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Heme Oxygenase-1 plays an important protective role in experimental autoimmune encephalomylitis Liu, Yingru 2003

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Heme Oxygenase-1 Plays an Important Protective Role in Experimental Autoimmune Encephalomylitis By Yingru Liu B.Med., Beijing Medical University, 1990 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In T H E F A C U L T Y OF GRADUATE STUDIES Graduate Program in Neuroscience We accept this thesis as conforming to the required standard  The University of British Columbia © Yingru Liu, 2003  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or  by  his or  her  representatives.  It  is  understood  that  copying  or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of t^dlAYOiCldn  ce  Cfrc*due4 e Trocprw*  The University of British Columbia Vancouver, Canada Date  DE-6 (2/88)  M*r*A  7, 2o*3___  ABSTRACT  Increasing evidence shows that oxidative stress plays an important role in the pathogenesis of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Heme oxygenase-1 (HO-1) is a heat shock protein induced by oxidative stress, and represents a powerful endogenous defensive mechanism against free radicals in many diseases. However, the role of this important enzyme in E A E remains unknown. The purpose of this thesis is to investigate the induction of HO-1 in E A E , and evaluate its effect on this disease. The expression of HO-1 in E A E was detected by Western blot analysis and immunohistochemistry. These studies revealed that HO-1 is strongly induced in E A E lesions, while none could be detected in the normal rat spinal cord. However, the level of HO-1 expression is different at various phases of E A E . The expression of HO-1 is weak at the onset of clinical signs. During the following days, HO-1 activity increases substantially as disease progresses, and reaches its maximum at the peak of clinical signs. Subsequently, I demonstrated that hemin, an inducer of HO-1, inhibited E A E effectively. In contrast, tin mesoporphyrin, an inhibitor of HO-1, markedly worsened E A E especially at the peak period of disease, correlating well with the level of HO-1 activity at this time. These data indicate that HO-1 plays an important protective role in E A E , and that targeted induction of HO-1 overexpression by pharmacological modulation may serve as a novel approach to therapeutic intervention in MS.  T A B L E OF CONTENTS ABSTRACT T A B L E OF CONTENTS LIST OF TABLES AND FIGURES ABBREVIATIONS  ii iii v vii  ACKNOWLEDGEMENTS  ix  Chapter 1 INTRODUCTION  1  1.1 Multiple Sclerosis (MS) 1.2 Animal Models of Multiple Sclerosis 1.2.1 Theiler's Murine Encephalomyelitis Virus (TMEV) Infection 1.2.2 Experimental Autoimmune Encephalomyelitis (EAE) 1.3 Pathogenesis of EAE 1.4 Antioxidant Systems of the Organisms 1.5 Heme Oxygenase (HO) , 1.5.1 Constituent HO (HO-2) 1.5.2 Inducible HO (HO-1) HO-1, a Potent Antioxidant Enzyme HO-1 and diseases 1.6 Hypothesis and Experimental Objectives Chapter 2 MATERIALS AND METHODS 2.1 Materials 2.1.1 Animals 2.1.2 Chemicals and Regents 2.2 Methods 2.2.1 Solution Preparation 2.2.2 Induction of EAE 2.2.3 Treatment Regimen 2.2.4 Tissue Preparation 2.2.5 Western Blot Analysis 2.2.6 Immunohistochemistry 2.2.7 Immunohistochemical Double-staining 2.2.8 Hematoxylin-eosin Staining 2.2.9 Statistical Analysis  1 7 7 10 16 20 22 24 25 25 28 29 31 31 31 31 32 32 34 35 37 38 40 41 42 43  iii  Chapter 3 RESULTS 3.1 3.2 3.3 3.4  Specificity of HO antibodies Expression of HO-2 in EAE Induction of HO-1 in EAE Protective Effects of HO-1 on EAE 3.4.1 Effects of Hemin and SnMP Treatment on Clinical EAE 3.4.2 Histopathological and Immunohistological Findings  Chapter 4 DISCUSSION AND SUMMARY 4.1 The Role of HO-1 in EAE 4.2 Summary 4.3 Cautions and Future Directions 4.3.1 Human Multiple Sclerosis and Rodent Experimental Autoimmnue Encephalomylitis 4.3.2 Duality of HO-1 in Antioxidant Defense 4.3.3 Future Directions REFERENCES  44 44 46 51 57 57 62 70 70 74 75 75 76 78 80  iv  LIST OF T A B L E S A N D FIGURES Tables:  Table 1.1. Comparison Between Multiple Sclerosis and EAE  15  Table 2.1. The Experimental Design  36  Table 3.1. Effects of Hemin and SnMP Treatment on EAE in Lewis Rats  60  Figures:  Figure 1.1. MS prevalence (per 10 ) in several Canadian locations 5  2  Figure 1.2. Clinical course of acute EAE and chronic relapsing EAE (CREAE)  12  Figure 1.3. The heme oxygenase enzyme reaction  22  Figure 3.1. Western blot analysis of the specificity of HO-1 and HO-2 antibodies  45  Figure 3.2. Expression of HO-2 in the normal Lewis rat spinal cord Immunohistochemical staining  47  Figure 3.3. Localization of HO-2 in the normal rat spinal cord Immunohistochemical double-staining  48  Figure 3.4. Expression of HO-2 in the lesions of EAE vs. in the normal rat spinal cord - Immunohistochemical staining  49  Figure 3.5. Western blot analysis of HO-2 expression in the lesions of EAE vs. in the normal rat spinal cord  50  Figure 3.6. Expression of HO-1 in the normal rat spinal cord and in the lesions of EAE - Immunohistochemical staining  52  Figure 3.7. Localization of HO-1 in the lesions of EAE - Immunohistochemical double-staining  53  Figure 3.8.1, Expression of HO-1 in different phases of EAE vs. in normal physiological conditions - Immunohistochemical staining. II, Correlation of HO-1 expression with the clinical signs of EAE  54  v  Figure 3.9. Western blot analysis of HO-1 expression in different phases of EAE vs. in normal physiological conditions Figure 3.10. Effects of hemin and SnMP treatment on EAE in Lewis rats  56 58  Figure 3.11. Comparison of histopathology in the spinal cords between hemintreated and control EAE rats - HE staining  63  Figure 3.12. Comparison of histopathology in the spinal cords between SnMPtreated and control EAE rats - HE staining  64  Figure 3.13. Comparison of HO-1 expression in the lesions between SnMPtreated and control EAE rats - Immunhistochemical staining  66  Figure 3.14. Comparison of HO-1 expression in the lesions between hemintreated and control EAE rats - Immunohistochemical staining  67  Figure 3.15. Comparison of histopathology and HO-1 expression in lymphoid nodes among normal, control, hemin-, and SnMP-treated rats Figure 4.1. Heme oxygenase in tissue protection and injury  68 77  vi  ABBREVIATIONS NO  nitric oxide  O2"  superoxide anion  •OH  hydroxyl radical  ABC  avidin-biotin complex  ADCC  antibody-dependent cell-mediated toxicity  BBB  blood-brain barrier  CFA  complete Freund's adjuvant  CNS  central nervous system  CO  carbon monoxide  CREAE  chronic relapsing experimental autoimmune encephalomyelitis  DAB  3,3'-diaminobenzidine  DAI  day after immunization  EAE  experimental autoimmune encephalomyelitis  Fe  iron  GP  X  glutathione peroxidase  HE staining  hematoxylin-eosin staining  HLA  human leukocyte antigen  H2O2  hydrogen peroxide  HO  heme oxygenase  ICAM  intercellular adhesion molecule  IFN  interferon  IL  interleukin  iNOS  inducible nitric oxide synthase  LT  lymphotoxin  MBP  myelin basic protein  MMP  matrix metalloproteases  MOG  myelin oligodendroglia glycoprotein  vii  MS  multiple sclerosis  ONOO"  peroxynitrite  PLP  proteolipid protein  PPMS  primary progressive multiple sclerosis  RRMS  relapsing-remitting multiple sclerosis  ROS  reactive oxygen species  SnMP  tin mesoporphyrin  SOD  superoxide dismutase  TCR  T-cell receptor  TMEV  Theiler's murine encephalomyelitis virus  TNF  tumor necrosis factor  VCAM  vascular cell adhesion molecule  ACKNOWLEDGEMENTS  I wish to express my deep gratitude to my supervisors, Dr. Max Cynader and Dr. Donald Paty, for their guidance, understanding and encouragement throughout. I will always appreciate the generous gifts of their time, advice, tolerance, and continuous support. I owe a great debt of gratitude to Bing Zhu and Lilian Luo for their help with the EAE model and many other important experimental techniques. I also enjoy the inspiring and pleasant discussion with them. I sincerely thank my supervisory committee members, Dr. Lome Kastrukoff, Dr. Joel Oger and Dr. Yu Tian Wang for their time, constructive criticism, helpful discussion and valuable suggestions. I am also very grateful to Dr. William Jia, Dr. Qiang Gu, Dr. Shiv Prasad, Xuefeng Wang, Jing Cui, Xiaohong Zhang, Tara Stewart, and Tim Blanche for their help, and for making the laboratory a fun and enjoyable working environment. Finally, I would like to express my appreciation to my wife Ping for her assistance with my experiments, and for providing me with encouragement and support over the years.  Chapter 1 INTRODUCTION  1.1 MULTIPLE SCLEROSIS  Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS), and is a common cause of disability in young adults (Paty et al., 1998). Thefirstreported case of MS in the medical literature is attributed to Charles Proper Ollivier in his 1824 monograph (Ollivier, 1824). Thefirstdescription of the pathology of demyelinating plaques was made by Carswell in 1838 and by Cruveilhier (1842) around the same period. The physician responsible for establishing MS as a clinical pathologic entity was Jean Martin Charcot at the Salpetriere in Paris in 1860s. From then on, MS became recognized in neurological clinics. By the beginning of the 20th century, a disease only a few years earlier meriting individual case reports had became one of the most common reasons for admission to a neurological ward. Now, MS is recognized throughout the world, with around 2 million affected individuals, including 300,000 ~ 350,000 individuals in North America (Conlon et al., 1999). These crude statistics conceal the harsh reality of a frightening and potentially disabling disease for young adults. Incidence and clinicalfeatures. MS has an incidence of about 2 per 100,000 every year, prevalence of around 30 per 100,000, and lifetime risk of one in 1,000 (Warren et al., 2001). The prevalence of MS varies considerably around the world. The prevalence is  1  94.0 Figure 1.1. MS prevalence (per 10 ) in several Canadian locations: British Columbia (1982); Saskatoon, Saskatchewan (1977); London, Ontario (1984); and Newfoundland (1985), and in one American location: Rochester, Minnesota (1985). (Adapted from Multiple Sclerosis, Edited by Donald W. Paty and George C. Ebers, 1998) 5  highest in northern Europe, northern United States, Canada (Fig. 1.1), southern Australia, and New Zealand, and is lowest in Asia and Africa. MS affects women more frequently than men (Duquette et al., 1992). The onset usually occurs between the ages of 20 and 40, rarely before the age of 15 or after 50 (Martyn, 1991; Sadovnick and Ebers, 1993). 80% of patients begin with relapsing-remitting MS (RRMS), which is characterized by discrete clinical "attacks" or "relapses" followed by subsequent improvement (Compston and Coles, 2002). Most RRMS patients eventually develop secondary progressive MS (SPMS), where there is progressive deterioration with or without occasional relapses. About 20%o patients have primary progressive MS (PPMS) characterized by progressive course from onset without relapses or remissions. MS is extremely variable insofar as the neurological symptoms and degree of disability are concerned. The most common symptoms include sensory disturbances, unilateral optic neuritis, diplopia, Lhermitte's  2  sign, limb weakness, clumsiness, gait ataxia, and neurogenic bladder and bowel symptoms. Eventually, cognitive impairment, depression, dysarthria, dysphagia, vertigo, progressive quadriparesis and sensory loss, ataxic tremors, pain, sexual dysfunction, and other manifestations of CNS dysfunction may become troublesome (Paty et al., 1998). 50% of patients with MS need help walking within 15 years after the onset of disease (Weinshenker et al., 1989). Environment and genetics. Although MS has been studied for many years, its cause has remained elusive, and up to now no definitive therapy is available for this disease. The possible etiologies are suggested by the results of epidemiological studies, and from a large body of laboratory investigations. The world geographic distribution of MS may provide a clue to environmental determinants. MS is rare in tropical and subtropical areas. Within temperate zones, disease rates increase with increasing latitude both in the Northern and Southern Hemispheres (Kurtzke, 1980). A north-south prevalence gradient has been detected in Europe, the United States, Japan, Australia, and New Zealand (Ebers and Sadovnick, 1998). The geographic differences in prevalence may be explained by environmental factors such as nutrition or exposure to different infectious agents. The apparent change in the frequency of MS among people who migrate to and from highprevalence areas before puberty is another factor that has been presented to support the existence of an environmental factor (Kurtzke, 1985). Further evidence stems from the analysis of" MS epidemics", in particular, that on the Faroe Islands, where MS was unknown until 1940 and broke out shortly after British soldiers landed on its shores (Kurtzke, 1985; Kurtzke and Hyllested, 1979). The appearance of cases in the vicinity of the British camps has been interpreted as indicating that an unknown pathogen, possibly a  3  virus, had been brought to the island by the troops. Parallels to infectious demyelination have further supported the idea that transmissible agents might be the cause of MS. Besides their possible role as risk factor in the etiology of MS, viral and bacterial infections have also been implicated as triggers of acute attacks in relapsing-remitting MS (Andersen et al., 1993; Rapp et al., 1995). In fact, strong interest in an infectious etiology has repeatedly emerged since Pierre Marie in 1884 first proposed that MS often starts as an infectious process (Marie, 1884). During the past century, more than a dozen viruses have been suggested to be associated with MS, including rabies, herpes simplex, scrapie, measles, simian virus V, coronaviruses, retroviruses, and recently human herpes virus 6 (HHV-6) (Kastrukoff and Rice, 1998). However, none of these claims has stood the test of time. Despite many attempts, an infectious agent has not been identified in MS. The issue of environmental determinants as a factor for development of MS remains unresolved. Although the finding of different prevalence rates of MS in relationship to latitude generally has been thought to reflect environmental factor, genetic differences of the populations living in these areas may also contribute (Ebers, 1983). Evidence that genetic factors have a substantial effect on susceptibility to MS is unequivocal. Caucassian populations show considerably higher prevalence than other ethnic groups sharing the same environment (Kurtzke et al., 1979). Japanese and other oriental people retain their relatively low susceptibility after immigrating to North America (Kurtzke, 1985). In a number of small ethnic groups, including Yakuts, Inuit, New Zealand Maoris that live in high-prevalence areas, MS is not observed at all (Waksman and Reynolds, 1984). Further evidence for the contribution of genetic factors to the pathogenesis of MS comes from  4  family and twin studies. First-degree relatives