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The role of macrophages in the age-dependent susceptibility to coxsackievirus B3 infection Girn, Jaskamal 1999

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THE ROLE OF MACROPHAGES IN THE AGE-DEPENDENT SUSCEPTIBILITY TO COXSACKIEVIRUS B3 INFECTION by Jaskamal Girn B.Sc. (Microbiology), University of British Columbia, 1995 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Pathology and Laboratory Medicine We accept the following thesis in conforming to the required standard: The University of British Columbia Apr i l , 1999 © Jaskamal Girn ,1111 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 pQrHWrry l OMYJ iMbpCOfayCU W e c i i d r i e The University of British Columbia Vancouver, Canada DE-6 (2/88) 11 ABSTRACT Group B coxsackieviruses (CVB) are etiological agents of a febrile exanthematous disease that may be complicated by viral myocarditis, pancreatitis, and encephalitis. C V B infections are notable for being extremely severe in the very young compared to adult hosts. Preliminary work in mice has shown that macrophages contribute to host resistance to CVB3, since depletion of macrophages in vivo resulted in a more exacerbated infection. Based on this finding, the overall objective of this study was to dissect the specific properties of macrophages which participated in resistance against CVB3 and to compare them between young and adult CD-I mice. It was found that macrophages from young CD-I mice were less efficient in inactivating CVB3 in vitro than adult mice. Comparison of NO production by macrophages from young and adult CD-I mice in vitro demonstrated that young mice were not impaired in their ability to produce this antiviral molecule; in fact, the younger animals produced significantly greater levels of NO following induction by IFN-y, or LFN-y plus TNF-ct. In conjunction with this, the importance of NO in CVB3 resistance in vivo was determined by treatment of young and adult CD-I mice with a NOS enzyme inhibitor, L - N M M A . The resulting diminished activity of NOS caused a more severe infection in both the young and adult CD-I mice, with the effects generally more pronounced in the younger animals. Comparison of TNF-cc production by macrophages from young and adult CD-I mice in vitro showed that the younger animals produced less of the cytokine following induction by IFN-y plus LPS. Strain variation was also found in macrophage functions between CD-I mice and B A L B / c mice (the latter being extremely susceptible to CVB3 resulting in mortality). It was found that virus inactivation by macrophages from young and adult Ill B A L B / c mice was markedly different, with macrophages from the younger mice being permissive to viral replication. In addition, TNF-oc was produced in greater quantities by macrophages from the young mice, contrary to the results obtained with the CD-I mice. NO production by macrophages from B A L B / c mice, however, followed a similar trend to the CD-I mice, with the younger animals producing more NO than the older animals. The mechanism for the extreme susceptibility (and mortality) in the B A L B / c may involve the relatively high levels of TNF-a and NO generated by the younger mice, which may produce detrimental effects, like shock. iv TABLE OF CONTENTS Abstract List of Figures List of Tables Abbreviations Acknowledgements INTRODUCTION 1 1. History 2. Classification and Taxonomy 2 3. Genome and Life Cycle 2 4. Infection Process In Vivo 3 5. CV and Myocarditis 3 5.1 Mechanism of Pathogenesis of Myocarditis 5 6. CV and Diabetes 6 7. Factors Affecting the Course of CV Infection 7 7.1 Host Genetics 8 7.2 Host Immune Status and Function 9 7.3 Hormones 10 7.4 Virus Factors 10 8. Effect of Host Age on CV Infection 11 9. Immune Responses to CV Infection 13 9.1 Innate Mechanisms of Resistance 14 9.2 Acquired Mechanisms of Resistance 16 10. The Role of Macrophages in CV