNAs in Lumbar Spinal
Cords of EAE Rats as Detected by RNase Protection Assay or Northern Blot Analysis
EAE was induced in Lewis rats and animals were scored for clinical symptoms as
described in chapter two. Total RNAs from control and EAE rat spinal cords (5 pg for
RNase protection assay and 10 pg per lane for Northern blot analysis) were subjected to
RNase protection assay (upper panel) and Northern blot analysis (lower panels) as
described in chapter 2. Exposure time for the RNase protection assay gel was 18 hours
(at -80 C). Exposure times for the Northern blots were 18 hours (at -80 C) for the
RBS11 and cyclophilin hybridized membranes, and 7 days (at -80 C) for the CXCR4
hybridized membrane.


100
development of therapeutic agents that target disease processes involving chemokines and
chemokine receptors.


33
Mycobacterium tuberculosis (H37Ra) was from Difco (Detroit, MI). Male Lewis rats
(200-250 g) and male Sprague Dawley rats (170-200) were obtained from Harlan
(Indianapolis, IN). Murine MCP-1, murine MIP-la, murine MIP-ip, human RANTES,
rat fractalkine, human TGF-P were from R&D system (Minneapolis, MN). NTB2
autoradiographic emulsion D19 developer and fixer were from Kodak (Rochester, NY).
TdT transferase and biotin-16-dUTP were from Boehringer Mannheim (Indianapolis, IN).
An 829-base pair cDNA containing sequences encoding rat cyclophilin was kindly
provided by Dr. A. John Maclennan (University of Florida, Gainesville, FL). Rat
genomic DNA libraries, Haelll partial digest (lambda Charon 4A) was a kind gift of Dr.
Kevin R. Lynch (University of Virginia, Charlottesville, VA).
Methods
Polymerase Chain Reaction (PCR) Amplification of Novel DNA Sequences
Two degenerate oligonucleotides 5-TAC CTS CTS AAC CTG GCC HTL JSY
GA-3 and 5-ATD AQR GGP TTP ALP CAG CHG TG-3 (where Y=T or C, S=G or C,
P=A or G, R=T or G, H=A/T, L=C/T/G, Q=T/C/A, J=A/T/G and D=A/G/C) were
synthesized and used as primers for the amplification of DNA fragments (using rat
genomic DNA as the template) encoding novel rat GCRs. The amino acid sequences
encoded by these oligonucleotides correspond to highly conserved regions of G-protein
coupled receptors (and specifically chemokine receptors). The conditions for the
amplification of DNA in the polymerase chain reaction (PCR) were: 94 C (1 minute),
45C (2 minutes), and 72C (3 minutes), 30 cycles. PCR products were subcloned to the


4-5. Calcium Mobilization Response to Different Forms of Fractalkine by CHO Cells
Expressing Rat CX3CR1 79
4-6. Dose-Dependent Increases in Rat CX3CR1 mRNA by Transforming Growth
Factor-P (TGF-P) Treated Microglia 80
4-7. In Situ Hybridization Analysis of CX3CR1 Expression in the Rat Facial Nucleus
After Motor Neuron Axotomy 81
4-8. Colocalization of Rat CX3CR1 mRNA With Microglial Cells 82
4-9: Fractalkine Expression in the Rat Facial Nucleus After Motor Neuron Axotomy 83
5-1. Expression of Rat CX3CR1 in C6 GliomaNorthern Blot Analysis 92
5-2. Expression of Rat CX3CR1 in C6 GliomaIn Situ Hybridization Analysis 93
5-3. Expression of Rat CX3CR1 in Infiltrating Microglia in C6 Gliomas 94
5-4. Lectin (GSA I-B4) Staining of RG2 (A, B) and C6 (C, D) Sections 95
5-5. TUNEL (Terminal Transferase dUTP Nick End-Labeling) Analysis in RG2 and
C6 Gliomas 96
x


106
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Receptors. J. Biol. Chem. 271:32770-32776.


CHAPTER 4
ROLE FOR NEURONALLY-DERIVED FRACTALKINE IN MEDIATING
INTERACTIONS BETWEEN NEURONS AND CX3CR1-EXPRESSING
MICROGLIAAN IN VIVO STUDY IN THE FACIAL MOTOR NEURON
REGENERATION PARADIGM
Introduction
Among the four subfamilies of chemokine peptides, the newly identified
chemokine termed fractalkine/neurotactin (Bazan et al., 1997 and Pan et al., 1997) has
some very unique properties. Its full-length amino acid sequence is comprised of five
regions: a signal peptide, a chemokine domain, a highly glycosylated mucin-like stalk
which tethers the chemokine domain with a transmembrane domain and a short
intracellular region. There are three amino acids in between the first two of the four
highly conserved cysteine residues in its chemokine domain which makes it the first
member of the CX3C chemokine subfamily. This peptide can exist as both secreted
soluble and membrane anchored forms. The soluble form is chemotactic for monocytes
and lymphocytes while the membrane anchored form can promote a strong adhesion of
these leukocytes to endothelial cells. Unlike most chemokine peptides, fractalkine is
expressed in non-hematopoietic tissues including brain, kidney, lung, heart and adrenal
gland.
An orphan rat G-protein coupled receptor termed RBS11 has a tissue
distribution pattern that is very similar to fractalkine (Harrison et al., 1994). It is widely
expressed in the adult rat. Although its amino acid sequence is most similar to other
65


TABLE OF CONTENTS
page
ACKNOWLEDGMENTS iii
LIST OF TABLES viii
LIST OF FIGURES ix
ABSTRACT xi
CHAPTERS
1. INTRODUCTION 1
Chemokine: Identification, Expression, and Function 1
Identification of Chemokine Peptides 2
Production, Expression, and Functions of Chemokines 4
Molecular Cloning and Expression of Chemokine Receptors 7
Molecular Cloning of CXC Chemokine Receptors 7
Molecular Cloning of CC Chemokine Receptors 9
Cloning of CX3CR1, XCR1 12
Signal Transduction Properties of Chemokine Receptors 13
Expression of Chemokines and Chemokine Receptors in the Central
Nervous System 15
Chemokines and Receptors Expression in EAE and Other Models for
MS 15
The Expression of Chemokines and Their Receptors in MS Patients 19
Chemokines and Receptors in Other Neuropathologies 20
Microgliathe Brain Immune Responsive Cells 23
Chemokines, Chemokine Receptors and HIV 24
Some Chemokine Receptors are HTV Entry Cofactors 24
Inhibition of HTV Entry by Chemokine Ligands 26
Chemokine Receptors and HIV in the CNS 27
Specific Aims 28
2. MATERIALS AND METHODS 32
Materials 32
Methods 33
v


14
CXCR2 can couple to many G-protein subtypes including Gi2, Gi3, G14, G15, and G16
(Wu et al., 1993). Kuang et al. also examined the selective G protein coupling by CC
chemokine receptors in cotransfected COS-7 cells (1995). Their data revealed that while
both CCR1 and CCR2a couple to G14, only CCR2a couples to G16. Neither CCR1,
CCR2a nor CCR2b couple to Gq/11. A similar study using both COS-7 and HEK-293
cell lines suggests that receptor-G-protein interactions are highly cell type specific (Arai
and Charo, 1996). The Gfiy subunits of Gi proteins have been shown to activate specific
isoforms of PLCP (Wu et al., 1993, Kuang et al. 1995) and mediate chemotaxis (Neptune
and Bourne, 1997 and Arai et al., 1997). When human neutrophils are activated by IL-8,
but not GRO-a or NAP-2, there is a stimulation in phospholipase D activity in these cells
which is accompanied by the increase of respiratory burst (LHeureux et al., 1995, Jones
et al., 1996). RANTES has also been shown to be able to stimulate phospholipase D
activity in Jurkat cells (Bacon et al., 1998). The inhibition of cAMP (Shyamala et al.,
1997), induction of arachidonic release (Locati et al., 1994) and stimulation of inositol
phosphate formation (Arai et al., 1996) have been well documented in chemokine
receptor transfected cell systems when exposed to the chemokine peptides. Bacon et al.
have demonstrated that the induction of mobilization of Ca2+ in T cells by RANTES is
mediated by both a G protein coupled pathway and activation of protein tyrosine kinases
(1995). Other studies have also provided evidence of the activation of a number of
different tyrosine kinases when the cells are treated with chemokine peptides (Schraw and
Richmond, 1995, Bacon et al., 1996, Ganju et al., 1998). Studies from two groups
revealed that chemokines can stimulate the activation of MAP-kinases (Jones et al., 1995,
Knall et al., 1996), phosphoinositide 3-kinases (Knall et al., 1997, Turner et al., 1995),


62
Figure 3-6. Rat CCR5 mRNA Tissue Distribution in Rat Solid Organs and Cells
Two fa.g of total RNA from rat tissues or cells were subjected to RNase protection assay
(see chapter two). The RNAs were hybridized simultaneously with [l2P]-radiolabeled 230
bp rat CCR5 and 114 bp GAPDH riboprobes. The dried gel was exposed to X-ray film at
-80 C for 10 hours.


93
Figure 5-2. Expression of Rat CX3CR1 in C6 GliomaIn Situ Hybridization Analysis
In situ hybridization analysis was carried out as described in chapter two. RG2 (A-F),or
C6 (G-L) tumors are probed with antisense (A, C, E, G, I, K) or sense (B, D, F, H, J, L)
riboprobes of CX3CR1 (A. B. G, H), fractalkine (C, D, I, J) or TGF-P (E. F, K. L).


123
Uehara, T., Baba, I., and Nomura, Y. (1998) Induction of cytokine-induced
neutrophil chemoattractant in response to various stresses in rat C6 glioma cells. Brain
Res. 790:284-292.
Uguccioni, M., Mackay, C.R., Ochensberger, B., Loetscher, P., Rhis, S., LaRosa,
G.J., Rao, P., Ponath, P.D., Baggiolini, M., and Dahinden, C.A. (1997) High expression
of the chemokine receptor CCR3 in human blood basophils. Role in activation by
eotaxin, MCP-4, and other chemokines. J. Clin. Invest. 100:1137-1143.
Valkovic, T., Lucin, K., Krstulja, M., Dobi-Babic, R., and Jonjic, N. 1998
Expression of monocyte chemotactic protein-1 in human invasive ductal breast cancer.
Pathol. Res. Pract. 194:335-340.
Vanguri, P., and Farber, J.M. (1994) EFN and virus-inducible expression of an
immediate early gene, crg-2/IP-10, and a delayed gene, I-Aa, in astrocytes and microglia.
J. Immun. 152:1411-1418.
Van Meir, E.G., Ceska, M., Effenberger, F., Walz, A., Grouzmann, E.,
Desbaillets, I., Frei, K., Fontana, A., and de Tribolet, N. (1992) Interleukin-8 is produced
in neoplastic and infectious diseases of the human central nervoous system. Cancer Res.
52:4297-4305.
Vicari, A.P., Figueroa, D.J., Hedrick, J.A., Foster, J.S., Singh, K.P., Menon, S.,
Copeland, N.G., Gilbert, D.J., Jenkins, N.A., Bacon, K.B., and Zlotnik, A. (1997) TECK:
a novel CC chemokine specifically expressed by thymic dendritic cells and potentially
involved in T cell development. Immunity 7:291-301.
Walz, D.A., Wu, V.Y., de Lamo, R., Dene, H., and McCoy, L.E. (1977) Primary
stucture of human platelet factor 4. Thromb. Res. 11:893-898.
Walz, A., Peveri, P., aschauer, H., and Baggiolini, M. (1987) Purification and
amino acid sequencing of NAF, a novel neutrophil-activating factor produced by
monocytes. Biochem. Biophys. Res. Commu. 149:755-761.
Weinberg, A.D., Wyrick, G., Celnik, B., Vainiene, M., Bakke, A., Offher, H., and
Vandenbark, A.A. (1993) Lymphokine mRNA expression in the spinal cords of Lewis
rats with experimental autoimmune encephalomyelitis is associated with a host recruited
CD45 hi/CD4+ population during recovery. J. Neuroimmunol. 48:105-118.
Weller, M., and Fontana, A. (1995) The failure of current immunotherapy for
malignant glioma. Tumor-derived TGF-beta, T-cell apoptosis, and the immune privilege
of the brain. Brain Res. Brain Res. Rev. 21:128-151.


28
Specific Aims
There is great interest in characterizing the expression, regulation, and functional
roles of chemokine receptors in the CNS. Thus, the specific aims of this dissertation
addressed three major areas.
1. The molecular cloning and functional characterization of rat CCR2, CCR5, and
examination of the expression of these receptors in the CNS both in vitro (cultured
microglia) and in vivo (EAE).
2. Characterization of the expression of a chemokine:receptor pair, rat
fractalkine:CX3CRl, in the CNS. Determination of expression site(s) of these
molecules in the facial motor neuron regeneration model.
3. Examination of expression of rat fractalkine and CX3CR1 in rat glioma models (RG2
and C6 gliomas).


45
cacodylate, 1 mM cobalt chloride) for 5 minutes, the tissue sections were incubated in
TdT buffer containing 2.5 U/jal terminal transferase and 10 pmol biotinylated 16-dUTP in
a humid atmosphere for 1 hour at 37C. The reaction was stopped by transferring the
slides into 300 mM sodium chloride, 30 mM sodium citrate for 20 minutes in room
temperature. After blocking the coverslips for 30 minutes in 2% bovine serum albumin
(BSA), they were incubated with the ABC kit for 30 minutes at room temperature and
developed with 3,3-diaminobenzidine as substrate. Negative controls were carried out
by omission of terminal transferase and biotinylated UTP.


98
Allergic Encephalomyelitis (EAE), was utilized to examine the expression of these
chemokine receptors during inflammation of the CNS. My results revealed elevation of
the expression of CCR5 and CCR2 mRNA levels in lumbar spinal cords of diseased rats.
These informations could be useful in developing reagents against MS by interrupting
signaling of these chemokine receptors.
To determine expression site(s) of chemokine receptors in the CNS, a non
inflammatory CNS injury modelfacial motoneuron regeneration model was adopted.
The advantage of this model is that one can stimulate a CNS reaction with out breaking
the blood brain barrier. Thus, only endogenous cells are responsible for any changes
during the reaction. Using this model, I determined expression sites of a newly identified
chemokine/receptor pair, fractalkine and CX3CR1. In situ hybridization analysis
revealed that while neurons express fractalkine, microglia are the principal sources of
CX3CR1 mRNA. Thus, neurons could activate a microglial reaction by providing certain
forms of fractalkine whereas microglia may regulate their expression levels of CX3CR1
to elicit their neuroprotective or neurotoxic functions in responce to changes of the
microenvironment. These results suggest a potential mechanism by which neurons and
microglia communicate with each other and play their functional roles under normal or
diseased situations.
Since chemokine peptides and receptors are immune relevant molecules and
development of neoplasms generally involve a host immune responses, I further
examined the expression of fractalkine and CX3CR1 in two rat glioma models, RG2 and
C6 gliomas with the attempt to begin to understand the functional roles of these
molecules in glioma development. Fractalkine was not detectable in either RG2 or C6


15
and protein kinase B (Tilton et al., 1997) in the chemokine receptor expressing cells.
Recently, Wong et al. reported the activation of STATs (Signal Transducers and
Activators of Transcription) in T cells by RANTES and MIP-la (Wong et al., 1998).
In conclusion, different chemokine peptides use different G proteins in different
cells to stimulate different signal transduction pathways for different cellular responses.
However, more studies are needed in order to understand, more completely, the signal
transduction pathways mediated by chemokines and chemokine receptors.
Expression of Chemokines and Chemokine Receptors in the Central Nervous System
While the expression and functional roles of chemokines and their receptors have
been widely explored in the periphery, there is very limited information available
regarding the expression and function(s) of chemokines and chemokine receptors in the
CNS. Most of this information has been collected from studies on experimental allergic
encephalomyelitis (EAE), the best known laboratory animal model of human multiple
sclerosis (MS).
Chemokines and Receptors Expression in EAE and Other Models for MS
EAE is an inflammatory demyelinating disease of the central nervous system,
which has many clinical and pathological features in common with multiple sclerosis
(MS). In 1933, Rivers and his coworkers discovered that immunization of susceptible
animals with brain tissue could induce an acute or chronic encephalomyelitis. This model
has evolved considerably over the last few decades. EAE can be produced in monkeys,
mice, rats, guinea pigs and rabbits using a variety of antigen preparations, including
whole spinal cord, MBP (myelin basic protein) (Kies et al., 1960), PLP (proteolipid


119
Raport, C.J., Gosling, J., Schweickart, V.L., Gray, P.W., and Charo, I.F. (1996)
Molecular Cloning and Functional Characterization of a Novel Human CC Chemokine
Receptor (CCR5) for RANTES, MIP-la, and MIP-lp.y. Biol. Chem. 271:17161-17166.
Raport, C.J., Schweickart, V.L., Eddy Jr., R.L., Shows, T.B., and Gray, P.W.
(1995) The orphan G-protein-coupled receptor-encoding gene V28 is closely related to
genes for chemokine receptors and is expressed in lymphoid and neural tissues. Gene
163:295-299.
Reinecker, H.C., Loh, E.Y., Tingler, D.J., Mehta, A., Roombeau, J.L., and
MacDermott, R.P. (1995) Monocyte-chemoattractant protein 1 gene expression in
intestinal epithelial cells and inflammatory bowel disease mucosa. Gastroenterology
108:40-50.
Ren, L., Gourmala, N., Boddeke, H.W.G.M., and Gebicke-Haerter, P.J. (1998)
Lipopolysaccharide-induced expression of IP-10 mRNA in rat brain and in cultured rat
astrocytes and microglia. Brain Res. Mol. Brain Res. 59:256-263.
Rivers, T.M., Sprunt, D.H., and Berry, G.P. (1993) Observations on attempts to
produce acute disseminated encephalomyelitis in monkeys. J. Exp. Med. 58:39-53.
Robinson, E., Keystone, E.C., Schall, T.J., Gillett, N., and Fish, E.N. (1995)
Chemokine expression in rheumatoid arthritis (RA): evidence of RANTES and
macrophage inflammatory protein (MIP)-l beta production by synovial T cells. Clin. Exp.
Immunol. 101:398-407.
Rollins, B.J., Stier, P., Ernst, T., and Wong, G.G. (1989) The human homolog of
the JE gene encodes a monocyte secretory protein. Mol. Cell Biol. 9:4687-4695.
Rollins, B.J., Walz, A., and Baggiolini, M. (1991) Recombinant human MCP-l/JE
induces chemotaxis, calcium flux, and the respiratory burst in human monocytes. Blood
78:1112-1116.
Roos, R.S., Loetscher, M., Legler, D.F., Clark-Lewis, I., Baggiolini, M., and
Moser, B. (1997) Identification of CCR8, the receptor for the human CC chemokine I-
309.JBiol. Chem. 272:17251-17254.
Rossi, D.L., Hardiman, G., Copeland, N.G., Gilbert, D.J., Jenkins, N., Zlotnik, A.,
and Bazan, J.F. (1998) Cloning and characterization of a new type of mouse chemokine.
Genomics 47:163-170.
Rucker, J., Edinger, A.L., sharron, M., Samson, M., Lee, B., Berson, J.F., Yi, Y.,
Margulies, B., Coliman, R.G., Doranz, B.J., Parmentier, M., and Dorns, R.W. (1997)
Utilization of chemokine receptors, orphan receptors, and Herpesvirus-encoded receptors
by diverse human and simian immunodeficiency viruses. J. Virol. 71:8999-9007.