have a 20-fold increased risk of developing MS compared with the population background (Sadovnick and Ebers, 1993). Nonrelated children adopted into MS families, however, retain the population-based risk of developing the disease, supporting the importance of genetics in MS susceptibility (Ebers et al., 1995). The concordance rate for MS in monozygotic twins is 25%-30%, in contrast to 3%-5% concordance between dizygotic twins and non-twin siblings and a population risk of 0.1%-0.4% (Sadovnick et al., 1996). Because the rate is considerably lower than 100%, it is believed that MS, like other autoimmune diseases, is not determined by a single gene but rather several genes that jointly contribute to susceptibility. The candidate genes include human leukocyte antigen (HLA), T-cell receptor (TCR), myelin basic protein (MBP), immunoglobulin (Ig), and tumor necrosis factor (TNF) genes (Fukazawa et al., 2000). Pathology and pathogenesis. The pathological hallmark of MS is inflammation, demyelination with various degrees of axonal damage, and gliosis (Moore, 1998). Although MS predominantly affects the CNS myelin, axonal loss may be the major determinant of permanent disability in this disease (Ferguson et al., 1997). MS lesions may occurs anywhere within the white matter but favor the periventricular regions, optic nerves, brain stem, cerebellum, and spinal cord (Noseworthy et al., 2000). Inflammatory cells are typically perivascular in location, but they may diffusely infiltrate the parenchyma. The composition of the inflammatory infiltrate varies depending on the stage of demyelination activity (Moore, 1997). In general, it is composed of lymphocytes and macrophages; the latter predominate in active lesions. The pathology of the MS lesion suggests that MS is an immunopathological disease. It is now widely believed that  5  an autoimmune response is involved in the pathogenesis of MS. Further evidence for the possible autoimmune nature of MS derives from its clinical course, female predominance, response to immunosuppressive therapies, association with HLA genes, and parallels with an animal model, experimental autoimmune encephalomyelitis (EAE) (Ebers, 1998). However, a definite autoimmune etiology for MS has not been unequivocally demonstrated. It seems from a large number of epidemiological, demographic, genetic and immunological studies that MS is a complex disease that has multiple etiologies, and that genetic, environmental and other factors interact to produce the disease.  Despite our inability to identify the cause of MS, our understanding of events involved in the evolution of the MS lesions is increasing. Recent research has focused on the inflammatory response that is detectable in the CNS of patients with MS to clarify the nature of the local immunopathological response and the possible targets. This, in turn, has led to a number of innovative therapies of MS, which target the processes involved in lesion development, rather than the precise cause of the disease. Over the past century, animal models of MS have proved extremely useful for investigating the pathophysiological phenomena observed in MS in the human, and are also used to develop new treatments for this disease. Several therapies approved for treatment of MS were developed preclinically based on their success in treating various MS models, including Theiler's murine encephalomyelitis virus (TMEV) infection and EAE (Steinman, 1999).  6  1.2 ANIMAL MODELS OF MULTIPLE SCLEROSIS  Due to the fact that the origin of MS has not yet been established, various MS animal models have been developed applying immunologic, virologic, toxic and genetic parameters in order to understand the pathogenesis of this major demyelinating disease. Viral and autoimmune models have been developed to investigate the virologic and immunologic features of MS. In some genetic models, the use of transgenic technology to over-express or prevent expression of genes encoding molecules related to inflammation has allowed direct examination of their role in the experimental demyelinating disease (Owens et al., 2001). Of the large body of MS animal models, TMEV infection and EAE are considered among the best ones. This is based on the extensive similarities among TMEV infection, EAE and MS in the clinical and histopathological features (Rodriguez et al., 1987), similar genetic susceptibility shared by TMEV infection (Lipton and Melvold, 1984; Rodriguez et al., 1991), EAE (Bernard, 1976; Villarroya et al., 1990) and MS (Fukazawa et al., 2000), and much evidence suggesting the viral (Krutzke, 1985; Kurtzke and Hyllested, 1979) and autoimmune etiology for MS (Ebers, 1998). In the last few decades, studies conducted with these two animal models of MS have provided insight into the immunopathological response and mechanisms of myelin destruction characteristic of that in MS.  1.2.1 Theiler's Murine Encephalomyelitis Virus (TMEV) Infection TMEV model was first documented in the 1930s. In 1934, TMEV was isolated by Max Theiler (1934) from the CNS of mice with spontaneous flaccid paralysis of the hind  7  limbs. It is a cardiovirus in the family Picornaviridae (Pevear et al., 1987). Based on different biological and pathological properties, TMEV strains are divided into two subgroups. The first subgroup, consisting of GDVII and FA viruses, is highly virulent and causes an acute fatal polioencephalomyelitis in animals (Theiler, 1937). The second one, called TO group, including the Daniels (DA) and BeAn, produced in susceptible strains of mice a biphasic disease of the CNS, resulting in inflammatory demyelination (Lipton, 1975, 1980). Among inbred mouse strains, there is a spectrum of susceptibility to the development of TMEV-induced demyelinating disease after infection (Lipton and Dal Canto, 1979). Strains such as SJL/J and DBA/1 are highly susceptible. Several specific gene loci have been identified as being involved in differential susceptibility: the class I major histocompatibility complex (MHC) locus H-2D (Clatch et al., 1985; Patick et al., 1990), the Tmevd-1 locus on chromosome 3 (Melvold et al., 1990), and the Tmevd2 locus on chromosome 6 (Melvold et al., 1987). In most susceptible strains acute disease appears less than 2 weeks after virus inoculation, and is not always clinically apparent (Lipton, 1975, 1978). 3-5 weeks after infection, mice develop late chronic demyelinating disease with clinical signs from a mild waddling gait to frank spastic hind limb paralysis and urinary incontinence. Both acute and chronic diseases depend on the strain, sex (Kappel et al., 1990), and age of the mouse (Steiner et al., 1984), as well as dose and strain of virus. Histopathological findings following TMEV infection with TO subgroup are consistent with a biphasic disease. During the acute phase, virus replicates predominantly within neurons of the hypothalamus, brain stem, and spinal cord (Rodriguez et al., 1983). Infection of white matter, meninges, choroid plexus or ependyma is not found. Little  8  demyelination or parenchymal inflammation is observed in the acute phase, even in mice susceptible to the late phase. Therefore, the early phase of TMEV infection resembles acute polio virus-induced encephalomyelitis with paralysis due to cytolytic infection of motor neurons (Daniels et al., 1952). In contrast, in the chronic phase, inflammation and demyelination increase in the white matter of the spinal cord. Lesions are most common in the lateral columns of the thoracic region and the largest may encompass the majority of the white matter (Dal Canto and Lipton, 1975). Lesions are characterized by the presence of lymphoid infiltrates and large numbers of macrophages that quickly populate the spinal cord columns (Dal Canto and Lipton, 1977). Two potential mechanisms have been proposed to explain the cause of demyelination in TMEV infection. Observations that would favor a direct effect of virus include: (1) persistence of infectious virus in the CNS for prolonged periods of time albeit at low titers during the chronic phase (Lipton and Dal Canto, 1976a, b). (2) Co-localization of demyelinating lesions with areas of ongoing CNS infection as determined by in situ hybridization and immunochemistry (Brahic et al., 1984; Chamorro et al., 1986). (3) Studies indicating that nude mice infected with DA strains of TMEV develop demyelination after lytic infection of oligodendrocytes in the presence of rising titers of virus and in the absence of functional T cells (Roos and Wollmann, 1984; Rosenthal et al., 1986). Other observations would favor an immune-mediated mechanism: (1) CNS infiltrates in the chronic phase of TMEV infection are composed mainly of macrophages and T cells (Clatch et al., 1990). (2) Clinical symptoms in many susceptible mice with TMEV infection show a good correlation with the T cell infiltration, rather than the CNS viral titers (Tsunoda et al., 1996). (3) Treatment of infected mice with antithymocyte  9  serum or cyclophosphamide diminishes mononuclear cell infiltration and demyelination (Lipton and Dal Canto, 1976a). (4) Susceptibility is controlled by the MHC gene in many mouse strains (Lipton and Melvold, 1984). The similarity of pathological alterations in TMEV-induced demyelinating disease and EAE, the classical model for autoimmune demyelination, suggested initially that demyelination in TMEV infection could follow a process of secondary anti-myelin autosensitization, thus resulting in a virally induced EAE-like model. However, recent evidence indicates that this is not the case. For example, in myelinating and myelinated organotypic cultures, while EAE sera and cells inhibited myelination and were able to demyelinate organotypic culture, no effect was noted with sera and cells from TMEV-infected animals (Barbano and Dal Canto, 1984). Demyelination could not be initiated by transfer of either serum or lymphoid cells from TMEV-infected donors to naive syngenic recipients (Barbano and Dal Canto, 1984). These studies strongly suggest that demyelination in TMEV infection is not due to an EAE-like mechanism.  1.2.2 Experimental Autoimmune Encephalomyelitis (EAE) EAE is the most intensively studied animal model of MS, and many believe the best (Ebers, 1998). Interestingly, it was started at the same period as TMEV model. The first time that EAE was induced was in 1933 when Rivers et al. observed a paralysis accompanied by demyelination in monkeys given repeated intramuscular injections of rabbit brain extract (River et al., 1933). From then on, this model has evolved considerably, and a number of new developments have made it increasingly relevant to MS. EAE can be produced in a variety of animal species using different CNS antigen  10  preparations, including whole spinal cord (Brown and McFarlin, 1981), myelin basic protein (MBP) (Fritz and McFarlin, 1989), proteolipid protein (PLP) (Trotter et al., 1987), and peptides of these proteins (Martin et al., 1992). With the development of complete Freund's adjuvant, EAE can be induced in susceptible animals following a single injection of CNS antigen emulsified in the adjuvant (Morgan, 1946; Kabat et al., 1947). In 1960, passive transfer of EAE by lymphocytes was introduced by Paterson (1960). From that time, passive transfer of EAE has been achieved by injection of activated encephalitogenic T cells, obtained from sensitized animals, and is now known as "passive or adoptive EAE". Although EAE can be induced in various animal species, it has been found inducible only in susceptible strains, according to diverse modalities (Swanborg, 1995). Susceptibility also depends on other contributing factors, such as age (Funjinami and Paterson, 1977), sex (Voskuhl and Palaszynski, 2001) and even commercial source of the animals. EAE susceptibility has been shown to be under genetic control in rats, mice, guinea pigs, and rabbits (Bernard, 1976; Geczy et al., 1984; Villarroya et al., 1990). The susceptible strains in the most-used species because of their high level of reproducibility are female SJL/J and PL/J mice (Fritz et al., 1983), and Lewis rat (Gasser et al., 1975). Genetic susceptibility in EAE is linked to the immune system through the T-cell response to a particular encephalitogenic epitope dependent upon the T-cell receptor (TCR) repertoire and MHC class II restricting elements available to the responder strain of animal (Swanborg, 1995). For example, the response of Lewis rats to MBP epitope 68-86 is restricted by RT-1B (the rat homologue of murine I-A), and the T cells preferentially use V 2:Vp8 in their TCR (Burns et al., 1989; Chluba et al., 1989), whereas the response a  11  of Lewis rats to MBP epitope 87-99 is restricted by RT-1D (the homologue of I-E) with utilization of diverse T cell V region gene products (Offher et al., 1989; Sun et al., 1992). Similar genetic associations have also been reported in populations with a high incidence of MS where HLA class II DR genes, the human homologue of I-E, are over-represented (Martin et al., 1992).  A.  Days after immunization 0  W M  5  TO' :i£ i*i;*iyi-.ri»»»«»««»«»»»•»»»'  B.  'M  35  :  40  i.i-*»  Days after immunization G  10,  15  20.  