Infection 17 10.1 Macrophage-Virus Interactions'. General Aspects 17 10.2 Macrophages and CV 23 11. Nitric Oxide 24 11.1 Biosynthesis of NO 26 ii vii viii ix xiii 11.2 Macrophage NO 11.3 Antiviral Role of NO 11.4 Nitric Oxide and CV RATIONALE AND OBJECTIVES MATERIALS METHODS 1. Cell Maintenance 2. CVB3(CG) Stock Preparation 3. CVB3(CG) Titration 4. CVB3 Infection of CD-I Mice 5. In-Situ Hybridization 5.1 Large Scale pCVB3-Rl Plasmid Preparation Using PEG Precipitation 5.2 RNA Probe Labelling By In Vitro Transcription 5.3 Quantification of DIG-Labelled Transcript: Dot Blot 5.4 In-Situ Hybridization Using DIG-Labelled RNA Probe 6. Histology and Histopathological Interpretation 7. Virus Titration of Mice Tissue 8. In Vitro Inactivation of CVB3 By Peritoneal Cells 9. Quantification of Nitric Oxide Production 10. CVB3 Infection of CD-I Mice in the Presence of a NOS Inhibitor 11. TNF-a Production In Vitro 12. TNF-a ELISA 13. Silica Depletion of Peritoneal Cells in BALB/c Mice 14. Calculation of Macrophage Content of Peritoneal Cell Populations 15. Statistical Analysis vi RESULTS AND DISCUSSION 57 1. Age-dependent Resistance To CVB3 57 1.1 CVB3 Infection of Young and Adult CD-1 Mice 57 2. Role of Macrophages in CVB3 Infection 65 2.1 Depletion of Peritoneal Cells With Subsequent CVB3 Infection 65 3. Comparison of Specific Macrophage Functions in Young and Adult Mice 67 3.1 Inactivation of CVB3 By Peritoneal Cells Isolated From Young and Adult CD-I Mice 67 3.2 The Role of Macrophage Nitric Oxide in Controlling CVB3 Infection 68 3.2.1 Inhibition of Nitric Oxide Synthase In Vivo With Subsequent CVB3 Infection 69 3.2.2 Intrinsic Capability of Peritoneal Cells From Young and Adult CD-1 Mice to Produce NO In Vitro 73 3.3 Intrinsic Capability of Peritoneal Cells From Young and Adult CD-1 Mice To Produce TNF-a In Vitro 11 4. Strain Variation: A Comparison With BALB/c Mice 78 4.1 Inactivation of CVB3 By Peritoneal Cells Isolated From Young and Adult BALB/c Mice 79 4.2 Intrinsic Capability of Peritoneal Cells From Young and Adult BALB/c Mice To Produce NO In Vitro 80 4.3 Intrinsic Capability of Peritoneal Cells From Young and Adult BALB/c Mice To Produce TNF-a In Vitro 81 CONCLUSIONS 84 REFERENCES 89 LIST OF FIGURES Fig. 1: World Health Organization global surveillance of viral infections related to cardiovascular disease (1975-1985) Fig. 2: Development of age-dependent resistance to CVB3 Fig. 3: Effects of transfer of peritoneal exudate cells (p.e.c.) on mortality of newborn mice infected with CVB3 Fig. 4: The various roles of macrophages in immunity and inflammation Fig. 5: Biosynthesis of NO from L-arginine and O2 Fig. 6: CVB3(CG) titres in heart, pancreas, and serum of young and adult CD-I mice Fig. 7: Histopathological analysis of CVB3(CG) infection of young and adult CD-I mice Fig. 8: In-situ hybridization of CVB3(CG) RNA in heart tissue of young and adult CD-I mice Fig. 9: In-situ hybridization of CVB3(CG) RNA in pancreas of young and adult CD-I mice Fig. 10: Effects of silica on CVB3(RK) infection in BALB/c mice Fig. 11: Timecourse of inactivation of CVB3(CG) by peritoneal cell cultures isolated from young and adult CD-I mice Fig. 12: Effect of inhibition of NOS on CVB3(CG) infection in young and adult CD-I mice. Analysis of tissue at day 2 p.i. Fig. 13: NO production by peritoneal cells from young and adult CD-I mice. Fig. 14: TNF-a production by peritoneal cells from young and adult CD-I mice Fig. 15: Timecourse of inactivation of CVB3(CG) by peritoneal cell cultures from young and adult BALB/c mice Fig. 16: NO production by peritoneal cells from young and adult BALB/c mice Fig. 17: TNF-a production by peritoneal cells from young and adult BALB/c mice LIST OF TABLES Table 1: Experimental factors that affect mononuclear phagocyte -virus interactions Table 2: Some viruses known to be permissive for macrophages Table 3: MP functions that may be altered by virus infection Table 4: