THE EFFECTS OF OPIOID GROWTH FACTOR (OGF) AND LOW-DOSE NALTREXONE (LDN) ON THE CD4 + T CELL-

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1 The Pennsylvania State University The Graduate School Department of Neural and Behavioral Sciences THE EFFECTS OF OPIOID GROWTH FACTOR (OGF) AND LOW-DOSE NALTREXONE (LDN) ON THE CD4 + T CELL- BASED IMMUNE RESPONSE TO MOG PEPTIDE A Thesis in Anatomy by Daniel P. McHugh 2011 Daniel P. McHugh Submitted in Partial Fulfillment Of the Requirements for the Degree of Master of Science May 2011

2 The thesis of Daniel P. McHugh was reviewed and approved* by the following: Patricia J. McLaughlin Professor of Neural and Behavioral Sciences Director of Graduate Program in Anatomy Thesis Advisor Robert H. Bonneau Professor of Microbiology and Immunology and Pediatrics Ian S. Zagon Distinguished Professor of Neural and Behavioral Sciences *Signatures are on file in the Graduate School. ii

3 ABSTRACT Multiple Sclerosis (MS) is an inflammatory, demyelinating disorder that affects the central nervous system (CNS). This disorder is classified as autoimmune in nature due to the elevated levels of CD4 + T cells that are specific for major myelin proteins. Though the cause of MS is unknown, it has been suggested that genetically susceptible individuals have a predisposition to develop MS after exposure to certain environmental factors. The four types of MS are relapsing-remitting (RR)-MS, primary progressive (PP)-MS, secondary progressive (SP)- MS, and primary relapsing (PR)-MS. (RR)-MS patients generally recover in between relapses and symptoms subside. However, over half of the (RR)-MS patients that do not receive treatments early in their disease course gradually develop secondary progressive (SP)-MS. Primary relapsing MS is an extremely rare form of MS that is characterized by neurological decline with exacerbations that occur later in life. Experimental autoimmune encephalomyelitis (EAE) is a frequently studied model of MS. EAE can be induced in many ways but a common method is to prime mice with specific myelin peptide epitopes in adjuvant. The specific myelin peptide used in this study was the myelinoligodendrocyte glycoprotein (MOG ). EAE produces a T cell-mediated autoimmune response that closely mimics MS. Due to the similarities between EAE and MS, EAE has become a favored model for evaluating the pathogenesis of and therapies for MS. Opioid growth factor (OGF) has the ability to suppress lymphocyte proliferation in cell cultures that are stimulated with PHA and in the secondary lymphoid organs of mice injected with MOG peptide. The action of OGF is mediated by OGFr, which is responsible for causing signaling events that upregulate the expression of the cyclin-dependent inhibitory kinases p16 and p21. By inhibiting the downstream signaling events that occur in a stimulated iii

4 and actively proliferating lymphocyte, OGF is able to suppress lymphocyte proliferation in the lymphoid organs of mice injected with MOG peptide. OGF not only suppresses proliferation of stimulated lymphocytes but also represses the incidence and progression of disease symptoms in EAE mice. Recently, OGF has been shown to act as an immunomodulatory agent on T lymphocytes in culture that were stimulated with phytohemagglutinin (PHA). OGF was able to suppress T lymphocyte proliferation in a dose-dependent manner but OGF required the lymphocytes to be stimulated and proliferating. These data demonstrated that the OGF-OGFr axis is capable of suppressing T lymphocyte proliferation in vitro. In order to determine if OGF could act as an immunosuppressive agent in the spleens and inguinal lymph nodes (ILNs) of MOG-induced EAE mice, the following animal study was performed. Mice were injected with MOG peptide that was emulsified in complete Freund s adjuvant in the right and left flanks on days 0 and 7, respectively. Mice were treated with OGF or LDN beginning on the day of disease induction (day 0) and were isolated from mice on days 5, 12, and 20. OGF and LDN were most effective at suppressing lymphocyte proliferation in the spleens of EAE mice 5 and 12 days post-initial injection with MOG peptide. On day 5, OGF and LDN caused a 40% and 26% decrease in the number of cells in the spleens of EAE mice, respectively. OGF suppressed CD4 + and CD8 + T cell proliferation more effectively than B cells. OGF and LDN were most effective at suppressing B and T cell proliferation in the ILNs of EAE mice 5 days post-initial injection with MOG peptide. On day 5, OGF and LDN treatment resulted in a 49% and 35% decrease in the number of cells in the draining ILNs of EAE mice, respectively. However, OGF treatment did not suppress B and T lymphocyte proliferation in the draining ILNs of EAE mice on days 12 and 20. iv

5 Moreover, OGF suppressed B and T lymphocyte proliferation in the spleens and ILNs of mice injected with CFA and pertussis toxin, without MOG peptide, but not the extent of suppression seen in EAE mice treated with OGF. In summary, these data demonstrate that OGF is effective at suppressing lymphocyte proliferation and repressing EAE symptoms early in the disease course. Thus, exogenous application of OGF is able to act as an immunosuppressive agent on cells that are stimulated in culture as well as in mice that are induced to have EAE through MOG peptide injection. v

6 TABLE OF CONTENTS Page Number List of Figures List of Abbreviations Acknowledgements viii x xiii Chapter 1. Introduction 1.1 Multiple Sclerosis Clinical Subtypes of Multiple Sclerosis T Cell-Mediated Immunity Enkephalins and the OGF-OGFr Axis Experimental Autoimmune Encephalomyelitis Myelin Oligodendrocyte Glycoprotein-Induced EAE Endogenous Opioid Peptides and T Cell Immunology T Cell Immunology in the EAE Model of Multiple Sclerosis The Effects of OGF on T Cell Proliferation in EAE Conclusions 10 Chapter 2. Objectives 11 Chapter 3. Materials and Methodology 3.1 Mice and Induction of EAE Behavior Score Treatment Groups 13 vi

7 3.3 Isolation of Lymphocytes Quantification of Lymphocytes by Flow Cytometry Quantification of Lymphocytes After Injections With CFA and Pertussis Toxin Statistical Analysis 16 Chapter 4. Results 4.1 Quantification of Lymphocytes in Non-Challenged and MOG-Induced EAE Mice Behavior Score of MOG-Induced EAE Mice Treated With OGF and LDN The Effects of OGF and LDN on Splenocyte Populations in MOG-Induced EAE Mice The Effects of OGF and LDN on Lymphocyte Populations in the Inguinal Lymph Nodes of MOG-Induced EAE Mice The Effects of OGF and LDN on Lymphocyte Populations in the Spleen and Inguinal Lymph Nodes of Non-Challenged Mice The Effects of OGF and LDN on the Number of Lymphocytes in Mice Treated With Complete Freund s Adjuvant and Pertussis Toxin 57 Chapter 5. Discussion 60 REFERENCES 69 vii

8 LIST OF FIGURES Figure 4.1 Figure 4.2 Quantification of Lymphocytes in the Spleens of Non-Challenged and EAE Mice 19 Quantification of Lymphocytes in the Inguinal Lymph Nodes of Non-Challenged and MOG-Induced EAE Mice 20 Figure 4.3 Behavior Score of EAE Mice Over Time 23 Figure 4.4 Figure 4.5 Figure 4.6 Figure 4.7 Figure 4.8 Figure 4.9 Figure 4.10 Figure 4.11 Figure 4.12 Figure 4.13 Figure 4.14 Figure 4.15 Effects of OGF and LDN on B and T Cell Percentages in the Spleens of MOG-Induced EAE Mice on Day 5 Post-Initial MOG Challenge 28 Effects of OGF and LDN on B and T Cell Percentages in the Spleens of MOG-Induced EAE Mice on Day 12 Post-Initial MOG Challenge 30 Effects of OGF and LDN on B and T Cell Percentages in the Spleens of MOG-Induced EAE Mice on Day 20-Post Initial MOG Challenge 32 The Effects of OGF and LDN on Lymphocyte Numbers in the Spleens of MOG-Induced EAE Mice 34 Histogram of the Number of Lymphocytes in the Spleens of Non- Challenged and MOG-Induced EAE Mice on Days 5, 12, and Effects of OGF and LDN on B and T Cell Percentages in the Right ILNs of EAE Mice on Day 5 Post-Initial MOG Challenge 42 Effects of OGF and LDN on B and T Cell Percentages in the Left ILNs of EAE Mice on Day 5 Post-Initial MOG Challenge 44 Effects of OGF and LDN on B and T Cell Percentages in the Right ILNs of EAE Mice on Day 12 Post-Initial MOG Challenge 46 Effects of OGF and LDN on B and T Cell Percentages in the Left ILNs of EAE Mice on Day 12 Post-Initial MOG Challenge 48 Effects of OGF and LDN on B and T Cell Percentages in the Right ILNs of EAE Mice on Day 20 Post-Initial MOG Challenge 50 Effects of OGF and LDN on B and T Cell Percentages in the Left ILNs of EAE Mice on Day 20 Post-Initial MOG Challenge 52 Histogram of the Number of Lymphocytes in the ILNs of Non- Challenged and MOG-Induced EAE Mice on Days 5, 12, and viii

9 Figure 4.16 Figure 4.17 The Effects of OGF and LDN on the Non-Stimulated Lymphocytes in the Spleens and ILNs of Non-challenged Mice 56 Histograms Comparing the Effects of OGF and LDN on Mice Immunized with CFA and Pertussis Toxin versus EAE Mice 59 ix