25  30-  35 :  40,  Figure 1.2. Clinical course of acute E A E and chronic relapsing E A E  (CREAE). Figure A shows a typical pattern of recovery from acute EAE. The severity of clinical symptoms peaks around Day 15, the animal then recovers completely without treatment by Day 18-20. Figure B shows a typical pattern of clinical symptoms from CREAE. The severity of symptoms peaks around Day 15, the animal then recovers completely but a relapse of symptoms occurs at Day 2325. (Adapted from Clinical and Experimental Immunology 100:344-51, 1995, Huitinga T)  12  Like MS, the clinical course of EAE can be quite variable. Acute EAE, normally produced in the Lewis rat, presents as a monophasic disease, characterized by inflammatory foci in the CNS, with limited demyelination (Bernard et al., 1992). Chronic relapsing EAE (CREAE), usually induced in mice by immunization with whole CNS tissue homogenate, presents as a chronic relapsing and remitting disease, which is characterized by both massive inflammation and demyelination in the CNS (Bernard et al., 1992). These clinical variations of EAE have served as animal models for the different clinical courses observed in individual patients with MS. Acute EAE offer the advantage of predictable time of onset and uniform severity of disease. Most animals spontaneously recover (Swanborg, 1988). CREAE resembles MS in human closely. However, a disadvantage is the relative resistance of mice to EAE, which is reflected in variable incidence and time of onset. In acute EAE, the clinical signs begin 10-12 days after immunization (Fig. 1.2A) (Huitinga et al., 1995). The clinical presentation is characterized by a waddling gait, rapidly progressing to ataxia, hind limb weakness, paralysis, and urinary incontinence. Most animals recover by 18-20 days postimmunization. In CREAE, the following relapse can be expected after 3-4 weeks of induction. More remissions and relapses may follow, although less predictably than the first relapse (Fig. 1.2B) (Huitinga et al., 1995). Pathological alterations of EAE reflect many of the features seen in MS. Inflammatory infiltrates precede white matter changes and consist of lymphoid infiltrates in meninges, perivascular spaces, and eventually the parenchyma itself. Macrophages are numerous, although rarely as numerous as in TMEV infection in SJL/J mice (Traugott et al., 1986; Lyman et al., 1989). Correspondence with clinical evidence of remitting-  13  relapsing activity is generally good, but pathological changes often precede the clinical presentation of signs by a few days. During the remitting phase of the disease, inflammation subsides and glial processes take much of the space left behind them. Remyelinating activity has been observed in CREAE lesions (Raine et al., 1984). The cytokine pattern displayed by encephalitogenic T cells in the CNS of EAE is similar as in MS, which is composed of IL-1, INF-y, TNF-a, and IL-2 etc (Noronha and Arnason, 1996). T cell receptor rearrangements indicative of T cells reactive to myelin can even be detected in demyelinating areas from spinal cords of mice with EAE (Steinman, 1996). Similarly, antibody responses to MBP and to myelin oligodendroglia glycoprotein (MOG) can be detected at the site of vesiculating myelin in EAE brain (Warren et al., 1995; Genainetal., 1999). Overwhelming evidence demonstrates that EAE is T cell-mediated autoimmune disease. Different from TMEV infection, the driving antigen in EAE is clearly derived from myelin itself. Such encephalitogenic antigens include MBP (Fritz and McFarlin, 1989; Goverman et al., 1993), PLP (Sobel et al., 1994), and MOG (Amor et al., 1994). It was Kabat (1946, 1947) who first suggested that EAE may have an autoimmune etiology and that the appropriate autoantigen was located in the white matter. He shrewdly observed that injection of fetal CNS tissue (before myelination) did not cause EAE. In addition, adoptive transfer of EAE by MBP-specific T cells unequivocally demonstrates that encephalitogenic T cells play a critical pathogenic role in EAE. In summary, the clinical, immunopathological, and histopathological features of EAE resemble those seen in MS patients (Table 1.1). Thus, the experimental disease has been widely utilized as a model with which to gain insight into the mechanisms underlying its  14  human counterpart. Since it is becoming increasingly convincing that an autoimmune process is involved in the pathogenesis of MS, and due to the extensive characterization,  Table 1.1 Comparison between Multiple Sclerosis and E A E Clinical Presentation Relapses and remissions Paralysis Ataxia Visual impairment Genetics MHC-linked susceptibility Females more susceptible Pathology in Lesions T cells reactive to myelin Antibodies to myelin a-4 integrin, complement TNF-a, IFN-y Demyelination Axonal dystrophy Therapy IFN-y, systemic anti-TNF-a, systemic IL-4 transduced T cells TNF-a transduced T cells Copaxone IFN-p  MS  EAE  present present present present  present present present present  yes yes  yes yes  present present present present present present  present present present present present, mild present  worsens worsens not done not done improves improves  cures cures cures worsens cures improves  * Table adapted from Neuron 24: 511-514, 1999, Steinman L.  detailed knowledge of the encephalitogenic epitopes on multiple myelin proteins in EAE, and the relative ease of induction, I employ this model of MS in my studies. In addition, as mentioned above, EAE can be induced in a variety of species and strains of animals. But rats of the Lewis strain offer the advantage of predictable time of onset and uniform susceptibility to MBP. Most animals spontaneously recover. I have chosen Lewis rats in my studies because the acute onset and spontaneous recovery offer a model with which to study the mechanisms that underlie the induction and remission phases of the disease.  15  1.3 PATHOGENESIS OF E A E  It is now well accepted that EAE is an autoimmune disease characterized by perivascular CD4 T cell and mononuclear cell inflammation in the CNS (Swanborg, +  1988). Although its pathogenesis is not fully understood, we have seen tremendous progress in the studies of the immune response in this disease. A widely accepted view of the process involved in EAE lesions reveals that T cells, macrophages, immunoglobulin, and complement play a role in pathogenesis. Adhesion molecules, cytokines, chemokines, and metalloproteases are critical participants in the development of the inflammatory response in the brain and spinal cord (Stuerzebecher and Martin, 2000). Initially, autoreactive myelin-specific CD4 T cells are activated in the periphery +  following immunization with MBP or other myelin antigen in complete Freund's adjuvant (Miller and Shevach, 1998). Interferon-gamma (IFN-y), tumor necrosis factoralpha (TNF-a), and other cytokines released in the inflammatory response, can induce the cerebrovascular endothelial cells to express vascular cell adhesion molecule (VCAM), intercellular adhesion molecule (ICAM), and major MHC class II molecules (Washington et al., 1994). Once the blood-brain barrier (BBB) is inflamed, ICAM/lymphocyte function-associated antigen-1 (LFA-1) and VCAM/very late antigen-4 (VLA-4, expressed by T lymphocytes) interactions, in conjunction with other factors such as CD4MHC class II binding, allow autoreactive T cell diapedesis and entry into the CNS (Baron et al., 1993; Steffen et al., 1994; Romanic et al., 1997). Penetration of the T cells into the CNS parenchyma is enhanced by increased activity of endogenous matrix metalloproteases (MMP) responsible for the breakdown of extracellular matrix material  16  (Yong et al., 1998). Within the CNS, autoreactive T cells may be activated further by recognizing myelin antigen presented by resident microglia, astrocytes, or blood-derived monocytic cells in the context of MHC class II (Stuerzebecher and Martin, 2000). These T cells, and the associated antigen presenting cells, then release proinflammatory cytokines (known as Thl type cytokine) and chemotactic factors that lead to activation of the endothelium and resident glial cells, resulting in the recruitment of additional lymphocytes and monocytes to sites of inflammation (Olsson, 1995; Steinman, 1996). On the other aspect, cytokines characteristic of Th2-type responses, such as interleukin-4 (IL-4) and interleukin (IL-10), as well as transforming growth factor p (TGF-P), are thought to participate in recovery from the inflammatory events by down-regulating the activated state of endothelial cells, and lymphocytes (Miller and Karpus, 1994; Olsson, 1995). It is thought that encephalitogenic CD4 T cells are pivotal to disease expression +  through the production of cytokines that promote inflammation and activate secondary inflammatory cells such as macrophage. Included in the cytokines produced by these T cells are lymphotoxin (LT), TNF-a, and IFN-y (Miller and Karpus, 1994). These substances can result in direct nervous cell injury (Selmaj and Raine, 1988; Selmaj et al., 1991), promote cell-mediated demyelination (Zajicek et al., 1992), and activate macrophages, astrocytes, and microglia, which in turn express TNF-a in active lesions (Farrar and Schreiber, 1993). IFN-y promotes the induction of MHC in glial cells, allowing them to potentially present antigens (Welsh et al., 1993; MaCarron et al., 1990). IFN-y can also induce Fas receptor expression on oligodendrocytes. This result  17  contributes to CD4 lymphocyte-mediated oligodendrocyte destruction by Fas/Fas ligand +  interactions (Pouly et al., 2000). Although CD4 T cells are essential for the production of EAE, most evidence +  indicates that additional mechanisms are necessary for disease. Because oligodendrocytes in vivo do not express MHC class II molecules, it is unlikely that they are directly killed by encephalitogenic CD4 T cells (Suzumura et al., 1986; Grenier et al., 1989). Recent +  studies have shown that cytotoxic CD8 T cells responses can be induced by various +  myelin peptides presented in the context of MHC class I, and that these T cells produce TNF-a, IFN-y, and other potentially toxic factors such as perforin and granzyme (Tsuchida et al., 1994). Since oligodendrocytes can be induced by cytokines to express MHC class I antigens, CD8 T cells could initiate myelin loss in the lesion (Grenier et al., +  1989; Tsuchida et al., 1994). B cells, demyelinating antibodies, and complements also have a role in EAE pathogenesis (Warren and Catz, 1992; Piddlesden et al., 1993; Wucherpfennig et al., 1997). Antibodies directed against myelin constituents can cause demyelination by several means. These include antibody-dependent cell-mediated toxicity (ADCC) (Brosnan et al., 1977); release of cytokines through Fc receptor stimulation on macrophages, natural killers, or mast cells (Raine and Scheinberg, 1988; Stangel and Compston, 2001); myelin opsonization, or complement activation (Hartung i  and Rieckmann, 1997). In addition, in immune reactions mediated by CD4 T cells in EAE, recruitment and +  activation of macrophages plays an important role. Recent evidence suggests that, apart from their antigen-presenting function, macrophages and their products, free radicals, have a pivotal role in the final effector phase of EAE (Benveniste, 1997). Consistent with  18  this is the observation that deletion of macrophages can protect animals against EAE (Huitinga et al., 1990), and that the presence of chemokines involved in macrophage recruitment correlates with disease expression (Berman et al., 1996). Furthermore, that macrophages represent the final common pathway for myelin removal and degradation is generally accepted (Smith, 2001). Activated macrophages release many factors that are toxic for neurons and oligodendrocytes, among which free radicals are important ones (Klegeris and McGeer, 1994). In recent years, increasing evidence demonstrates that oxidative stress plays an important role in the pathogenesis of MS and EAE. First, it has been shown that the CNS, notably the oligodendrocyte, is extremely vulnerable to oxidative damage due to many risk factors, including a high iron content, extensive elaborations of membranes, and relatively low levels of antioxidant defenses (Smith et al., 1999; Hollensworth et al., 2000). Second, there is convincing evidence to demonstrate that oxidative stress is a prominent feature of inflammatory demyelinating disease. Free radicals are produced massively both in EAE and MS (Smith et al., 1999). In the center and edge of active EAE lesions, the presence of both ROS and RNS has been clearly documented, strongly implicating a role for these volatile oxidants in lesion formation (Guy et al., 1993; Ruuls et al., 1995). It has also been indicated that free radicals play an important role in the pathogenesis of disruption of the BBB in EAE (Guy et al., 1994; Merrill and Murphy, 1997), and that these factors are required for the phagocytosis of myelin by macrophages (van der Goes et al., 1998). The use of several antioxidants has proved effective in the treatment of EAE (Ruuls et al., 1995; Zhao et al., 1996). Free radicals of primary concern are the superoxide anion ('O2"), hydroxyl radical (OH), hydrogen peroxide  (H2O2),  peroxyl radical (ROO), nitric oxide ( NO), and peroxynitrite -  19  (ONOO"). Free radicals can damage the CNS by various cellular effects, including lipoperoxidation, DNA oxidation and protein oxidation (Gate et al., 1999).  1.4 ANTIOXIDANT SYSTEMS OF T H E ORGANISMS  Oxidative stress is an unavoidable consequence of life in an oxygen-rich atmosphere. Free radical production occurs continuously in all cells as part of normal cellular function (Halliwell and Gutteridge, 1999a). However, excess free radical production originating from endogenous or exogenous sources might play a role in many diseases. Oxidative injury is increasingly recognized as an important pathological factor in a wide variety of human diseases, including many neurological disorders, such as cerebrovascular disease, mitochondrial disorders, amyotrophic lateral sclerosis, Huntington's disease, Alzheimer's disease, and epilepsy (Delanty and Dichter, 1998). Organisms employ a multitude of antioxidant systems to defend against the deleterious effects of oxidative stress. These systems can be divided into four main groups: antioxidant enzymes, transition metal binding protein, heat shock proteins, and small molecule antioxidants (Halliwell and Gutteridge, 1999b). (1) Antioxidant enzymes catalytically remove free radicals and other "reactive species". Examples are superoxide dismutase (SOD), catalase, and glutathione peroxidase (GP ). SOD catalyze the dismutation of 0 ~: 20 " + 2H X  2  2  +  H 0 + 0 . The toxic product 2  2  2  H 0 is usually removed in mammalian cells by catalase and GP . Both catalase and GP 2  2  X  X  detoxify H 0 by reducing it to water and oxygen (Halliwell and Gutteridge, 1999b). 2  2  These enzymes are present in almost all cell types, indicating a universal requirement of  20  them for organisms (Halliwell and Gutteridge, 1999b). (2) Transition metal binding proteins, including ferritin, transferrin, lactoferrin, and caeruloplasmin, act as a crucial component of the antioxidant defense system by sequestering iron and copper so that they are not available to drive the formation of the hydroxyl radical (Gutteridge and Stocks, 1981; Halliwell and Gutteridge, 1984, 1990; Harrison and Arosio, 1996). (3) Heat shock proteins are evolutionary highly conserved molecules classified according to molecular weight that occur in most cell types and that are expressed usually as a protective mechanism by cells in response to heat shock and stress (Young, 1990). These proteins act to preserve, or recover the function of other proteins during and after stress (Kaufmann, 1994). (4) In addition, organisms contain a complex mixture of small molecule antioxidants. These low-molecular-mass agents can directly react with free radicals and disarm them (Ozben and North Atlantic Treaty Organization. Scientific Affairs Division., 1998). Examples are glutathione, a-tocopherol, bilirubin, and uric acid. Some low-molecular-mass antioxidants come from the diet, especially ascorbic acid and a-tocopherol. There is an intimate relationship between nutrition and antioxidant defense (Halliwell and Gutteridge, 1999c). The constituent antioxidant defenses are often overwhelmed in free radical-induced diseases. Many enzymes and proteins comprising these protective systems are inducible under conditions of oxidative stress adaptation, in which the expression of over 40 mammalian genes is upregulated (Davies, 2000). Among these a key induced activity is HO-1. For example, it was confirmed in IMR-90 and HeLa cells by Northern blot analysis that the NO-mediated induction of HO-1 is much more substantial than of many other antioxidant enzymes and proteins, such as a tyrosine/threonine phosphatase  21  (CL100/MKP-1) and manganese containing SOD (Marquis and Demple, 1998). HO-l is now considered to be one of the most powerful cellular defensive mechanisms against oxidative stress due to many its properties, which I will discuss in the following.  