Anti-viral mediators produced by macrophages ABBREVIATIONS ADCC AP BCG BH4 Ca 2 + cGMP CMI CMV CTL CV CVB CVB3 CVB3(CG) CVB3(RK) CVB4 dH 20 DEPC DIG DMEM DNA EBV EDTA antibody-dependent cellular cytotoxicity alkaline phosphatase Bacillus Calmette-Guerin tetrahydrobiopterin calcium (2+) ion cyclic GMP cell-mediated immunity cytomegalovirus cytotoxic T lymphocyte coxsackievirus coxsackievirus group B coxsackievirus group B serotype 3 coxsackievirus group B serotype 3 (Charles Gauntt) coxsackievirus group B serotype 3 (Reinhard-Kandolf) coxsackievirus group B serotype 4 distilled water diethylpyrocarbonate digoxygenin Dulbecco's modified essential medium deoxyribonucleic acid Epstein-Barr virus ethylenediamine tetraacetic acid ELISA FBS Fc FAD FMN GM-CSF HIFBS HIV H 2 0 2 hr(s) HSV Ig IFN-ct, -p, -y IL ip IRES L D 5 0 animals L-NMMA LPS M-CSF MHC min(s) enzyme-linked immunosorbent assay fetal bovine serum crystallizable fragment flavin adenine dinucleotide flavin mononucleotide granulocyte-macrophage colony stimulating factor heat-inactivated fetal bovine serum human immunodeficiency virus hydrogen peroxide hour(s) herpes simplex virus immunoglobulin interferon-alpha, -beta, -gamma interleukin intraperitoneal internal ribosome entry site lethal dose of virus that will kill 50% of the inoculated N^monomethyl-L-arginine acetate salt lipopolysaccharide macrophage-colony stimulating factor major histocompatibility complex minute(s) multiplicity of infection mononuclear phagocytes nicotinamide adenine dinucleotide phosphate, reduced nitroblue-tetrazolium chloride/5 -bromo-4-chloro3-indolyl phosphate, p toluidine salt nuclear factor-kappa B nitric oxide nitrite nitrate nitric oxide synthase natural killer oxygen superoxide anion hydroxyl radical peroxynitrite phosphate buffered saline peritoneal exudate cells paraformaldehyde plaque forming units prostaglandin E2 post-infection polyriboadenylic acid ribonucleic acid XII R P M R/T SCID SNAP TNF-a, -p UV VPg revolutions per minute room temperature severe combined immunodeficiency S-nitroso-N-acetyl penicillamine tumor necrosis factor-alpha, -beta ultra-violet genome-linked viral protein ACKNOWLEDGEMENTS XUl I would like to thank my supervisor, Dr. Janet Chantler, for all of her support and direction throughout the work on this thesis. I am grateful for all that you have done for me, Jaki. I feel lucky to have been given the privilege to work in your lab and to contribute to one of the most interesting fields around. I would like to thank my research committee members: Dr. Richard Hegele, Dr. David Speert, and Dr. James Hudson for their suggestions and input into my project. Thank you for taking time out of your busy schedules to meet with me. I am extremely appreciative to the people from the labs of Dr. Jan Ochnio, Dr. Richard Stokes, Dr. David Speert, and Dr. John Schrader for their help in various techniques that I used in my project. I would also like to thank the Animal Unit personnel of the B.C. Research Institute For Children's and Women's Health for their help in my animal experiments (especially when working with those nasty CD-I beasts!). I am grateful to the students and technician in our lab: Ellamae Stadnick, Karen Lund, and Xiaoyun Wang for providing me with assistance when I needed it. Thanks for all of the fun times in and out of the lab. Thank you to my family: Mom and Dad, Manjinder, Jaspal, and Manpreet for all of your support and patience when I was plugging away on the computer, not lifting a finger to help out at home. Last, and by far not the least, I would like to thank Navraj Heran for just being a part of my life. Thanks for inspiring me to be the best that I can be. 1 INTRODUCTION 1. History In 1948, during a time of epidemics of paralytic poliomyelitis in the United States, Gilbert Dalladorf and Grace Sickles isolated a virus from the stools of two children exhibiting signs resembling poliomyelitis [Dalldorf, 