10 LIST OF ABBREVIATIONS ANOVA APC APC β BSA analysis of variance antigen presenting cell allophycocyanin beta bovine serum albumin C degrees Celsius CD CFA CSF CNS δ EAE et al FACS FITC G 1 HLA IACUC IFN ILNs IMDM i.p. cluster of differentiation complete Freund s adjuvant cerebrospinal fluid central nervous system delta experimental autoimmune encephalomyelitis and others Fluorescence Activated Cell Sorting fluorescein isothiocyanate gap 1 phase of cell cycle human leukocyte antigen Institutional Animal Care and Use Committee interferon inguinal lymph nodes Iscove s-modified Dulbecco s Media intraperitoneal x

11 κ kg LDN mg/kg MHC kappa kilogram low-dose naltrexone milligrams per kilogram major histocompatibility complex ml mm MOG MOG MOG-EAE milliliter millimolar myelin oligodendrocyte glycoprotein myelin oligodendrocyte glycoprotein amino acid sequence MOG-induced EAE mice µ mu µg microgram µl microliter MS NTX ng OGF OGFr PE PBS multiple sclerosis naltrexone nanogram opioid growth factor opioid growth factor receptor phycoerythrin phosphate buffered saline % percent xi

12 PHA PPE S TGF-β Th v/v w/v phytohemagglutinin preproenkephalin gene synthesis phase of cell cycle transforming growth factor-beta helper T cell volume concentration (volume/volume) mass concentration (weight/volume) xii

13 ACKNOWLEDGEMENTS I would like to thank Dr. McLaughlin, my thesis advisor, for her guidance during my years at Penn State Hershey. She was extremely understanding and supportive during class work and research periods. Dr. McLaughlin was always available to discuss any concerns and to answer any questions that I had in order to complete my thesis. After completing my work here at PSU-Hershey, I realize how difficult it would have been without good advisors like Dr. McLaughlin and Dr. Zagon. In addition to Drs. McLaughlin and Zagon, I would like to thank Dr. Bonneau, who played a critical role on my committee and served as an excellent mentor over the past three years of my life. Dr. Bonneau was always available to answer questions about immunology and the future directions of my project. His invaluable academic advice carried me through my graduate school years at Penn State. I would also like to thank Renee Donahue and Anna Campbell for all of their help and advice during the course of my thesis work. The initiation and completion of this thesis wouldn t have been possible without the help of Renee. Though she was busy finishing her own dissertation, she was always willing to go above and beyond her daily expectations and help me with any task or answer any of my questions. Anna was also available to answer any questions and provided suggestions on numerous research topics throughout the past two years. Lastly, I would like to thank my parents and extended family members for their continuous support and patience throughout my entire life. I was fortunate to have an amazing support system throughout my undergraduate and graduates years of education. Without my family and friends none of this would have been possible. xiii

14 Chapter 1 INTRODUCTION 1.1 Multiple Sclerosis Multiple sclerosis (MS) in an inflammatory autoimmune demyelinating disorder that affects the central nervous system (CNS) and usually presents between 20 and 40 years of age [Sospedra et al., 2005]. MS affects over 400,000 people in the United States alone and 2 million people globally, making it the most common neurological disorder in young adults [Forte et al., 2007; Irvine, and Blakemore, 2008]. Jean-Martin Charcot, a French neurologist, first described the disease in 1868 as an accumulation of inflammatory cells within the brain and spinal cord white matter of patients with intermittent episodes of neurologic dysfunction [Charcot, 1868]. Proteolytic digestion of the blood-brain barrier and myelin protein by serine proteases is known to contribute to the development and progression of MS [Terayama et al., 2005]. The inflammatory infiltrates that enter the central nervous system, an immune privileged site, may be the primary cause of demyelination. Alternatively, it is thought that these immune infiltrates may accumulate at sites of prior injury and contribute to the progressive tissue damage [Ercolini et al., 2006]. MS is considered autoimmune in nature because it is associated with elevated levels of CD4 + T cells specific for the major myelin proteins, as well as for the presence of myelin-specific antibodies [Ercolini et al., 2006]. Although MS remains an incurable disease and its induction is unknown, there are multiple factors believed to contribute to the pathogenesis of MS, including genetics and additional environmental triggers. MS is believed to occur in genetically susceptible individuals after exposure to undefined environmental factors [Oksenberg et al., 2005]. The general population prevalence of MS varies 1

15 between /100,000 in Northern Europe and North America, and 6-20/100,000 in low-risk areas such as Japan [Sospedra et al., 2005]. The prevalence of disease substantially increases in family members of MS patients; first-degree relatives of affected individuals have a 2%-5% higher risk to develop MS and monozygotic twins have a concordance rate of 25% [Sospedra et al., 2005]. Investigators have searched for individual susceptibility genes and the data are strongest for one or more genes on chromosome 6p21 in the area of the major histocompatibility complex (MHC) [Sospedra et al., 2005]. A region at or near the HLA-DRB1 locus in the MHC, influences the risk of MS [Ramagopalan et al., 2009]. Certain environmental factors have been found to contribute to the etiology of MS. Also, individuals of Northern European descent are more likely to develop the disease due to genetic predispositions. Insufficient exposure to sunlight has been proposed as a key environmental factor for the disease [Ramagopalan et al., 2009]. Studies have shown that MS patients are deficient in vitamin D [Nieves et al., 1994] and dietary vitamin D intake reduces disease risk [Munger et al., 2006]. There are high prevalence rates for the disease in regions that have a large number of Northern European descendents [Sospedra et al., 2005]. The dominant haplotype of Northern Europe is HLA-DRB1 * 1501 and it increases the risk of MS by 3-fold [Ramagopalan et al., 2009]. Recent studies have linked genetic susceptibility to the environmental risk factor of insufficient vitamin D exposure [Ramagopalan et al., 2009] Subtypes of Multiple Sclerosis There are four major forms of MS: relapsing-remitting (RR)-MS, primary progressive (PP)-MS, secondary progressive (SP)-MS, and primary relapsing (PR)-MS. (RR)-MS is the most common form of the disease and occurs in 85%-90% of patients [Sospedra et al., 2005]. 2

16 This form of MS affects women about twice as often as men [Sospedra et al., 2005]. Over half of (RR)-MS patients who do not receive treatment early in their disease courser later develop secondary progressive (SP)-MS. Approximately 10%-15% of patients are diagnosed with (PP)- MS that is characterized by a subtle disease onset but a steady progression of disease severity [Sospedra et al., 2005]. Primary relapsing MS is an extremely rare form of MS that is characterized by neurological decline with exacerbations that occur later in life. Although the disease has four different courses it can take, it is unclear as to which factors are responsible for the ambiguity. 1.2 T Cell-Mediated Immunity T lymphocytes are components of the adaptive immune system that start and complete their development in the thymus. Once development is completed, they enter the bloodstream as naïve T cells, which are mature recirculating T cells that have not yet encountered their specific antigen [Dustin et al., 2003]. If naïve T cells encounter their specific antigen, they are induced to proliferate and differentiate into armed effector T cells [Tseng and Dustin, 2002]. Antigenpresenting cells (APCs) express costimulatory molecules on their surfaces that are specifically designed to present antigens and to prime naïve T cells [Kasper and Shoemaker, 2010]. The interaction between the APC and the T cell is an essential step in mounting an adaptive immune response [Kasper and Shoemaker, 2010]. Dendritic cells, monocytes, macrophages and B cells are all antigen-presenting cells that are capable of presenting antigens to naïve T cells [Kasper and Shoemaker, 2010]. There are two major classes of T cells that have different effector functions and are distinguished by the expression of the cell-surface proteins CD4 and CD8 [Zamoyska, 1998]. 3

17 CD4 + and CD8 + T cells recognize different classes of MHC molecules [Wang et al., 2002]. CD4 binds to the MHC class II molecule and CD8 binds to the MHC class I molecule [Wang et al., 2002]. The peptides that stimulate T cells to proliferate are recognized only when bound to an MHC molecule [Baker et al., 2002]. During differentiation, CD4 + T cells can become polarized in response to exposure to specific interleukins [Kasper and Shoemaker, 2010]. A CD4 + naïve T cell exposed to IL-12 will be polarized to a Th1 cell and will begin to secrete interferon gamma (IFN- ). The same CD4 + naïve T cell exposed to IL-4 will be polarized to a Th2 cell and one exposed to TGF-, IL-1, IL-6, IL-21 and IL-23 will be polarized into a Th17 cell [Kasper and Shoemaker, 2010]. Th17 cells are responsible for producing IL-17 and IL-22, which are involved in the migration and trafficking of neutrophils [Kasper and Shoemaker, 2010]. On acute and chronic MS plaques, IL-17 receptors have been identified immunohistochemically in patients with MS [Kasper and Shoemaker, 2010]. Th1 cells are involved in MS pathogenesis and Crohn s disease, whereas Th2 cell involvement has been implicated in asthma and ulcerative colitis [Kasper and Shoemaker, 2010]. 1.3 Enkephalins and the OGF-OGFr Axis Endogenous opioids are internally derived peptides that bind to classical and nonclassical opioid receptors [Gutstein and Akil, 2001; Wang et al., 2008; Wollemann and Benyhe, 2004]. The endogenous opioid system, originally found to be involved in neurotransmission, has a variety of biological functions, including regulation of cell proliferation, inflammation, and immunity [Gutstein and Akil, 2001; Wang et al., 2008; Wollemann and Benyhe, 2004]. All of the endogenous opioid peptides are derived from three precursor proteins: proopiomelanocortin, proenkephalin, and prodynorphin [Przewlocki et al., 2001; Peterson et al., 4