1.5 H E M E OXYGENASE (HO)  co  iron  NADP Heme Oxygenase  Heme  > Biliverdin NADPH NADH  NADPH  Biliverdin Reductase  NADP NADH  Bilirubin  Figure 1.3. The heme oxygenase enzyme reaction. Scheme of catalytic conversion of heme into bilirubin, carbon monoxide (CO), and iron. (Adapted from Biochemical Pharmacology 60:1121-1128, 2000, Immenschuh S)  Heme oxygenase is the rate-limiting enzyme in the catabolism of heme molecules to biliverdin, carbon monoxide (CO), and free iron (Tenhunen et al., 1968) (Fig. 1.3). Biliverdin is subsequently converted to bilirubin by biliverdin reductase (Kikuchi and Yoshida, 1983). Although it has been known for more than a hundred years that bile pigments are the products of the physiological degradation of heme, it is only 30 years since the enzyme responsible for this metabolic process was first recognized. HO was originally identified in 1968 and 1969 by Tenhunen et al. in a series of papers where they  22  characterized the enzyme HO as well as its cellular localization (Tenhunen et al., 1968, 1969). To date, three isoforms of HO, HO-1, HO-2, and HO-3, have been reported (Maines, 1988; McCoubrey et al., 1992; McCoubrey et al., 1997). They are products of different genes (Maines, 1999). HO-1 is a 32-kDa protein that is inducible by numerous stimuli, and has been shown to be identical to heat shock protein 32 (Maines, 1999). HO2, a 36-kDa protein, is constitutively active but unresponsive to any of the inducers of HO-1 (Shibahara et al., 1993; McCoubrey and Maines, 1994). It exists primarily in the brain and testes. HO-3, a recently cloned gene product 33-kDa in size, is nearly devoid of catalytic capability and may function chiefly as a heme-sensing or a heme-binding protein (McCoubrey et al., 1997). Its property awaits further characterization. The HO system was initially considered only as enzymes for degradation of the heme and production of toxic waste products. However, in the past decades, concomitant with the discovery of the essential role of another toxic gas, NO, for generation of cGMP, there has been an explosion of new information regarding these enzymes. It is believed that the HO enzyme reaction is physiologically significant because HO degrades the prooxidant heme and produces equimolar amounts of bilirubin, a potent antioxidant, CO, a transmitter, and iron, which have important regulatory and protective functions of their own (Llesuy and Tomaro, 1994; Weiss et al., 1994; Maines, 1997). It has been shown that HO plays a key role in the response of cells and organisms to oxidative stress and that its second form, HO-2, has physiological functions in the brain (Maines, 1999). HO has now found relevance in a number of experimental injuries and human diseases ranging from inflammation to neurodegenerative diseases (Maines, 1999).  23  1.5.1  Constituent H  O (HO-2)  Constituent HO, HO-2, predominates under normal physiological conditions. It is widely expressed in endothelium and neurons, with high concentrations in the brain and testes, where its distribution closely parallels that of soluble guanylate cyclase (Zakhary et al., 1996). HO-2 is crucial to the normal functioning and cellular homeostasis in the CNS and other systems (Maines, 2000). This would involve its generating products, particularly the CO that are of importance to signal transduction. HO-derived CO has been recognized to be an important cellular messenger with various physiological functions (Marilena, 1997). The signaling functions of CO resemble that of the signaling gas NO. In contrast to NO, however, which form peroxynitrite with superoxide, CO does not form radicals. The pioneering work of Snyder et al. and Morita has clearly shown that CO generated from HO can regulate vasomotor tone by promoting vasorelaxation (Morita and Kourembanas, 1995; Snyder et al., 1998). CO has been implicated in the control of cerebral blood flow in the brain, a tissue with a great capacity to generate CO from HO-2 (Montecot et al., 1998). In brain, there is also evidence that CO is involved in long-term potentiation (Stevens and Wang, 1993; Zhuo et al., 1998), which plays a key role in memory and learning. HO inhibitors prevent induction of long-term potentiation in the CAI region of hippocampal slices (Hawkins et al., 1994). In addition, CO has been shown to be implicated in neuroendocrine control of corticotropin-releasing hormone from the hypothalamus (Pozzoli et al., 1994). Consistent with this finding, HO-2 mutant mice were reported to show decreased responsiveness to electrical field stimulation (Zakhary et al., 1997). All these effects of CO are mediated through the activation of guanylate cyclase on the binding of CO to the heme moiety of this enzyme and  24  subsequent cGMP generation (Zigmond, 1999). But the function of CO is thought to be 50-100 times less potent than that of NO based on the capacity of both molecules to activate guanylate cyclase. However, the relative inefficiency of CO in binding guanylate cyclase may be largely neutralized because although NO is extremely reactive and labile, CO is chemically very stable. Unlike NO, CO reacts exclusively with heme and thus can accumulate in the cell to levels that are presumably much higher than those of NO. In spermatozoa, it appears that HO-2 expression is linked to the maturation and expression of factors that regulate sperm development (McCoubrey et al., 1995). HO-2 plays an important role in male reproductive behavior (Burnett et al., 1998). HO-2 is notably concentrated in the innervation of the bulbospongiosus, which mediates ejaculation. Reflex activity of this muscle is abolished in HO-2"" mice. Moreover, 7  ejaculation is substantially reduced in intact HO-2 " mice (Burnett et al., 1998). Lastly, 7  recent evidence also favors a neuroprotective role for HO-2. Oxygen toxicity in brain culture is markedly augmented in HO-2" mice (Dore et al., 1999a). Augmented A  neurotoxicity is associated with a selective increase in apoptotic death and is rescued by HO-2 transfection (Dore et al., 2000). HO-2 " animals also display greatly increased _/  neural damage after middle cerebral artery occlusion (Dore et al., 1999b).  1.5.2 Inducible HO (HO-1) HO-1, a potent antioxidant enzyme. In contrast to HO-2, HO-1 is the inducible isoform of HO. With the exception of the spleen where senescent erythrocytes are destroyed and HO-1 expression is constitutively high, under physiological conditions HO-1 expression is low (Braggins et al., 1986). However, in response to numerous stress  25  stimuli, HO-1 is highly inducible in various cells and tissues (Maines, 1999). In the CNS, the protein is primarily expressed at high levels in the glial cell population and macrophages in response to stress (Ewing et al., 1992). HO-1 is induced by heme and also by a variety of non-heme products such as: ultraviolet irradiation (Ossola and Tomaro, 1998), heavy metals (Eyssen-Hernandez et al., 1996), inflammatory cytokines (Terry et al., 1998), endotoxin (Carraway et al., 1998), heat shock (Shibahara et al., 1987), oxidative stress (Keyse and Tyrrell, 1989), hypoxia (Lee et al., 1997) and hyperoxia (Lee et al., 1996). One common feature of these inducers is their capacity to generate free radicals, which not only demonstrates that HO-1 can be induced by agents causing oxidative stress but also supports the speculation that HO-1 functions as a cytoprotective molecule against oxidative stress. Indeed, ample evidence currently supports the notion that HO-1 represents a powerful endogenous defensive mechanism against oxidative stress in vitro and in vivo. The important function of HO-1 has been confirmed by observations in a series of studies. For example, it has been shown that induction of HO-1 protects endothelial cells from oxidant-mediated injury (Motterlini et al., 2000). This cytoprotective effect is considerably attenuated by tin protoporphyrin IX, an inhibitor of HO activity. Cultured cells from HO-1 knockout mice are highly susceptible to heme- or hydrogen peroxide-mediated toxicity (Poss and Tonegawa, 1997). In addition, exposure of HO-1-deficient mice to endotoxin results in increased hepatocellular necrosis and in higher mortality from endotoxic shock as compared to control animals (Poss and Tonegawa, 1997). A recent report demonstrating the first identified case of HO-1-deficient human patient lends additional support to the evolving paradigm that HO-1 serves to provide cytoprotection against oxidative stress (Yachie et  26  al., 1999). This patient exhibits growth retardation, anemia, and increased sensitivity to oxidative damage. Although the mechanism mediating HO-1-induced cytoprotection is still incompletely understood, recent data suggests that the antioxidant function of HO-1 is due not only to degradation of the pro-oxidant heme but also to the formation of the catalytic by-products (iron/ferritin, CO, and bilirubin), which may represent the potential mediators. As the aforementioned, CO plays an important role in regulating vasomotor tone by promoting vasorelaxation (Snyder et al., 1998). The vasorelaxation may allow maintenance of blood flow at sites of oxidative stress, thus advance the clearance of free radicals. CO may also possess anti-inflammatory effects because vasal relaxation can counter those effects of coagulation and thrombosis that lead to anoxia and tissue necrosis (Otterbein and Choi, 2000). Bilirubin, another by-product of heme catabolism, has long been regarded as a potentially cytotoxic waste product that needs to be excreted. However, in recent years, bilirubin has been demonstrated to be a powerful antioxidant substance in vitro and also be a very effective physiological antioxidant in vivo (Marilena, 1997). Bilirubin and its serum albumin complex are superoxide scavengers and peroxyl radical-trapping antioxidants, its potency increasing with decreasing pH (Stocker et al., 1987). It has been shown that bilirubin suppresses oxidation more strongly than many other antioxidants, including a-tocopherol, SOD, and catalase (Stocker et al., 1987; Wu et al., 1991). Bilirubin added to the cultures is markedly neuroprotective. As little as lOnM bilirubin concentration protects against 10,000 times higher concentrations of hydrogen peroxide (Dore et al., 1999a). Recent observations suggest that bilirubin exerts its powerful neuroprotective effects by redox cycling (Baranano and Snyder, 2001): when bilirubin  27  acts as an antioxidant, it is itself oxidized to biliverdin, which is in turn immediately reduced back to bilirubin by the tissue excess of biliverdin reductase. Synder and Baranano have been able to demonstrate this process utilizing mixtures of the key chemicals and enzymes (Snyder and Baranano, 2001). In addition, bilirubin exhibits several other physiological effects, such as antimutagenic and anticomplement actions (Marilena, 1997). On the other aspect, enzymatic degradation of heme by HO also produces iron, which is cytotoxic via the production of hydroxyl radical by Fenton chemistry. It has been suggested that coinduction of ferritin, a protein that could sequester the redox-active iron, may counteract iron release (Balla et al., 1992). Vile and Tyrrell have shown that  ^  synthesis of ferritin is unregulated depending on increased HO-1 activity during the cellular stress response (Vile and Tyrrell, 1993). The HO-1-dependent release of iron also results in the increase in ferritin expression (Eisenstein et al., 1991). Furthermore, another recent report demonstrated that over-expresison of HO-1 upregulates and interacts with an iron ATPase present in the endoplasmic reticulum (Baranano et al., 2000), which is closely linked to cellular iron extrusion mechanisms. This would limit intracellular iron content and prevent iron generated by HO from damaging cells. A protein associated with iron extrusion has recently been cloned (Donovan et al., 2000). In summary, the antioxidant effect of HO-1 is multifactoral and dependent on the diverse dimensions of HO activity. All these properties contribute to the potent antioxidant effect of HO-1. HO-1 and diseases. HO-1 is induced in a number of experimental injuries and diseases of various organs, including carrageenin-induced pleurisy (Willis et al., 1996),  28  congestive heart failure (Raju et al., 1999), kidney reperfusion injury (Raju and Maines, 1996) , caerulein-induced pancreatitis (Sato et al., 1997), atherosclerotic lesions (Juan et al., 2001), traumatic brain injury (Fukuda et al., 1996), cerebral hemorrhage (Matz et al., 1997) , ischemic stroke (Takizawa et al., 1998), and neurodegenerative diseases (Pappolla et al., 1998; Riedl et al., 1999). It has been demonstrated that activation of the endogenous HO-1 gene may be protective against the deleterious effects of stressmediated injury in various pathological conditions. For example, Takizawa et al. have shown in vivo that induction of HO-1 by hemin protects cortical neurons against transient forebrain ischemia, whereas specific inhibition of HO enzyme activity by tinmesoporphyrin LX tends to decrease viable neurons in the cortex (Takizawa et al., 1998). Similarly, a carrageenin-induced complement-dependent pleurisy is attenuated by induction of HO-1 activity, while inhibition of HO-1 activity potentiates this inflammatory response (Willis et al., 1996). Others have shown that HO-1 inhibits the development of atherosclerosis in apolipoprotein E-deficient mice (Juan et al., 2001). Also, a recent study demonstrated that HO-1 induction attenuates the experimental Parkinson's disease (Riedl et al., 1999). These observations in different organs along with the findings that HO-1-deficient mice are more susceptible to oxidative damage provide strong evidence that HO-1 may play a protective role in diseases associated with oxidative stress. However, few studies have reported the expression of HO-1 in EAE, and the role of this important enzyme in EAE remains unresolved.  1.6 HYPOTHESIS AND EXPERIMENTAL OBJECTIVES  29  Since oxidative stress plays an important role in pathogenesis of EAE, and HO-1 represents a powerful inducible antioxidant mechanism in many other stress-mediated diseases, it is reasonable to make a hypothesis that HO-1 may be induced and play a role in EAE. In this research, I will detect the induction of HO-1 in EAE, and evaluate its effect on this disease. In addition, I will investigate whether HO-2 is constituently expressed in the spinal cord as in the brain.  30  Chapter 2 MATERIALS AND METHODS  2.1 MATERIALS  2.1.1 Animals A total of 77 adult male Lewis rats with body weights between 175 and 200 g were obtained from Charles River Laboratories (Laval, Canada). The rats were housed in the animal facility of Vancouver General Hospital, and were cared for in accordance with the Animal Welfare Act of the University of British Columbia. The rats were bred at least one week before being used for experiments.  