25
al.. 1996). Evidence from Choe et al. (1996) demonstrated that 1) CCR5, when expressed
along with CD4, allowed cell lines resistant to most primary HIV-1 isolates to be
infected, 2) CCR3 facilitated infection by a more restricted subset of primary virus, 3)
eotaxin, one of the ligands for CCR3, inhibited infection by these isolates, and 4) the
sequence of the third variable region of the HTV gpl20 exterior envelope glycoprotein is
the important region for HIV-chemokine receptor interaction. Doranz et al. (1996)
demonstrated that besides CCR5 and CXCR4, the receptors CCR3 and CCR2b are entry
cofactors for a dual-tropic HIV-1 isolate (89.6). Later, V28 (human CX3CR1), and
CCR8 were identified as coreceptors by certain strains of HTV-1 (Rucker et al., 1997,
Combadiere et al., 1998, Jinno et al., 1998).
The role of CCR5 as a cofactor for HIV-1 entry became unequivocal when Liu et
al. (1996) identified two individuals that had been multiply exposed to HIV-1 but
remained uninfected, had inherited a homozygous defective CCR5. The encoded
defective CCR5 cannot be detected at the cell surface. Thus, These cells are resistant to
HIV infection.
In order to determine the important regions for the fusion property of CCR5, a
number of studies using chimeras have been carried out. Rucker et al. (1996) determined
regions in CCR5 and CCR2b that set HIV-1 tropic specificity by making CCR5/CCR2b
chimeras. They found that residues 2-5 in the N-terminus of CCR5 are important in M-
tropic virus fusion whereas residues 6-9 are important for dual-tropic (89.6) virus fusion.
Atchison and colleagues (1996) demonstrated that various segments of extracellular
regions of human CCR5 are sufficient to reconstitute fusion in the human CCR5/mouse


3
isolated using heparin-sepharose chromatography. Its amino acid sequence was
determined by treatment of this peptide with different peptidases and ultimately analyzed
by protein sequencing. PF-4 is a 70-residue peptide with a highly negatively charged N-
terminal region, characteristic of most of the chemokine peptides.
Ten years after the discovery of PF4, human IL-8 was identified by three
independent groups. Walz et al. (1987) and Yoshimura et al. (1987) purified this peptide
from conditioned media of human blood monocyte cultures treated with E.coli
lipopolysaccharide (LPS). Simultaneously, Schmid and Weissman (1987) isolated an IL-
8 cDNA clone from a cDNA library of SEA (staphylococcal enterotoxin A)-treated
human peripheral blood leukocytes by the technique of differential colony hybridization
(St. John and Davis 1979). IL-8 was found to be 35% identical to PF-4 in their amino
acid sequences.
Among the CC chemokine subfamily, MIP-la (Obaru et al., 1986) and MIP-1 P
(Lipes et al., 1988) were the first to be identified. A cDNA encoding human MIP-la was
isolated from a cDNA library constructed from the mRNA of TPA (12-o-tetradecanoyl
phorbol-13-acetate) or PHA (phytoemagglutinin)-stimulated human tonsillar lymphocytes
by differential colony hybridization. MIP-1 P was isolated in a similar manner from an
activated T-cell library. MCP-1 is the most characterized CC chemokine. It was isolated
by several groups using either protein purification and sequencing or sequence homology
screening techniques (Yoshimura et al., 1989a-c, Matsushima et al., 1989, Furutani et al.,
1989, Rollins et al., 1989). In the past ten years, more than 40 CXC and CC chemokine
peptides have been identified by techniques including protein purification and
sequencing, sequence homology screening, or signal sequence trapping (Yoshie et al.,


80
Figure 4-6. Dose-Dependent Increases in Rat CX3CR1 mRNA by Transforming Growth
Factor-p (TGF-p) Treated Microglia
Northern analysis of rat CX3CR1 mRNA in primary cultures of rat microglia treated with
TGF-p. Cultured microglia were incubated for eight hours with DMEM (control) or
DMEM containing the specified concentration of TGF*p. At the end of the incubation,
total RNA was isolated from the culture dishes using the Trizol Reagent (Gibco/BRL).
Ten pg of total RNA from each group was subject to Northern analysis. Immobilized
RNA was hybridized with "P-labelled 1.1 kbp CX3CR1 cDNA fragment by the method
of Church and Gilbert. Final wash temperature was 65C. The positions of migration of
18 S and 28 S ribosomal RNA are shown. Exposure to Kodak X-AR film was for 4 days
(-80C). The lower panel depicts a subsequent hybridization of the blot with j2P-labelled
DNA encoding rat cyclophilin and was used to control for lane loading. Exposure time
for the cyclophilin hybridized membrane was 18 hours.


72
orthologs, rat fractalkine gene encodes a protein that has a chemokine domain followed
by a highly glycosylated mucin-like stalk, a transmembrane domain and a short
intracellular domain. Its mRNA can be found in some non-hematopoietic tissues
including lung, heart, kidney, and adrenal gland. However, its expression is most
abundant in the brain where it is expressed principally by neurons.
By in situ hybridization analysis, we demonstrated that the expression of CX3CR1
mRNA was transiently up regulated in the facial motor nucleus (FMN) after axotomy.
The time course of this up-regulation parallels the transient increase in microglial cell
numbers after axotomy. It is also known that, at this time point, the majority of
axotomized motoneurons are tightly ensheathed by the perineuronal microglial cells
(Kreutzberg 1996, Streit 1996). In an in vivo study, Kiefer et al. (1993) demonstrated
that TGF-P mRNA level was up regulated transiently in the lesioned FMN. It peaked
between days 2-7 and gradually comes down after that and returned to normal by three
weeks post-lesion. Our in vitro study indicated that cultured microglia constitutively
express rat CX3CR1. The expression is up regulated in a dose dependent manner after
TGF-P treatment. Collectively, it can be speculated that TGF-P may play an important
role in regulation of microglial expression of CX3CR1 in vivo as well.
Compared to the other CNS inflammatory models such as EAE, the motor neuron
regeneration model provides the advantage of confined cell population within the CNS
because of the intact blood brain barrier. This brings a great advantage in our effort to
identify the cellular localization of the genes that we are interested in. Our data
unequivocally demonstrated that in the FMN, microglia are the principal source of
CX3CR1 whereas fractalkine is primarily expressed by motor neurons. Our collaborator


68
Results
Molecular Cloning of Rat Fractalkine
A rat brain cDNA library (Strategene, CA) was screened by hybridization using a
rat factalkine cDNA probe. The hybridization probe was generated by PCR using primers
derived from a mouse cDNA (Genbank accession number R75309). The rat fractalkine
cDNA was sequenced by standard methods and conceptualized protein sequence is shown
in Figure 4-1. The conceptualized amino acid sequence derived from the cDNA predicts
a 392 amino acid protein with a molecular mass of 42,161 Da. The amino acid sequence
identities of rat fractalkine with human and murine forms are 64% and 81%, respectively.
Analogous to these human and murine proteins, rat fractalkine contains a signal peptide,
chemokine module, mucin-like stalk, transmembrane spanning region, and a short
intracellular C-terminus.
Tissue Distribution of Rat Fractalkine
In collaboration with Dr. Lili Feng (The Scripps Research Institute), we carried
out RNase protection assays in order to determine the tissue distribution of rat fractalkine.
Analogous to its human and murine orthologs, rat fractalkine is expressed in a number of
non-hematopoietic tissues including kidney, lung, heart, and adrenal gland. Among the
tissues that weve examined, brain was identified as the most abundant source of rat
fractalkine (Figure 4-2).
Distribution of Rat Fractalkine in the Brain
In situ hybridization analysis revealed that rat fractalkine is highly expressed in
discrete regions of rat brain including cortex, hippocampus, caudate putamen, thalamus,


Polymerase Chain Reaction (PCR) Amplification of Novel DNA
Sequences 33
Isolation of Rat Genomic Clones 34
Southern Blot Analysis of Rat Genomic DNA 34
Transfection of HEK293 Cells 34
Measurement of Intracellular Calcium Levels 35
Expression of Rat CCR5 in Xenopus Laevis Oocytes 36
RNA Isolation, RNase Protection Assay, and Northern Analysis 36
Preparation of Primary Cultures of Rat Microglia 37
Induction of Rat EAE 38
Molecular Cloning and Sequencing of Rat Fractalkine cDNA 39
Expression and Functional Characterization of Rat CX3CR1 in
Transfected Cells 39
In situ Hybridization Analysis 40
Lectin Staining and Immunohistochemical Analysis 42
Facial Nerve Transections 43
Tumor Inoculation 44
Terminal Transferase-Mediated UTP Nick End Labeling (TUNEL-
Labeling) 44
3. CHEMOKINE RECEPTOR EXPRESSION IN CULTURED GLIA AND RAT
EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS 46
Introduction 46
Results 48
Isolation of Rat Genomic DNA Containing Sequences Encoding Novel
G-Protein Coupled Receptors 48
Signal Transduction Properties of Rat CCR2 and CCR5 49
Genomic Analysis 50
Tissue Specific Expression of Rat CCR2 and CCR5 50
Expression and Regulation of the Rat CCR2 and CCR5 in Cultured
Microglia 51
Modulation in Rat EAE 51
Discussion 52
4. ROLE FOR NEURONALLY-DERIVED FRACTALKINE IN MEDIATING
INTERACTIONS BETWEEN NEURONS AND CX3CR1-EXPRESSING
MICROGLIAAN IN VIVO STUDY IN THE FACIAL MOTOR NEURON
REGENERATION PARADIGM 65
Introduction 65
Results68
Molecular Cloning of Rat Fractalkine 68
Tissue Distribution of Rat Fractalkine 68
Distribution of Rat Fractalkine in the Brain 68
Identification of RBS11 as the Receptor for Rat Fractalkine 69
vi


LIST OF FIGURES
Figure page
1 -1. Amino Acid Alignment of Representative Members of the Human Chemokine
Superfamily 29
1-2. Lectin (GSA I-B4) Staining of Facial Motor Nucleus Four Days After Facial
Nerve Transection 31
3-1. Sequence Comparison of Rat and Murine C-C Chemokine Receptors 56
3-2. Calcium Mobilization Response to MCP-1 By HEK293 Cells Expressing Rat
CCR2 58
3-3. Functional Responses of Cells Expressing Rat CCR5 59
3-4. Southern Blot Analysis of Restriction Endonuclease-Digested Rat Genomic
DNA 60
3-5. Rat CCR2 RNA Tissue Distribution in Rat Solid Organs and Cells 61
3-6. Rat CCR5 mRNA Tissue Distribution in Rat Solid Organs and Cells 62
3-7: Dose-Dependent Increase in Rat CCR5/2 mRNA by Interferon-y (IFNy) Treated
Microglia 63
3-8. Expression of CCR2, CCR5, CXCR4, and RBS11 mRNAs in Lumbar Spinal
Cords of EAE Rats as Detected by RNase Protection Assay or Northern Blot
Analysis 64
4-1.Amino Acid Sequence of Rat Fractalkine (Genbank accession number R75309)..75
4-2. Nase Protection Analysis of Fractalkine mRNA in Various Tissues of Adult Rat 76
4-3. Ddistribution of Rat Fractalkine mRNA in Adult Rat Brain 77
4-4. Whole Cell Radioligand Binding Analysis of CHO-CX3CR1-Expressing Cells..78
IX


ACKNOWLEDGMENTS
I would like to thank my mentor, Dr. Jeffrey K. Harrison, who was courageous
enough to let me (someone who didnt have much experience in research) to work in his
lab to build up my career in medical research science. During the past four years, he
patiently guided me in every step along the way of my research from pipetting to
scientific thinking, writing and presentation. His constant challenge prompted me to try
harder and be better which made my years in graduate school very rewarding. I would
also like to express my appreciation to present and former members of the laboratory for
providing an enjoyable working environment. A special thank goes to Mina Salafranca
for teaching me many of the techniques necessary to complete this dissertation.
I would also like to thank every member of my committee, Drs. Wolfgang J.
Streit, Thomas C. Rowe, John M. Petitto, and Joel Schiffenbauer. I am grateful for their
understanding and support. Special thanks to Dr. Streit and members of his laboratory for
generously giving me unlimited access to their knowledge, facilities and friendship.
I want to express my gratitude to all of the faculty members, secretary staffs and
fellow graduate students of the Department of Pharmacology and Therapeutics for
making my years in graduate school a truly pleasant experience. I would like to show my
appreciation to Lynn Raynor for her friendship, encouragement and enlightening
conversations, especially during difficult days of my life.
in


I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
K. Harrison, Chair
it Professor of Pharmacology and
Therapeutics
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy, j /
xA -A
Wolfgang J. Streit
Associate Professor of Neuroscience
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
lomas C. Rowe
Associate Professor of Pharmacology and
Therapeutics
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Joel Schiffenbauer
Associate Professor of Molecular
Genetics and Microbiology
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Associate Professor of Neuroscience


51
macrophages (Fig. 3-6). However, in addition, CCR5 mRNA was detected in extracts of
whole rat brain.
Expression and Regulation of the Rat CCR2 and CCR5 in Cultured Microglia
The expression and regulation of the CCR2 and CCR5 gene in the cultured rat
microglia was investigated by Northern blot analysis shown in Figure 3-7. RNA from
microglial cells treated with various concentrations of IFN-y was isolated and a Northern
blot was generated as described in chapter 2. This blot was subsequently probed with a
1.5 kbp Clal/BamHI fragment of rat CCR5. Steady state levels of CCR5 mRNA present
in the cultured microglia were dose-dependently increased by IFN-y treatment. CCR2
mRNA was not detected in primary cultures of rat microglia (either control or IFN-y-
treated. Figure 3-5).
Modulation in Rat EAE
A number of cytokines, including IFN-y and chemokine peptides, are known to be
elevated in spinal cords from EAE animals (Merrill et al., 1992; Weinberg et al., 1993;
Hulkower et al., 1993; Ransohoff et al., 1993; Godiska et al., 1995; Miyagashi et al.,
1995; Berman et al., 1996; Glabinski et al., 1997). In order to examine the expression of
the receptors for chemokine peptides in vivo, in collaboration with Dr. Wolfgang J. Streit,
we examined for the expression of chemokine receptor genes in rat EAE. EAE was
induced in Lewis rats by hind footpad injection of myelin basic protein. Total RNA from
the lumbar enlargements of the spinal cords from animals at various stages of the disease
were examined for the presence of chemokine receptor mRNAs. Figure 3-8 depicts an
analysis of multiple chemokine receptor (CCR2, CCR5, CXCR4, and RBS11) mRNA


105
Cocchi, F., DeVico, A., Garzino-Demo, A. Arya, S., Gallo, R., and Lusso, P.
(1995) Identification of RANTES, MIP-la, and Mff-ip as the major HTV-suppresive
factors produced by CD8+ T cells. Science 270:1811-1815.
Coldtz, I., Zwahlen, R., Dewald, B., and Baggiolini, M. (1989) In vivo
inflammatory activity of neutrophil-activating factor, a novel chemotactic peptide derived
from human monocytes. Am. J. Pathol. 134:755-760.
Cole, K.E., Strick, C.A., Paradis, T.J., Ogbome, K.T., Loetscher, M.., Gladue,
R.P., Lin, W., Boyd, J.G., Moser, B., Wood, D.E., Sahagan, B.G., and Neote, K. (1998)
Interferon-inducible T cell alpha chemoattractant (I-TAC): Anovel Non-ELR CXC
chemokine with potent activity on activated T cells through selective high affinity binding
to CXCR3. J. Exp. Med. 187:2009-2021.
Combadiere, C., Ahuja, S.K., and Murphy, P.M. (1995a) Cloning and functional
expression of a human eosinophil CC chemokine receptor. J. Biol. Chem. 270:16491-
16494.
Combadiere, C., Ahuja, S.K., and Murphy, P.M. (1995c) Cloning, chromosomal
localization, and RNA expression of a human (3 chemokine receptor-like gene. DNA Cell
Biol. 14:673-680.
Combadiere, C., Ahuja, S.K., Tiffany, H.L., and Murphy, P.M. (1996) Cloning
and functional expression of CC CKR5, a human monocyte CC chemokine receptor
selective for MIP-la, Mip-ip, and RANTES. J. Leukoc. Biol. 60:147-152.
Combadiere, C., Ahuja, S.K., Van Damme, J., Tiffany, H.L., Gao, J.L., and
Murphy, P.M. (1995b) Monocyte chemoattractant protein-3 is a functional ligand for CC
chemokine receptors 1 and 2B. J. Biol. Chem. 270:29671-29675.
Combadiere C., Salzwedel K., Smith E.D., Tiffany H.L., Berger E.A., and Murphy
P.M. (1998) Identification of CX3CR1. A chemotactic receptor for the human cx3c
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186:1793-1798.