18 1998]. Enkephalins, endorphins, endomorphins and dynorphins are the four types of endogenous opioids that have a high degree of sequence homology but that differ in action and receptor affinity [Drolet et al., 2001; Okada et al., 2002]. [Met 5 ]-enkephalin is a native opioid peptide that is encoded by the preproenkephalin A (PPE) gene and is negative growth regulator [Akil et al., 1984; Noda et al., 1982]. Investigators initially believed that this native peptide was only found in neural cells [Akil et al., 1984], but subsequent studies revealed that [Met 5 ]-enkephalin regulates neural and non-neural cell proliferation [Zagon et al., 2002]. Due to its large distribution and variety of biological roles, this peptide has been termed the opioid growth factor (OGF) [Zagon et al., 2002]. In a variety of cells and tissues examined from both humans and animals, OGF was the primary opioid peptide involved with cell growth [McLaughlin et al., 2000; McLaughlin et al., 1999]. All other opioid peptides that altered the growth of specific tissues were other products of the PPE gene that encodes OGF [Zagon et al., 2002]. The action of OGF is mediated by the OGF receptor (OGFr), which is localized on the outer nuclear envelope of eukaryotic cells [Zagon et al., 2002]. Although the OGF-OGFr axis fulfills the pharmacological properties of opioid peptides that bind with classical opioid receptors, OGFr has genomic and proteomic properties that differ from the classical opioid receptors (Zagon et al., 2002). The OGF-OGFr axis upregulates the cyclin-dependent inhibitory kinase pathway, specifically p16 and p21 [Cheng et al., 2007a,b, 2009a] and delays the G 1 /S transition of the cell cycle [Cheng et al., 2007a]. Through the use of nuclear localization signals that are encoded on the OGFr for guidance by karyopherin through the nuclear pore, the OGF- OGFr complex is able to undergo nucleocytoplasmic transport [Zagon et al., 2005a; Cheng et al., 2009b]. Though OGF-OGFr interactions are able to inhibit cell proliferation and maintain 5

19 homeostasis of cellular renewal, the blockade of OGF from OGFr through continuous exposure to opioid antagonists (e.g., Naltrexone) or neutralization of OGF by antibodies causes an increase in cancer cell proliferation but has no affect on lymphocytes that are not actively proliferating [Zagon et al., 2009a and 2010b]. Numerous reports have provided evidence that there is a connection between the OGF- OGFr axis and autoimmune diseases [Zagon et al, 2010a]. In a recent study, OGF administered to mice with myelin oligodendrocyte-induced experimental autoimmune encephalomyelitis (EAE) repressed the onset and progression of EAE [Zagon et al, 2010a]. Lymphoid cells that were isolated from the spleen and then stimulated by phytohemagglutinin (PHA) were depressed in number by OGF treatment [Zagon et al., 2010b]. However, PHA stimulation is not a natural biological process and does not accurately represent an in vivo stimulation. Numerous investigators have studied the effects of opioid peptides on T lymphocytes and have presented conflicting results [Zagon et al., 2010b]. [Met 5 ]-enkephalin has been found to increase, decrease or have no effect on T lymphocyte proliferation [Zagon et al., 2010b]. 1.4 Experimental Autoimmune Encephalomyelitis EAE is a popular and frequently studied model of MS. There are multiple ways that investigators have induced EAE in mice and these methods have allowed investigators to study the immunopathology of the disease as well as the effects of treatments. EAE can be induced by priming mice with whole myelin proteins or specific myelin peptide epitopes in adjuvant [Ercolini et al., 2006], through adoptive transfer of myelin-specific CD4 + T cells [Ercolini et al., 2006], or through a viral infection [Tsunoda et al., 2007]. Actively induced EAE produces a T cell-mediated autoimmune response that closely mimics MS. Demyelination and paralytic 6

20 episodes are associated with the infiltration of myelin-specific inflammatory CD4 + T cells into the CNS [Ercolini et al., 2006]. EAE and MS are similar pro-inflammatory diseases because levels of certain cytokines are upregulated and both are characterized by demyelination and neurodegeneration [Kennedy et al, 1992]. An uncommitted CD4 + T cell that becomes polarized to Th1 cells will begin to express and secrete IFN- [Kasper and Shoemaker, 2010]. Th2 cells, which become polarized in the presence of IL-4, immediately begin to secrete IL-4 and IL-13 [Kasper and Shoemaker, 2010]. Th17, a recently identified helper T cell subset, becomes differentiated in the presence of transforming growth factor (TGF)-, IL-1, IL-6, IL-21, and IL- 23 and immediately begins to secrete IL-17 [Kasper and Shoemaker, 2010]. Because of the similarities between EAE and MS, EAE has become a favored model for evaluating the pathogenesis of and therapies for MS Myelin Oligodendrocyte Glycoprotein-Induced EAE Myelin oligodendrocyte glycoprotein (MOG) is a myelin protein found on the outer surface of the oligodendrocytes. Although MOG makes up less than 0.1% of the CNS myelin, its location on the oligodendrocyte makes it an easy target for the immune system [Rodriguez, 2007]. MOG-induced EAE is a common murine model used by investigators because MOG- EAE produces a chronic disease course [Gold et al., 2006] that contains the three main phases of MS, inflammation, demyelination, and neurodegeneration [Jones et al., 2008]. Not only does the model produce the three main phases of MS, but it also elicits a similar immune response found in MS patients. The EAE model in C57BL/6 mice produces a T and B lymphocyte response that demonstrates the cognate interactions involved in a T-cell dependent immune response. 7

21 1.5 Endogenous Opioid Peptides and T Cell Immunology It has been demonstrated that all 3 classical opioid receptors,,, and are present on immune cells [Sharp et al., 1998] and that opioid peptides can have affects on immune cell function [Wybran et al., 1979]. Numerous investigators have studied the effects of opioid peptides on T lymphocytes; however their reports present conflicting results [Carr et al., 1996; McCarthy et al., 2001]. Some of the differences in the methods used in these studies included whether the peptides were natural or synthetic, the concentration and/or class of peptide and whether or not the cells were stimulated. -endorphin and [Met 5 ]-enkephalin (OGF) have diverse affects on T lymphocyte proliferation and have received the most focus. In the case of - endorphin, studies report an increase [Hemmick and Bidlack, 1990; Gilmore and Weiner, 1989] and decrease [Marchini et al., 1995; Hough et al., 1990] in T cell number. Investigators found that [Met 5 ]-enkephalin (OGF) caused an increase [Kowalski et al, 1998; Hucklebridge et al., 1989, 1990], a decrease [Kamphius et al., 1998; Shahabi et al., 1995], or had no effect [Wybran et al, 1995; Gilman et al., 1982] on T lymphocyte number. A recent study conducted by Zagon et al, revealed that only exogenously delivered OGF at concentrations ranging from 10-4 to 10-7 M altered T lymphocyte proliferation, but the cells required stimulation and had to be actively proliferating [Zagon et al., 2010b]. Because the OGF-OGFr axis can be modulated by exogenous OGF, the axis is thought to serve as a means for inducing immunosuppression facilitated by native biological components and physiological components. 1.6 T Cell Immunology in the EAE Model of Multiple Sclerosis After investigators observed that the transfer of myelin-specific CD4 + T cells could induce EAE, investigators demonstrated that EAE could be directly induced with autoreactive T 8

22 cells transferred to naïve animals [Sospedra et al., 2005]. However, unlike other autoimmune diseases such as myasthenia gravis, the transfer of antibodies cannot induce EAE. This evidence allowed investigators to conclude that MS is likely to be a T cell-mediated autoimmune disease [Sospedra et al., 2005]. Evidence suggests that Th1-polarized CD4 + T cells are the effector cells in MS that contribute to immune pathogenesis as they do in the EAE model [Kasper and Shoemaker 2010]. Evidence on the induction and maintenance of MS still favors CD4 + autoreactive T cells as an essential factor for the pathogenesis of MS due to the following evidence-based conclusions: CD4 + T cells contribute to the CNS- and CSF-infiltrating inflammatory cells in MS, there is a genetic risk conferred by HLA-DR and HLA-DQ molecules, and many other components of the adaptive and innate immune function are partly controlled by CD4 + helper T cells [Sospedra et al., 2005]. Th17 CD4 + T cells, which produce IL-17, are another cell type involved in the disease progression of MS [Kasper and Shoemaker, 2010]. IL-17 receptors, which are expressed at high levels on MS endothelial cells, have been identified immunohistochemically in patients with acute and chronic MS [Kasper and Shoemaker, 2010]. IL-17 and IL-22 are involved in the recruitment of neutrophils to sites of inflammation. This evidence suggests that Th17 T cells are involved in the promotion of CNS inflammation [Kebir et al., 2007]. CD8 + T cells, which are also found in MS lesions, can mediate suppression on CD4 + lymphoproliferation through the secretion of perforin [Kasper and Shoemaker, 2010]. Perforin is cytotoxic and leads to CD4 + cell inactivation, which suggests that CD8+ T cells have a regulatory function in MS disease progression [Kasper and Shoemaker, 2010]. Even though CD8 + cells have the ability to suppress injury, they can also transect axons, induce oligodendrocyte death, and promote vascular permeability at the sites of MS lesions. [Kasper and 9