1948 #1]. This virus was recognized as unique since it was not neutralized by antisera against the polioviruses, and more importantly, could be isolated upon infection of newborn mice (polioviruses were not known to grow in mice) [Crowell, 1997 #2]. This isolate constituted a previously unidentified group of enteroviruses that was named coxsackievirus in recognition of the small village of Coxsackie in the state of New York where the children lived [Crowell, 1997 #2]. Subsequent observations revealed that some coxsackievirus isolates caused generalized skeletal muscle destruction leading to a rapid fatal paralysis of the infected newborn mouse, and this histopathology was distinct from other coxsackieviruses, which caused a slow, spastic paralytic death [Gifford, 1951 #3]. The different viruses were placed into two groups, A and B, depending on their pathogenicity in newborn mice. The group A viruses caused generalized myositis, infecting skeletal and heart muscle, whereas the group B viruses caused focal myositis and a more widespread infection, damaging the exocrine pancreas, heart, liver, spleen, kidney, central nervous system and brown fat [Hyypia, 1993 #4]. Coxsackieviruses were also distinguished from each other by the ease in which they could be propagated in tissue culture, with group B viruses growing more readily in culture than group A [Sickles, 1959 #6; Sickles, 1955 #5]. 2 2. Classification and Taxonomy Coxsackieviruses belong in the family Picornaviridae and genus enterovirus. The differences in pathogenesis (and histopathology) in newborn mice described above divide coxsackieviruses into either group A or B. Group A is composed of serotypes A1-A22 and A24 (A23 turned out to be the same virus that had previously been identified as echovirus 9, while group B is composed of serotypes B1-B6. 3. Genome and Life Cycle Coxsackieviruses are small (24-30 nm), nonenveloped viruses with icosahedral symmetry. Similar to the other picornaviruses, the genome is (+) sense, single-stranded RNA and approximately 7400 bases in length. A small protein, VPg, is covalently-linked to the 5' end of the genome and a poly A-tail is at the 3' end. The VPg on the 5' end is unlike the normal 5' methyl cap found on most eukaryotic RNA. Thus, CV undergoes a unique translation mechanism involving an internal ribosome entry site (IRES). The capsid contains four proteins (VP1, VP2, VP3, VP4), which together, form a "canyon" structure that serves as the attachment site to cell surface receptors during infection. Following entry into the cell, the virus genome acts directly as messenger RNA for the synthesis of a large polyprotein which is subsequently cleaved into functional proteins: the structural proteins (capsid) and non-structural proteins (including virus-encoded proteases and replicase). Replication generates a (-) sense RNA which acts as a template for the synthesis of new viral genomic (+) sense RNA, which becomes packaged into capsid 3 structures to yield new infectious virus progeny. The entire replicative cycle occurs within the cell cytoplasm. The virus particles then exit following cell lysis. 4. Infection Process In Vivo The portal of entry of coxsackieviruses is the alimentary tract following ingestion. The incubation period is approximately 1 to 2 weeks but may be up to 35 days. Primary multiplication of virus occurs locally in tonsils and Peyer's patches, and due to its resistance to low pH (~ pH 3.0), the virus readily traverses the acidity of the stomach to eventually reach the small intestine where additional replication occurs in the mucosal lining. Virus enters the deep cervical and mesenteric lymph nodes and spreads into the blood (viremic phase) allowing for virus spread to target organs via infection of the reticuloendothelial system [Melnick, 1996 #7]. As virus infects intestinal cells, it is shed in feces, thus, the route of transmission of coxsackieviruses is considered to be fecal-oral. In regions with higher standards of sanitation, aerosol transmission may provide an additional mode of entry [Baboonian, 1997 #8]. 