2.1.2 Chemicals and Reagents The following items were obtained from Amersham Pharmacia Biotech (Baie d'Urde, Quebec): ECL™ western blot analysis system, horseradish peroxidase (HRP) conjugated anti-rabbit IgG, hydrophobic polyvinylidene difluoride (PVDF) membrane. The following items were purchased from Bio-Rad laboratories (Hercules, CA): protein assay, N, N, TV, /Y-tetramethylene-ethylenediaine (TEMED), sodium dodecyl sulfate (SDS), ammonium persulfate (APS), 2-mercaptoethanol. The following items were obtained from Frontier Scientific (Logan, UT): hemin, tin mesoporphyrin (SnMP). The following items were obtained from Sigma (Saint Louis, MO): ammonium nickel sulfate, bovine serum album (BSA), 3,3'-diaminobenzidine (DAB), eosin Y, guinea pig  31  myelin basic protein (MBP), hydrogen peroxide (H2O2), imidazole, incomplete Freund's adjuvant, paraformaldehyde (PFA), Triton X-100. The following items were obtained from StressGen (Victoria, BC): rabbit anti-HO-1 polyclonal antibody (Product #: SPA-895), rabbit anti-HO-2 polyclonal antibody (Product #: OSA-200) The following items were obtained from Vector Labs (Burlingame, CA): avidinbiotin complex (ABC), biotinylated goat anti-rabbit IgG, biotinylated goat anti-mouse IgG, normal goat serum. BenchMark™ Protein Ladder was purchased from GIBCO BRL (Grand Island, NY). Euthanyl was obtained from Bimeda-MTC (Cambridge, ON). Harris' hematoxylin was obtainedfromFisher Scientific (Nepean, ON). Heat-inactivated Mycobacterium tuberculosus H37Ra was obtained from Difco (Detroit, MI). Kodak BioMax MR film was purchased from Eastman Kodak Company (Rochester, NY). Mouse anti-EDl monoclonal antibody was obtained from Accurate (Westbury, NY). Mouse antineurofilaments monoclonal antibody was obtained from Sternberger (Lutherville, MD).  2.2 METHODS  All procedures included in this study were approved by the Animal Care Committee of the University of British Columbia (Protocol #: AO-0153).  2.2.1 Solution Preparation  32  0.1 M Phosphate buffered saline (PBS) (pH 7.4): 2.89 g sodium dihydrogen orthophosphate (NaH2P04 H 0), 11.5 g disodium hydrogen orthophosphate -  2  anhydrous (T^HPC^), and 9 g sodium chloride were dissolved in distilled H2O (dH 0) to make 1 liter PBS. 2  4% paraformaldehyde (PFA): 20% stock PFA was made by dissolving 20 g PFA in 100 ml dH 0 at 60°C. 5 N NaOH was then added until the solution became 2  clear. Stock PFA was filtered and stored at 4°C. Before use, 20% PFA was diluted with 0.1 M PBS to 4% working PFA solution. Triple detergent: it contains 50mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 0.1% SDS, l%NP-40, 0.5% sodium deoxycholate, 100 ug/ml PMSF, and 1 u.g/ml aprotinin. PMSF and aprotinin were added immediately before use. Before PMSF and aprotinin were added, the solution was stored at room temperature. 30% Acrylamide Mix: 29.2 g acrylamide and 0.8 g TV, TV-methylene-bisacrylamide were dissolved in 100 ml dH20, and kept at 37°C for 5-10 minutes to improve dissolution. Solution was filtered and stored at 4°C in a dark container before use. Stacking gel: 0.33 ml 30% Acrylamide Mix, 0.25 ml 1.0 M Tris (pH 6.8), 0.02 ml 10% SDS, 0.02 ml 10% APS, and 0.002 ml TEMED were added into 1.4 ml dH 0 2  and mixed well to make 2 ml stacking gel. 10% APS and TEMED were added just before use. Separating gel (10%): 1.7 ml 30% Acrylamide mix, 1.3 ml 1.5M Tris (pH 8.8), 0.05 ml 10% SDS, 0.05 ml 10% APS, and 0.002 ml TEMED were added into 2.0  33  ml  (JH2O  and mixed well to make 5 ml 10% separating gel. 10% APS and TEMED  were added just before use. Electrophoresis buffer: 9 g Tris-base, 43.2 g glycine, and 3 g SDS were dissolved in dE^O to make 600 ml 5X stock solution, which was stored at 4°C. .  Sample buffer: 0.6 ml 1 M Tris-HCl (pH 6.8), 5 ml 50% glycerol, 2 ml 10% SDS, 0.5 ml 2-mercaptoethanol, 1 ml 1% bromophenol blue, and 0.9 ml dE^O were mixed well to make 10 ml 5X sample buffer. It is stable for weeks at 4°C or for months at-20°C. Transfer buffer: 5.82 g Tris, 2.93 g glycine, 0.0375 g SDS, and 200 ml of methanol were dissolved in dH^O to make 1 liter transfer. 0.05 M Tris buffer (pH 7.6): 6.06 g Tris-hydrochloride, 1.39 g Tris-Base, and 9 g sodium chloride were dissolved in dEbO to make 1 liter Tris buffer.  .  Nickel DAB: 4 g ammonium nickel sulfate (Ni(NH )2S04-6H 0) and 20 ml 1 M 4  2  imidazole was dissolved in 380 ml 0.05 M Tris buffer. 0.07% DAB was added before use and mixed well. Acidified alcoholic eosin: 2.0 g water-soluble eosin Y was dissolved in 40 ml dHaO. 160 ml 95% ethanol was then added and mixed well. The working eosin solution was made by adding 30 ml dH^O, 120 ml 100% ethanol, and 0.5 ml acetic acid into 50 ml above solution and mixing well.  2.2.2 Induction of E A E A. Antigen preparation 1. 5mg lyophilized guinea pig MBP was dissolved in 5ml dH^O.  34  2. 5ml modified complete Freund's adjuvant (CFA) was prepared by adding heatinactivated Mycobacterium tuberculosus H37Ra to incomplete Freund's adjuvant to a final concentration of 10 mg/ml. 3. The 5ml MBP was drawn into a 5-ml Luer-Lok tuberculin syringe, and the 5ml modified CFA was drawn into another 5-ml Luer-Lok tuberculin syringe. The two syringes were then connected by a special 18-G steel needle. MBP/CFA was emulsified by repeatedly drawing the mixture between the syringes through the needle until the emulsion had thickened. A stable emulsion should not disperse when a drop was placed on an aqueous surface. B. Rats immunization 1. The rats were anesthetized with ketamine/xylazine (5:1, v/v) at a dose of 100 ul/lOOg by i.m. injection. Abdomen skin was sterilized by 75% ethanol. 2. The emulsion was drawn into a 1-ml tuberculin syringe with a 26-G needle. The rats were then immunized with a s.c. injection of 50 ul emulsion in each side of abdomen near the inguen. The rats were weighed and monitored daily after immunization. Clinical EAE was scored in a "blinded" manner as follows: 0, no disease; 1, tail limpness; 2, hind limb paraparesis with clumsy gait; 3, hind limb paralysis; 4, tetraplegia; 5, moribund or dead. During EAE, any animal deemed to be in a moribund state was immediately euthanized.  2.2.3 Treatment Regimen As shown in Table 2.1, the rats were divided into three groups: 30 served as controls, 20 were treated with 40 umol/kg hemin, an HO-1 inducer, once daily from 7 to 17 days  35  Z o  o o  GO 3  2  H •a  3  3  i  00  o c •a  CD CL OQ  O  c  T3  H  OQ rt O  O C 13  5'  < CT  -<  3".  1  o  tt  cn  crq_ o"  s. o o 3 •a  a 3" B Eo" o & o 3 Sot?  §• s: & s S o I2.  z  o  3 CL C o  3 CL  CL  o CT CL  o  <'  <T  O  O  3  ^ cr  cr  CT CL  < CT 3"  3* 3 7?  a.  CT '<  1 — 1  5'  £1cr CT  CT ft CT CL  CL — En" O C L CT -t CT w ft O)  3  o  oo  _ tt  o  o o CL  a 3i £3  CT CL  3> » S 3 ft  3<  ft.  ""• 13 C t CT 3"  g  03  CT  33  P  ft ^  V„  2.  gas. £2.  oo ON  oo  CL  —  i—> cn  -> 8 CL  tt.  CT 3  C L CT -t  CT to c/l  ft  CT  GO  z  o  3 CT  O  CT  &  O  H -  S- 5'  w tt  •~ 2. D g»  > CT 9  rt 3"  13 W  CT oo  tt  CT  O  rt  CL w  cn CT /-^ tt CT"  O  c  CT  3  o  & s CT tt oo  •  00  cr CT o & C  tt  K) CT  3  O  3  13  CT  CT £.  zo  CL  —i tt O Eo' CT tt > CD a > CT ^  3 SS" o  CT^ rt O  5-  CL  3  on  13  a* o  o  3  CT  >  co  00  * > CT o  3  »  3 CL  5 <' 3 <& 3  CT* CT  tt ^  tf>0Q CT  3  3  co  e  tfOQ  9 3  3" W  tt ^  CT  z  »  3 Eo- o 9 -. fto 3 CT  "o2-9,W>  (  o" tt  tt  z rt a 3  urse on  o  3 13  3  O  3. -i  •o—  oo"  tS  O rt-  2. 3.  ca  cn  ft  CT tt CT CL  &  O  >^  tt  cT" tt  CT 3  CL  tt  S.I y "  ^ rtH  00 CT  rj ft  tt rt,  CT O  tt_ oo C n t° rt, v: 'pT cl 2. cr p, C L C L O  2O  3 qs tt e.  >  oo _.  CT tt 3 00  a-  3" CT  cyi  CK  rt tt  Vi  CT  3  ft  ft T= CT *r* tt  CT  CT"  o" i CT" CT g CL ft 0) tt  3) ,.  after immunization (DAI), and 20 were treated with 40 umol/kg SnMP, an HO-1 inhibitor, once daily from 7 to 17 DAI. Drugs were dissolved in 0.1 N NaOH and adjusted to pH 7.4 with 1 N HCI before use, and administered by i.p. injection. Control rats received injections with the same volume of saline on the same schedule. The volume injected in each rat was 0.5 ml. In each group, 10 rats chosen randomly were followed for the full clinical course until 30 DAI. In control group, 9 rats chosen randomlyfromthe remaining 20 rats were sacrificed at onset of disease (around 11 DAI), 9 rats were sacrificed at peak of disease (around 14 DAI) for histological studies (7 rats for histopathological and immunohistochemical studies, and 2 rats for western blot analysis in each case), and 2 rats were sacrificed after alleviation of disease for histopathological and immunohistochemical studies. In hemin- and SnMP-treated groups, all the remaining rats were sacrificed at peak of disease for histological studies. Hemintreated rats without clinical EAE were sacrificed at the same time point. In addition, 7 normal rats were sacrificed for histological comparative study.  2.2.4 Tissue Preparation Rats were sacrificed with an i.p. injection of 1.0 ml euthanyl. For western blot analysis, the rats were perfused transcardially briefly with 0.1 M PBS, and the lumbosacral spinal cords were immediately removed. The spinal cords were stored at 80°C until use. For immunohistochemistry, the rats were perfused with 300-500 ml 0.1 M PBS, followed by 100 ml 4% PFA. The lumbosacral spinal cords and inguinal lymph nodes were immediately removed, embedded in Tissue-Tek and frozen with 2methylbutane at -80°C for 5 minutes. The tissues were stored at -80°C until sections  37  were prepared. Serial sections were cut at 10 um on a Reichert-Jung 2800 Frigocut cryostat. The slices were stored at -20°C before staining.  2.3.5 Western Blot Analysis  A. Preparation Of Protein Samples Ten volumes of ice-cold triple detergent were added to one unit per weight of frozen brain tissue (10 ml triple detergent per gram tissue). The brain tissue was teased apart using a pair of forceps and the solution was incubated on ice for 30 minutes. High molecular weight chromosomal DNA was sheared by sonication on ice for 1 minute at maximum speed. The solution was then centrifuged at 15,000 g for 5 minutes at 4°C. The supernatant was aliquoted and placed into a -20°C freezer for short-term storage or -80°C for long-term storage.  B. SDS-Polyacrylamide Gel Electrophoresis The procedures for SDS-polyacrylamide gel electrophoresis include pouring a gel, preparing and loading samples, running the gel, and transferring to the PVDF membrane. The procedures for pouring a gel are as follows: 1) Carefully introduce 10% separating gel into gel sandwich using a pipette. Make sure there were no air bubbles in the gel. 2) Gently add about l-5mm of water on top of the separating gel solution, and wait for about 40 minutes at room temperature to allow the separating gel to polymerize. 3) Pour off the water covering the separating gel and pipet the stacking gel solution onto the separating gel until solution reaches top of front plate. 4) Carefully insert comb into gel sandwich until the bottom of teeth reach top of front plate. Make sure no bubbles were trapped on ends of the teeth. 5) Wait for about 30 minutes to allow the stacking gel  38  to polymerize. 6) Remove comb carefully. 7) Place the gel into electrophoresis chamber. 8) Add electrophoresis buffer to inner and outer reservoir. In order to load the same amounts of protein in each well, the protein samples were added to 1:7 diluted protein assay (5 ul protein sample was added to 1 ml diluted protein assay), and then the relative protein concentrations were measured from absorbance at 595 nm by spectrophotometer. After combining the protein sample with sample buffer, they were boiled for 5 minutes to denature the protein. Following spin down protein solution for 10 seconds, each well was introduced equal amounts of protein, about 30 ul of the protein sample according to the relative concentration. 10 ul protein molecular weight ladder was loaded to one well. The gel was run under 200 V for about 40 minutes until the dye front migrated to about 5mm from the bottom of the gel. The gel then was washed with transfer buffer for 15 minutes. The dry PVDF membrane was soaked in 100% methanol for 5 seconds, washed in dF^O for 5 minutes, and then incubated in transfer buffer for 10 minutes with shaking. At the same time, two thick filter papers were also soaked in transfer buffer. Then, a blotting membrane/filter paper sandwich was assembled by putting one filter paper, PVDF membrane, gel, and the other filter paper in the order from bottom to top. Proteins were electrophoretically transferred to the membrane under 20 V and 135 mA for about 30 minutes. C. Immunoblotting The PVDF membrane with protein was placed into a heat sealable plastic bag containing 5% BSA in PBS-T and incubated at 4°C overnight with shaking. All the washes and rinses in this procedure were done with PBS-T. The next day, the membrane  39  was quickly rinsed twice, washed once with shaking for 15 minutes and twice more for 5 minutes each with shaking. After the washes, the membrane was incubated in a heat sealed bag with primary rabbit anti-HO-1 (1:1000), or rabbit anti-HO-2 (1:1000) for 1 hour at room temperature with shaking. Unbound primary antibody was washed off by two quick rinses, followed by a 15-minute wash with shaking and then two more washes for 5 minutes each with shaking. The membrane was then incubated in HRP conjugated anti-rabbit IgG (1:1000) for 1 hour with shaking. Unbound secondary antibody was washed off by two quick rinses, followed by a 15-minute wash with shaking and then two more washes for 5 minutes each with shaking. The excess liquid from the washed membrane was drained onto a paper towel and the membrane was placed protein side up on a piece of Saran wrap. The remainder of the procedures was performed in the dark room. The membrane was then covered with the detection solution (consisting of equal volumes of solution 1 and solution 2 from Amersham ECL™ Western blotting analysis system) for exactly 1 minute. The membrane was removed from the detection solution and wrapped in a fresh piece of Saran wrap. The membrane was put in a cassette with the protein side up and a Kodak BioMax MR film was placed on the membrane. The exposure times for the film variedfrom30 seconds to 2 minutes. The ECL signal was detected by developing the film.  2.2.6 Immunohistochemistry The procedures for immunohistochemical staining are as follows: 1) The slides were arranged in a suitable bottomless glass slide carrier, and were air-dried for 30 minutes. 2) The sections werefixedin acetone for 10 minutes. 3) After two 5 minute washes in 0.1 M  40  PBS, the sections were immersed in 0.6 % H2O2 for 10 minutes to block endogenous peroxidase, followed by three 5 minute washes in 0.1 M PBS. 4) To block non-specific binding, the sections were incubated in 5% normal goat serum for 30 minutes. 5) The sections were then incubated with primary rabbit anti-HO-1 (1:500), or rabbit anti-HO-2 (1:500) in 0.1 M PBS with 0.4% Triton at 4°C overnight. 6) After three 5 minute washes in 0.1 M PBS on the next day, the sections were incubated with affinity purified biotinylated goat anti-rabbit IgG (1:500) for 1 hour. 7) Following three 5 minute washes in 0.1 M PBS, the sections werefinallyincubated in ABC (1:400) for 1 hour, and washed three times for 5 minutes each. 8) The bound antibodies were visualized with 0.07% DAB and 0.03% H 0 in PBS. 9) The sections were then washed in dH 0, gradually 2  2  2  dehydrated in increasing graded ethanol, cleared in xylene, and coverslipped. Immunohistology of HO-1 cells in spinal cord sections was evaluated +  microscopically by a semiquantitative score by evaluating number and distribution of H O - l cells: 0, no H O - l cells; +, singular H O - l cells; ++, accumulation of H O - l cells +  +  +  +  surrounding blood vessels; +++, many H O - l cells in the inflammatory infiltrate +  surrounding the blood vessel with H O - l cells spreading into the surrounding +  parenchyma.  