4
1997). These peptides are generally 90-99 amino acids long with a 20-25 amino-acid
signal sequence.
The only member of the C chemokine subfamily, lymphotactin, was isolated from
a complementary DNA library generated from activated mouse pro-T cells (Kelner et al.,
1994). Its gene is located on human chromosome one. While Kelner reported its
chemotactic activity for lymphocytes, Yoshida et al, (1998) experienced difficulties in
reproducing these results. The functional activity of this peptide has yet to be determined.
Fractalkine, a newly identified chemokine, is the first member of the CX3C
chemokine subfamily (Bazan et al., 1997, Pan et al., 1997, Rossi et al., 1998) and has
some very unique properties. The full-length amino acid sequence is comprised of five
regions: a signal peptide, a chemokine domain, a highly glycosylated mucin-like stalk
which tethers the chemokine domain with a transmembrane domain and a short
intracellular region. There are three amino acids in between the first two of the four
highly conserved cysteine residues in its chemokine domain. This peptide can exist as
both secreted soluble and membrane anchored forms. The soluble form is chemotactic
for monocytes and lymphocytes while the membrane anchored form can promote a strong
adhesion of these leukocytes to endothelial cells. Unlike most chemokine peptides,
fractalkine is expressed in non-hematopoietic tissues including brain, kidney, lung, heart
and adrenal gland.
Production, Expression, and Functions of Chemokines
A wide variety of cells can produce chemokine peptides including
monocytes/macrophages, T cells, B cells, fibroblasts, endothelial cells, epithelial cells,
keratinocytes, platelets, neutrophils, smooth muscle cells and a number of tumor cell


2
residues in their amino acid sequences (Figure 1-1). By far, CC and CXC chemokine
subfamilies are the largest among these four subfamilies. Members of both subfamilies
have four highly conserved cysteine residues. The first cysteine forms a disulfide bond
with cysteine 3 and the second cysteine is linked to cysteine 4 by a disulfide bridge. In
the CXC chemokine subfamily, the first two cysteines are separated by one amino acid
whereas in CC chemokines, the first two cysteines are adjacent to each other. Many CXC
chemokines are chemotactic for neutrophils while most CC chemokines have chemotactic
activity toward monocytes, macrophages, lymphocytes, eosinophils and basophils.
Representative members of CXC chemokine subfamily are IL-8 (interleukin 8),
PF-4 (platelet factor 4), GRO (growth-regulated oncogene) -a, *p, -y, IP-10 (interferon-
inducible protein 10) and SDF-1 (stromal cell-derived factor 1). Representative members
of the CC chemokine subfamily include the MCPs (monocyte chemoattractant protein-1,
-2, -3, -4, -5), the MIPs (macrophage inflammatory protein-la, lp, and -3), RANTES
(regulated upon activation normal T cell expressed and secreted) and 1-309.
Lymphotactin is the only chemokine, identified to date, that has only two
cysteines in its amino acid sequence. It is chemotactic for lymphocytes and is designated
as the first member of C chemokine subfamily (Kelner et al., 1994). Recently, a novel
chemokine peptide, termed fractalkine, has been characterized that has three amino acids
between the first two cysteine residues. It is designated as the first member of CX3C
chemokine subfamily (Bazan et ah, 1997, Pan et ah, 1997).
Identification of Chemokine Peptides
The first identified chemokine peptide was PF-4 (Deuel et ah, 1977, Hermodson
et ah, 1977, Walz et ah, 1977). Because of its high affinity for heparin, this peptide was


50
did not show a detectable change in [Ca2"]¡ in the presence of either murine MCP-1 or
MIP-la. Electrophysiological experiments were carried out to examine the calcium
mobilization of CCR5 expressing Xenopus laevis oocytes in response to ligand
application. Figure 3-3 demonstrates traces of Ca'-activated C1 current recorded from
Xenopus laevis oocytes microinjected with CCR5 (c)RNA and exposed to MEP-la or
MIP-1B. No response can be detected when the cells were exposed to the buffer only
(data not shown).
Genomic Analysis
Southern blot analysis of restriction-digested rat genomic DNA probed with
radiolabeled DNA encoding rat CCR5 showed consistent hybridization to two distinct
sets of genomic fragments (Fig. 3-4). These hybridizing restriction fragments were of
identical size to the hybridizing restriction fragments from the isolated rat genomic
bacteriophage clones. These two sets of the hybridizing signals can be seen even under
more stringent wash conditions as shown in the left panel of Figure 3-4 where the blot
was washed at 65C as compared to the right panel with the wash temperature at 50C.
Tissue Specific Expression of Rat CCR2 and CCR5
The tissue and cell distribution of rat CCR2 and CCR5 mRNAs were determined
by RNase protection assay. Analysis of total RNA from solid rat organs and cells probed
with an antisense riboprobe of rat CCR2 revealed that adult rat lung, spleen, kidney,
thymus, and macrophages are sources of CCR2 (Fig. 3-5). CCR2 mRNA was not
detected in rat liver or rat brain (data not shown). Similar to that of rat CCR2, rat CCR5
mRNA was also found to be expressed in rat lung, spleen, kidney, thymus, and


34
Smal site of pGEM7Zf(+) (Promega) and subjected to DNA sequence analysis (Sanger
dideoxy chain termination, Sequenase kit).
Isolation of Rat Genomic Clones
Approximately 1 x 106 independent recombinants of a Haelll partially digested
Sprague Dawley rat genomic DNA library (lambda Charon 4A) was screened with the
:P-radiolabeled 0.7-kbp (pCrecl3) PCR products. Fifteen pCrecl3 hybridizing positive
signals were plaque purified and phage DNAs purified by standard methods (Sambrook et
al., 1989). Restriction-digested recombinant rat genomic DNAs hybridizing to the
pCrecl3 DNA were subcloned to pGEM7Zf(+) and subjected to partial DNA sequence
analysis (Sanger dideoxy chain termination, Sequenase kit).
Southern Blot Analysis of Rat Genomic DNA
Rat genomic DNA (15 pg, from normal adult Wistar rat liver) was restriction
endonuclease-digested and subsequently electrophoresed through 0.85% agarose gel and
subjected to Southern blot analysis (Sambrook et al., 1989). The transferred DNAs were
hybridized to the j2P-radiolabeled 1.0 kbp Clal/BamHl fragment of the rat CCR5 gene
using the method of Church and Gilbert (1984). Final wash temperatures were 50C and
65C.
Transfection of HEK293 Cells:
The open reading frame (orf) of rat CCR2 was amplified using the PCR and the
cloned genomic DNA as the template. Two oligonucleotides were synthesized and used
in the PCR (30 cycled of 94C for 1 minute, 55C for 2 minutes, and 72C for 3 minutes).
The oligonucleotides used were 5-TTC TCT GTC CAC AGA ATC and 5-CTA CTT


Finally, I would like to thank my family, in particular, my husband, who has
always been there for me throughout our ten years of marriage, and my mother, who
taught me a principle that carries my life to a truly blessed onea principle that says it is
fine if you are not the best but it is detrimental if you dont try your best.
IV


113
Kiefer, R., Lindholm, D., and Kreutzberg, G.W. (1993) Interleukin-6 and
transforming growth factor-bl mRNAs are induced in rat facial nucleus following
motoneuron axotomy. Eur. J. Neurosci. 5:775-781.
Kies, M.W., Murphy, J.B., and Alvord, E.C. (1960) Fractionation of guinea pig
brain proteins with encephalitogenic activity. Fed. Proc. 19:207.
Kitaura, M., Nakajima, T., Imai, T., Harada, S., Combadiere, C., Tiffany, H.L.,
Murphy, P.M., and Yoshie, O. (1996) Molecular cloning of human eotaxin, an
eosinophil-selective CC chemokine, and identification of a specific eosinophil eotaxin
receptor, CC chemokine receptor 3 .J. Biol. Chem. 271:7725-7730.
Knall, C., Worthen, G.S., and Johnson, G.L. (1997) Interleukin 8-stimulated
phosphatidylinositol-3-kinase activity regulates the migration of human neutrophils
independent of extracellular signal-regulated kinase and p38 mitogen-activated protein
kinases. Proc. Natl. Acad. Sci. U.S.A. 94:3052-3057.
Knall, C., Young, S., Nick, J.A., Buhl, A.M., Worthen, G.S., and Johnson, G.L.
(1996) Interleukin-8 regulation of the Ras/Raf/mitogen-activated protein kinase pathway
in human neutrophils. J. Biol. Chem. 271:2832-2838.
Koch, A.E., Kunkel, S.L., Harlow, L.A., Mazarakis, D.D., Haines, G.K., Burdick,
M.D., Pope, R.M., Walz, A., and Stricter, R.M. (1994) Epithelial neutrophil activating
peptide-78: a novel chemotactic cytokine for neutrophils in arthritis. J. Clin. Invest.
94:1012-1018.
Koike, K., Sakamoto, Y., Kiyama, H., Masuhara, K., Miyake, A., and Inoue, M.
(1997) Cytokine-induced neutrophil chemoattractant gene expression in the rat
hypothalamus by osmotic stimulation. Mol. Brain Res. 52:326-329.
Kostulas, N., Kivisakk, P., Huang, Y., Matusevicius, D., Kostulas, V., and Link,
H. (1998) Ischemic stroke is associated with a systemic increase of blood mononuclear
cells expressing interleukin-8 mRNA. Stroke 29:462-466
Kreutzberg, G.W. (1996) Microglia: a sensor for pathological events in the CNS.
Trends Neurosci. 19:312-318.
Kuang, Y., Wu, Y., Jiang, H., and Wu, D. (1995) Selective G-protein coupling by
C-C chemokine receptors. J. Biol. Chem. 271:93975-3978.
Kuna, P., Reddigari, S.R., Rucinski, D., Oppenheim, J.J., and Kaplan, A.P. (1992)
Monocyte chemotactic and activating factor is a potent histamine-releasing factor for
human basophils. J. Exp. Med. 175:489-493.


13
Finally, there is an interesting chemokine receptor termed Duffy antigen receptor
for chemokines (DARC) which interacts with most of the CC and CXC chemokines but
does not signal (Chaudhuri et al., 1993, Horuk et al., 1993). This gene encodes a 338-
amino-acid protein that also contains seven hydrophobic transmembrane regions
conserved in all G-protein coupled receptors. Its function is likely to serve as reservoir of
chemokines in the body.
The murine and rat orthologs of many of these human chemokine receptors have
also been identified (Bozic et al., 1994, Cerretti et al., 1993, Harada et al., 1994, Heinrich
and Brano, 1995, Lee et al., 1995, Dunstan et al., 1996, Nagasawa et al., 1996, Heesen et
al., 1996, Gao and Murphy, 1996, Hoogeworf et al., 1996, Post et al., 1995, Boring et al.,
1996, Kurihara and Brano, 1996, Gao and Murphy, 1995, Meyer et al., 1996, Schweickart
et al., 1994, Zingoni et al., 1998), (Table 1). These murine and rat orthologs will be useful
in studying the expression, regulation and functional role of chemokines and chemokine
receptors in vivo under physiological and pathological conditions using a number of
murine or rat animal models of human diseases.
Signal Transduction Properties of Chemokine Receptors
Chemokines act on their receptors to stimulate a number of signal transduction
pathways to elicit their functions. Almost all receptors stimulate a transient intracellular
calcium increase upon activation. In most cases, except for MCP-2, this response can be
abolished by pertussis toxin pretreatment (Baggiolini et al., 1997, Sozzani et al., 1994)
suggesting a Gi protein involvement in their signal transduction pathways.
Complementation experiments in cotransfected COS-7 cells indicated that CXCR1 and


CHAPTER 3
CHEMOKINE RECEPTOR EXPRESSION IN CULTURED GLIA AND RAT
EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS
Introduction
Chemokines are a growing family of peptides that are involved in leukocyte
recruitment and activation. These peptides can be divided into four subfamilies (CC,
CXC. C, and CX3C subfamilies) based on the relative positions of highly conserved
cysteine residues in their amino acid sequences.
Some of the receptors for chemokines have been identified by molecular cloning
techniques and they are members of the rhodopsin superfamily of G-protein coupled
receptors (GCRs). These receptors are characterized by their seven transmembrane
spanning helices in their amino acid sequences. Although most chemokine receptors
interact with more than one chemokine peptides, in general, they can only be stimulated
by chemokines from the same subfamily. To date, the primary structures of at least
sixteen functional human chemokine receptors have been determined. Five of these
receptors bind, and are activated by, C-X-C chemokines nine receptors are activated by
C-C chemokine peptides, one receptor binds the C chemokine lymphotactin and one
receptor is bound and activated by the CX3C chemokine fractalkine. Genes encoding
chemokine receptors have also been identified in the mouse and rat. It has been
demonstrated that human CXCR4, CCR5, CCR2, CCR3, CCR8, and CX3CR1 act as
cofactors for entry into cells of primary isolates of HTV. While the expression and
46


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101


99
gliomas whereas CX3CR1 rtiRNA was found in C6 but not RG2 glioma. In situ
hybridization analysis indicated that CX3CR1 is expressed in microglia. The differential
expression of CX3CR1 in these gliomas is suggestive of a role for CX3CR1 in regulating
microglial function in responding to tumor growthin the CNS.
Future directions based on studies in this dissertation could be aimed at studying
signal transduction pathways that these chemokine receptors use to elicit their functional
roles; analyzing the regulation of expression of ffactalkine and CX3CR1 mRNA as well
as protein in vitro and in vivo; examining the regulation of expression and functional
roles of different forms of ffactalkine peptides in normal as well as disease processes;
investigating the effect of these molecules on regulating the expression of other
intracellular components.
To test whether TGF-P, ffactalkine and CX3CR1 are involved in microglia
apoptosis in these glioma models, we can treat cultured microglia with TGF-P plus or
minus ffactalkine and look for the different percentage of apoptotic cells in each group of
treatment via TUNEL labeling. More convincing evidence has to come from in vivo
systems. Studies in ffactalkine or CX3CR1 knock out animals will be very useful in
testing potential roles of these molecules in regulating microglia function.
In conclusion, among rat chemokine receptors and peptides that I examined, rat
CCR5, CX3CR1 and ffactalkine are constitutively expressed in the CNS. Expression
levels were found to be regulated under pathological circumstances. The expression and
regulation of these molecules in the CNS indicates important functional roles in
neuropathology as well as neurophysiology. These investigations could ultimately lead to


CCR5, have been isolated. While both are expressed in a number of peripheral tissues of
adult rat. CCR5 mRNA is also found in rat brain, spinal cord, and cultured microglia.
IFN-y treatment of cultured microglial upregulates CCR5 expression in a dose-dependent
manner. While CCR5 mRNA expression was elevated in EAE rats, CCR2 mRNA
becomes detectable in the spinal cord of these diseased rats.
We also studied the expression of RBS11, a chemokine receptor-like gene, which
has recently been identified as receptor for a newly characterized chemokine termed
fractalkine. Both fractalkine and RBS11 (CX3CR1) are constitutively expressed in a
wide variety of regions of the brain. In situ hybridization analysis revealed that rat
CX3CR1 is expressed primarily in microglia whereas its ligand, fractalkine, is expressed
in neurons. Interactions between this ligand and receptor may provide a mechanism by
which neurons communicate with microglia.
Lastly, we examined the expression of fractalkine and CX3CR1 in two rat glioma
models: C6 and RG2 gliomas. Although rat fractalkine was not detectable in either of the
gliomas, rat CX3CR1 mRNA was detected in C6 but not RG2 gliomas. The differential
expression of CX3CR1 in these gliomas suggests that CX3CR1 expression may be one of
the markers of the functional state of microglia in the CNS.
t
xii


91
Desbaillets et al., 1997, Uehara et al., 1998) in glioma cell lines or glioma tissues.
Polynuclear cells that have the morphology constant with neutrophils were found in my
data generated by lectin or TUNEL stainin (Figure 5-4b and 5-5b); this observation leads
to an alternative explanation of the result. It is possible that TUNEL positive islands in
RG2 tumors are necrotic foci in fast growing RG2 tumors as a result of inflammation and
oxygen deprivation within tumor tissue. High levels of H2O2 inside neutrophils
responded to HRP-congugated lectin and presented false lectin positive signals. To test
this possibility, double staining of these glioma section with neutrophil specific marker
and TUNEL labeling could help to identify infiltrating cells and/or apoptotic cells.
Taken together, this is the first study demonstrating differential expression of
ffactaikine receptor, CX3CR1, in rat glioma models. The different CX3CR1 expression
may represent different functional roles or different survival states of microglia in
different rat glioma tissues. Future studies based on these observations could provide
insights into glioma biology as well as functions of fractalkine and CX3CR1 in tumor
development.


CC chemoklne family
12
MXV-1A APMGSDPPTA CCT8YTARKL
KIP-la ....ADTPTA CCTSYTSRQI
RANTXS SPYSSDT.TP CCFAYIARPL
MCP-1 QPDAINAPVT CCYNFTNRKI
1-309 .KSMVQPFSR CCFSFAEQEI
3
PRNFWDYY. ET3SLCSQPA WFQTKRSKQ
PQNFIADYF. ET8SQCSKPG VIFLTKRSRQ
PRAHIKEYF. YTSGKCSNPA WTVTRKNRQ
SVQRLASYRR IT8SKCPKEA VXFKTIVAKE
PLRAILCYR NTSSICSNEG LIFKLKRGKE
4
VCADPSESWV QEYVYDLELN
VCADPSEEWV QKYVSDUtLS A
VCANPEKKHV REY IKS LEMS
ICADPKQKWV QDSMDHLDKQ TQTPKT
ACALDTVGHV QRHRKMLRHC PSKRK .
CXC chemokine family
12 3 4
ATO GKEESLDSDL YAELKCMCZX TT8.GIHPKN ZQSLEVIGKG THCNQVXVIA TL.XDGRKIC LDPDAPRIKK IVQKKLAGDE SAD.
GRO-a ASV ATELRCQCLQ TLQ.GIHPKN ZQSVNVKSPG PHCAQTEVIA TL.KNGRKAC LNPASPIVXX IIEKMLNSDK SN..
PF4 EAEE DGDLQCLCVX TT8.QVRPRH IT8LE7IRAG PBCPTAQLZA TL.KNGRKIC LDLQAPLYKX ZIKXLLES
IL-I S AKBLRCQCZK TYS1CPFBPKF ZKELRVZESG PHCANTEIIV KL.SDGRELC LDPKENWVQR WEKFLKRAE NS..
IP-10 VPL SRTVRCTCXS ISNQPVNPRS LEKUCIIPAS QFCPRVEIIA TMKKKGEKRC LNPESKAIKN LLKAVSKEMS KRSP
C chemoklne family
1 2
Ltn EGVGTEVL EESSCVNLQT QRLPVQKIKT YIIWEGAMRA VIFVTKRGLK ICADPEAKWV LAAIKTVDGR ASTRKNMAET VPGTGAQRST STAITLTG
K)
CX3C chemoklne family
12 3 4
Fractallclna QHHGVTKCNI TCSKMTSKIP VALLIHYQQN QASCGKRAII LETRQHRLFC ADPKEQNVKD AMQHLDRQAA ALTRNGGTFE KQIGEVKPRT
TPAAGGMDES WLEPEATGE SSSLEPTPSS QEAQRALGTS PELPTGVTGS SGTRLPPIPK AQDGGPVGTE LFRVPPVSTA ATNQSSAPHQ
PGPSLNAEAK TSEAPSTQDP STQASTASSP APEENAPSEG QRVWGQGQSP RPENSLEREE MGPVPAHTDA FQDWGPGSMA HVSWPVSSE
GTPSREPVAS GSWTPKAEEP IHATMDPQRL GVLITPVPDA QAATRRQAVG LLAFLGLLFC LGVAMFTYQS LQGCPRKMAG EMAEGLRYIP
RSCGSNSYVL VPV
Figure 1-1: Amino Acid Alignment of Representative Members of the Human Chemokine Family
The line indicates the location of predicted membrane spanning segment of fractalkine. Arabic numbers above the sequence refers to
the four highly conserved cysteine residues.