23 Shoemaker, 2010]. Because CD8 + T cells have the capability of suppressing and/or facilitating the immune pathogenesis of MS, the mechanisms involving CD8 + function are essential to understanding their precise role in MS. 1.7 The Effects of OGF on T Cell Proliferation in EAE There is little evidence on the effects of OGF on T cell proliferation in the EAE model of MS. In a study conducted by Zagon et al, splenic-derived lymphocytes that were stimulated in vitro with phytohemagglutinin (PHA) and treated daily with OGF showed a reduction in cell number [Zagon et al, 2010a]. The administration of exogenous OGF to MOG-induced EAE mice suppressed DNA synthesis and resulted in a subsequent decrease in T lymphocyte proliferation [Zagon et al, 2010a]. Investigators concluded that the decrease in T lymphocyte number had prevented or attenuated the disease onset and progression, respectively. 1.8 Conclusions MS remains an incurable disease and is considered to be autoimmune in nature because it is associated with elevated levels of CD4 + T cells specific for the major myelin proteins. Numerous investigators have studied the effects of opioid peptides on T lymphocytes and have presented conflicting results. [Met 5 ]-enkephalin has been found to increase, decrease, or have no effect on T lymphocyte proliferation. In the recent studies, the lymphocytes required stimulation and had to be actively proliferating for OGF to have an effect. The following experiments are the in vivo correlate of in vitro studies previously performed in our laboratory [Zagon et al, 2010a]. 10

24 Chapter 2 OBJECTIVES The hypothesis of this research is that endogenous opioid peptides regulate B and cell populations in the secondary lymphoid organs of MOG-induced EAE mice. It is also hypothesized that endogenous opioids regulate the expansion of B and T lymphocytes in the secondary lymphoid organs of mice challenged with CFA and pertussis toxin. Specific Aim 1: To quantify the number of CD4 + T cells, CD8 + T cells, and B cells in the spleens and inguinal lymph nodes of MOG-induced EAE mice. Specific Aim 2: To determine whether daily administration of OGF or LDN alters the percentage of CD4 + T cells, CD8 + T cells, and B cells isolated from the spleen and inguinal lymph nodes of MOG-induced EAE mice. Specific Aim 3: To determine whether daily administration of OGF or LDN alters the total number of lymphocytes in the spleens and inguinal lymph nodes of mice injected with complete Freund s adjuvant and pertussis toxin. 11

25 Chapter 3 MATERIALS AND METHODOLOGY 3.1 Mice and Induction of EAE Six to eight week-old C57BL/6 female mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were housed in a controlled temperature room (22-25 C) and standard rodent diet and water were available ad libitum. All experiments were conducted in accordance with protocol approved by the Penn State Hershey Institutional Animal Care and Use Committee. To induce EAE, a total of 800 g of mouse MOG (Penn State College of Medicine Core Research Facilities, 99% purity) was injected into mice after being dissolved in phosphatebuffered saline (PBS) and emulsified in complete Freund s adjuvant (CFA, DIFCO Laboratories, Lawrence, KS), and supplemented with 500 g heat-inactivated Mycobacterium tuberculosis (DIFCO Laboratories). On day 0, 400 g of MOG peptide was injected subcutaneously in the right flank and an additional 400 g of MOG peptide was injected in the left flank on day 7. Intraperitoneal (i.p.) injections of 500 ng of pertussis toxin (List Biological Laboratory, Campbell, CA) dissolved in 200 l PBS were administered on days 0 and Behavior Score The behavior scores of EAE mice from each of the experimental groups were assessed beginning on day 9 post-initial MOG injection. Each mouse was placed on a flat surface and tail tonicity and righting reflex were evaluated. The mice were placed on a wire-mesh screen and 12

26 their ability to grip was noted along with any observed foot faults. The behavior of each mouse was scored using a modified scale [Suen et al., 1997; Zagon et al., 2009, 2010] of The scores for each category were additive along with gradations for intermediate behavior: a 0 was given if there were not any clinical signs of disease; 1 point was assigned for each of the different gaits that could be observed (slightly abnormal, wobble or severe); 0.5 points were given if there was a slow righting reflex and an additional 0.5 points was given if the righting reflex was absent. Each hindlimb was observed while the mice were inverted on a mesh screen: 0.5 points were given for any faults/slips, 0.5 points were given if the hindlimb was near paralysis and an additional 0.5 points was given if the hindlimb was completely paralyzed. Mice that experienced forelimb paralysis were given a score of 9 and any mouse that died from EAE was assigned a score of Treatment Groups The experiments within each specific aim contained different groups of mice. In the first specific aim, one group of mice did not receive any immunizations and served as the normal control group. A second group of mice was challenged with MOG peptide only. These two groups served as baseline controls for the normal expression of lymphocytes in normal mice and mice with EAE, respectively. During the experiments for the second and third specific aims, there were four treatment groups that were observed daily for behavior. In the second specific aim, mice were challenged with MOG peptide. However, in the third specific aim, mice were challenged with supplemented CFA and pertussis toxin only. The first group of mice served as a control for both specific aims and consisted of normal, unchallenged C57BL/6 mice; these mice did not receive 13

27 any daily injections. The second group of mice received daily i.p. injections of 200 l of PBS [Zagon et al., 2009]; this group was referred to as the MOG+Vehicle group. The third group of mice received daily i.p. injections of 10 mg/kg of OGF (Sigma Chemicals, St. Louis, MO) beginning on the initial day of injections (day 0); this group of mice was referred to as the MOG+OGF. The mice in the fourth experimental group received i.p. injections of 0.1 mg/kg of naltrexone (Sigma Chemicals, St. Louis, MO) dissolved in PBS. This treatment is referred to as low-dose Naltrexone (LDN). Saline, OGF, or LDN injections began on the initial day of MOG injection. 3.3 Isolation of Lymphocytes Mice (3/group) were euthanized by cervical dislocation on days 5, 12, and 20 after initial injection with MOG peptide. The spleens were removed and mechanically dissociated using a 60-mesh stainless steel screen. The splenocytes were collected and the red blood cells from the spleen were lysed with a hypotonic saline solution (17 mm Tris, 0.14 mm NH 4 Cl, ph=7.65) for 5 minutes in a 37 C water bath. Lymphocytes were isolated from inguinal lymph nodes of each mouse and mechanically dissociated using a 60-mesh stainless steel screen. Lymph nodes isolated from normal mice were pooled and dissociated together. However, lymph nodes from mice injected with MOG peptide were processed and counted separately. The inguinal lymph nodes were not treated with the hypotonic saline solution. The resulting lymphocytes were resuspended in Iscove s-modified Dulbecco s media (IMDM) and were counted with a hemacytometer after the addition of trypan blue dye. The IMDM was supplemented with 10% (v/v) fetal bovine serum, 0.075% (w/v) sodium bicarbonate, % (v/v) -mercaptoethanol, 100 units/ml penicillin, and 100 g/ml streptomycin sulfate. 14

28 The splenic and inguinal lymph node-derived lymphocytes were counted and used immediately for standard flow cytometry. 3.4 Quantification of Lymphocytes by Flow Cytometry To determine the number of CD4 + T cells, CD8 + T cells, and B cells present, standard flow cytometric analysis of cell surface markers on splenic and inguinal lymph node-derived lymphocytes was performed. Lymphoid cells were isolated and quantified from each of the four different treatment groups on days 5, 12, and 20. Fc receptors were blocked with an anti- CD16/32 antibody obtained from 2.4G2 hybridoma cell culture supernatants [Bonneau et al., 2006] supplemented with 10% mouse serum (Sigma Aldrich). The cell surface expression of CD4, CD8 and B220 were detected using anti-cd4 PE-conjugated antibody (clone GK1.5; ebioscience), anti-cd8a APC-conjugated antibody (clone ; ebioscience), and anti-cd45r FITC-conjugated antibody (clone RA3-6B2; ebioscience), respectively. Following washes with FACS buffer (Hank s-buffered saline solution supplemented with 1% (w/v) BSA), cells were resuspended in FACS buffer and immediately analyzed by flow cytometry. Flow cytometric analysis was conducted on a FACScan flow cytometer (Becton Dickinson, San Diego, CA). Fifty thousand events were collected per sample. The dot plots and histograms were analyzed using FlowJo software (TreeStar, Inc., Ashland, OR) Quantification of Lymphocytes After Injections with CFA and Pertussis Toxin Mice were injected with supplemented CFA and pertussis toxin. The number of lymphocytes in the spleens and inguinal lymph nodes of these mice were counted and analyzed on day 5 post-initial injection with CFA and pertussis. The CFA was supplemented with 500 g 15

29 heat-inactivated Mycobacterium tuberculosis and emulsified in an equal volume of PBS. Subcutaneous injections were given in the right flank on day 0 and intraperitoneal injections of 500 ng of pertussis toxin dissolved in 200 l PBS were administered on days 0 and 2. Comparisons were made against mice that were injected with MOG peptide. 3.5 Statistical Analysis All data were analyzed (GraphPad Prism Software, La Jolla, CA) using one-way analysis of variance, with subsequent comparisons made using Newman-Keuls tests. P values less than 0.05 were considered statistically significant. 16