2.2.7 Immunohistochemical double-staining Since in many other pathological conditions, the expression of HO-1 is prominent in activated macrophages (Mautes et al., 1998; Wang et al., 1998; Beschorner et al., 2000), and in the brain under physiological conditions, the expression of HO-2 is prominent in neurons (Yamanaka et al., 1996), I further investigated whether HO-1 is primarily  41  expressed in infiltrated macrophages in EAE, and whether HO-2 exists primarily in neurons in the spinal cord. Immunohistochemical double-staining technique was employed to define the cellular distribution of HO-1 and HO-2 in the rat spinal cords. The slides were fixed and processed as described in the immunohistochemistry protocol. After the sections were incubated with 5% normal goat serum for 30 minutes, one of the antibodies to be used in the double staining procedure, rabbit anti-HO-1 (1:500) or rabbit anti-HO-2 (1:500), was added to the sections. The immunohistochemistry procedures were carried out as described above. Instead of coverslipping the sections at the end, procedures were repeated from step 3) for the second staining. The sections were incubated with 0.6% H2O2 for 10 minutes to deactivate the activity of peroxidase enzyme remaining from the initial immunohistochemistry step. Following three 5 minute washes in 0.1 M PBS, the sections were blocked for 30 minutes with 5% goat normal serum and then the second antibody to be used in the double-staining, mouse anti-EDl (1:500) or mouse anti-neurofilaments (1:500), was added. The immunohistochemistry procedures were carried out as before. However, in step 6), the biotinylated goat anti-mouse IgG (1:800) was used according to the primary antibodies. And in step 8), the bound antibodies were visualized with nickel DAB containing 0.03% H 0 . 2  2  2.2.8 Hematoxylin-eosin (HE) staining The sections were stained with hematoxylin-eosin for histopathological examination. The procedures for HE staining are as follows: 1) The slides were air-dried for 30 minutes. 2) The sections were then immersed in 100%, 95%, 50% ethanol, and dH20  42  consecutively for 1 to 2 minutes in each case. 3) The sections were stained in Harris' hematoxylin for 4 to 8 minutes. 4) After a quick wash in water, the sections were differentiated by three or four dips (about 3 to 10 seconds) in 1% acid alcohol, followed by 1 minute wash in water. 5) To make the staining more bluish, the sections were immersed in 1.5% NaHCO"3 for 30 seconds. 6) The sections were washed well in running water. 7) The sections were then stained in acidified alcoholic eosin for A to 2 minutes, l  depending on intensity desired. To differentiate the eosin staining, the sections were washed in running water for Vi to 2 minutes. 8) The slides were gradually dehydrated in increasing graded ethanol, cleared in xylene, and coverslipped. Severity of inflammation in the spinal cord sections was graded semiquantitatively in blinded fashion as follows: 0, no inflammation; 1, mild meningeal inflammation and/or rare parenchymal infiltration; 2, moderate meningitis, sub-meningeal infiltration and small scattered perivascular infiltration; 3, severe meningitis, parenchymal infiltration and/or multiple perivascular infiltraion; 4, foci of necrosis and/or neutrophilic infiltration (Cao et al., 2000).  2.2.9 Statistical Analysis All data are expressed as mean ± s.e.m. Data on the effects of hemin and SnMP treatment shown in Figure 3.10 were analyzed using two-factor ANOVA. When significant differences were obtained, Fisher's PLSD post hoc tests were carried out for multiple comparisons. Data in Table 3.1 were analyzed using one-tailed Mann-Whitney test, p < 0.05 was considered statistically significant.  43  Chapter 3 RESULTS  3.1 SPECIFICITY OF HO ANTIBODIES  First, I investigated the specificity of the HO-1 and HO-2 antibodies by western blot analysis. Both of them were obtained from StressGen (Victoria, BC), and are polyclonal antibodies with the rabbit as the host. It was reported that anti-HO-1 and anti-HO-2 polyclonal antibodies could detect HO-1 and HO-2, respectively in samples from human, monkey, mouse (weakly), rat, rabbit and guinea pig (Technical specifications from StressGen: http://www.stressgen.com/reagents). In addition, anti-HO-1 antibody has shown very little cross activity with HO-2, and anti-HO-2 antibody has shown very little cross activity with HO-1. Figure 3.1 A shows the western blot analysis performed using anti-HO-1 polyclonal antibody on the spinal cords of rats with EAE at the peak of clinical signs. Figure 3. IB shows the similar analysis performed on the spinal cords of normal rats using anti-HO-2 polyclonal antibody. One main band at 32 kDa in the first case, and one main band at 36 kDa in the second case were detected confirming the fact that the anti-HO-1 and anti-HO2 antibodies from StressGen react well with the rat HO-1 and HO-2 proteins, respectively. Although both antibodies could recognize additional protein bands, the nonspecific bands were much weaker compared to the HO-specific band in each case. Therefore, these two HO antibodies appear to specifically recognize their target proteins in the rat, and are suitable for performing immunohistochemistry in the rat spinal cord to  44  Figure 3.1. Western blot analysis of the specificity of HO-1 and HO-2 antibodies. Panel A shows the western blot analysis performed on the spinal cords of Lewis rats with E A E at the peak of clinical signs using anti-HO-1 polyclonal antibody purchased from StressGen. The antibody mainly recognized a band of 32 k D a in the spinal cord protein preparations. Panel B shows the western blot analysis performed on the spinal cords of normal Lewis rats using anti-HO-2 polyclonal antibody purchased from the same company. The antibody mainly recognized a band of 36 k D a in the spinal cord protein preparation. Although both antibodies could recognize additional protein bands, the nonspecific bands were much weaker compared to the HO-specific band in each case.  45  detect the expression and to localize the distribution of HO-1 and HO-2 proteins, respectively. Since in western blot analysis, equal amounts of protein are loaded into each well, the result is semi-quantitative. I used it to determine the level of HO activity in combination with immunohistochemical studies.  3.2 EXPRESSION OF CONSTITUENT HO (HO-2) IN E A E  I measured the expression of HO-2 in the lesions of EAE at two time points, the onset and the peak of the illness, and compared the level of its activity with that in the normal rat spinal cord under physiological conditions. The results showed that, as in the brain, HO-2 is intensely expressed in the normal rat spinal cord (Fig. 3.2). The expression of HO-2 was most pronounced in the gray matter of the spinal cord, where anti-HO-2 antibody specifically stained large anterior horn motoneurons and many smaller neurons including probably interneurons. The immunostaining was characteristically cytoplasmic. Typically, intensely stained cytoplasm surrounding an unstained nucleus was observed. Subsequent double staining confirmed that HO-2 was predominantly restricted to spinal cord neurons (Fig. 3.3). And most neurons in the spinal cord displayed HO-2 immunoreactivity. However, the level of HO-2 activity in EAE did not increase significantly compared to the control level under normal physiological conditions. As shown in Figure 3.4, the cellular distribution and intensity of HO-2 immunoactivity in the spinal cords of rats with EAE at different phases (the onset and the peak of EAE) were similar as in the spinal  46  Figure 3.2. Expression of H O - 2 in the normal Lewis rat spinal cord -  HO-2 is intensely expressed in the normal rat spinal cord. The immunoreactivity of HO-2 was expressed primarily in the large anterior horn motoneurons and many smaller neurons including interneurons in the gray matter. The immunostaining was confined within the neuronal cytoplasm, although occasionally, the nerve cell nucleus was also stained. The scale bar represents 100 (jm. Immunohistochemical staining.  47  Figure 3.3. Localization of HO-2 in the normal rat spinal cord -  Immunohistochemical double-staining. Localization of HO-2 in the normal rat spinal cord was investigated using immunohistochemical double-staining technique. Spinal cord sections were incubated first with anti-HO-2 followed by antibody against neurofilaments. The first primary antibody was visualized with DAB (brown staining), while the secondary primary antibody was visualized with nickel DAB (dark blue staining). The results revealed that HO-2 (brown) was predominantly restricted to spinal cord neurons (dark blue), and most neurons (dark blue) in the spinal cord displayed HO-2 immunoreactivity (brown). The scale bar represents 40 \im.  48  Figure 3.4. Expression of HO-2 in the lesions of E A E vs. in the normal rat spinal cord - Immunohistochemical staining. The expression of HO-2 in the lesions of EAE  at two time points, the onset and the peak of the illness, was detected and compared with the expression under normal physiological conditions. (A) shows the typical expression of HO-2 in the normal rat spinal cord. (B), (C) show the expression of HO-2 in the spinal cords of rats with EAE at the onset (clinical grade 1) and at the peak of clinical signs (clinical grade 3), respectively. The cellular distribution and intensity of HO-2 immunoactivity in the rat spinal cords were similar among all three cases, indicating that HO-2 activity did not increase in EAE. The scale bars represent 100 u.m.  A  B  C  Figure 3.5. Western blot analysis of HO-2 expression in the lesions of EAE vs. in the normal rat spinal cord. Panel A shows the western blot analysis performed on the spinal cord of normal rats using anti-HO-2 antibody. Panel B, C show the western blot analysis performed using anti-HO-2 antibody on the spinal cords of rats with E A E at the onset and at the peak of clinical signs, respectively. There is no significant change in the expression of HO-2 in the rat spinal cords among all three cases, confirming the results achieved by immunohistochemistry in Figure 3.4.  50  cords of normal rats. This result was confirmed by western blots, showing similar amounts of HO-2 in spinal cord preparations obtained from normal rats and from rats with EAE at the onset and at the peak of clinical signs (Fig. 3.5). All these results indicate that HO-2 indeed exists in the rat spinal cord. However, as many other constituent antioxidant enzymes, the activity of HO-2 does not increase significantly in EAE.  3.3 INDUCTION OF HO-1 IN E A E  Different from HO-2, no HO-1 could be detected in normal rat spinal cords (Fig. 3.6A). By contrast, in all control rats with developed clinical EAE, HO-1 was strongly expressed in perivascular cells in the inflamed lesions of spinal cords (Fig. 3.6B). In most cases, meningeal accumulation of HO-1 protein with parenchymal infiltration was observed. By immunohistochemical double staining, the HO-1 was shown to be expressed mainly in the infiltrating macrophages (Fig. 3.7). The HO-1 staining and the macrophage differentiation antigen EDI staining coincided well with each other. Since increasing evidence suggests that free radicals play an important role in the pathogenesis of EAE, especially in thefinaleffector phase of this disease (Huitinga et al., 1990; Hooper et al., 1997), I further investigated the induction levels of HO-1 at different time points of EAE, including the onset and the peak of the illness. The results showed that the levels of HO-1 expression at different phases of EAE were very different. HO-1 activity substantially increased as EAE progressed (Fig. 3.8.1 and II). At day 10 to 11 after immunization, at the onset of clinical signs, only singular H O - l cells were +  51  A  B  4  '•  la  '* » »  Figure 3.6. Expression of HO-1 in the normal rat spinal cord and in the lesions of EAE - Immunohistochemical staining. The expression of HO-1 was measured in the spinal cord sections obtained from normal rats (A) and from the rats with E A E at the peak of clinical sings (B, clinical grade 3). No HO-1 could be detected in the normal rat spinal cord. By contrast, HO-1 was strongly expressed in perivascular cells in the inflamed lesions of E A E . The scale bars represent 60 u;m.  52  Figure 3.7. Localization of HO-1 in the lesions of E A E - Immunohistochemical double-staining. Localization of HO-1 in the lesions of EAE was investigated using immunohistochemical double-staining technique. Spinal cord sections were incubated first with anti-HO-1 followed by antibody against macrophage differentiation antigen EDI. Thefirstprimary antibody was visualized with DAB (brown staining), while the secondary primary antibody was visualized with nickel DAB (dark blue staining). The results revealed that HO-1 (brown) was expressed mainly in the infiltrating macrophages (dark blue). Only individual H O - l cells (brown) were not stained by anti-EDl antibody. The scale bar represents 1 5 p:m. +  5  3  Figure 3.8.1, Expression of HO-1 in different phases of E A E vs. in normal physiological conditions - Immunohistochemical staining. The expression of HO-1 in the lesions of EAE at three time points, the onset (B, clinical grade 1), the peak of the illness (C, clinical grade 3) and after alleviation of disease (D, clinical grade 1), was detected and compared with the expression under normal physiological conditions (A). The levels of HO-1 expression at different phases of EAE were different. HO-1 activity substantially increased as EAE progressed. (A) No HO-1 could be detected in the normal rat spinal cord. (B) At the onset of clinical signs, only singular H O - l cells were observed in the lesions. (C) HO-1 activity reached its maximum at the peak of clinical EAE. (D) After the clinical signs of EAE remitted, H O - l cells decreased when the inflammatory infiltrates in the lesions of the spinal cord attenuated. The scale bars represent 60 um. II, Correlation of HO-1 expression with the clinical signs of E A E . The degree of clinical signs was scored as: 0, no disease; 1, tail limpness; 2, hind limb paraparesis with clumsy gait; 3, hind limb paralysis; 4, tetraplegia; 5, moribund or dead. Immunohistology of HO-1 cells in spinal cord sections was evaluated microscopically by a semiquantitative score by evaluating number and distribution of HO-1 cells: 0, no H O - l cells; +, singular H O - l cells; ++, accumulation of H O - l cells surrounding blood vessels; +++, many H O - l cells in the inflammatory infiltrate surrounding the blood vessel with H O - l cells spreading into the surrounding parenchyma. +  +  +  +  +  +  +  +  +  54  Clinical course of EAE — • — HO-1 induction in EAE 5T  Days after immunization  Figure 3.9. Western blot analysis of H O - 1 expression in different phases of E A E vs. in normal physiological conditions. Panel A shows the western blot analysis performed on the normal rat spinal cord using anti-HO-1 antibody. Panel B, C show the western blot analysis performed using anti-HO-1 antibody on the spinal cords of rats with E A E at the onset (clinical grade 1) and at the peak of clinical signs (clinical grade 3), respectively. The expression of HO-1 was strong in the lesions of E A E at the peak of clinical signs, but weak at the onset of clinical signs, while none could be detected in the normal spinal cord. These results confirm the results achieved by immunohistochemistry in Figure 3.8.  