125
Yoshida, T., Imai, T., Kakizaki, M., Nishimura, M., Takagi, S., Yoshie, O. (1998)
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20
Chemokines and Receptors in Other Neuropathologies
The expression of chemokines was also characterized in a number of other
neuropathological conditions. A traumatic injury to adult mammalian central nervous
system results in reactive astrogliosis and the migration of hematogenous cells into the
damaged neural tissue. Several groups looked at expression of chemokines in models of
mechanical injury of the brain based on the hypothesis that chemokines might play an
important role in this cellular accumulation after the injury. Glabinski et al. (1996)
identified the expression of MCP-1 mRNA in mouse brain after penetrating mechanical
injury within 3 hours postinjury of the brain. MCP-1 mRNA levels stayed high 12 hours
after injury and came back to base line around 24 hours postinjury. Using ELISA, they
revealed that MCP-1 protein level was elevated in the homogenates of the lesioned tissue
by 12 hours postinjury and approached baseline by 48 hours. Their in situ hybridization
analysis again showed that MCP-1 was expressed by GFAP (glial fibrillary acidic
protein) expressing astrocytes. In another study, Berman et al. (1996) also found the
upregulation of MCP-1 mRNA and protein level in the rats subjected to stab wounds of
the cortex. However their immunohistochemistry data indicated that MCP-1 was
expressed by macrophages and endothelial cells from morphological assessment.
Ghimikar et al. (1996) found the expression of two other chemokines; namely RANTES
and MEP-1 p, in the rat stab wound brain by immunohistochemical staining. Double
labeling studies showed that MIP-ip, but not RANTES, was expressed by astrocytes and
macrophages near the lesion site.
Hausmann et al. (1998) compared the type of chemokine expression between
cortical lesions under sterile conditions or with LPS (lipopolysaccharide) injection. Only


19
The Expression of Chemokines and Their Receptors in MS Patients
A number of studies have focused on examining the expression of chemokines in
CNS tissue and cerebrospinal fluids (CSF) of MS patients or cell lines derived from MS
patients. Miyagishi et al. (1995) observed an increase of MIP-la concentration in CSF of
MS patients by ELISA. Based on this evidence, Bennetts et al. (1997) looked at the
potential protection effect of CCR5 (one of the receptors for MIP-la) deletion mutation
against MS. Two CCR5 deletion mutation individuals have been diagnosed with MS.
This suggested that the absence of CCR5 is not protective against MS. It is likely that
other MIP-la receptors are responsible for MIP-la related pathogenesis in MS patients.
Since the demyelination process that occurs in MS patient is, in part, due to an
inflammatory response in which CD4+ and CD8+ T cells and macrophage infiltrate white
matter, Biddison et al. (1997, 1998) did a series of in vitro studies demonstrating that
CD8+ T cell lines derived from MS patients are capable of secreting MIP-la, MIP-ip,
and IP-10. Soluble products of these CD8+ T cells are chemotactic for CD4+ T cells.
Antisera treatment revealed that the majority of this chemotactic activity is mediated by
IP-10. Another study using post-mortem tissue samples of MS patients revealed that the
expression of RANTES mRNA level in MS brain is higher than control brain. In situ
hybridization analysis indicated that RANTES mRNA localization was associated with
perivascular T cells (Hvas et al., 1997). These data further suggested that chemokines
play a proinflammatory role in the pathogenesis of MS.


44
were prepared for in situ hybridization and immunohistochemistrv analysis as described
above.
Quantitation of areal grain densities of bilateral facial motor nuclei was performed
on emulsion-dipped slides under dark field conditions using a video imaging system
(Microcomputer Imaging Device) and image analysis program (Imaging Research Inc.)
interfaced via a video camera (Sony XC77) connected to a Zeiss microscope (Axioscop
20). Statistical comparisons (one-way analysis of variance) were made between
ipsilateral and contralateral values within each post-lesion time point.
Tumor Inoculation
The RG-2 or C6 glioma cell lines were maintained in a monolayer culture in
DMEM with 10% FBS. Female Fisher rats were anesthetized with sodium pentobarbital.
Tumor cells (5xl05/5pl) were injected stereotactically into the right cerebral hemisphere
through a burr hole. Five pi of saline solution was injected into a group of rats that
served as controls.
Terminal Transferase-Mediated UTP Nick End Labeling (TUNEL-Labeling)
TUNEL-labeling was performed following the protocols by Gavrieli and
colleagues (1992) with some adjustments. RG2 or C6 cell inoculated Fisher rats were
sacrificed, fixed and cryoprotected as described for in situ hybridization analysis. After
rinse in water 2x5 minutes, endogenous peroxidase was inactivated by incubating the
slides in 3% water for 10 minutes at room temperature. Tissues were then exposed to
0.2% triton X-100/PBS for 5 minutes in room temperature. Slides were incubated in
water at 60C for one hour and cooled down in room temprature before TdT treatment.
After pre-incubating in TdT buffer (30 mM Tris-HCl, pH 7.2, 140 mM sodium


CHAPTER 2
MATERIALS AND METHODS
Materials
DNA modifying enzymes, RNA polymerases and the random primer labeling kit
were purchased from Promega (Madison, WI) with the exception of DNA polymerase
(phi) which was purchased from Stratagene (La Jolla, CA). Reverse transcriptase, TRIzol
Reagent, DMEM, trypsin, penicillin/streptomycin, LipofectAMINE and rat recombinant
interferon-y were from Life Technologies, Inc. (Grand Island, NY). In vitro transcription
kit was purchased from Ambion (Austin, TX). [Alpha-j2P]dCTP (3000 Ci/mmol) and
[alpha-'_'P]dATP (2000 Ci/mmol) were from DuPont/NEN (Wilmington, DE). [Alpha-
JJP]rUTP (2500 Ci/mmol) was from Amersham (Arlington Heights. IL). DNA
sequencing kit (Sequenase, v. 2.0) was from U.S. Biochemical Corp. (Cleveland, OH).
HEK293 cells were from ATCC (Rockville, MD). FURA2-A/M was from Molecular
Probes (Eugene, OR). Xenopus laevis were purchased from Nasco (Fort Atkinson, WI).
The mammalian expression vectors, pcDNA3 and pCLDN-10b, were from Invitrogen
(San Diego, CA) and SmithKline Beecham (King of Prussia, PA), respectively. Guinea
pig myelin basic protein, complete Freunds adjuvant (CFA), lectin (GSA I-B4), and
monoclonal-GFAP antibody were from Sigma (St. Louis, MO). Biotinylated anti-mouse
IgG and horseradish peroxidase-avidin D were from Vector (Burlingame, CA).
32


75
Rat Fractalkine (CX3C)
MAP
A G
Q H L
L L I
Q H R
H Q T
G K F
P T A
P R S
STS
AAV
E E K
P K Q
K S P
S T V
S G S
V F I
A V G
SQL
GMT
H Y Q
H F C
A A L
E K R
L Q S
P K A
K A Q
S T T
ATE
N V G
G P E
H P S
Q A P
T P V
L L A
AWL
K C N
L N Q
A 0 P
T R N
V D N
L S Q
M G T
DAG
I W P
S P P
S E G
E T N
V A P
K V E
P D S
F L G
L R L
I T C
ESC
K E K
G
V T P
P Q W
S Q E
L A A
S S A
TIA
Q P P
P V H
T S S
E P I
Q A A
L L
A A F
H K M
G K R
W V Q
R
K
P
K
V
L
W
T
E
H
T
F H L
T S P
All
DAM
C T L
I P V
LET
K H L
I T S A
T L L W
P A A V
P Q S T
Y Q S G
S T Q A
V Q E Q
D I F Q
K T P S
ATAD
R R Q
L G V A
T R G L
N R L L
T G S S
G I S E
S S L W
S T T S
D S T P
D R G P
P E L V
P Q K L
M F
L R
AYQSLQGCPRKMAGEMVEG
VPRSCGSNSYVLVPV
L
T
R
D
S
F
P
V
A
S
E
G
A
S
Y
Figure 4-1. Amino Acid Sequence of Rat Fractalkine (Genbank accession number
R75309)
A rat brain cDNA library (Strategene, CA) was screened by hybridization using a rat
factalkine cDNA probe. The hybridization probe was generated by PCR using primers
derived from a mouse cDNA (Genbank accession number R75309). The rat fractalkine
cDNA was sequenced by standard methods (Sanger dideoxy chain termination,
Sequenase kit).


117
Napolitano, M, Zingoni, A., Bemardini, G., Spinetti, G., Nista, A., Storlazzi,
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56
1 60
ratCCRS MDFQGSI PTYIYDIDYS MSAPCQKVNV KQIAAQLLPP
murCCRS MDFQGSV PTYSYDXDYG MSAPCQKINV KQIAAQLLPP
huCCRS MDYQVSS P..IYDIMYY TSEPCQKINV KQIAARLLPP
ratCCR2 MEDSHMLPQF IHGILSTSHS LTPRSIQELD EGATTPYDYD DGEPCHKTSV KQIGANILPP
murCCR2 MEDNNMLPQF IHGILSTSHS LTTRSIQELD EGATTPYDHD DGEPCHKTSV KQIGANILPP
huCCR2b MLSTSRS RFIRNTNESG EEVTTITDYD YGAPCHKFDV KQIGAQLLEE
ratCCRS
murccrS
huCCRS
ratCCR2
murCCR2
huCCR2b
ratCCR5
murccr5
huCCR5
ratCCR2
murCCR2
huCCR2b
ratCCRS
murccrS
huCCR5
ratCCR2
murCCR2
huCCR2b
ratCCRS
murccrS
huCCRS
ratCCR2
murccr2
huCCR2b
ratCCRS
murccr5
huCCRS
ratccr2
murCCR2
huCCR2b
ratCCRS
murccr5
huCCRS
ratCCR2
murCCR2
huCCR2b
61
LYSLVFIFGF VGNMMVFLIL ISCKKLKSMT
LYSLVFIFGP VGNMMVFLIL ISCKKLKSVT
LYSLVFIFGT VGNMLVILIL INCKRLKSMT
LYSLVFIFGF VGNMLVIIIL ISCKKLKSMT
LYSLVFIFGF VGNMLVIIIL IGCKKLKSMT
LYSLVFIFGF VGNMLWLIL INCKKLKCLT
X
121
FGNIMCKLFT GLYHIGYFGG IFFIILLTID
FGNIMCKVFT GLYHIGYFGG IFFIILLTID
FGNTMCQLLT GLYFIGFFSG IFFIILLTID
FGNIMCKLFT GLYHIGYFGG IFFIILLTID
FGNIMCKVFT GLYHIGYFGG IFFIILLTID
FGNAMCKLFT GLYHIGYFGG IFFTILI.TTD
III
181
AVFVSLPEII FMRSQKEGSH YTCSPHFLHI
AVFASLPEII FTRSQKEGFH YTCSPHFPHT
AVFASLPGII FTRSQKEGLH YTCSSHFPYS
AVFASLPGII FTKSEQEDDQ HTCGPYFP..
AVFGSLPGII FTKSKQDDHH YTCGPYTT..
AVFASVPGII FTKCOKEDSV YVCGPYFP..
IV
241
GILNTLFRCR NEKKRHRAVR LITAIMIVYF
GILHTLFRCR NEKKRHRAVR LIFAIMIVYF
GILKTLLRCR NEKKRHRAVR LIFTIMIVYF
GILHTLFRCR NEKKRHRAVR LIFAIMIVYF
GILHTLFRCR NEKKRHRAVR LIFAIMIGYF
GILKTLLRCR NEKKRHRAVR VIFTIMIVYF
301
DQAMQVTETL GMTHCCLNPV IYAFVGEKFR
DQAMQATETL GMTHCCLNPV IYAFVGEKFR
DQAMQVTETL GMTHCCINPI IYAFVGEKFR
DQAMQVTETL GMTHCCVNPI IYAFVGEKFR
DQAMQVTETL GMTHCCINPV IYAFVGEKFR
DOATOVTETL GMTHCCINPI IYAFVGEKFR
VII
361 377
SSVYTRSTGE QEVSTGL
SSVYTRSTGE HEVSTGL
SSVYTRSTGE QEISVGL
SSTFTPSTGE QEVSVGL
SSTFTPSTGE QEVSVGL
TSTNTPSTGE QEVSAGL
DIYLFNLAIS DLLFLLTLPF
DIYLLNLAIS DLLFLLTLPF
DIYLLNLAIS DLFFLLTVPF
DIYLFNLAIS DLLFLLTLPF
DIYLLNLAIS DLLFLLTLPF
DTYLLNLAIS DLLFLITLPL
XX
RYLAIVHAVF AFKARTVTFG
RYLAIVHAVF ALKVRTVNFG
RYLAWHAVF ALKARTVTFG
RYLAIVHAVF AFKARTVTFG
RYLAIVHAVF ALKARTVTFG
RYLAIVHAVF ALKARTVTFG
QYRFNKHFQT LKMVILSLIL
QYHFNKSFQT LKMVILSLIL
QYQrNKNFQT LKIVILGLVL
..TINKNFQT IMRNILSLIL
. QLNKNFQT IMRNILSLIL
..RGNNNFHT IMRNILGLVL
V
LFNTPYNIVL LLTTFQEYFG
LFWTPYNIVL LLTTFQEFFG
LFWAPYNIVL LLNTFQEFFG
LFNTPYNIVL FLTTFQEFLG
LFNTPYNIVL FLTTFQESLG
I.FNTPYNIVI LLNTFQEFFG
VI
NYLSVFFRKH IVKRFCKHCS
SYLSVFFRKH MVKRFCKRCS
NYLLVFFQKH IAKRFCKCCS
RYLSIFFRKH IAKNLCKQCP
RYLSIFFRKH IAKRLCXQCP
RYLSVFFRKH ITKRFCKQCP
120
MAHYAANENV
MAHYAANEWV
NAHYAAAQND
MAHYAANENV
MAHYAANENV
WAHSAANEMV
180
VITSWTNW
VITSWTNAV
WTSVITNW
VITSWTNW
VITSWTNW
WTSVTTMLV
240
PLLVMVICYS
SLLVMVICYS
PLLVMVICYS
PLLVMVICYS
PLLVMVICYS
PIAIMYICYS
300
LNNCSSSNRL
LNNCSSSNRL
LNNCSSSNRL
MSNCWDMHL
MSNCVIDKHL
LSNCESTSQL
360
IFQQVNPDRV
IFQQONPDRA
IFQQEAPERA
VFYRETADRV
VFYRETADRV
VFYRETVDGV
Figure 3-1. Sequence Comparison of Rat and Murine C-C Chemokine Receptors
Multiple alignment of two rat CC chemokine receptor amino acid sequences (ratCCR2
and ratCCR5) with the two functional human CC chemokine receptor (huCCR2 and
huCCR5) and the two murine CC chemokine receptor (murCCR2 and murCCR5).
Alignment was achieved using the PILEUP algorithm of the GCG package (39). Lines
denote putative transmembrane regions. Genbank Accession Numbers:
(huCCR2b:U03905, huCCR5:X91492, murCCR2:U47035, murCCR5:U47036,
ratCCR2:U77349, ratCCR5:U77350).


82
Figure 4-8. Colocalization of Rat CX3CR1 mRNA With Microglial Cells
Rat CX3CR1 mRNA hybridization signals (silver grains) from anti-sense [33P]-UTP-
labeled riboprobe hybridized-sections are found principally over lectin (GSA-I-B4)
staining microglia in the neuropil of the unoperated facial motor nucleus (B). Several
CX3CR1 mRNA containing microglial cells are found perineuronally [arrows in (A)]
surrounding axotomized motomeurons (N: motor neuron cell body). (C) Sense [33P]-
UTP-labeled riboprobe-hybridized section showing only a few scattered silver grains.


36
placed in a SLM 8000 model spectrofluorimeter (driven by an IBM PC). FURA-2
fluorescence was monitored at 510nm, with automatic slewing between excitation
wavelengths at 340nm and 380nm. Ratios at these two excitations were automatically
calculated.
Expression of Rat CCR5 in Xenopus Laevis Oocytes
Plasmid (pcDNA3) constructs containing the open reading frames of rat CCR2
and rat CCR5 were linearized, and complementary RNAs were synthesized in an in vitro
transcription reaction using T7 RNA polymerase. The transcription products were
confirmed by Northern blot analysis. Xenopus laevis oocytes were injected with 50 ng of
RNA in a total volume of 25 nl per oocyte. Electrophysiological recordings were made 3-
4 days after injection in oocytes superfused with Oocytes Ringers solution at room
temperature under voltage-clamped conditions using two microelectrodes (both filled
with 3 M KC1), with the membrane potential routinely clamped at -50 mV using a TEV-
200 voltage clamp instrument. Test chemokines were dissolved in distilled water and
diluted to a final concentration of 250 nM in Oocytes Ringers solution. Thirty pi of each
chemokine was applied directly onto voltage-clamped oocytes for 120s, and the current
induced was monitored on a Tektronix 5113 dual beam storage oscilloscope linked to an
IBM-PC.
RNA Isolation. RNase Protection Assay, and Northern Analysis
Total RNA was isolated using TRIzol Reagent according to the manufacturer's
recommended procedure. The RNase protection assay was performed as previously
described (Feng et al., 1994). The antisense riboprobe for rat CCR2 was generated from
an in vitro transcription reaction using a plasmid construct containing the Dral/EcoRI


120
Rucker, J., Samson, M., Doranz, B.J., Libert, F., Berson, J.F., Yi, Y., Smyth, R.J.,
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27
tansduction pathway that induced tyrosine phosphorylation of the protein tyrosine kinase
Pyk2 of CXCR4+ or CCR5+ target cells. These effects of HIV-1 on target cells through
chemokine receptors are helpful for better understanding the pathogenesis of HTV-1.
Chemokine Receptors and HIV in the CNS
In the CNS, microglia are the principal target cells for HTV infection. Several
groups examined the coreceptors that were responsible for HIV-1 infection and got some
contradictory data. He, et al. (1997) reported the expression of CCR3 and CCR5 in
microglia of primary human brain cultures. Their data suggested that anti-CCR3 and
anti-CCR5 antibody can both block M-tropic and brain-tropic HIV-1 isolates infection to
these microglia, indicating that CCR3 and CCR5 are cofactors for some strains of HTV-1
infection in microglia. Another study carried out by Shieh et al. (1998) suggested that
predominantly CCR5, and to a much less extent CCR3 or CXCR4 are coreceptors for
some of the HTV-1 isolates in cultured human microglia infection. However, while
Ghorpade et al. confirmed the expression of CCR3 and CCR5 in cultured human
microglia, their usage of antibodies to CCR3 or CCR5 failed to demonstrate similar
levels of blocking of HTV-1 infection or replication. They concluded that there might be
other chemokine receptors in microglia that are responsible for HIV-1 infection.
Elucidation of the roles of chemokine receptors in HTV infection will be very
useful in developing therapeutic regimes in AIDS treatment.