30 Chapter 4 RESULTS 4.1 Quantification of Lymphocytes in Non-Challenged and MOG-Induced EAE Mice Splenic and inguinal lymph node-derived lymphocytes were isolated from nonchallenged mice and MOG-induced EAE (MOG-EAE) mice on days 5, 12 and 20. The lymphocytes from both experimental groups were quantified at each time point. On day 5 (Figure 4.1A), the average number of lymphocytes in control C57BL/6 mice was 4.9 ± 0.4 x 10 7 cells and the average number of lymphocytes in MOG-EAE mice was 6.9 ± 0.3 x 10 7 cells. This finding represents a 42% increase in the number of lymphocytes in the mice treated with the MOG peptide on day 5. The average number of lymphocytes in control and MOG-EAE mice on day 12 (Figure 4.1B) was 5.8 ± 0.7 x 10 7 cells and 1.0 ± 0.05 x 10 8 cells, respectively; this represents a 74% increase relative to non-challenged mice. On day 20 (Figure 4.1C), the average number of lymphocytes in control and MOG-EAE mice were 6.2 ± 0.7 x 10 7 cells and 5.9 ± 0.2 x 10 7 cells, respectively; this represents a 6% increase from non-challenged mice on day 20. Inguinal lymph nodes (ILNs) were removed from each mouse and the numbers of lymphocytes from each sample were quantified on days 5, 12, and 20. On day 5 (Figure 4.2A), the average number of lymphocytes in normal, non-challenged ILNs was 4.1 ± 3.8 x However, the average number of lymphocytes in the right ILNs of MOG-EAE mice was 1.1 ± 0.7 x 10 6 and the average number of lymphocytes in left ILNs of EAE mice was 3.6 ± 3.3 x Though the error bars were small, there was a 2.5-fold increase in the number of lymphocytes in the draining ILNs on the same side in which the MOG peptide was injected. On day 12 17

31 (Figure 4.2B), the average number of lymphocytes in normal ILNs was 3.4 ± 0.2 x 10 5 cells. However, the right ILNs of MOG-EAE mice had 4.0 ± 1.5 x 10 6 cells and the left ILNs had 3.2 ± 1.1 x 10 6 cells. There was an 11-fold increase in the number lymphocytes on the right side in which MOG peptide was injected on day 0 and a 9-fold increase in the number of lymphocytes on the left side that was injected with MOG peptide on day 7. On day 20 (Figure 4.2C), the average number of lymphocytes in normal ILNs was 4.1 ± 0.06 x The average number of lymphocytes in the right ILNs of MOG-EAE mice was 7.6 ± 1.8 x 10 6, which represented a 17-fold increase compared to the control mice. The average number of cells in the left ILNs of MOG-EAE mice was 6.4 ± 0.8 x 10 6 cells, which represented a 15-fold increase. 18

32 Figure 4.1 Quantification of lymphocytes in the spleens of non-challenged and MOG-induced EAE mice. Histograms represent the number of lymphocytes in EAE mice and mice that were not challenged with MOG peptide. (A) Lymphocytes were isolated from non-challenged and EAE mice on day 5, (B) day 12, and (C) day 20. Values represent the means ± SEM for 3 mice per experimental group. Significantly different from normal spleens at p < 0.05 (*). 19

33 Figure 4.2 Quantification of lymphocytes in the inguinal lymph nodes of non-challenged and MOG-induced EAE mice. Histograms represent the number of lymphocytes in inguinal lymph nodes of MOG-induced EAE mice and mice that were not injected with MOG peptide. (A) Lymphocytes were isolated from non-challenged and EAE mice on day 5, (B) day 12, and (C) day 20. Values represent the means ± SEM for 3 mice per experimental group. Significantly different from normal lymph nodes at p < 0.05 (*) or p < (***) and significantly different from right inguinal lymph nodes of challenged mice at p < (+++). 20

34 4.2 Behavior Score of MOG-Induced EAE Mice Treated With OGF or LDN MOG-induced EAE mice were treated with OGF, LDN or saline and were observed daily for behavior. Generally, disease symptoms were not apparent until 8 days after the disease had been induced (day 0). Disease scores were recorded daily and a point system (Section 3.1.1) was used to calculate a daily point value. The daily averages and standard error values for mice from each experimental group were used to plot the disease score over time (Figure 4.3). On days 10 and 11, EAE mice treated with vehicle, OGF, or LDN displayed a slightly abnormal gait as an initial sign of EAE behavior. The average disease scores for EAE mice treated with vehicle, OGF or LDN were only 2.0 ± 0.3, 2.0 ± 0.2, and 1.0 ± 0.1, respectively. On days 11-13, mice from all treatment groups began to display foot faults when inverted on the mesh grid. However, EAE mice in the LDN and vehicle treatment groups had a slower righting reflex than mice treated with OGF. EAE mice treated with LDN and vehicle experienced a more severe disease course and reached peak disease symptoms on day 19. On day 13, EAE mice treated with LDN began to display a wobbly gait and distal tail paralysis as they walked. The average disease score for EAE mice treated with LDN was 2.0 ± 0.2. EAE mice treated with vehicle or LDN had a more severe onset of disease and by day their hindlimbs were partially paralyzed and their tails did not have any tone. On day 15, the average disease scores for mice treated with vehicle or LDN were 4.5 ± 0.3 and 5.5 ± 0.3, respectively. On day 17, the average disease scores for EAE mice treated with vehicle or LDN increased to 6.0 ± 0.2 and 6.5 ± 0.3, respectively. Mice in the vehicle and LDN treatment groups reached peak disease on day 19 with an average disease score of 7.5 ± 0.3 and 7.5 ± 0.2, respectively. These mice had complete paralysis of their hindlimbs, lacked tail tonicity, and had righting reflexes that were very slow or absent. 21

35 EAE mice in the OGF treatment group displayed a slightly abnormal gait and distal tail paralysis beginning on days 11 and 12. The average disease score for the OGF treatment group on days 11 and 12 was 2.5 ± 0.3. However, only minor foot faults were observed on the mesh grid on days for this treatment group. On day 16, EAE mice treated with OGF lost tone in their tails and the average disease score for these mice was 3.5 ± 0.3. By day 17, OGF-treated mice began to display a wobbly gait and their righting reflexes were slower than on previous days, resulting in an average disease score of 4.5 ± 0.5. Contrary to the LDN and vehicle treatment groups, EAE mice treated with OGF did not experience complete hindlimb paralysis and their gait remained wobbly until day 20. OGF-treated mice experienced a less severe disease course compared to the LDN and vehicle experiment groups and the peak disease score for the OGF treatment group was only 5.0 ± 0.5. The disease score of EAE mice treated with OGF was significantly lower than EAE mice treated with vehicle or LDN during the 20-day behavior study. 4.3 The Effects of OGF and LDN on Splenocyte Populations in MOG-Induced EAE Mice MOG-induced EAE mice received daily injections of OGF (10 mg/kg), LDN (0.1 mg/kg), or an equivalent volume of saline (vehicle) beginning on the day of disease induction (day 0). On days 5, 12, and 20, spleens were dissected from each of the experimental groups. The percentages of CD4 + T cells, CD8 + T cells and B cells were measured by flow cytometry and representative flow cytometry analysis for these days is presented in Figures 4.4, 4.5 and 4.6, respectively. 22

36 Figure 4.3 The behavior of MOG-induced EAE mice was observed daily and behavior recordings began on day 9 when behavior was apparent. EAE mice from each of the experimental groups were observed for EAE behavior and these findings were used to calculate a daily disease score. EAE mice received daily injections of OGF (10 mg/kg), LDN (0.1 mg/kg) or an equivalent volume of saline (vehicle). Disease scores ranged from 0 (no disease symptoms) to 10 (death from EAE). The daily summation of points was used to calculate the average score for each experimental group. The means and standard error values were used to plot the disease score over time. Significantly different from EAE mice treated with vehicle at p < 0.05 (*), p < 0.01 (**), or p < (***) and significantly different from EAE mice treated with LDN at p < 0.05 (^), p < 0.01 (^^), or p < (^^^). 23

37 On day 5, the average number of lymphocytes in the spleens of normal mice was 8.1 ± 0.5 x 10 7 (Figure 4.7A). The percentage of B cells in the spleens of normal mice (Figure 4.4 A,B) was 51.8% (3.2 ± 0.3 x 10 7 cells), the percentage of CD4 + T cells in the spleens of control mice was 30.3% (1.9 ± 0.1 x 10 7 cells) and the percentage of CD8 + T cells was 17.9% (1.4 ± 0.1 x 10 7 cells). In the spleens of MOG-EAE mice that received daily injections of vehicle, the average number of lymphocytes was 1.1 ± 0.02 x 10 8 cells (Figure 4.7A). The percentage of B cells in the spleens of vehicle-treated MOG-EAE mice (Figure 4.4 C,D) was 55.7% (3.8 ± 0.2 x 10 7 cells). However, the percentage of CD4 + T cells was 26.9% (1.8 ± 0.1 x 10 7 cells), which was a 10% decrease in the number of CD4 + T cells from non-challenged mice. The percentage of CD8 + T cells was 17.3% (1.1 ± 0.1 x 10 7 cells), which was a 21% decrease in the number of CD8 + T cells from normal mice. The spleens of MOG-EAE mice that were treated daily with OGF (Figure 4.4 E,F) had an average of 6.6 ± 0.7 x 10 7 lymphocytes (Figure 4.7A), which was a 40% decrease in the total number of lymphocytes compared to MOG-EAE mice treated with vehicle. The percentage of B cells found in the spleens of EAE mice treated with OGF was 64.9% (2.8 ± 0.3 x 10 7 cells), the percentage of CD4 + T cells was 21.4% (1.1 ± 0.2 x 10 7 cells) and 13.6% (6.8 ± 0.7 x 10 6 cells) of the splenocytes were CD8 + T cells. When compared to EAE mice treated with vehicle, there was a 26% decrease in the number of B cells, a 39% decrease in the number of CD4 + T cells and a 38% decrease in the number of CD8 + T cells in MOG-EAE mice that were treated daily with OGF. In the spleens of MOG-EAE mice that were treated daily with LDN (Figure 4.7A), the average number of lymphocytes was 8.1 ± 1.1 x 10 7 cells, which was a 26% decrease in the total number of lymphocytes from MOG-EAE mice treated with vehicle. In the spleens of mice treated with LDN (Figure 4.4 G,H), the percentages of lymphocytes were: 68.1% (3.9 ± 0.7 x 10 7 cells) B cells, 20.3% (1.2 ± 0.1 x 10 7 cells) CD4 + T 24