56  observed in perivascular infiltrates (Fig. 3.8.IB). During the following days as disease progressed, the number of HO-1 cells increased massively. HO-1 activity reached its +  maximum at the peak of clinical EAE, when much HO-1 immunoreactivity was observed in the spinal cord lesions with parenchymal infiltration (Fig. 3.8.IC). However, after alleviation of clinical signs, H O - l cells decreased because the inflammatory infiltrates in +  the spinal cord including the macrophages were attenuated (Fig. 3.8.ID). These results were also confirmed by western blot analysis (Fig. 3.9), which demonstrated that the expression of HO-1 were strong in the lesions of EAE at the peak of clinical signs, but weak at the onset of clinical signs, while none were observed in normal spinal cords. The strong induction of HO-1 in EAE, especially at the peak of the illness, suggests that this antioxidant enzyme may have an important role during lesion formation and resolution in EAE.  3.4 P R O T E C T I V E E F F E C T S O F H O - 1 O N E A E  To evaluate the role of HO-1 in EAE, I investigated the effects of an HO-1 inducer, hemin, and an HO-1 inhibitor, SnMP, on the clinical outcome and the histological reaction in EAE in Lewis rats.  3.4.1 Effects of hemin a n d S n M P treatment on clinical E A E  In each group, 10 rats chosen randomly were followed for the full clinical course until 30 DAI. Rats were monitored daily for clinical signs and weight and the results are presented in Figure 3.10. Weight was expressed as a percent of weight before treatment  57  Figure 3.10. Effects of hemin and SnMP treatment on EAE in Lewis rats. (A) Effects on clinical course of disease. (B) Effects on rat weight loss. Rats were immunized as described in Materials and Methods. Animals were given either 40 umol/kg hemin or 40 umol/kg SnMP by i.p. injection once daily from 7 to 17 days after immunization. Control rats received saline injections on the same schedule. The degree of clinical signs was scored as: 0, no disease; 1, tail limpness; 2, hind limb paraparesis with clumsy gait; 3, hind limb paralysis; 4, tetraplegia; 5, moribund or dead. Weight is expressed as a percent of weight before treatment. Number in parentheses indicates the number of rats studied. Values are the mean ± s.e.m. Data in both (A) and (B) were analyzed using two-way ANOVA followed by Fisher's PLSD post hoc tests. The results showed that hemin suppressed EAE, and SnMP exacerbated EAE significantly (F(l,162) = 145.47,/? < 0.001; F(l,162) = 90.31,/? < 0.001, respectively). Weight loss in hemin-treated group was significantly lessened (F( 1,234) = 123.26,/? < 0.001). *p < 0.001 subsequent Fisher's PLSD post hoc tests revealed significant differences from controls at each time.  58  A  Days after immunization  B  59  Table 3.1 Effects of hemin and SnMP treatment on E A E in Lewis rats Parameter Incidence Onset Duration of illness (days) Maximum clinical severity (scalefrom0 to 5) Maximum weight loss (% of body weight) Histological grade  Control 10/10 10.2 ± 0 . 2 (10)  Hemin-treated 6/10 12.3 ± 0.2 (6)**  SnMP-treated 10/10 10.6 ± 0 . 3 (10)  7.1 ± 0 . 1 (10) 3.0 ± 0.1 (10)  5.0 ± 0.2 (6)**  7.4 ± 0 . 3 (10)  1.1 ±0.4(10)**  4.6 ± 0 . 3 (10)**  19.2 ± 1.0(10)  9.3 ± 2 . 6 (10)**  19.5 ± 1.1 (10)  2.6 ± 0.2 (7)  1.3 ± 0 . 4 (10)*  3.2 ± 0 . 2 (10)*  Table 3.1. Effects of hemin and SnMP treatment on E A E in Lewis rats. Data are expressed as mean ± s.e.m. except the incidence of clinical EAE. Number in parentheses indicates the number of rats studied. In the hemin-treated group only those rats with clinical EAE were included for the evaluation of onset and duration of illness. Statistical comparison with controls was made using one-tailed Mann-Whitney test. *p < 0.01; **p < 0.001, statistical analyses indicated significant differences from the control group.  60  to normalize for differences in weight between rats. In addition, I use several other parameters to assess the effects of hemin and SnMP treatments and the results are summarized in Table 3.1. Statistical analyses showed that both hemin and SnMP treatment produced significant effects on clinical EAE compared to vehicle controls (F(l,162) = 145.47,/? < 0.001; F(l,162) = 90.31,p < 0.001, respectively). In the control group, as expected, all 10 rats developed EAE at 10-11 DAI. The severity of symptoms peaked at 13-14 DAI. All control rats suffered complete hind limb paralysis at the peak of disease. The maximum weight loss for control rats was 19.2 ± 1.0% (n = 10, Table 3.1). The rats then recovered completely without treatment by 17-18 DAI with 7 days duration of illness on average. By contrast, as shown in Figure 3.1 OA and Table 3.1, hemin suppressed EAE effectively and SnMP worsened EAE greatly. In the hemin-treated group, the onset of EAE was significantly delayed (p < 0.001) and maximum clinical symptoms were significantly attenuated (p < 0.001). In 4 of 10 hemin-treated rats, the EAE was completely inhibited. Only 6 rats in this group exhibited any clinical signs of EAE with a lower maximum clinical score (1.8 ± 0.2, n = 6) than that of control rats (3.0 ± 0.1, n = 10) (p < 0.001). In addition, the duration of illness was significantly reduced in hemin-treated vs. control rats (p < 0.001). Weight loss associated with disease was also lessened. The average weight loss was 19.2%) for controls and 15.5% for hemin-treated rats with clinical EAE. The strong therapeutic effect of hemin in EAE was observed without severe side effects. However, it was noted that in the earliest days of hemin treatment, the treated rats suffered 5.0 ± 1.1% (n = 10) weight loss, which was not observed in SnMP and vehicletreated rats (Fig. 3.10B).  61  In the SnMP-treated group, contrary to expectation, SnMP did not advance the onset of EAE. In two cases, EAE was even delayed 2 days. The clinical course of EAE in this group before the normal peak of disease (about 14 DAI) was similar to the control group. However, from then on, in contrast to controls, which recovered fast from disease, only 2 SnMP-treated rats recovered slowly and completely at last. At about 6-8 days after onset, 6 of 10 SnMP-treated rats developed tetraplegia before euthanasia and two died of severe EAE quickly. The maximum clinical symptoms of SnMP-treated rats were markedly aggravated compared with those of control rats (p < 0.001). Although there was no significant differences in maximum weight loss between SnMP-treated rats before euthanasia and control rats, during the peak period of the illness, most SnMP-treated rats were obviously in worse conditions, and tube feeding with supplemental nutrition was necessary. In order to exclude any toxicity of SnMP, we administered SnMP daily on the same schedule to two normal rats for 15 days. No adverse reactions were observed.  3.4.2 Histopathological and immunohistological findings Seven to 10 rats in each group were sacrificed at the peak of their illness for assessment of histological inflammation. The severity of inflammation in the spinal cord was graded in a double-blinded fashion. The results showed that all control rats suffered a similar degree of inflammation in spinal cords (Fig. 3.11 A). There was extensive meningeal inflammation with extension into the parenchyma. However, in the hemintreated rats with clinical EAE, only moderate meningeal and mild parenchymal infiltration was detected (Fig. 3.1 IB). It is noted that the cellular infiltration was mainly located perivascularly or in the submeningeal area. In the hemin-treated rats without  62  Figure 3.11. Comparison of histopathology in the spinal cords between hemintreated and control E A E rats - HE staining. Rats were sacrificed at the peak of clinical signs. Hemin-treated rats without clinical E A E were sacrificed at the same time point. The spinal cord sections were stained with hematoxylin-eosin for histopathological examination. (A) shows the typical inflammatory manifestation in the spinal cords of control rats with grade 3.0 E A E . There was extensive meningeal inflammation with extension into the parenchyma. (B) In the hemin-treated rats with clinical E A E , only moderate meningeal and mild parenchymal infiltration was detected. (C) In the hemintreated rats without clinical E A E , no obvious inflammation in the spinal cords was observed. The scale bars represent 250 |im.  63  Figure 3.12. Comparison of histopathology in the spinal cords between SnMPtreated and control E A E rats - H E staining. Rats were sacrificed at the peak of clinical signs. The spinal cord sections were stained with hematoxylin-eosin for histopathological examination. (A) shows the typical inflammatory manifestation in the spinal cords of control rats with grade 3.0 EAE. There was extensive meningeal inflammation with extension into the parenchyma. (B) In most cases, SnMP aggravated the inflammation in SnMP-treated rat spinal cords, in which severe meningitis with prominent parenchymal infiltration and/or multiple perivascular infiltration (arrows) were observed. The scale bars represents 250 u,m.  64  clinical EAE, no obvious inflammation in the spinal cords was observed (Fig. 3.1 IC). In contrast, all SnMP-treated rats suffered severe meningitis with prominent parenchymal infiltration and/or multiple perivascular infiltration in the spinal cords (Fig. 3.12B). The average histological grade for control, hemin-treated and SnMP-treated rats was 2.6 ± 0.2 (n = 7), 1.3 ± 0.4 (n = 10) and 3.2 ± 0.1 (n = 10), respectively (Table 3.1). Both the histological grades for hemin-treated and for SnMP-treated rats were significantly different from that for control rats (p < 0.001). The inflammation in SnMP-treated rats could be more severe, but according to the Animal Welfare Act, we should euthanize the rats soon once they developed tetraplegia. I then confirmed that hemin and SnMP took effects in EAE by modulating HO-1 activity. Immunohistochemical studies were carried out to investigate the effects of hemin and SnMP on the immunoactivity of HO-1 in the lesions of EAE. Same spinal cord sections as above were used. The results clearly showed that HO-1 expression was lower in the spinal cords of SnMP-treated rats (Fig. 3.13B), even though the inflammatory infiltrates were more extensive (Fig. 3.12B). HO-1 expression also decreased in the spinal cords of hemin-treated rats because the inflammatory infiltrates were attenuated (Fig. 3.14B). In the spinal cords of hemin-treated rats without clinical EAE or inflammation, little expression of HO-1 was observed (Fig. 3.14C). In order to investigate whether hemin could increase the activity of HO-1,1 examined the induction of HO-1 in inguinal lymph nodes at the same time. The results showed that the expression of HO-1 in inguinal lymph nodes of hemin-treated rats (Fig. 3.15C) was increased significantly compared with that in control rats (Fig. 3.15B), while SnMP decreased the expression of HO-1 in SnMP-treated rats (Fig. 3.15D). As in the spinal  65  B -4  Figure 3.13. Comparison of HO-1 expression in the lesions between SnMP-treated and control EAE rats - Immunhistochemical staining. The expression of HO-1 was detected in the spinal cord sections obtained from control and SnMP-treated rats sacrificed at the peak of their illness. (A) shows the typical expression of HO-1 in the spinal cords of control rats with grade 3.0 EAE. (B) HO-1 level in SnMP-treated rat spinal cords was reduced significantly, although the inflammation was more extensive (shown in Fig. 3.12), indicating that SnMP is an effective inhibitor of HO-1. The scale bars represent 60 fim.  Figure 3.14. Comparison of HO-1 expression in the lesions between hemin-treated and control E A E rats - Immunohistochemical staining. The expression of HO-1 was detected in the spinal cord sections obtained from control and hemin-treated rats sacrificed at the peak of their illness. Hemin-treated rats without clinical E A E were sacrificed at the same time point. ( A ) shows the typical expression of HO-1 in the spinal cords of control rats with grade 3.0 E A E . (B) HO-1 expression in the spinal cords of hemin-treated rats decreased when the inflammatory infiltrates including the macrophages attenuated. (C) In the spinal cords of hemin-treated rats without clinical sings or inflammation, little expression of HO-1 was observed. The scale bars represent 60 pm.  Figure 3.15. Comparison of HO-1 expression in lymphoid nodes among normal, control, hemin-, and SnMP-treated rats. Lymphoid node sections were obtained from normal rats (A), control (B), hemin- (C) and SnMP-treated rats (D) sacrificed at the peak of their illness. HO-1 expression was detected by immnohistochemical staining. As in the spinal cord, little expression of HO-1 could be detected in the normal rat inguinal lymph node without inflammation (A). The expression of HO-1 in the inguinal lymph nodes of hemin-treated rats (C) was much higher than that in control rats (B), while SnMP decreased the expression of HO-1 significantly in SnMP-treated rats (D), which indicates that hemin and SnMP are an effective inducer and an effective inhibitor of HO-1, respectively. The scale bars represent 15 (im.  68  cord, little expression of HO-1 was detected in the normal rat inguinal lymph node (Fig. 3.15A). These results demonstrated that hemin and SnMP are an effective inducer and an effective inhibitor of HO-1, respectively. Considering HO-1 has a protective role in EAE, the results correspond well to the effects of hemin and SnMP treatment described above.  