26
CCR5 chimera. In addition, data from studies of CCR5/CCR2 chimeras suggests that
either the N-terminus or the rest of CCR5 are sufficient for M-tropic HIV fusion.
Inhibition of HTV Entry by Chemokine Ligands:
Since Cocchi et al. first reported the inhibitory effect of MEM a, MIP-ip,
RANTES on HTV infection, there have been several studies examining the role of
chemokine ligands in suppressing HIV infection. With the identification of MCP-2 as a
ligand for CCR5, Gong et al. (1998) demonstrated that this peptide blocks CD4/CCR5-
mediated HIV-1 entry/replication. Evidence from other groups proved the ability of
inhibitors for either CCR5 or CXCR4 in blocking HTV-1 entry of the respective strain
(Simmons et al., 1997, Howard et al., 1998). While the mechanisms by which
chemokines and coreceptor inhibitors block HIV-1 infection are not fully elucidated,
studies from Amara et al. (1997) and Howard et al. (1998) indicated that these molecules
induced coreceptor internalization.
Interactions between HIV and coreceptors have been shown to inhibit the binding
between chemokine peptides and their receptors. Studies from Wu et al. (1996) and
Trkola et al. (1996) suggested that CD4 could induce the interaction between gpl20
glycoprotein of HIV-1 and CCR5 resulting in inhibition of the binding of MIP-la and
MIP-ip to CCR5. On the other hand, studies carried out by Dolei et al. (1998) suggested
that ligands for CCR5, MIP-la, MIP-1 p, and RANTES, increased the replication of some
T-cell-tropic HIV-1 strains and induced expression of the CXCR4 coreceptor.
To further explore the effect of HTV envelope protein on target cells after binding
to these chemokine receptors, Davis and colleagues (1997) work indicated that HTV-1
envelope glycoproteins from both T-tropic and M-tropic strains activated a signal


35
ACT TTA CAA CCC. The PCR product was reacted with the Klenow fragment of DNA
poll in the presence of dNTPs and cloned subsequently to the EcoRV site of pcDNA3.
The resultant expression plasmid construct was subject to DNA sequence analysis to
verify the fidelity of the Pfu DNA polymerase used in the PCR. The Dral/BamHI
fragment containing the rat CCR5 orf was cloned to the HindElI and BamHI sites of
pCLDN-10b; the Hindm site was made blunt by the action of the Klenow fragment of
DNA poll in the presence of dNTPs. A KpnI/BamHI fragment of this pCLDN-10b
construct was subcloned subsequently to the same sites of pcDNA3. HEK293 cells were
grown in DMEM supplemented with 10% heat inactivated fetal bovine serum (FBS) and
penicillin/streptomycin (complete media). HEK293 cells (2 x 105 cells) were transfected
with 1 Opg of plasmid DNA using LipofectAMINE reagent according to the
manufacturers recommended procedure. Approximately 72 hours post transfection, cells
were replaced in complete media containing the neomycin analog G418 (0.8 mg/ml).
Clonal cells resistant to G418 were isolated and propagated for further analysis. Cells
expressing (c)RNA hybridizing to G418 were isolated and propagated for further
analysis. Cells expressing (c)RNA hybridizing to the Clal/BamHI DNA fragment of rat
CCR5 were characterized further.
Measurement of Intracellular Calcium Levels
HEK293 cells (~5x 106) which stably express rat CCR2 were dissociated from the
culture dish and diluted subsequently into 10 ml MEM (serum- and antibiotic-free). The
suspension was made in 5 mM of FURA-2/AM and was incubated for 30 minutes in the
dark at 37C. The cells were then washed twice with PBS buffer and subsequently
resuspended in 2 ml HBSS buffer. The cell suspension was transferred to cuvettes and


71
Quantitative analysis indicated that, in the control FMN, only 30% of the lectin staining
microglia were CX3CR1 positive (a cluster of>10 silver grains over a single cell) whereas
in the lesioned FMN, there is approximately 60% of the lectin staining microglia
expressing CX3CR1. In addition, the intensity of CX3CR1 mRNA signal, represented by
numbers of silver grains found over the microglia, appear to be comprable between the
control and lesioned FMN. This suggests that CX3CR1 mRNA expressed by each single
microglia did not change after facial nerve axotomy.
Expression of Rat Fractalkine in FMN After Facial Nerve Transection
The expression of fractalkine mRNA in the facial motor nucleus following
axotomy was also examined. In situ hybridization analysis demonstrated that the
motoneurons were the primary source of fractalkine mRNA (Figure 4-9 A-D).
Fractalkine positive cells were not colocalized with either lectin positive microglia or
anti-GFAP positive astrocytes. A decrease in the hybridization signal in motoneurons
four days after axotomy was also evident. Western blot analysis done by Elizabeth Wees
in our laboratory demonstrated that lower molecular mass forms of rat fractalkine protein
levels were upregulated in the lesioned FMN.
Discussion
The expression of some of the chemokine and chemokine receptors in the CNS
has been reported by a number of different groups. In this study, we isolated the rat
ortholog of human and murine fractalkine by homology screening technique and
examined the expression and regulation of rat fractalkine and its receptor rat CX3CR1, in
the facial motor neuron regeneration paradigm. Consistent with its human and murine


102
Arenberg, D.A., Polverini, P.J. Kunkel, S.L., Shanafelt, A., Hesselgesser, J.,
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53
chemokine receptors (Ahuja and Murphy, 1993), there is a preponderance of acidic amino
acid residues in the conceptualized amino acid sequences of the respective N-termini, a
region that is likely to participate in binding with the positively charged N-termini of
chemokine peptides. A serine rich region is found in the carboxy-terminal domain of
both receptors, suggestive of potential phosphorylation regulation of these proteins. The
amino acid sequences also carry potential N-linked glycosylation sites (N-X-S/T) for both
receptors. The overall amino acid sequence identity between human and rat CCR2 and
CCR5 are 79% and 83% respectively. The high amino acid sequence identity prompt us
to hypothesize that these genes encode rat orthologs of the two human chemokine
receptors.
Functional analysis of these rat chemokine receptors in receptor-expressing-cell
systems finalized their identity as rat CCR2 (MCP-1 receptor) and CCR5 (receptor for
MIP-la, MIP-ip and RANTES), respectively. Similar to its human ortholog, rat CCR2
can be stimulated by MCP-1 in low nanomolar concentrations. Although we failed to
demonstrate a transient intracellular Ca2+ increase in rat CCR5 expressing HEK cells, we
were able to detect a Ca"~ activated Cl" current in rat CCR5 expressing oocytes in
response to murine MIP-la, MIP-ip and human RANTES. The identical ligand
specificity between rat and human orthologs indicates that the rat can serve as a good
model in studying the functional roles of human CCR2 and CCR5 under physiological
and pathological circumstances.
Rat CCR2 and CCR5 are found in a number of cells and tissues that can either
produce or respond to chemokine peptides. In addition to the peripheral cells and tissues
that express both rat CCR2 and CCR5, rat CCR5 is also constitutively expressed in the


67
antigenic profiles have changed dramatically indicative of an activated state of these cells.
Increase of CR3 is found as early as 24 hours after axotomy (Graeber et al., 1988a). De
novo synthesis of MHC (major histocompatibility complex) class I occurrs around 3-4
days after the lesion followed by the expression of MHC class II and CD4 antigens (Streit
et al, 1988, Streit et al., 1989,). These findings demonstrate that microglia can express
molecules involved in communication with other immune responsive cells. However,
since there is no inflammation and leukocyte infiltration in this paradigm, the relevance
of these molecules in the regeneration process is not apparent.
After facial nerve transection, the production of a number of cytokines,
neurotrophic factors and cell matrix proteins by microglia in the lesioned FMN is
elevated including interleukin IL-6 (Kiefer et al., 1993), TGF-pi (Kiefer et al., 1993),
vimentin (Graeber et al., 1988b), and a polymeric ECM protein TSP (thrombospondin,
Moller et al., 1996). TSP has been shown to promote neuritic outgrowth from neurons in
the in vitro system (Lawer et al., 1986). The up-regulation of these molecules may play
an important role for microglia to elicit their function as neuroprotective cells in the
motoneuron regeneration paradigm.
Since microglia are immunoresponsive cells in the CNS and chemokine peptides
and receptors are immunologically relevant molecules, we hypothesized that chemokines
and/or their receptors are expressed in microglia and play a role in microglia function. In
this study, we examined the expression of rat fractalkine and its receptor CX3CR1 in the
FMN after facial nerve transection.


24
endogenous cells are responsible for the change. This model is very useful in our studies
examining the expression of chemokines and chemokine receptors in the CNS.
In conclusion, by far, most studies in regard to chemokines and chemokine
receptors in the CNS still remain at a descriptive level. However, these data implicate
important role(s) of chemokines and their receptors in a broad variety of
neuropathological conditions.
Chemokines, Chemokine Receptors and HIV
In the past two years, chemokines and chemokine receptors have been brought to
the spotlight largely because of the discovery of the functional role chemokines and
chemokine receptors play in HIV (Human immunodeficiency virus) infection.
Some Chemokine Receptors Are HIV Entry Cofactors
The primary interaction site between HIV-1 and infectable cells is the CD4
molecule. This interaction is mediated by the viral surface glycoprotein gpl20. In 1995,
Cocchi et al. demonstrated that chemokine peptides, MIP-la, MIP-ip, RANTES, were
the major HIV suppressive factors produced by CD8+ T cells. One year later, a G-protein
coupled receptor, termed fusin, was characterized as an HIV-1 entry cofactor based on the
fact that CD4+ cells made to express this receptor became susceptible to T cell-tropic
HIV-1 infection (Feng et al., 1996). Fusin was identified later as the receptor for a unique
CXC chemokine, SDF-1.
Five independent groups published their work identifying a CC chemokine
receptor, CCR5, as the co-receptor for Macrophage-tropic HTV-1 entry into CD4+ cells
(Deng et al., 1996, Dragic et al., 1996, Alkhatib et al., 1996, Doranz et al. 1996, Choe et


18
while other groups failed to demonstrate any expression of MIP-2, a CXC chemokine
peptide, Adamus et al. (1997) were able to detect MIP-2 mRNA expression in Lewis rats
at day 10 after MBP injection, the time of disease onset. Their in situ hybridization data
suggested that MCP-1 was expressed in astrocytes and infiltrating cells in the white
matter.
The involvement of chemokines in EAE development became evident when
Karpus et al. (1995) demonstrated that injection (i.p.) of an antibody towards MIP-la to
SJL/J mice passively transferred with EAE can prevent the development of clinical
disease as well as infiltration of mononuclear cells into the CNS. Antibody therapy could
also be used to ameliorate the severity of ongoing clinical disease of these EAE mice.
However, in their report, they didnt see the production of MCP-1 and MIP-2 peptides by
ELISA in diseased animal spinal cord, anti-MCP-1 failed to prevent the disease
development. In another study with the attempt to intervene the development of EAE,
Wojcik et al. (1996) revealed that intrathecal administration of antisense oligonucleotides
to MBP-injected Lewis rats could significantly reduce EAE scores at the height of clinical
disease.
Recently, Lane et al. (1998) examined the expression of a number of chemokines
using another model, virus-induced encephalomyelitis. Transcripts of CRG-2 (cytokine-
response gene-2)/IP-10, RANTES, MCP-1, MCP-3, MEP-1(3, and MIP-2 were seen in the
brain of mouse hepatitis virus-infected mice 3 days postinfection. These transcripts were
increased markedly in brains and spinal cords at day 7, which coincides with the
occurrence of acute viral encephalomyelitis. Their in situ hybridization data confirmed
that CGR-2/IP-10 was expressed by astrocytes.


118
Pan, Y., Lloyd, C., Zhou, H., Dolich, S., Deeds, I, Gonzalo, J.A., Vath, J.,
Gosselin, M., Ma, J., Dussault, B., Woolf, E., Alperin, G., Culpepper, J., Gutierrez-
Ramos, J.C., and Gearing, D. (1997) Neurotactin, a membrene-anchored chemokine
upregulated in brain inflammation. Nature 387: 611-617.
Paterson, P. (1960) Transfer of allergic encephalomyelitis in rats by means of
lymph node cells. J. Exp. Med. 111:119-135.
Pettinelli, C.B., Fritz, R.B., Chou, C.-H.J., and Mcfarlin, D.E. (1982)
Encephalitogenic activity of guinea pig myelin basic protein in the SJL mouse. J.
Immunol. 129:1209-1211.
Peveri, P., Walz, A., Dewald, B., and Baggiolini, M. (1988) a novel neutrophil
activating factor produced by human mononuclear phagocytes. J. Exp. Med. 167:1547-
1559.
Ponath, P.D., Qin, S., Post, T.W., Wang, J., Wu, L., Gerare. N.P., Newman, W.,
Gerard, C., and Mackay, C.R. (1996) Molecular cloning and characterization of a human
eotaxin receptor expressed selectively on eosinophils. J. Exp. Med. 183:2437-2448.
Post, T.W., Bozic, C.R., Rothenberg, M.E., Luster, A.D., Gerard, N., and Gerard,
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J. Immunol. 155:5299-5305.
Power, C.A., Church, D.J., Meyer, A., Alouani, S., Proudfoot, A.E.I., Clark-
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encoding cytokines IP-10 and JE/MCP-1 in experimental autoimmune
encephalomylelitis. FASEB 7:592-600.


76
Figure 4-2. RNase Protection Analysis of Fractalkine mRNA in Various Tissues of Adult
Rat
Five pg of total RNA from rat tissues or cells were subjected to RNase protection assay
(see Materials and Methods). The RNAs were hybridized simultaneously with 32P-
radiolabeled 400-base pair Ncol fragment of rat fractalkine cDNA and 93-base pair
ribosomal RNA (L32) riboprobes. The dried gel was exposed to Kodak X-RP film at -80
C for 72 hours.


40
transfected using LipofectAMINE and 10 pg of rat or human fractalkine encoded
DNA/100 mm plate and used three days post-transfection.
Synthetic chemokines were generated by native chemical ligation of peptides
synthesized by solid-phase methods (Applied Biosystems 430A Peptide Synthesizer),
purified by reverse-phase high-performance liquid chromatography, and characterized by
electrospray mass spectrometry (Dawson et al., 1994). Purified synthetic chemokines
were reconstituted in phosphate buffered saline (PBS) and stored frozen, until use, at
minus 70C.
Whole cell binding analysis was performed on three million PBS-rinsed cells per
35 mm well. Cells were incubated (1 hour at room temperature) in 1 ml HBSS/0.1%
BSA, l25I-labeled human fractalkine/chemokine domain (100 Ci/mmol) and various
concentrations of cold competing chemokine ligands. At the end of the incubation, the
cells were rinsed 3 x 2 ml with ice-cold HBSS/0.1% BSA. Total radioactivity retained in
the wells was removed with 0.2 N NaOH and quantitated by gamma spectroscopy.
In Situ Hybridization Analysis
In situ hybridization was carried out according to previously published procedures
(McNamara and Routtenberg, 1995). Radiolabled [j3P] (c)RNA probes were prepared by
in vitro transcription from T7 or Sp6 RNA polymerase promoters in the pGEM7Zf(+)
plasmid system. For RBS11 probes, a full length cDNA clone in pGEM7Zf(+) was
digested with Kpnl and a resultant 450 bp fragment was isolated and subcloned to the
Smal site of pGEM7Zf(+). The antisense probe for RBS11 was prepared by linearizing
the pGEM7Zf(+) plasmid with Hindlll. For preparation of the sense strand probe for
RBS11 the plasmid was digested with EcoRI. For rat fractalkine probes, a cDNA clone


37
fragment (365-base pair) of rat CCR2. The antisense probe for rat CCR5 was made in a
similar manner using an EcoRV/Bgin restriction fragment (310-base pair) of rat CCR5.
The antisense probe for rat fractalkine and RBS11 were made using total RNA (five pg)
and '"P-UTP-labeled riboprobes generated from in vitro transcription reactions utilizing
plasmid construct templates containing either a 0.4 kbp Ncol fragment of rat fractalkine
cDNA or a 0.45 kbp Smal/Kpnl fragment of RBS11 cDNA.
Total RNA (10 pg/lane) extracted from various cell and tissue preparations was
electrophoresed through denaturing 1.2% agarose and subjected to Northern blot analysis
(Church and Gilbert, 1984). For hybridization probes, the following DNAs were used:
the 1.5-kbp Clal/BamHI fragment of the rat CCR5 gene, a 1.1-kbpRBSll protein coding
DNA sequence, a 700 base pair cDNA fragment for rat fractalkine, and an 829-base pair
cDNA encoding rat cyclophilin, all of which were j2P-radiolabeled by the random primer
method (1-2 x 109 dpm/pg).
Preparation of Primary Cultures of Rat Microglia
Microglial cultures were prepared as previously described, with minor
modifications (Giulian and Baker, 1986). In brief, brains were removed from neonatal
rats and placed in an isotonic salt solution containing 100 U of penicillin G, 100 pg of
streptomycin and 0.25 pg of amphotericin B (Fungizone) per ml (pH 7.4). All blood
vessels and pia mater were removed, and the diencephalon were dissected out and
chopped into approximately 2 to 3 mm chunks. The brain pieces were then dissociated
by trituration at 37C in 20 ml of isotonic saline solution (pH 7.4) containing 0.25%
trypsin (wt/vol) and DNase-I (8 pg/ml). The dissociated cell suspension was added to an
equal volume of DMEM containing 10% FBS; the suspension was then centrifuged at


54
brain as well as cultured microglia. This observation is in agreement with the finding that
the human gene is expressed in human microglia present in primary brain cultures (He et
al., 1997). In vivo studies will be necessary to prove unequivocally that rat CCR5 is
expressed in microglial cells in the CNS.
My data revealed that steady state levels of rat chemokine receptor mRNA were
regulated in cultured microglia in vitro as well as in an EAE animal model in vivo. After
IFN-y treatment, the expression of rat CCR5 mRNA is up-regulated in cultured microglia.
On the other hand, the lesion of EAE involves in infiltration of the IFN-y producing T
lymphocytes due to the break down of the blood brain barrier (Merrill et al., 1992,
Weinberg et al., 1993). Taken together, it is conceivable that in normal rat spinal cord,
the CCR5 hybridizing signal was contributed by endogenous microglia. The microglial
expression of CCR5 is promoted by these EFN-y producing T lymphocytes during EAE.
This hypothesis is supported by my data demonstrating the increased expression of CCR5
mRNA in the spinal cords of EAE rats. While constitutive expression of CCR2 is absent
in rat spinal cord, its expression is detectable in EAE rats. It is known that infiltrating
macrophages are also found in the spinal cords of EAE rats (Huitinga et al., 1995).
Cultured macrophages are capable of expressing CCR2, CCR5 and another chemokine
receptor gene, RBS11, in vitro (Combadiere et al., 1995b; Raport et al., 1995). Thus,
macrophages are likely to be the sources of CCR2 hybridizing signal and part of the
increased CCR5 and RBS11 hybridizing signals in the spinal cords of diseased rats.
Studies combining in situ hybridization and histochemical analysis should clarify the cell
type expression of each of these receptors in the EAE model.