38 cells and 11.6% (7.3 ± 0.3 x 10 6 cells) CD8 + T cells. When compared to MOG-EAE mice that were treated with saline, there was a 3% increase in the number of B cells, a 33% decrease in the number of CD4 + T cells and a 34% decrease in the number CD8 + T cells in EAE mice that were treated daily with LDN. On day 12, the average number of lymphocytes in the spleens of non-challenged mice (Figure 4.7B) was 6.6 ± 0.7 x 10 7 cells with 55.6% (3.3 ± 0.5 x 10 7 cells) B cells (Figure 4.5 A,B). The percentages of CD4 + T cells and CD8 + T cells (Figure 4.5 A,B) were 28.2% (1.8 ± 0.1 x 10 7 cells) and 14.6% (9.5 ± 0.8 x 10 6 cells), respectively. The average number of lymphocytes in the spleens of MOG-EAE mice that received daily injections of vehicle (Figure 4.7B) was 9.7 ± 0.7 x 10 7 cells with 56.8% (3.6 ± 0.4 x 10 7 cells) B cells (Figure 4.5 C,D). In the spleens of EAE mice treated with saline, the percentages of CD4 + T cells and CD8 + T cells (Figure 4.5 C,D) were 27.6% (2.2 ± 0.5x10 7 cells) and 15.5% (1.2 ± 0.01x10 7 cells), respectively. MOG-EAE mice treated with OGF (Figure 4.7B) had an average of 6.7 ± 0.5 x 10 7 splenic-derived lymphocytes on day 12, which was a 31% decrease in the total number of splenic-derived lymphocytes as compared to MOG-EAE mice treated with vehicle. The percentage of B cells, CD4 + T cells and CD8 + T cells in MOG-EAE mice treated with OGF (Figure 4.5 E,F) were 64.2% (2.7 ± 0.5 x 10 7 cells), 23.9% (1.1 ± 0.2 x 10 7 cells) and 11.8% (5.1 ± 0.6 x 10 6 cells), respectively. In the spleens of MOG-EAE mice treated with OGF, there was a 25% decrease in the number of B cells, a 50% decrease in the number of CD4 + T cells and a 58% decrease in the number of CD8 + T from vehicle-treated MOG-EAE mice. On day 12, the average number of lymphocytes in the spleens of MOG-EAE mice treated with LDN (Figure 4.7B) was 7.4 ± 0.2 x 10 7 cells, which was a 24% decrease in the total number of splenic-derived lymphocytes from EAE mice treated with vehicle. The average percentage of B cells, CD4 + T cells and CD8 + T 25

39 cells in the spleens of MOG-EAE mice treated with LDN (Figure 4.5 G,H) were 56.8% (2.7 ± 0.4 x 10 7 cells), 28.9% (1.4 ± 0.1 x 10 7 cells) and 14.3% (6.3 ± 0.6 x 10 6 cells), respectively. In the spleens of LDN-treated MOG-EAE mice there was a 25% decrease in the number of B cells, a 36% decrease in the number of CD4 + T cells and 48 %decrease in the number of CD8 + T cells from EAE mice treated with vehicle. On day 20, the average number of lymphocytes in the spleens of normal mice (Figure 4.7C) was 4.6 ± 0.9 x 10 7 cells. The percentages of B cells, CD4 + T cells, and CD8 + T cells in the spleens of normal mice (Figure 4.6 A,B) were 44.7% (2.1 ± 0.6 x 10 7 cells), 35.3% (1.2 ± 0.2 x10 7 cells), and 19.9% (7.4 ± 0.6 x 10 6 cells), respectively. The average numbers of lymphocytes in the spleens of MOG-EAE mice treated with vehicle, OGF (10 mg/kg), or LDN (0.1 mg/kg) on day 20 (Figure 4.7C) were 5.9 ± 0.7 x 10 7 cells, 4.7 ± 0.9 x 10 7 cells, and 4.7 ± 0.4 x 10 7 cells, respectively. These values were not statistically significant from one another; however, OGF and LDN did cause a 20% decrease in the total number of lymphocytes when compared to the total number of lymphocytes found in the spleens of EAE mice treated with vehicle. The average percentages of B cells, CD4 + T cells, and CD8 + T cells in the spleens of MOG-EAE mice treated with vehicle (Figure 4.6 C,D) were 51.8% (1.5 ± 0.2 x 10 7 cells), 33.7% (9.8 ± 1.4 x 10 6 cells) and 14.4% (4.3 ± 0.4 x 10 6 cells), respectively. When compared to normal mice, there was a 29% decrease in the number of B cells, an 18% decrease in the number of CD4 + T cells and a 42% decrease in the number of CD8 + T cells in the spleens of EAE mice treated with vehicle. In the spleens of MOG-EAE mice treated with OGF (Figure 4.5C), the average percentage of B cells, CD4 + T cells, and CD8 + T cells (Figure 4.6 E,F) were 55.4% (1.8 ± 0.4 x 10 7 cells), 31.1% (1.2 ± 0.8 x 10 7 cells) and 13.4% (4.8 ± 0.6 x 10 6 cells), respectively. In the spleens of MOG-EAE mice treated with OGF for 20 days, there was a 20% increase in the 26

40 number of B cells, a 22% increase in the number CD4 + T cells and a 12% increase in the number of CD8 + T cells from vehicle-treated MOG-EAE mice. The average percentages of B cells, CD4 + T cells and CD8 + T cells in the spleens of MOG-EAE mice treated with LDN (Figure 4.6 G,H) were 53.7% (1.6 ± 0.2 x 10 7 cells), 28.5% (1.1 ± 0.8 x 10 7 cells), and 7.1% (6.1 ± 0.3 x 10 6 cells), respectively. When compared to the lymphocyte populations in the spleens of MOG-EAE mice treated with vehicle, there was a 7% increase in the number of B cells, a 12% increase in the number of CD4 + T cells, and a 42% increase in the number of CD8 + T cells in the spleens of EAE mice treated with LDN. Figure 4.8 demonstrates the effects of OGF and LDN on the number of lymphocytes in the spleens of EAE mice over time. The suppression of B and T cell proliferation was apparent at all time points. However, by day 20, mice that were treated with vehicle experienced a decrease in total cell counts as well as OGF and LDN-treated mice. Therefore, cell counts in all EAE mice treated with OGF, LDN, or an equivalent volume of saline had decreased to a number that was similar to normal, unchallenged mice. 27

41 Figure 4.4 The effects of OGF and LDN on the percentages of B and T cells in the spleens of EAE mice on day 5 post treatment with MOG peptide. The cell surface expressions of CD4, CD8, and B220 were detected by conjugated antibodies that recognized these cell surface markers and then analyzed by standard flow cytometry. Lymphocyte percentages from normal mice (A,B) or from MOG-induced EAE mice that were treated with an equivalent volume of saline (C,D), OGF (10 mg/kg, daily) (E,F), or LDN (0.1 mg/kg, daily) (G,H). 28

42 29

43 Figure 4.5 The effects of OGF and LDN on the percentages of B and T cells in the spleens of EAE mice on day 12 post treatment with MOG peptide. The cell surface expressions of CD4, CD8, and B220 were detected by conjugated antibodies that recognized these cell surface markers and then analyzed by standard flow cytometry. Lymphocyte percentages from normal mice (A,B) or from MOG-induced EAE mice that were treated with an equivalent volume of saline (C,D), OGF (10 mg/kg, daily) (E,F), or LDN (0.1 mg/kg, daily) (G,H). 30

44 31

45 Figure 4.6 The effects of OGF and LDN on the percentages of B and T cells in the spleens of EAE mice on day 20 post treatment with MOG peptide. The cell surface expressions of CD4, CD8, and B220 were detected by conjugated antibodies that recognized these cell surface markers and then analyzed by standard flow cytometry. Lymphocyte percentages from normal mice (A,B) or from MOG-induced EAE mice that were treated with an equivalent volume of saline (C,D), OGF (10 mg/kg, daily) (E,F), or LDN (0.1 mg/kg, daily) (G,H). 32