69  Chapter 4 DISCUSSION A N D S U M M A R Y  4.1 T H E ROLE OF HO-1 IN E A E  The results clearly show that HO-1 is strongly expressed in EAE, mainly in the macrophages present in the lesions, possibly because activated macrophages are the principle cells responsible for production of reactive oxygen species (ROS) (Griot et al., 1989). During the initiation of EAE, CD4 T cells activate macrophages through the +  production of proinflammatory cytokines, which we think, in combination with ROS and other factors, induce the expression of HO-1 at the same time. The apparent paradox of molecules causing both cytotoxicity and cytoprotection is recapitulated in other systems. For example, the complement membrane attack complex induces cell lysis but also induces changes that protect against lysis (Piatt and Nath, 1998); activation of NF-kB induces expression of proinflammatory and proimmunity genes but also forestalls apoptosis; interleukin-2 promotes activation of effector T cells but also induces apoptosis of autoreactive T cells (Piatt and Nath, 1998). Contrary to my expectation, HO-1 was not induced at high levels from the beginning to the end of EAE. The levels of HO-1 expression at different phases of disease were very different. The expression of HO-1 was weak at the onset of clinical signs. During the following days of neurological disease, HO-1 activity substantially increased and reached its maximum at the peak of clinical signs, which suggests that oxidative stress plays a more important role in the final effector phase of EAE.  70  As in other diseases, HO-1 plays a protective role in EAE. Hemin, the inducer of HO1, significantly ameliorated the clinical signs of EAE, delayed the onset and reduced the disease duration. The strong therapeutic effect of hemin in EAE was observed without severe side effects. However, it is noteworthy that in the earliest days of treatment, hemin induced obvious weight loss in the treated rats compared to SnMP and control treatments (Fig. 3.1 OB). As indicated before (Marks, 1994), I postulate that weight loss was due to hemin induction of HO-1 and increased formation of CO, which could serve as a signal transduction mechanism in the medial hypothalamus to suppress appetite. This result further demonstrates that hemin acted as an effective inducer of HO-1 in this study. In contrast, all SnMP-treated rats developed EAE and the severity of clinical signs was worsened severely at the peak period of disease. The use of SnMP in normal rats for 15 days excluded the possibility that any SnMP-related toxicity could aggravate EAE. All these results demonstrate that HO-1 plays an important protective role in EAE. However, beyond the expectation, SnMP did not advance the onset of EAE. Surprisingly, in 2 of 10 rats it postponed the onset slightly (Table 3.1). Although SnMP is one of the best HO-1 inhibitor presently available, it has been found to inhibit inducible nitric oxide synthase (iNOS) activity to some extent (Meffert et al., 1994). Because iNOS-derived NO plays a role in inflammatory BBB dysfunction (Giovannoni et al., 1998), it is considered that this may be among the mechanisms underlying the unexpected phenomenon. On the other hand, the results have observed that HO-1 activity substantially increases as EAE progresses. I think that HO-1 plays a more important role as the disease progresses to its peak period. This may also explain why in these experiments the detrimental effect of SnMP treatment did not reach its maximum until peak EAE. In recent years, there has  71  been controversy about the effect of SnMP on diseases. Since SnMP is a metalloporphyrin, it possesses some anti-inflammatory properties. Some research has reported the beneficial effect of SnMP and other similar metalloporphyrins on diseases. For example, Nagai et al. (1992) showed that ZnPP inhibited experimental arthritis in mice. However, our results suggest that any drugs that inhibit endogenous HO-1 should be used very cautiously in EAE and MS. The results are consistent with the previous findings concerning the function of HO-1. In recent years, HO-1 is considered to be one of the most powerful cellular defensive mechanisms against oxidative stress, and plays a protective role in many stress-mediated diseases, such as inflammation (Willis et al., 1996), ischemic stroke (Takizawa et al., 1998), and neurodegenerative diseases (Riedl et al., 1999). As described in chapter Introduction, the antioxidant effect of HO-1 is multifactoral and dependent on the diverse dimensions of HO activity. The protection afforded by HO-1 seems to be mainly due to degradation of the preoxidant heme and the production of the catalytic by-products including biliverdin and bilirubin, which have been shown to be potent antioxidants. However, other activities of HO-1 may also be involved in its effect on EAE. For example, CO, another heme metabolite, which is considered as a gaseous messenger, has recently been shown to have anti-inflammatory effect (Otterbein et al., 2000). It was demonstrated that CO at low concentrations differentially and selectively inhibited the expression of the pro-inflammatory cytokines such as TNF-a, IL-1 p and increased the expression of the anti-inflammatory cytokine IL-10. CO mediates these antiinflammatory effects through a pathway involving the mitogen-activated protein kinases. On the other hand, the availability of sufficient heme is essential for the formation of  72  functional iNOS dimers and catalytic activity (Stuehr, 1997). Thus, HO-l-meidated heme degradation appears to be a negative feedback regulation for the production of iNOSderived NO, and ameliorates cytotoxicity caused by the free radical NO and its reactive derivative peroxynitrate (ONOO") (Foresti and Motterlini, 1999). In addition, HO-1 and its activity products also exhibit other actions. For example, overexpression of HO-1 is known to modulate cellular immune functions, which include inhibition of natural killer cell-mediated cytotoxicity, lymphoproliferative response and inhibition of T cellmediated cytotoxicity (Willis et al., 1996; Woo et al., 1998). In vitro, bile pigments have been shown to display anticompliment activity (Nakagami et al., 1993; Haga et al., 1996). All these properties contribute to the powerful protective effect of HO-1 in EAE. A central question is why HO-1 is important in EAE. Oxidative stress resulting from toxic effects of ROS has come to occupy a remarkably common position in the mechanisms of a wide variety of diseases (Andreoli, 2000). Recently, it has been demonstrated that oxidative injury is important in the etiology of EAE because the CNS, notably the oligodendrocyte, is extremely vulnerable to oxidative damage (Smith et al., 1999). Oxidative stress contributes significantly to the BBB disruption in EAE (Guy et al., 1994; Merrill and Murphy, 1997). More importantly, increasing evidence suggests that free radicals and their principal source, the macrophages, may play an essential role in the autoimmune effector pathway of EAE and MS. In vivo depletion of macrophages by mannosylated liposomes containing dichloromethylene diphosphonate, which have no effect on lymphocytes, has been shown to prevent demyelination (Huitinga et al., 1990). In relapsing-remitting EAE progression, it was observed that macrophages in the CNS increased dramatically in comparison to number of CD4 T cells, suggesting that +  73  macrophages may play a greater role in the later stages of disease (Begolka et al., 1998; Zhu et al., 1999). Similarly, in a recent experiment, Hooper et al. (1997) demonstrated that monocytes expressing iNOS were concentrated in regions immediately surrounding the plaque areas of post mortem brain tissue from patients with MS. Their data suggest that activated macrophages cause these lesions through the production of peroxynitrate, a potent toxic intermediate formed by the combination of NO and superoxide. On the other hand, there is convincing evidence to demonstrate that ROS production is a prominent feature of inflammatory demyelinating disease. ROS are produced massively both in EAE and MS. Damage to lipid membranes by ROS has been demonstrated in EAE and MS (Ruuls et al., 1995). Since the CNS has relatively low levels of constituent antioxidant defenses (Smith et al., 1999), it is not surprising that HO-1, the powerful inducible antioxidant enzyme, plays an important defensive role in EAE.  4.2 S U M M A R Y  The following conclusions can be drawn from the experiments. 1) As in the brain, HO-2 is constituently expressed in the spinal cord neurons. But its activity level does not increase in EAE. 2) Heme oxygenase-1 is induced in EAE and plays an important protective role in this disease, especially at the peak period of the illness. 3) Targeted induction of HO-1 overexpression by pharmacological modulation may serve as a novel approach for therapeutic intervention in multiple sclerosis, while any drugs that inhibit HO-1 should be used very cautiously in this disease.  74  4.3 CAUTIONS AND FUTURE DIRECTIONS  4.3.1 Human Multiple Sclerosis and Rodent Experimental Autoimmune Encephalomyelitis As described in chapter Introduction, EAE is the most intensively studied animal model of MS, and many believe the best (Ebers, 1998). Over the past century, this induced animal model has proved extremely useful for studying the spontaneous, and at present etiologically undefined, human disease MS. Several therapies approved for treatment of MS were developed preclinically based on their success in treating EAE (Steinman, 1999). One promising therapeutic approach to come out of the EAE model is Copaxone, a synthetic polymer analog of MBP, which has been successful in reducing the relapse rate in MS in clinical trials and is currently approved for treatment of MS (Arnon, 1996). IFN-P was demonstrated to be effective both clinically in relapsing remitting MS and in EAE, though in the treatment of EAE severe relapses were seen when IFN-P was discontinued (Ruuls et al., 1996). However, to say that EAE is rodent MS is certainly an open question. The central unresolved issue bearing on the utility of EAE as an object of study is the fact that human MS is spontaneous, progressive, long lasting, and generally terminal, while EAE is induced, and in most cases is acute, self-limiting, and not fatal. In EAE model, the immune inducing compound is directly taken from the CNS, which is most probably not the case in MS, for which the influence of environmental factors has been evidenced by epidemiologic studies (Kurtzke, 1980; Ebers and Sadovnick, 1998) and occurrence of relapses has been linked to infections (Andersen et al., 1993; Rapp et al., 1995). Though  75  EAE has many features that reflect what is known about the pathophysiology of MS, there are many differences between the pathology of EAE and MS (Petry et al., 2000). Therefore, extrapolations must be made with caution when predicting what might happen in MS, based on results obtained in the EAE model. For example, as shown in Tabel 1.1, there is discrepancy between the results with inhibition of TNF-a in EAE versus MS. A clinical trial was performed to test the efficacy of inhibition of TNF-a in MS. Results showed that treatments with monoclonal anti-TNF antibody and soluble TNF receptor actually exacerbated disease (van Oosten et al., 1996), and the trial was halted while in progress. Paradoxically, in EAE systemic administration of anti-TNF-a antibody protects from paralysis (Korner et al., 1997; Wildbaum and Karin, 1999). So far, the development of the approved MS drug, Copaxone, based on its efficacy in EAE and the success of IFN-J3 in treating EAE must be balanced with the failures of anti-TNF-a in MS, after its successful use in various models of EAE. It is important to remember that caution must be practiced in extrapolating finding from EAE to MS.  4.3.2 Duality of HO-1 in Antioxidant Defense If HO-1 protects cells from oxidative damage, why must this molecule be induced? Why is this molecule not expressed constitutively at a high level? The answer might be that this "protective" molecule can, under certain circumstances, also induce tissue injury (Fig. 4.1). Among the consequences of heme degradation by HO-1 is the liberation of free iron, a potent oxidant. HO-1 generates bilirubin, which has antioxidant properties in vitro but which has been implicated in tissue injury in kernicterus (Hansen, 2002), and in renal injury that accompanies hepatic failure (Sural et al., 2000). Another product of HO  76  • Fe-ferritin (nor toxic) Fe  • oxidant — • tissue injury  CQ-  » protection or tissue l.Tjury  vp0i protecfrw elfecls •tissue:injury  ONA fragmentaoon mitochondrial dysJwnctton  Figure 4.1. Heme oxygenase in tissue protection and injury. Under conditions of oxidative stress, heme, a Fe-containing prosthetic group, may be released from heme proteins whereupon it induces cellular injury. Heme oxygenase, induced by endotoxin, oxidants and cytokines, protects against tissue injury by: metabolizing heme; fostering the synthesis of the Fe-sequestering protein ferritin; producing carbon monoxide (CO), which is vasodilatory; promoting the production of bilirubin, an anti-oxidant; and triggering cellular protection against toxicants through pathways as yet undefined. However, each of the products generated by the action of heme oxygenase on heme - Fe, CO and bilirubin - may cause injury in certain circumstances. For example, Fe catalyzes oxidant reactions, CO may poison heme proteins, and bilirubin may injury lipid bilayers. (Adapted from Nature Medicine 4: 1364-1365, 1998, PattJL)  activity, CO, may defend against tissue ischemia by promoting vasodilation; however, it may also stimulate the generation of oxidants by mitochondria (Zhang and Piantadosi, 1992; Piantadosi et al., 1995). It would seem that molecules such as HO-1 are probably not exclusively cytoprotective. Indeed, some studies have reported damaging effects of HO-1 overexpression in oxidative stress. Dennery et al. (1997) have demonstrated that, when grown in normoxia, confluent monolayers of O2R.95 cells had increased immunoreactive HO-1 protein levels. However, further overexprssion of HO-1 by transfection with HO-1 cDNA in a prC/CMV vector to increase HO-1 expression did not confer further resistance to oxygen toxicity. Subsequent evaluation of heme and iron  77  content showed that in the HO-1 cDNA transfected cells, cellular heme was lowered as compared with controls; however, cellular iron levels were increased, perhaps abrogating the beneficial effects of heme sequestration by increasing release of redox-active iron (Dennery et al., 1997). It has been suggested that coinduction of ferritin, a protein that could sequester the redox-active iron released from heme degradation, may counteract iron release (Balla et al., 1992) but this reaction does not always accompany HO-1 induction, as demonstrated by Dennery et al. (Dennery et al., 1996; Tom et al., 1996). Therefore, it is conceivable that there may be circumstances under which HO-1 is not beneficial or even detrimental. In fact, the concept of an oxidant-antioxidant function is not unique to HO-1, and it has been observed with other antioxidants as well. For example, increased expression of SOD and allopurinol provides protection against oxidative injury, but higher doses do not prove protective and even exacerbate injury (Bernier et al., 1989; Nelson et al., 1994). It will be important to understand the range and manner of overexpression of HO-1 that will be beneficial. In summary, although we believe that in most circumstances, including EAE, HO-1 plays an protective role in oxidative stress, we also believe that prior to considering therapeutic maneuvers to enhance HO-1, a complete understanding of the physiologic consequences of HO-1 induction and associated reactions, in each particular setting, will be crucial.  4.3.3 Future Directions In the future, I would like to do more studies on the effect of HO-1 in the human disease MS. For example, if post mortem spinal cord tissue from patients with MS is available, I will study the expression of HO-1 in the lesions of MS. If HO-1 is found to be  78  strongly induced in MS as in EAE, it would support the view that HO-1 may also have an important role in human disease MS. Furthermore, I would like to study HO-1 expression in the peripheral blood cells including macrophages collected from MS patients. 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