UNIVERSITY OF FLORIDA


9
recently (Zou et al., 1998, Tachibana et al., 1998) suggest that CXCR4 plays a very
important role in embryonic development of the cerebellum as well as vascularization of
the gastrointestinal tract.
Human CXCR5 was also isolated as an orphan receptor that was expressed in B
cells and Burkitts lymphoma tissue (Dobner et al., 1992). Forster et al. (1996) provided
evidence indicating the involvement of this receptor in B cell migration and localization
by using CXCR5 knock out mice. Recently, Gunn et al. (1998) and Legler et al. (1998)
characterized a CXC chemokine BLC (B-lymphocyte chemoattractant)/BCA-1 (B cell-
attracting chemokine 1) that can bind and stimulate Ca"* mobilization and chemotaxis of
cells transfected with the CXCR5 gene.
Molecular Cloning of CC Chemokine Receptors
To date, ten human CC chemokine receptors have been characterized by different
groups. Neote et al. (1993) and Gao et al. (1993) discovered human CCR1 gene using
sequence homology cloning techniques in their attempt to isolate chemokine receptors
that are closely related to IL-8 receptors and the fMLP receptor. Specific binding and
Ca'" mobilization analysis revealed that RANTES, MIP-la are ligands of this receptor.
CCR1 mRNA was detected in B-lymphocytes and some human myeloid precursor cell
lines e.g. HL-60, U937 and THP-1. Additional chemokine peptides have been
characterized in recent years; MCP-2 (Gong et al., 1997), MCP-3 (Combadiere et al.,
1995b), Leukotactin-1 (Youn et al., 1997), and Hemofiltrate C-C chemokine (HCC)-l
(Tsou et al., 1998) have since been identified as ligands of CCR1.
Human CCR2a and CCR2b are MCP-1 receptors with alternatively spliced
carboxyl tails (Charo et al., 1994). RT-PCR technique was performed using mRNA from


95
Figure 5-4. Lectin (GSA I-B4) Staining of RG2 (A, B) and C6 (C, D) Sections
Lectin staining, to identify infiltrating microglia in RG2 or C6 gliomas, was carried out as
described in chapter two. Pictures were taken at different (A, C, lOOx, B, D: 4000x)
magnifications. Figure 5-4A illustrates one of many fields where lectin positive cells
clustered to form isolated islands.


63
IFN-y
28 S
18 S
cyclophilin
Figure 3-7: Dose-Dependent Increase in Rat CCR5/2 mRNA by Interferon-y (IFNy)
Treated Microglia
Northern analysis of rat CCR5/2 mRNA in primary cultures of rat microglia treated with
IFNy. Cultured microglia were incubated for six hours with DMEM (control) or DMEM
containing the specified concentration of IFNy. At the end of the incubation, total RNA
was isolated from the culture dishes using the Trizol Reagent (Gibco/BRL). Ten pg of
total RNA from each group was subject to Northern analysis. Immobilized RNA was
hybridized with 32P-labelled CCR5 (Clal/BamHI fragment) by the method of Church and
Gilbert. Final wash temperature was 65C. The positions of migration of 18 S and 28 S
ribosomal RNA are shown. Exposure to Kodak X-AR film was for 3 days (-80C). The
lower panel depicts a subsequent hybridization of the blot with 32P-labelled DNA
encoding rat cyclophilin and was used to control for lane loading. Exposure time for the
cyclophilin hybridized membrane was 18 hours.


CHAPTER 5
DIFFERENTIAL EXPRESSION OF CX3CR1 IN TWO RAT GLIOMA MODELS
ROLE FOR TGF-P AND CX3CR1 IN MICROGLIAL CELL DEATH WITHIN RAT
BRAIN TUMORS
Introduction
It has been known for sometime that tumor growth in vivo can create an
immunosuppressive environment (Sawamura and De Tribolet, 1990). What role
chemokines and their receptors play in this environment is a question that needs to be
addressed.
Some tumor cell lines and tumors have been found to express chemokine
peptides. For instance, IL-8 was found to be expressed in lung carcinoma, osteosarcoma,
melanoma, renal carcinoma, fibrosarcoma, astrocytoma and glioblastoma cell lines; Gro
peptides were found in melanoma, renal carcinoma and bladder carcinoma cell lines;
MCP-1 expression was seen in fibrosarcoma, glioma, histocytoma, melanoma,
osteosarcoma and sarcoma cell lines. In vivo studies revealed that MCP-1 is expressed in
ovarian cancer (Negus et al., 1995), prostate adenocarcinoma (Mazzucchelli et al., 1996),
and human invasive ductal breast cancer (Valkovic et al., 1998). In these studies, ovarian
cancer cells and breast cancer cells were found to be the sources of MCP-1 whereas
prostate adenocarcinoma cells were not. The expression and function of CXC
chemokines in tumor development have been studied rather extensively mainly based on
their angiogenic or antiangiogenic functions. IL-8 and IP 10 have been found in human
84


121
HTV-1 infectivity in macrophages and lymphocytes by a novel CCR5 antagonist. Science
276:276-279.
Soto, H., Wang, W., Stricter, R.M., Copeland, N.G., Gilbert, D.J., Jenkins, N.A.,
Hedrick, J., and Zlotnik, A. (1998) The CC chemokine 6Ckine binds the CXC chemokine
receptor CXCR3. Proc. Natl. Acad. Sci. USA 95:8205-8210.
Sozzani, S., Zhou, D., Locati, M., Riieppi, M., Prooost, P Magazin, M., Vita, N.,
Damme, J.V., and Mantovani, A. (1994) Receptors and transduction pathways for
monocyte chemotactic protein-2 and monocyte chemotactic protein-3. Similarities and
differences with MCP-1. J. Immunol. 152:3615-3622.
Spanaus, K.S., Nadal, D., Pfister, H.W., Seebach, J., Widmer. U., Frei, K., Gloor,
S., and Fontana, A. (1997) C-X-C and C-C chemokines are expressed in the cerebrospinal
fluid in bacterial meningitis and mediate chemotactic activity on peripheral blood-derived
polymorphonuclear and mononuclear cells in vitro. J. Immunol. 158:1956-1964.
Spleiss, O., Gourmala, N., Boddeke, H.W., Sauter, A., Fiebich, B.L., Berger, M,
and Gebicke-Haerter, P.J.( 1998) Cloning of rat HTV-1-chemokine coreceptor CKR5 from
microglia and upregulation of its mRNA in ischemic and endotoxinemic rat brain. J.
Neurosci. Res. 53:16-28.
Sprenger, H., Lloyd, A.R., Lautens, L.L., Bonner, T.I., and Kelvin D.J. (1994)
Structure, genomic organization, and expression of the human interleukin-8 receptor B
gene. J. Biol.Chem. 269:11065-111072.
St John, T.P., and Davis, R.W. (1979) Isolation of galactose-inducible DNA
sequences from Saccharomyces cerevisiae by differential plaque filter hybridization. Cell
16:443-452.
Streit, W.J. (1990) An improved staining method for rat microglial cells using the
lectin from Griffonia simplicifolia (GSA I-B4). J. Histochem. Cytochem. 38:1683-1686.
Streit, W.J., Graeber, M.B., and Kreutzberg, G.W. (1988) Functional plasticity of
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Streit, W.J., Graeber, M.B., and Kreutzberg, G.W. (1989) Expression of la antigen
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Neurol. 105:115-126.
Stricter, R.M., Polverini, P.J., Kunkel, S.L., Arenberg, D.A., Burdick, M.D.,
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functional role of the ELR motif in CXC chemokine-mediated angiogenesis. J. Biol.
Chem. 270:27348-27357.


12
Choe et al. (1998) have presented data demonstrating that human CCR9 can serve
as a coreceptor for HIV-1 and SIV entry. However, they did not present in detail how this
receptor was cloned and its amino acid sequence, neither did they discuss the tissue
distribution pattern of this receptor. Human CCR10 was identified by screening a human
lung cDNA library with a ,:P labeled probe containing known EST (Expressed Sequence
Tag) #149079 sequence (Bonini et al., 1997). The resulting cDNA contains a coding
region of 1,152bp for a 384-amino-acid protein. Its amino acid sequence is around 30%
identical to the other CC chemokine receptors. MCP-1, MCP-3, MCP-4 and RANTES
can bind to CCR10 expressing COS-7 cells in nanomolar concentration level. However,
binding of MCP-3 to the receptor did not stimulate a transient increase of the intracellular
Ca2~ levels. Thus, the real ligand(s) for CCR10 remains to be determined.
Cloning of CX3CR1. XCR1
The receptor for fractalkine, designated as CX3CR1, was identified recently (Imai
et al., 1997b). Previously, it was considered to be an orphan receptor (Harrison et al.,
1994, Raport et al., 1995, Combadiere et al., 1995c). It was found in many lymphoid cell
lines and is broadly expressed in a variety of tissues including many discrete regions of
the brain.
Most recently, the receptor for lymphotactin, designated as XCR1, has been
identified by Yoshida et al. (1998). It is expressed in placenta and weakly in spleen and
thymus among various human tissues. Lymphotactin binds to XCR1 and triggers an
intracellular Ca* increase and stimulates chemotaxis of XCR1 expressing cells. The
functional target of lymphotactin remains to be determined because of the controversial
results between two groups (Kelner et al., 1994, Yoshida et al., 1998).


81
F
Figure 4-7. In Situ Hybridization Analysis of CX3CR1 Expression in the Rat Facial
Nucleus After Motor Neuron Axotomy
In situ hybridization was performed on rat brainstem sections from animals sacrificed at 1
(A), 4 (B), 7 (C), 14 (D), and 21 (E) days post nerve transection using [35S]-UTP-labeled
riboprobe. Panel F depicts a quantitative analysis of the hybridization signal on the
operated () and contralateral (o) sides. Results are presented as the mean + S.E.M. of
data derived from at least three sections from three different animals. The hybridization
signal was specific for CX3CR1 as no increase in signal was observed using a sense
riboprobe (H) when compared to a corresponding anti-sense riboprobe hybridized-
section (G).


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
EXPRESSION OF CHEMOKINE RECEPTORS IN RAT CENTRAL
NERVOUS SYSTEM
By
Yan Jiang
December, 1998
Chairman: Jeffrey K. Harrison
Major Department: Pharmacology and Therapeutics
Chemokines (Chemoattractive cytokines) are a fast growing family of more than
40 structurally related proteins that are responsible for leukocyte chemoattraction and
activation during inflammatory processes. Receptors for the chemokine peptides are
members of the G-protein coupled receptor superfamily.
While the expression and functional roles of chemokine and chemokine receptors
in peripheral systems have been well characterized, related information regarding their
role in the central nervous system (CNS) is very limited. A number of chemokine
peptides and receptors have been found to be expressed or up regulated in
neuropathological circumstances such as EAE (Experimental Allergic
Encephalomyelitis), an animal model of human multiple sclerosis.
This study examined expression characteristics of chemokine receptors in the rat
CNS. Two genes of the rat orthologs of human CC chemokine receptors, CCR2 and
xi


Table 1-1. Ligand Specificity of Chemokine Receptors
Human Genes
Ligands
Human Genes
Ligands
CCRs
CXCRi
CCR1
MIP-lo, RANTES, MCP-2,
CXCR1
IL-8, GCP-2
MCP-3, Lkn-1, HCC-1
CXCR2
IL-8, GRO (a, B, y),
CCR2A
MCP-1
NAP-2, GCP-2, ENA-78
CCR2B
MCP-1, -2, -3, -4
CXCR3
IP-10, MIG, I-TAC
CCR3
Eotaxin-1, Eotaxin-2, RANTES,
CXCR4
SDF-1
MCP-2, -3, -4
CXCR5
BLC
CCR4
TARC, MDC
CCR5
MIP-lo, -IB, RANTES, MCP-2
CX3CR
CCR6
Exodus- 1/L ARC/MIP-3a
CX3CR1
Fractalkine
CCR7
MIP-3I1/ELC,
CCR8
SLC/Exodus-2/6Ckine
X£R
XCRl
Lymphotactin
1-309


23
disease (AD, Horuk et al., 1997). Immunohistochemical analysis has revealed that CCR3
and CCR5 are expressed on microglia in normal and AD brains (Xia et al., 1998).
However, not all ligands of these two receptors are found in the brain. MIP-10 was found
predominantly in a subpopulation of active astrocytes, which were more widespread in
AD than control brains. MIP-la appeared to be constitutively expressed at low levels by
neurons and microglia in both AD and control brain. Eotaxin, RANTES and MCP-3
immunoreactivity were not detected. Recently, Spleiss et al. (1998) found induction of
CCR5 mRNA in the lesioned region of rats that underwent brain ischemia or LPS
injection.
Microgliathe Brain Immune Responsive Cells
Microglia are the immune responsive cells in the central nervous system. Thus, it
is conceivable that function of chemokines and receptors in the CNS could relate to their
expression in microglia. There is evidence demonstrating the expression of some
chemokines and chemokine receptors in microglia. These molecules include IL-8
(Lipovski et ah, 1998), IP-10, MCP-1 (Sun et ah, 1997), MIP-la, MIP 1-0, RANTES
(lanabi et ah, 1998), SDF-0 (Ohtani et ah, 1998), CCR3, CCR5, and CXCR4 (Shieh et
ah, 1998). To study microglia activation in vivo, the facial nerve regeneration model is a
very useful animal model and has been well characterized. A transection of facial nerve
in the periphery stimulates a response in the facial motor nucleus. This response involves
microglia proliferation and activation. Facial motoneuron cell bodies are ensheathed by
these activating microglia (Figure 1-2). The advantage of this model is that one can
generate a microglia response without breaking the blood brain barrier so that only the


EXPRESSION OF CHEMOKINE RECEPTORS IN RAT CENTRAL
NERVOUS SYSTEM
By
YAN JIANG
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1998


122
Sun, D., Hu, X., Liu, X., Whitaker, J.N., and Walker, W.S. (1997) Expression of
chemokine genes in rat glial cells: the effect of myelin basic protein-reactive
encephalitogenic T cells. J. Neurosci. Res. 48:192-200.
Szaflarski, J., Ivacko, J., Hong Liu, X., Warren, J.S., S. and Silverstein, F. (1998)
Excitotoxic injury induces monocyte chemoattractant protein-1 expression in neonatal rat
brain. Brain. Res. Mol. Brain i?e.s.55:306-314.
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THIS DISSERTATION IS DEDICATED TO ALL THAT I LOVE


16
protein) (Madrid et al., 1982), and peptides of these proteins (Pettinelli et al., 1982,
Touhy, 1994). EAE can also be transferred via intravenous injection of lymphocytes
derived from sensitized donors (Paterson, 1960) or MBP-reactive cells (Ben Nun et al.,
1981).
Pathological findings of EAE animals include intense inflammation and some
demyelination of brain and spinal cord. Clinical signs occur within 9-15 days after
injection, which consist of weight loss, lethargy, loss of tail tonicity, different degrees of
paraparesis, and incontinence.
Because of the extensive inflammation involved in EAE, it has become a very
useful animal model for studying the expression and function of cytokines, including
chemokines, in the CNS. In 1993, Ransohoff et al. demonstrated expression of the
chemokine peptides MCP-1 and IP-10 in the spinal cords of EAE mice using RT/PCR
and in situ hybridization analysis. Their data suggested that astrocytes were the source of
MCP-1 and IP-10 mRNA. At the same time, Bermans group demonstrated expression of
MCP-1 mRNA and protein in spinal cords of EAE rats. MCP-1 mRNA levels were
elevated immediately before the onset of clinical signs, peaked with the height of clinical
disease, and declined with resolution of the disease (Hulkower et al., 1993). Later, they
defined the localization of MCP-1 by immunohistochemistry and in situ hybridization
(Berman et al., 1996). Their data indicated that at the onset of inflammation, prior to
clinical signs, MCP-1 was detectable in lymphocytes and endothelial cells in
subarachnoid locations whereas after the onset of the clinical signs, MCP-1 was widely
distributed in the spinal cord in lymphocytes, macrophages, astrocytes and endothelial
cells.


49
as rat CCR2 and rat CCR5. Figure 3-1 depicts a multiple alignment of rat, human and
murine CCR2 and CCR5. Rat CCR2 encodes a protein of 373 amino acids with a
calculated molecular mass of 42,763 Da, while rat CCR5 is a protein of 354 amino acids
and a calculated molecular mass of 41,017 Da. Both receptors contain structural
characteristics of CC chemokine receptors. There is a preponderance of acidic amino
acid residues in their amino acid sequences of the respective N-termini. Putative N-
linked glycosylation sites (N-X-S/T) can also be identified. The C-termini of each of
these rat receptors are serine rich, suggesting possible receptor regulation by
phosphorylation.
A comparison of amino acid identities of the rodent, murine and human receptors
is shown in Table 3-1. Rat CCR2 is 79% and 93% identical to human and murine CCR2
respectively while rat CCR5 is 83% and 92% identical to human and murine CCR5,
respectively. The most variable regions between rat and human orthologs were found in
putative extracellular portions (N-termini and loops). The high amino acid identity
between rat and human or murine CC chemokine receptors suggested the identity of these
novel rat chemokine receptors and allowed us to designate these rat receptors according
to nomenclature standardized for the chemokine receptor family.
Signal Transduction Properties of Rat CCR2 and CCR5
In order to confirm the identities of rat CCR2 and CCR5, functional assays were
carried out on cells expressing each of these receptor genes. Application of murine MCP-
1 stimulated a transient elevation in intracellular calcium ([Ca2+],) levels in rat CCR2
expressing HEK293 cells (Fig. 3-2) in a dose dose-dependent manner. The EC50 value
for this effect is approximately 0.4 nM. However, rat CCR5 expressing HEK293 cells


116
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cells to experimental rat glioma. Glia 6:75-79.
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Matsushima, K.. Yoshida, N., Springer, T.A., and Kishimoto, T. (1996) Molecular
cloning and characterization of a murine pre-B-cell growth-stimulating factor/stromal
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entry coreceptor fusin. Proc. Natl. Acad. Sci. 93:14726-14729.
Nagasawa, T., Tachibana, K., and Kishimoto, T. (1998) A novel CXC chemokine
PBSF/SDF-1 and its receptor CXCR4: their functions in development, hematopoiesis and
HTV infection. Semin. Immunol. 10:179-185.
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potent chemoattractant for lymphocytes and mapped to chromosome 9pl3. J. Biol. Chem.
272:19518-19524.