46 33

47 Figure 4.7 The histograms represent the number of lymphocytes in the spleens of MOGinduced EAE mice that received injections of OGF (10 mg/kg, daily), LDN (0.1 mg/kg, daily), or an equivalent volume of saline (daily) starting on the day of disease induction (day 0). The values represent the means ± SEM for splenocytes on days 5 (A), 12 (B), and 20 (C) post-initial injection with MOG peptide. Significantly different from normal spleens at p < 0.05 (*) and significantly different from spleens treated with vehicle (MOG+Veh) at p < 0.05 (+), p < 0.01 (++). 34

48 Figure 4.8 The histogram represents the number of lymphocytes in the spleens of normal and EAE mice on days 5, 12 and 20. The mice were treated with OGF (10 mg/kg, daily), LDN (0.1 mg/kg, daily), or an equivalent volume of saline (daily) on the day of disease induction (day 0). The values represent mean cell counts ± SEM for 3 mice per treatment group. Statistical analysis on each day was performed using one-way ANOVA; significantly different from normal spleens at p < 0.05 (*), and significantly different from spleens treated with vehicle at p < 0.05 (+) and p < 0.01 (++). 35

49 4.4 The Effects of OGF and LDN on Lymphocyte Populations in the Inguinal Lymph Nodes of MOG-Induced EAE Mice As discussed in section 4.2, MOG-induced EAE mice received daily injections of OGF (10 mg/kg), LDN (0.1 mg/kg), or an equivalent volume of saline (vehicle) beginning on the day of disease induction (day 0). On days 5, 12, and 20, inguinal lymph nodes (ILNs) were dissected from each of the experimental groups. The percentages of lymphocytes present in each sample were measured by flow cytometry and comparisons were made against mice from other treatment groups. On day 5 (Figure 4.15A), the average number of lymphocytes in the ILNs of nonchallenged mice was 4.7 ± 0.8 x 10 5 cells. In a normal mouse (Figure 4.9 A,B), the average percentages of B cells, CD4 + T cells, and CD8 + T cells were 29.9% (4.1 ± 0.7 x 10 5 cells), 40.1% (5.8 ± 0.1 x 10 5 cells), and 30.0% (4.4 ± 0.1 x 10 5 cells), respectively. The average number of lymphocytes in the right ILNs of MOG-EAE mice (Figure 4.15A) treated with vehicle was 7.0 ± 0.3 x 10 6 cells, which was a 15-fold increase from the lymph nodes of non-challenged mice. In the right ILNs of MOG-EAE mice treated with vehicle (Figure 4.9 C,D), the percentages of B cells, CD4 + T cells, and CD8 + T cells were 50.8 % (3.0 ± 0.1 x 10 6 cells), 32.1% (1.8 ± 0.04 x 10 6 cells), and 17.0% (9.9 ± 0.6 x 10 5 cells), respectively. There was a 7-fold increase in the number of B cells, a 3-fold increase in the number of CD4 + T cells and a 2.25-fold increase in the number of CD8 + T cells in right ILNs of MOG-EAE mice treated with vehicle. In the left (non-draining) ILNs of MOG-EAE mice treated with vehicle (Figure 4.15A), the average number of lymphocytes was 1.1 ± 0.5 x 10 6 cells and the percentages of B cells, CD4 + T cells, and CD8 + T cells (Figure 4.10 C,D) were 23.8% (2.1 ± 0.7 x 10 5 cells), 46.8% (4.5 ± 2.0 x 10 5 cells), and 29.4% (2.8 ± 0.1 x10 5 cells), respectively. The right ILNs of MOG-EAE mice treated with OGF (Figure 4.15A) had an average of 3.6 ± 1.1 x 10 6 lymphocytes, which was a 49% 36

50 decrease in the total number of lymphocytes from vehicle-treated MOG-EAE mice. The percentages of B cells, CD4 + T cells, and CD8 + T cells in the right ILNs of OGF-treated mice (Figure 4.9 E,F) were 54.9% (1.6 ± 0.5 x 10 6 cells), 28.9% (8.9 ± 0.3 x 10 5 cells), and 16.1% (5.1 ± 0.1 x 10 5 cells), respectively. There was a 47% decrease in the number of B cells, a 51% decrease in the number of CD4 + T cells, a 48% decrease in the number of CD8 + T cells from the right ILNs of vehicle-treated MOG-EAE mice. The left ILNs of MOG-EAE mice treated with OGF (Figure 4.15A) had an average of 9.5 ± 0.5 x 10 5 cells and the percentages of B cells, CD4 + T cells, and CD8 + T cells (Figure 4.10 E,F) were 34.8% (2.1 ± 0.2 x 10 5 cells), 40.6% (2.8 ± 0.1 x 10 5 cells), and 24.5% (1.8 ± 0.2 x 10 5 cells), respectively. In the left ILNs of MOG-EAE mice treated with OGF, there was a 38% decrease in the number of CD4 + T cells and a 36% decrease in the percentage of CD8 + T cells when compared to left ILNs of vehicle-treated MOG-EAE mice. The right ILNs of MOG-EAE mice treated with LDN (Figure 4.15A) had 4.6 ± 1.1 x 10 6 lymphocytes, which was a 35% decrease in the total number of lymphocytes from the right ILNs of vehicle-treated EAE mice. The percentages of B cells, CD4 + T cells, and CD8 + T cells in the right ILNs of these mice (Figure 4.9 G,H) were 55.3% (2.1 ± 0.6 x 10 6 cells), 27.9% (1.2 ± 0.3 x 10 6 cells), and 16.7% (6.7 ± 0.2 x 10 5 cells), respectively. LDN treatment resulted in a 30% decrease in the number of B cells, and a 33% decrease in the number of CD4 + T and a 32% decrease in the number of CD8 + T cells when compared to the right ILNs of vehicle-treated MOG-EAE mice. The left ILNs of MOG-EAE mice treated with LDN (Figure 4.15A) had an average of 1.1 ± 0.4 x 10 6 cells and the percentages of B cells, CD4 + T cells, and CD8 + T cells (Figure 4.10 G,H) were 32.2% (1.5 ± 0.0 x 10 5 cells), 43.3% (1.9 ± 0.0 x 10 5 cells), and 24.5% (1.1 ± 0.0 x 10 5 cells), respectively. When compared to the ILNs of MOG-EAE mice treated 37

51 with vehicle, there was a 29% increase in the number of B cells, a 58% decrease in the number of CD4 + T cells, and a 61% decrease in the percentage of CD8 + T cells. On day 12 (Figure 4.15B), the average number of lymphocytes in the ILNs of nonchallenged mice was 4.5 ± 0.5 x 10 5 and the percentages of B cells, CD4 + T cells, and CD8 + T cells (Figure 4.11 A,B) were 32.9% (4.2 ± 0.1 x 10 5 cells), 43.4% (5.6 ± 0.2 x 10 5 cells), and 23.6% (3.2 ± 0.1 x 10 5 cells), respectively. The average number of lymphocytes in the right ILNs of MOG-EAE mice treated with vehicle (Figure 4.15B) was 5.1 ± 0.8 x 10 6 cells, which was an 11-fold increase in the total number of lymphocytes from the ILNs of non-challenged mice. In the right ILNs of MOG-EAE mice treated with vehicle (Figure 4.11 C,D), the average percentages of B cells, CD4 + T cells, and CD8 + T cells were 60.8% (2.7 ± 0.5 x 10 6 cells), 23.7% (1.2 ± 0.2 x 10 6 cells), and 15.3% (7.9 ± 0.1 x 10 5 cells), respectively. There was a 6-fold increase in the number of B cells, a 3-fold increase in the number of CD4 + T cells and a 2.5-fold increase in the number of CD8 + T cells. The left ILNs of MOG-EAE mice treated with vehicle (Figure 4.15B), had an average of 3.9 ± 0.6 x 10 6 cells and the percentages of B cells, CD4 + T cells, and CD8 + T cells (Figure 4.12 C,D) were 53.3% (1.9 ± 0.3 x 10 6 cells), 31.2% (1.1 ± 0.2 x 10 6 cells), and 15.6% (6.2 ± 0.4 x 10 5 cells), respectively. On day 12, the left ILNs of MOG- EAE mice treated with vehicle had a 5-fold increase in the number of B cells, a 2-fold increase in the number of CD4 + T cells, and a 2-fold increase in the number of CD8 + T cells. The right ILNs of MOG-EAE mice treated with OGF (Figure 4.15B) had 6.8 ± 1.2 x 10 6 cells, which was a 33% increase in the total number of cells from the right ILNs of vehicle-treated MOG-EAE mice. The percentages of B cells, CD4 + T cells, and CD8 + T cells (Figure 4.11 E,F) were 56.5% (3.8 ± 1.2 x 10 6 cells), 28.4% (1.4 ± 0.1 x 10 6 cells), and 15.1% (8.0 ± 0.4 x 10 5 cells), respectively. There was a 41% increase in the number of B cells, a 17% increase in the number 38