57
Table 3-1. Amino Acid Identities of the Rat, Murine and Human Chemokine Receptors,
CCR2 and CCR5
Murine ortholog
Human ortholog
Rat CCR2
93%
79%
Rat CCR5
92%
83%


66
chemokine receptors, functional screening assays using a variety of CC and CXC
chemokine peptides failed to identify its ligand. The similar tissue distribution pattern of
fractalkine and RBS11 prompted us to hypothesize that RBS11 is the receptor for rat
fractalkine. The human ortholog of rat RBS11, termed V28, was also identified (Raport
et al., 1995 and Combadiere et al., 1995). Recently, studies identifying V28 to be a
receptor for human fractalkine have been reported (frnai et al., 1997, Combadiere et al.,
1998). V28 is designated human CX3CR1.
In our continuous effort to characterize the role(s) of chemokines and chemokine
receptors in the CNS, we employed an animal modelthe facial motor neuron
regeneration modelto explore the expression site(s) of the chemokines and chemokine
receptors in the CNS. A transection of the facial nerve in the periphery can trigger a
dramatic response in the facial motor nucleus (FMN) of the CNS. This response includes
microglia activation and proliferation as well as astrocyte activation. Ultimately, the
damaged motoneurons are regenerated. The advantage of this model is that one can
challenge the brain tissue without breaking the blood brain barrier. Therefore, only
endogenous cells are responsible for any reactions in the facial motor nucleus in the CNS.
The other phenomenon that is associated with this model is so called synaptic stripping
where activated microglia occupy perineuronal positions, forming a sheath around the
motoneurons and displacing synaptic terminals from the surface of regenerating
motoneurons (Blinzinger and Kreutzberg., 1968, Graeber and Streit, 1990a, Graeber et
al., 1990b).
Microglia activation in FMN after facial nerve transection was determined not
only by morphological but also immunohistochemical studies. After axotomy, microglia


BIOGRAPHICAL SKETCH
Yan Jiang was bom in May 1964 in Shanghai. Peoples Republican of China.
After finished her 11 years of precollege education, she went to medical school of
Shanghai Medical University in 1981 and got her medical degree six years later. In 1987,
she started her first post-graduate education as a resident in Department of radiation
oncology in Shanghai Cancer Hospital. During 5 years of her residency, she learned
everything needed to be a good doctor and enjoyed the experience. However, close to the
end of her residency, she realized that there was not enough challenge in the future if she
stayed in the same field as a radiation oncologist; it was time to try something new. She
decided to come to the States with her husband and start a new career as a medical
research scientist. The begining was painful as she put herself in a foreign country with
very little background in research and spoke very little English. But she managed to
survive as time went by. In 1994, she was accepted and went to graduate school in
department of Pharmacology and Therapeutics of the College of Medicine, University of
Florida, pursuing a Ph.D. degree. She has had a fun, frustrating but rewarding four and
half years during which she became a Gatorized Chinese. Now, its time for her to
move on.
126


115
Liu, R., Paxton, W.A., Choe, S., Ceradini, D., Martin, S.R., Horuk, R.,
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I.(1996) Chemokine receptor specific for IP 10 and Mig: structure, function, and
expression in activated T-lymphocytes. J. Exp. Med. 184:963-969.
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inflammation. N Engl J Med. 388: 436-445.
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D.D. (1982) Lipophilin-induced experimental allergic encephalomyelitis in guinea pigs. J.
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J., and Matsushima, K. (1997) Prevention of cerebral edema and infarct in cerebral
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Laissue, J.A., and Mueller, C. (1996) Monocyte chemoattractant protein-1 gene
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prevents kainate induction of protein FI (GAP-43) mRNA in hippocampal granule cells
and subsequent mossy fiber sprouting in the rat. Mol. Brain Res. 33, 22-28.


31-
Control
Axotomized
Figure 1-2. Lectin (GSA I-B4) Staining of Facial Motor Nucleus Four Days After Facial
Nerve Transection
Microglia are stained by lectin from griffonia simplicifolia. Upper panel shows facial
motor nucleus of the unoperated side and lower panel shows facial motor nucleus four
days after facial nerve transection. Figure was kindly provided by Dr. Wolfgang J. Streit.


7
Molecular Cloning and Expression of Chemokine Receptors
Many of the receptors for these chemokine peptides have been characterized by
molecular cloning techniques (Table 1-1). They are members of the rhodopsin family of
the G-protein coupled receptor superfamily, which are characterized by their putative
seven hydrophobic transmembrane helices in their amino acid sequences.
Molecular Cloning of CXC Chemokine Receptors
To date, five human CXC chemokine receptors have been identified. Two
receptors for IL-8 were the first chemokine receptors to be identified. In 1991, William
Woods group characterized a receptor by expression cloning of a human neutrophil
cDNA library that was transfected into COS-7 cells as pools and examined for IL-8
binding activity (Holmes et al., 1991). The pure clone obtained encodes a 350-amino-
acid protein that not only binds to, but also responds to IL-8, to form a transient
intracellular calcium increase in transfected cells. Its amino acid sequence was found to
be most closely related to two other G-protein coupled receptors, the fMLP and C5a
receptors. On the other hand, Philip Murphys group (Murphy and Tiffany, 1991)
identified another IL-8 receptor gene in an attempt to isolate the human ortholog of a
rabbit N-formyl peptide receptor (F3R). They designed an oligonucleotide probe
corresponding to nucleotides of the cDNA sequence of F3R to screen the cDNA library of
a leukemia cell line (HL-60). The isolated gene contained a 1065-bp open reading frame
(orf), encoding a 355-amino-acid protein. It is 77% identical to the other IL-8 receptor. It
can bind to IL-8 and several other CXC chemokines including MGSA (melanoma growth
stimulatory activity)/GRO and NAP (neutrophil activation protein)-2. Application of


17
Godiska and colleagues examined the expression of a number of chemokines in
EAE mice using RT/PCR and Northern blot analysis and found that mRNAs encoding
RANTES, MIP-la, MlP-lli, TCA3, IP-10, MCP-1 KC/GRO and MCP-3 were expressed
in the spinal cords of EAE mice. Also, SDF was expressed in normal as well as diseased
spinal cords and no expression of M1P-2 was detected in either normal or diseased tissue
(1995). Using in situ hybridization analysis combined with immunohistochemistry,
Miyagishi et al. identified the cellular localization of RANTES and MIP-lii in rat EAE.
Their evidence indicated that mRNAs for RANTES and MIP-lli were mostly expressed
by CD3-positive T-cells located around blood vessels. These mRNAs were also co
localized with GFAP-positive astrocytes as well as OX42-positive
macrophages/microglia (1997). They were able to detect MIP-la expression in the spinal
cord by day 10 post injection, but they failed to identify its cellular colocalization due to
the fact that the combination of immunohistochemistry and in situ hybridization was
much less sensitive using MIP-la probes.
Glabinski and colleagues examined the time course of the expression of five
chemokines (MCP-1, MIP-la, KC/GRO-a, IP-10, and RANTES), during spontaneous
relapses of chronic EAE mice (1997), using RT-PCR, ELISA, and in situ hybridization
analysis. They found that all five chemokines were simultaneously up regulated during
relapses in the brain and spinal cord, in addition, expression was confined to the CNS. In
situ hybridization revealed that IP-10 and MCP-1 are expressed by GFAP-positive
astrocytes. The nuclear morphology and location of KC-expressing cells suggested
astrocytes as the cellular expressions of this chemokine. MIP-la and RANTES are
produced exclusively by mononuclear leukocytes in perivascular cuffs. Interestingly,


39
enlargements of the spinal cords removed. Total RNA was isolated and subsequently
subjected to either RNase protection assay or Northern blot analysis as described above.
Molecular Cloning and Sequencing of Rat Fractalkine cDNA
A rat brain cDNA library (Strategene, CA) was screened by hybridization using a
rat factalkine cDNA probe. The hybridization probe was generated by PCR using primers
derived from a mouse cDNA (Genbank accession number R75309). The rat fractalkine
cDNA was sequenced by standard methods (Sanger dideoxy chain termination,
Sequenase kit).
An EcoRI/Sall DNA fragment containing sequences encoding full length rat
fractalkine protein was subcloned to the mammalian cell expression vector, pCDM8
(Invitrogen). DNA containing human fractalkine protein coding sequence was generated
by PCR and subcloned into the same vector. To generate peptidase-resistant membrane-
anchored fractalkine, both human and rat fractalkine mutant forms were also prepared by
PCR, in which the putative dipeptidase site Arg-Arg, adjacent to the transmembrane
spanning domain, was mutated into Ser-Ala.
Expression and Functional Characterization of Rat CX3CR1 in Transfected Cells
A cDNA containing the open reading frame of RBS11 was cloned to the EcoRV
site of pcLDN-10b. CHO cells (grown in Hams FI2/10% heat inactivated fetal bovine
serum (FBS)/penicillin/streptomycin) were transfected with 10 pg of plasmid DNA using
LipofectAMINE reagent (Life Technologies, Inc.). G418-resistant clonal cells expressing
(c)RNA hybridizing to RBS11 cDNA were characterized further in binding and
functional assays. HEK293T cells (grown in DMEM/10% FBS/pen/strep) were


96
Figure 5-5. TUNEL (Terminal Transferase dUTP Nick End-Labeling) Analysis in RG2
and C6 Gliomas
TUNEL analysis was carried out on sections containing RG2 (A) or C6 (B) gliomas as
described in chapter two. Panel A is a representative field where TUNEL positive cells
clustered to form isolated islands in RG2 tumors. Panel B illustrates that very little
TUNEL positive cells can be seen in C6 tumors.


92
i ^ /
&'&# f
B
cf ^
s£
^ cr ^ ^
Cc *4^ s\j 'S?
uc uv o 0 jv
& & Figure 5-1. Expression of Rat CX3CR1 in C6 GliomaNorthern Blot Analysis
Northern blot analysis was carried out to examine the expression of rat fractalkine and
CX3CR1 in cultured RG2 and C6 cells as well as RG2 and C6 gliomas. Tumor tissues
were dissected out on an ice cold surface and normal tissues were removed. RNA
isolation was carried out using Trizol reagent. Ten pi of total RNA were loaded in each
lane. The hybridizations were carried out using the Church and Gilbert (1984) method.
The blots were hybridized with both 400bp and 750bp fractalkine cDNA fragment (A) or
a 1.2kbp rat CX3CR1 cDNA fragment (B). Hybridizing blot was exposed to the film for
four days at -80C. The lower panel depicts a subsequent hybridization of the blot with
"P-labelled DNA encoding rat cyclophilin and was used to control for lane loading.
Exposure time for the cyclophilin hybridized membrane was 18 hours


48
Towards our goal of defining chemokine receptor expression in the CNS, I have
isolated, by molecular cloning techniques, rat genes encoding the chemokine receptors
CCR2 and CCR5. I describe expression patterns of these genes in cells and tissues of the
CNS and report regulation of chemokine receptor expression in vitro in cultured rat
microglia and demonstrate in vivo modulation of chemokine receptor expression in EAE
rats.
Results
Isolation of Rat Genomic DNA Containing Sequences Encoding Novel G-Protein
Coupled Receptors
Degenerate oligonucleotide primers were designed based on the highly conserved
regions (TM2, TM3, and TM7) of the known human chemokine receptors (huEL-8RA,
huIL-8RB, huCCRl, and huCCR2 receptors) and the orphan rat chemokine receptor-like
gene, RBS11 (Holmes et al., 1991; Murphy et al., 1991; Neote et al., 1993; Gao et al.,
1993; Charo et al., 1994; Harrison et al., 1994). DNA fragments encoding novel rat
chemokine receptors were generated by PCR reactions using these oligonucleotides and
rat genomic DNA as the template. Consequently, these PCR products were utilized for
further screening of a Sprague Dawley rat genomic library under high stringency
conditions in order to isolate the full length DNA sequenses. Genomic library screening
using one of the fragments, pCrecl3, resulted in 15 plaque pure bacteriophage clones.
Restriction endonuclease digestion and DNA sequence analysis of each of these clones
revealed two distinct sets of bacteriophage containing open reading frames (orfs)
encoding novel G-protein coupled receptors with high sequence identity to other known
chemokine receptors. Hereafter, we designate the orfs containing the pCrecl3 sequences


52
levels in the lumbar enlargement of the EAE rats, as assessed by either RNase protection
analysis or Northern blot analysis. For each receptor, increases in the levels of mRNA
were observed in animals displaying clinical symptoms of the disease. In animals that did
not display symptoms, either no increases in mRNA (as for CCR2, CCR5, and CXCR4)
or minimal increases (as for RBS11) were seen. A correlation between the increase in
receptor RNA and the degree of clinical symptoms was observed for CCR2 and CCR5,
i.e. a greater increase in mRNA for these receptors was evident in the animal displaying
complete hind limb paralysis.
Discussion
In the past few years, many research groups have carried out studies to determine
the expression of chemokine peptides and chemokine receptors in the CNS. This project
launched our series of studies in an ongoing effort to characterize the expression,
regulation and functional roles of chemokine peptides and receptors in the central nervous
system. Our results demonstrate that the rat genome contains orthologous genes encoding
the chemokine receptors CCR2 and CCR5. Unlike rat CCR2, which is not constitutively
expressed in the brain or cultured microglia, rat CCR5 mRNA is constitutively expressed
in these tissues. In addition, steady state levels of chemokine receptor mRNA(s) are
regulated both in vitro in the cultured microglia and in vivo in rat EAE.
Two very closely related CC chemokine receptor genes, rat CCR2 and CCR5 have
been identified via sequence homology techniques. The deduced amino acid sequences
contain putative seven hydrophobic transmembrane alpha helices, which confirmed that
they are members of the G-protein coupled receptor superfamily. Similar to many other


88
approximately 3-4 weeks before being developed. Our data indicated that rat CX3CR1
mRNA is expressed in microglia within C6 gliomas as clustered silver grains were
evident over lectin binding positive cells (Figure5-3A). In RG2 gliomas rat CX3CR1
mRNA is expressed by microglia on the border of the tumor but not lectin positive cells
within the tumor (data not shown).
Different Microglial-Infiltrating Patterns Between C6 and RG2 Gliomas
Lectin (GSA I-B4) staining of both C6 and RG2 gliomas were carried out to
characterize microglial-infiltrating patterns of these two gliomas. Figure 5-4
demonstrates that within RG2 tumors, lectin positive microglia tend to cluster together to
form isolated islands (A); many cells have pyknotic nucleione of the characteristics of
apoptotic cells (B); many areas within RG2 tumors have none or very little microglial
infiltration (data not shown). On the other hand, inside C6 gliomas, infiltrating microglia
are evenly distributed throughout the tumor (C); these cells conserve the morphology of
normal microglial cells (D).
Microglial Apoptosis Within C6 and RG2 Gliomas
We further examined apoptosis within C6 and RG2 gliomas by TUNEL staining
(Terminal Transferase dUTP Nick End-Labelling) as described in chapter two. TUNEL
is a staining assay that labels cells with fragmented DNA ends, characteristic of apoptotic
cells. Figure 5-5 demonstrates that within RG2 tumors, TUNEL staining positive cells
are clustered to form isolated islands similar to what we see in RG2 tumors with lectin
staining (A). However, in the C6 gliomas that I tested, there are only a few TUNEL
positive cells (data not shown) and the major areas of the tumor mass were TUNEL
negative (B).


83
Figure 4-9: Fractalkine Expression in the Rat Facial Nucleus After Motor Neuron
Axotomy
In situ hybridization was performed on rat brainstem sections from animals sacrificed 4
days post nerve transection. Hybridization analysis of the contralateral (A and B) and
ipsilateral (C and D) facial motor nuclei using anti-sense (A and C) and sense (B and
D) [JP]-UTP-labeled riboprobes generated from rat fractalkine cDNA. Hybridization
signals are found principally over the motor neurons.


69
and olfactory bulb (Figure 4-3) and was significantly lower or absent in cerebellum and
white matter regions including corpus collosum and fimbria/fomix.
Identification of RBS11 As the Receptor for Rat Fractalkine
Figure 4-4 demonstrates the ability of radiolabeled chemokine domain of
fractalkine bind to RBS11-transfected CHO cells. Wild type CHO cells did not display
any significant binding. Inhibition binding constants (IC50s) of 2 and 3 nM for the
chemokine domains of rat and human fractalkine, respectively, were determined. Other
CC or CXC chemokine peptides that we tested, including MlP-la, RANTES, MCP-1
MIP-2, and SDF-1, did not displace the binding of labeled fractalkine to RBS11
expressing CHO cells. This evidence further corroborated that the binding between
fractalkine and RBS11 transfected cells was specific. Figure 4-5 depicts the functional
properties of RBS11 in the transfected CHO cells. Application of either soluble or
membrane-tethered forms of fractalkine stimulated a transient elevation of intracellular
calcium levels in the RBS11 expressing CHO cells. These data, collected by Dr. Jeffrey
Harrison, unequivocally identified that RBS11 is a functional receptor for rat fractalkine.
Expression and Regulation of Rat CX3CR1 mRNA in Cultured Microglia
To identify the expression site(s) of rat CX3CR1, Northern blot analysis was
carried out. Primary cultures of rat microglia were incubated for eight hours with DMEM
(control) or DMEM containing increasing concentrations of TGF*p. At the end of the
incubation, total RNA was isolated from these microglia and subjected to Northern blot
analysis. Figure 4-6 depicts constitutive expression of rat CX3CR1 mRNA in cultured
microglia. Steady state levels of CX3CR1 mRNA present in the cultured microglia were
dose-dependently increased by TGF-P treatment.


11
A number of different groups (Samson et al., 1996a, Raport et al., 1996,
Combadiere et al., 1996) isolated CCR5 by RT-PCR technique. These investigators were
able to identify MIP-la, MIP-1(3, and RANTES as its ligands. Gong et al. (1998)
provided evidence demonstrating that MCP-2 is also a ligand for CCR5. This receptor
was found to play an important role in macrophage-tropic HTV-1 infection as a fusion
cofactor (Deng et al., 1996, Dragic et al., 1996, Alkhatib et al., 1996).
Human CCR6 (Zaballos et al., 1996, Liao, et al., 1997a), CCR7 (Schweickart et
al., 1994), and CCR8 (Zaballos et al., 1996, Napolitano et al., 1996, Samson et al.,
1996b) were isolated via RT-PCR and initially reported as orphan receptors. LARC
(liver and activation-regulated chemokine)/MIP-3a/Exodus-l was found to be the ligand
of CCR6 (Baba et al., 1997, Power et al., 1997, Greaves et al., 1997, Liao et al., 1997b).
This receptor is expressed in spleen, lymph node, gut, pancreas and peripheral blood
lymphocytes, monocytes/macrophages. Two CC chemokine peptides, MIP-3p/CKb-
11/ELC (EBI1 -ligand chemokine) and SLC (secondary lymphoid-tissue chemokine), can
specifically bind to CCR7 expressing cells and stimulate a transient increase of the
intracellular Ca2* level (Yoshida et al., 1997, Willimann et al., 1998). A number of
lymphoid organs such as tonsil, spleen, and lymph node constitutively express CCR7.
This receptor was also found in several B- and T-cell lines but not in the myeloid cell
lines. Northern blot analysis was carried out on different tissue samples to determine the
expression of CCR8. CCR8 mRNA was detectable in spleen, lymph nodes and very
weakly in appendix and lymphocytes. To date, 1-309 is the only ligand found to be able
to interact with CCR8 expressing cells and stimulate Ca2+ mobilization (Roos et al., 1997,
Goya et al., 1998).


41
encoding chemokine domain of rat fractalkine was digested with Ncol. An
approximately 400 bp fragment was purified and subcloned to the Smal site of
pGEM7Zf(+). Antisense probe was generated by digesting this construct with EcoRI and
sense strand probe was obtained with BamHI digestion. In vitro transcription and
labeling of RNA probes was performed as described (McNamara and Routtenberg, 1995)
with mild modifications. Briefly, lOOpCi of [alpha-'P]-rUTP was added in a total 10 pi
reaction [40 mM Tris; 6 mM MgC12, 2 mM spermidine; 10 mM NaCl; 4 mM
dithiothreitol; 0.8 U/ml RNase inhibitor; 0.5 mM each ATP, CTP, GTP; linearized
template equivalent to 0.5 pg of whole plasmid; and 10 U of either T7 or Sp6 RNA
polymerase]. The reaction was allowed to proceed for 60 minutes at 37C. The DNA
template was removed by RNase-free DNase (10 U) for 15 minutes at 37C. Proteins and
free ribonucleotides were removed by NH4Ac and ethanol precipitation. Brain tissues
were embedded in O.C.T. compound (Miles Inc., Elkhart, EN) and 12-20 pm thick frozen
sections were made and placed on Superfrost/Plus microscope slides (Fisher Scientific,
Pittsburgh, PA). After fixed in 4% paraformaldehyde buffered PBS, pH 7.4. for 10 min
in 4C, tissue sections were washed in DEPC treated water and acetylated in a freshly
prepared solution of 0.1 M triethanolamine with acetic anhydride, pH 8.0, for 10 minutes.
After washing in 2x SSC, sections were dehydrated through graded ethanols. Sixty pi of
the hybridization mixture (50% formamide, 0.3 M NaCl, 20 mM Tris, pH 8.0, 5 mM
EDTA, 1 x Denhardf s, 10% Dextran Sulfate, 10 mM DTT) containing 1.5x 106 cpm
counts of radiolabeled RNA probe was applied to each slid and covered with a siliconized
coverslip. Hybridization was carried out for 16-18 hours at 60-65C in a humidified
chamber. The excess radioactivity was removed by washing the slides with two 20-min


59
murMIP-la murMIP-ip
(500 nM) (500 nM)
l l
Figure 3-3. Functional Responses of Cells Expressing Rat CCR5
Xenopus laevis oocytes expressing rat CCR5 were prepared as described in chapter two.
These oocytes were exposed to 500nM murine MIP-la (A) or 500 nM murine MIP-1 (3
(B). The current induced was monitored on a dual beam storage oscilloscope linked to an
IBM-PC.


77
Figure 4-3. Distribution of Rat Fractalkine mRNA in Adult Rat Brain
Hybridization analysis using anti-sense" (A) and sense (B) [35S]-UTP-labeled
riboprobes generated from rat fractalkine cDNA. Hybridized horizontal sections were
exposed to film for 18 hours (abbreviations used: Olf: olfactory bulb; ctx: cortex; cc:
corpus collosum; CPu: caudate putamen; Th: thalamus; ca3: ca3 region of the
hippocampus; gc: granule cell layer of the hippocampus; CG: central gray; cbm:
cerebellum).


74
microenvironment. However, the physiological relevance of these relative intracellular
calcium levels in both transfected cells and microglia still remains to be determined. We
hypothesize that the soluble form of fractalkine stimulates an unknown signaling pathway
which results in microglial chemotaxis towards facial motor neurons whereas the
membrane anchored form elevates intracellular calcium levels which functions to
promote the adhesion of microglia to these neurons and causes the ensheathement of
neurons by microglia, known as synaptic stripping.
In contrast to the up regulation of CX3CR1 mRNA in FMN after nerve
transection, the mRNA level of fractalkine is down-regulated four days after the lesion.
However, our Western blot analysis indicates the up-regulation of some lower molecular
mass forms of fractalkine protein in the lesioned FMN (data not shown). This
discrepancy between mRNA and protein levels of rat fractalkine in FMN after nerve
transection is suggestive of the dynamic regulation of fractalkine expression in the CNS.
The constitutive expression of fractalkine and CX3CR1 in discrete regions of the
CNS indicates that they are involved in fundamental processes of communication
between neurons and microglia under both pathological as well as physiological
circumstances. A better understanding of the functional roles of these molecules will
eventually lead to the development of therapeutic reagents that can interrupt disease
processes that these molecules are involved in.


21
MCP-1 expression was detectable in the sterile lesion. However, in the lesioned tissues
of LPS injection, they found expression of mRNAs of all chemokines examined,
including MCP-1, MIP-la, MIP-1 p, RANTES, IP-10 and KC. They also examined the
leukocytes and macrophage densities after the lesion and revealed that
macrophage/microglia accumulation occurred more rapidly; activated microglia further
from the lesion border; and more cells accumulated in LPS injected injury than sterile
injury. This indicated that chemokines are instrumental in the initiation of repair
processes following brain injury. Infusion of MCP-1 antisense oligonucleotides to the
stab wound injured rats not only reduced MCP-1 protein expression but also decreased
macrophage accumulation in the lesioned area (Ghimikar, et al., 1998). This data more
directly suggested a functional role of MCP-1 in macrophage chemoattraction after
mechanical brain injury.
Focal cerebral ischemia of rat induced by middle cerebral artery occlusion
(MCAO) is a frequently used animal model for ischemic stroke. Ischemic brain injury
secondary to an arterial occlusion is characterized by acute local inflammation. Using
this model, Liu et al. (1993) detected CINC/GRO/KC mRNA expression in the ischemic
tissue. Gourmala et al. (1997) identified MCP-1 mRNA expression in the ischemic tissue
of the rat after MCAO. Recently, Kostulas et al. (1998) examined expression of IL-8 in
the peripheral blood of healthy individuals or patients with ischemic stroke. They found
that plasma IL-8 concentration was increased in patients that suffered a stroke. This
correlated positively to IL-8 mRNA expression in mononuclear cells of examined
patients.


60
12
w#- v | j'vzsK
12
| .
7
#
5
3.2
3.2
3
* 2.9 ^
3
2.7
2.7
1.1
65 C 50 C
Figure 3-4. Southern Blot Analysis of Restriction Endonuclease-Digested Rat Genomic
DNA.
Wistar rat genomic DNA (15 pg) was completely digested with the indicated restriction
endonucleases and then subjected to Southern blot analysis using the 1.5 kbp [32P]-
radiolabeled Clal/BamHI fragment of rat CCR5 and the method of Church and Gilbert.
Final wash temperatures were 65C (A) and 50C (B). The blots were exposed to Kodak
XAR film with an intensifying screen at -80 C for 1 -3 days. The sizes of the hybridizing
bands are indicated (in kbp) and were estimated by comparison to a GIBCO/BRL 1 -kbp
ladder.


42
rinses of standard saline citrate (SSC) buffer (0.3 M NaCl, 30 mM sodium citrate), and a
30-min digestion in RNAse A at 37C in buffer containing 500 mM NaCl, 10 mM Tris,
pH 8.0, and 1 mM EDTA. After two more rinses with SSC, sections were stringently
washed in 0.1 X SSC at 60C for two hours, followed by two 10-min rinses in 0.5X SSC
at room temperature and dehydrated in graded ethanols containing 300 mM ammonium
acetate. Sections hybridized with sense radiolabelled riboprobe served as a control.
Lectin Staining and Immunohistochemical Analysis
Following in situ hybridization and prior to emulsion autoradiography, some
sections were stained with either isolectin from Griffonia simplicifolia (GSA I-B4, to
identify microglia) according to previously published procedures (Streit, 1990) or with
anti-GFAP (glial fibrillary acidic protein, to identify astrocytes) in order to determine
cellular localization of RBS11 or ffactalkine.
For lectin staining, sections were soaked in PBSC (PBS containing 0.3% Triton
X-100 and 0.1 mM CaCh, MgC^, and MnC^ ) at 4C for 1 hour before being incubated
with GS I-B4-HRP (10 pg/ml) in PBSC overnight at 4C. The sections were briefly
rinsed with PBS and the lectin binding sites were visualized using 3,3 -
diaminobenzidine-H202.
For GFAP staining, sections were rehydrated in PBS for 10 minutes and blocked
with PBS/10% goat sera for 30minutes before incubating overnight in 4C with anti-
GFAP antibody diluted 1:1000 in PBS/10% goat sera. After washing in PBS 3x 10
minutes, sections were then incubated with biotinylated anti-mouse-IgG (from goat) for
45 minutes in room temperature. With another extensive wash using PBS, 3x 10


112
Imai, T., Hieshima, K., Haskell, C., Baba, M., Nagira, M., Nishimura, M.,
Kakizaki, M., Takagi, S., Nomiyama, H., Schall, T.J., and Yoshie, O. (1997b)
Identification and molecular characterization of fractalkine receptor CX3CR1, which
mediates both leukocyte migration and adhesion. Cell 91:521-530.
Imai, T., Yoshida T., Baba, M, Nishimura, M., Kakizaki, M., and Yoshie, O.
(1996) Molecular cloning of a novel T cell-directed CC chemokine expressed in thymus
by signal sequenc trap using Epstein-Barr virus vector. J. Biol. Chem. 271:21514-21521.
Janabi, N., Di Stefano, M., Wallon, C., Hery, C., Chiodi, F., and Tardieu, M.
(1998) Induction of human immunodeficiency virus type 1 replication in human glial
cells after proinflammatory cytokines stimulation: effect of IFNgamma. ILlbeta, and
TNFalpha on differentiation and chemokine production in glial cells. Glia 23:304-315.
Jinno, A., Shimizu, N., soda, Y., Haraguchi, Y., Kitamura, T., and Hoshino, H.
(1998) Identification of the chemokine receptor TER1/CCR8 expression in brain-derived
cells and T cells as a new coreceptor for HTV-1 infection. Biochem. Biophys. Res.
Commun. 243:497-502.
Jones, S.A., Moser, B., and Thelen, M. (1995) A comparison of post-receptor
signal transduction events in Jurkat cells transfected with either IL-8R1 or IL-8R2.
Chemokine mediated activation of p42/p44 MAP-kinase (ERK-2). FEBS Lett. 364:211-
214.
Jones, S.A., Wolf, M., Qin, S., Mackay, C.R., ang Baggiolini, M. (1996) Different
functions for the interleukin 8 receptors (IL-8R) of human neutrophil leukocutes:
NADPH oxidase and phospholipase D are activated through IL-8R1 but not IL-8R2.
Proc. Natl. Acad. Sci.U.S.A. 93: 6682-6686.
Karpus, W.J., Lukcs, N.W., McRae, B.L., Stricter, R.B., Kunkel, S.L., and
Miller, S.D. (1995) An important role for the chemokine macrophage inflammatory
protein-la in the pathogenesis of the T cell-mediated autoimmune disease, experimental
autoimmune encephalomyelitis. J. Immunol. 155:5003-5010.
Kasahara, T., Mukaida, N., Yamashita, K., Yagisawa, H., Akahoshi, T. and
Matsushima, K. (1991) EL-1 and TNF-alpha induction of IL-8 and monocyte chemotactic
and activating factor (MAC) mRNA expression in a human astrocytoma cell line.
Immunology, 74:60-67.
Kelner, G.S., Kennedy, J., Bacon, K.B., Kleyensteuber, S., Largaespada, D.A.,
Jenkins, N.A., Copeland, N.G., Bazan, J.F., Moore, K.W., Schall, T.J., and Zlotonik, A.
(1994) Lymphotactin: a cytokine that represents a new class of chemokine. Science
266:1395-1399.


86
The experimental rat glioma models are useful in studying chemokine and
chemokine receptor expression and function in brain tumors. RG2 and C6 gliomas are
two very widely used rat models in experimental neuro-oncology (Barth et al., 1998).
Both gliomas are chemically induced with different immunogenic responses. RG2
glioma appears to be either weakly or non-immunogenic. This tumor is refractory to a
variety of therapeutic modalities and has an invasive growth pattern as well as uniform
lethality. Compared to RG2 glioma, C6 glioma has a potential to evoke an alloimmune
response partly because it is not syngeneic to any inbred strain. Morioka et al. (1992) has
reported that in RG2 gliomas, increased numbers of microglia were formed around and
infiltrated into the tumor. This indicated that tumor growth in the brain causes microglial
activation and proliferation. However, the question of whether or not these microglial
cells are functional as tumor surveillance cells remains unanswered. The microglial
response in C6 glioma has not been carefully studied.
Our data from the previous chapter demonstrated that CX3CR1 mRNA is
expressed in microglia in vivo. We then set out to determine if this is also true in tumor
tissues. The specific aim of this study was to determine the expression and to specify the
cellular localization of fractalkine and CX3CR1 in experimental rat glioma tissues (RG2
and C6 gliomas). Results generated by this study may provide more information in
understanding brain tumor biology as well as some aspects of the function of fractalkine
and CX3CR1 in the CNS.


5
lines. In the CNS, microglia, astrocytes as well as neurons have been shown to synthesize
chemokine peptides including IL-8, MCP-1, MIP-la, M3P-ip, TCA3 (T-cell activation-
3), and IP-10; expression has been demonstrated from both in vitro and in vivo studies
(Van Meir et al., 1992, Ransohoff et al., 1993, Vanguri and Farber, 1994, Huurwitz et al.,
1995, Hayashi et al., 1995, Glabinski et al., 1997, Xia et al., 1998). A number of brain
tumor tissues can also make chemokine peptides (Desbaillets et al., 1994, Takeshima et
al., 1994).
Chemokines act on different types of cells such as monocytes/macrophages, T
cells, basophils, fibroblasts, endothelial cells, keratinocvtes. melanoma cells, and
neutrophils to elicit their functions. The biological functions of chemokines include
chemotaxis (Colditz et al., 1989, Rampart et al., 1989); changing shape of the target cells
that is important for cell motion (Omann et al., 1987); release of histamine, elastase, p-
glucuronidase, myeloperoxidase, and other enzymes (Kuna et al., 1992, Walz et al., 1987,
Peveri et al., 1988); promotion of adherence (Detmers et al., 1991); induction of the
respiratory burst (Rollins et al., 1991); and increased cell growth (Bordoni et al., 1990).
In addition, chemokines play some roles in normal cellular physiology as well. They
have been shown to be involved in such diverse processes as development, hematopoiesis
(Nagasawa et al., 1998), angiogenesis (Stricter et al., 1995) and apoptosis (Hesselgesser
et al., 1998). In the past few years, numerous evidence has indicated that a number of
chemokines including MIP-la, MIP-ip, RANTES (Cocchi et al., 1995), SDF-a (Oberlin
et al., 1996, Bleul et al., 1996), MCP-2 (Gong et al., 1998), MDC (macrophage-derived
chemokine) (Pal et al., 1997) and eotaxin (Choe et al., 1996) can inhibit HIV entry and/or
viral replication within host cells.


10
a monocytic cell line as the template to isolate these receptor genes. Degenerate primers
were designed based on conserved sequences of CCR1, CXCR1 and CXCR2. Northern
blot analysis indicated that both CCR2 receptors are expressed in monocytes and THP-1
and MonoMac 6 cell lines. While MCP-1 is the only known ligand for CCR2a, MCP-2,
MCP-3 and MCP-4 are found to be additional ligands for CCR2b (Gong et al., 1997,
Combadiere et al., 1995b, Godiska et al., 1997).
Similarly, CCR3 was cloned by several different groups (Combadiere et al.,
1995a, Ponath et al., 1996, Daugherty et al., 1996, Kitaura et al., 1996). This receptor is
expressed in eosinophils and basophils (Uguccioni et al., 1997) and weakly in monocytes
and neutrophils. It is the only receptor, to date, for eotaxin-1 and eotaxin-2 (White et al.,
1997). However, RANTES, MCP-1 (Daugherty et al., 1996), MCP-2 (Heath et al.,
1997), MCP-3 (Ponath et al., 1996), MCP-4 (Garcia-Zepeda et al., 1996a) Leukotactin-1
(Youn et al., 1997) are also ligands for CCR3.
Human CCR4 was isolated from a basophil cell line, whose amino acid sequence
shares only 49% identity to CCR1 (Power et al., 1995). It is expressed at high levels in
thymus, T cells, B cells, monocytes, platelets, and to a lower level in spleen. The
identification of its ligand(s) has been controversial. While Power et al. demonstrated
detectable Ca2+ dependent Cl' currents in CCR4 expressing oocytes by MIP-la, MCP-1
and RANTES application, Imai et al. (1997a) failed to stimulate Ca"~ mobilization in
CCR4 expressing 293/EBNA cells with these ligands. However, they successfully
identified TARC (thymus and activation-regulated chemokine) as a specific ligand for
CCR4. Recently, Imai et al. (1998) demonstrated that MDC (macrophage-derived
chemokine) is also a ligand for CCR4.