52 of CD4 + T cells, and a 2% increase in the number of CD8 + T cells. The left ILNs of MOG-EAE mice treated with OGF (Figure 4.15B) had an average of 3.9 ± 0.9 x 10 6 lymphocytes and the average percentages of B cells, CD4 + T cells, and CD8 + T cells (Figure 4.12 E,F) were 61.2% (2.1 ± 0.6 x 10 6 cells), 25.9% (8.5 ± 0.3 x 10 5 cells), and 12.8% (4.1 ± 0.2 x 10 5 cells), respectively. There was an 11% increase in the number of B cells, a 23% decrease in the number of CD4 + T cells and a 34% decrease in the number of CD8 + T cells from the left ILNs of MOG- EAE mice treated with vehicle. The right ILNs of MOG-EAE mice treated with LDN (Figure 4.15B) had an average of 4.5 ± 0.4 x 10 6 cells, which was a 12% decrease in lymphocytes from right ILNs of vehicle-treated MOG-EAE mice. In the right ILNs of the MOG-EAE mice treated with LDN, the average percentages of B cells, CD4 + T cells, and CD8 + T cells (Figure 4.11 G,H) were 62.1% (2.3 ± 0.2 x 10 6 cells), 23.9% (1.1 ± 0.3 x 10 6 cells), and 13.8% (5.7 ± 0.1 x 10 5 cells), respectively. When compared to the right ILNs of EAE mice that were treated with vehicle, there was a 15% decrease in the number of B cells, an 8% decrease in the number of CD4 + T cells, and a 28% decrease in the number of CD8 + T cells. The left ILNs of MOG-EAE mice treated with LDN (Figure 4.15B) had an average of 3.1 ± 0.2x10 6 cells, which was a 21% decrease in total number of lymphocytes from the left ILNs of vehicle-treated MOG-EAE mice. The average percentages of B cells, CD4 + T cells, and CD8 + T cells in the left ILNs of MOG- EAE mice that were treated with LDN (Figure 4.12 G,H) were 53.8% (1.7 ± 0.3 x 10 6 cells), 30.4% (8.0 ± 0.3 x 10 5 cells), and 15.8% (4.0 ± 0.1 x 10 5 cells), respectively. When compared to the left ILNs of the vehicle treated mice, there was an 11% decrease in the number of B cells, a 27% decrease in the number of CD4 + T cells and a 35% decrease in the number of CD8 + T cells. On day 20, the average number of lymphocytes in the ILNs of non-challenged mice was 4.9 ± 0.4 x 10 5 cells (Figure 4.15C). The percentages of B cells, CD4 + T cells, and CD8 + T cells 39

53 in ILNs of non-challenged mice (Figure 4.13 A,B) were 31.1% (2.4 ± 0.3 x 10 5 cells), 38.7% (2.7 ± 0.7 x 10 5 cells), and 30.2% (2.1 ± 0.5 x 10 5 cells), respectively. The average number of lymphocytes in the right ILNs of MOG-EAE mice treated with vehicle (Figure 4.15C) was 2.6 ± 0.2 x 10 6 cells, which was a 5-fold increase from the ILNs of non-challenged mice. The average percentages of B cells, CD4 + T cells, and CD8 + T cells in the right ILNs of MOG-EAE mice treated with vehicle (Figure 4.13 C,D) were 51.5% (1.0 ± 0.1 x 10 6 cells), 29.5% (6.6 ± 0.1 x 10 5 cells), and 18.9% (4.5 ± 0.1 x 10 5 cells), respectively. When compared to the percentages of lymphocytes in the ILNs of non-challenged mice, there was a 4-fold increase in the number of B cells, a 2.5-fold increase in the number of CD4 + T cells, and a 2-fold increase in the percentage of CD8 + T cells. The left ILNs of MOG-EAE mice treated with vehicle had an average of 1.2 ± 0.1 x 10 6 cells (Figure 4.15C) and the average percentages of B cells, CD4 + T cells, and CD8 + T cells (Figure 4.14 C,D) were 41.6% (3.6 ± 0.6 x 10 5 cells), 35.4% (3.6 ± 0.04 x 10 5 cells), and 22.9% (2.3 ± 0.01 x 10 5 cells), respectively. There was a 6% increase in the number of B cells, a 33% increase in the number of CD4 + T cells and a 10% increase in the number of CD8 + T cells from the left ILNs of non-challenged mice. The right ILNs of MOG-EAE mice treated with OGF (Figure 4.15C) had an average of 3.1 ± 0.0 x 10 6 cells (n=1), which was a 19% increase in the total number of lymphocytes compared to the right ILNs of MOG-EAE mice treated with vehicle. The average percentages of B cells, CD4 + T cells, and CD8 + T cells in the right ILNs of MOG-EAE mice treated with OGF (Figure 4.13 E,F) were 44.8% (1.6 ± 0.9 x 10 6 cells), 30.8% (1.3 ± 0.8 x 10 6 cells), and 24.4% (1.1 ± 0.7 x 10 6 cells), respectively. When compared to the right ILNs of MOG-EAE mice treated with vehicle, there was a 60% increase in the number of B cells, a 96% increase in the number of CD4 + T cells, and a 144% increase in the number of CD8 + T cells. The left ILNs of MOG-EAE mice treated with OGF (Figure 4.15C) had an average of 40

54 1.3 ± 0.5 x 10 6 cells and the average percentages of B cells, CD4 + T cells, and CD8 + T cells (Figure 4.14 E,F) were 27.5% (4.1 ± 0.0 x 10 5 cells), 42.1% (6.3 ± 0.0 x 10 5 cells), and 30.4% (4.5 ± 0.0 x 10 5 cells), respectively. There was a 14% increase in the number of B cells, a 75% increase in the number of CD4 + T cells, and a 96% increase in the number of CD8 + T cells compared to the left ILNs of MOG-EAE mice treated with vehicle. The right ILNs of MOG- EAE mice treated with LDN (Figure 4.15C) had an average of 2.4 ± 0.1 x 10 6 cells, which was a 5-fold increase in the total number of lymphocytes compared to the total number found in the ILNs of unchallenged mice. The average percentages of B cells, CD4 + T cells, and CD8 + T cells in the right ILNs of MOG-EAE mice treated with LDN (Figure 4.13 G,H) were 44.1% (8.0 ± 1.0 x 10 5 cells), 32.4% (6.3 ± 0.8 x 10 5 cells), and 23.5% (4.4 ± 0.6 x 10 5 cells), respectively. There was a 20% decrease in the number of B cells, a 5% decrease in the number of CD4 + T cells, and a 2% increase in the number of CD8 + T cells. In the left ILNs of MOG-EAE mice treated with LDN (Figure 4.15C), the average number of lymphocytes was 2.5 ± 0.1 x 10 6 cells. The average percentages of B cells, CD4 + T cells, and CD8 + T cells in the left ILNs of MOG-EAE mice treated with LDN (Figure 4.14 G,H) were 48.5% (9.2 ± 0.3 x 10 5 cells), 28.9% (6.3 ± 0.1 x 10 5 cells), and 22.5% (4.8 ± 0.1 x 10 5 cells), respectively. When compared to the left ILNs of MOG- EAE mice treated with vehicle, there was a 155% increase in the number of B cells, a 75% increase in the number of CD4 + T cells, and a 108% increase in the number of CD8 + T cells. 41

55 Figure 4.9 The effects of OGF and LDN on the percentages of B and T cells in the right ILNs of MOG-induced EAE mice on day 5 post initial treatment with MOG peptide. The cell surface expressions of CD4, CD8 and B220 were detected by using conjugated antibodies that recognized these cell surface markers and then analyzed by standard flow cytometry. Lymphocyte percentages from normal mice (A,B) or from EAE mice that were treated with an equivalent volume of saline (C,D), OGF (10 mg/kg, daily) (E,F), or LDN (0.1 mg/kg, daily) (G,H). 42

56 43

57 Figure 4.10 The effects of OGF and LDN on the percentages of B and T cells in the left ILNs of MOG-induced EAE mice on day 5 post initial treatment with MOG peptide. The cell surface expressions of CD4, CD8 and B220 were detected by using conjugated antibodies that recognized these cell surface markers and then analyzed by standard flow cytometry. Lymphocyte percentages from normal mice (A,B) or from EAE mice that were treated with an equivalent volume of saline (C,D), OGF (10 mg/kg, daily) (E,F), or LDN (0.1 mg/kg, daily) (G,H). 44

58 45

59 Figure 4.11 The effects of OGF and LDN on the percentages of B and T cells in the right ILNs of MOG-induced EAE mice on day 12 post initial treatment with MOG peptide. The cell surface expressions of CD4, CD8 and B220 were detected by using conjugated antibodies that recognized these cell surface markers and then analyzed by standard flow cytometry. Lymphocyte percentages from normal mice (A,B) or from EAE mice that were treated with an equivalent volume of saline (C,D), OGF (10 mg/kg, daily) (E,F), or LDN (0.1 mg/kg, daily) (G,H). 46

60 47

61 Figure 4.12 The effects of OGF and LDN on the percentages of B and T cells in the left ILNs of MOG-induced EAE mice on day 12 post initial treatment with MOG peptide. The cell surface expressions of CD4, CD8 and B220 were detected by using conjugated antibodies that recognized these cell surface markers and then analyzed by standard flow cytometry. Lymphocyte percentages from normal mice (A,B) or from EAE mice that were treated with an equivalent volume of saline (C,D), OGF (10 mg/kg, daily) (E,F), or LDN (0.1 mg/kg, daily) (G,H). 48

62 49

63 Figure 4.13 The effects of OGF and LDN on the percentages of B and T cells in the right ILNs of MOG-induced EAE mice on day 20 post initial treatment with MOG peptide. The cell surface expressions of CD4, CD8 and B220 were detected by using conjugated antibodies that recognized these cell surface markers and then analyzed by standard flow cytometry. Lymphocyte percentages from normal mice (A,B) or from EAE mice that were treated with an equivalent volume of saline (C,D), OGF (10 mg/kg, daily) (E,F), or LDN (0.1 mg/kg, daily) (G,H). 50

64 51