5. CV and Myocarditis Myocarditis is defined as inflammation of the myocardium accompanied with cell necrosis/injury in a pattern differing from damage caused by ischaemic heart injury [Aretz, 1987 #115]. Many agents can cause myocarditis, including toxins, pharmacological substances, and a variety of infectious agents, of which, viruses are the most common 4 [McManus, 1988 #133]. Although a number of viruses are known to cause myocarditis, coxsackieviruses of group B are the most frequently involved (Fig. 1) [Grist, 1993 #132]. Most cases of viral myocarditis are asymptomatic (subclinical), but occasionally the disease may develop into overt symptoms such as transient inflammation or may have a fulminant course with an array of manifestations that may include heart failure, arrhythmias, and sudden death [See, 1991 #80]. In addition, the disease may progress to dilated cardiomyopathy, a disorder associated with enlargement of the heart, and remains the most common indication for cardiac transplantation [Abelmann, 1989 #81]. Effective treatment is limited due to a variety of reasons, such as poor understanding of the pathogenesis of the disease, delayed or nonspecific diagnosis, and a lack of effective antiviral agents [See, 1991 #80]. • Coxsackie B • Influenza B • Influenza A 0 Incidence Rate per 1000 20 40 • Coxsackie A • Cytomegalovirus • Parainfluenza • Echovirus • Poliovirus • Rhinovirus • Coronavirus 11 Adenovirus • Epstein-Barr virus • Varicella-Zoster • Respiratory syncytial virus • Herpes simplex • Mumps B Rubella • Hepatitis B virus • Rotavirus • Hepatitis A virus Fig. 1. World Health Organization global surveillance of viral infections related to cardiovascular disease (1975-1985). (Adaptedfrom reference [Grist, 1993 #132]). 5 5.1 Mechanism of Pathogenesis of Myocarditis Two opposing hypotheses attempt to explain the pathogenesis of CVB3-induced myocarditis, one of which stresses the importance of the immune system in causing disease, and the other which stresses the direct role of virus. The original hypothesis, which stresses the role of aberrant immune responses in the pathogenesis of myocarditis, was proposed in the 1970's [Woodruff, 1974 #134]. This hypothesis suggests that CVB3 infection of the myocardium induces immune responses in which the cellular constituents of the immune system (i.e., CTLs) recognize and lyse not only infected myocytes in vitro, but non-infected ones as well [Rose, 1996 #116; Huber, 1984 #105]. In addition, autoantibodies are generated due to molecular mimicry, in which anti-viral antibodies cross-react with normal heart tissue antigens [Gauntt, 1995 #118; Neumann, 1994 #117]. Thus, myocardial damage (which results in myocarditis) is the result of immunopathology and autoimmunity which is triggered by virus infection. The second hypothesis was proposed in the late 1980's and emphasizes the direct role of virus in causing damage to myocardial cells. The mechanism suggested is that low levels of virus present as a persistent infection within the myocytes is the cause of injury to the heart tissue, contrary to the original hypothesis implicating immune cells in heart damage [Klingel, 1992 #120; McManus, 1993 #119]. This continued low-level of CVB3 persistence within myocytes elicits a chronic anti-viral inflammatory response [Carthy, 1997 #121]. Strong evidence to support this second hypothesis came from a study demonstrating more severe disease following CVB3 infection of SCUD mice [Chow, 1992 #82]. In this study, a histopathological analysis of the myocardium showed the presence of cardiomyocyte necrosis well before the recruitment of an inflammatory infiltrate. 6 Moreover, in other studies, a decrease in disease severity was seen when the immune system was enhanced [Takada, 1995 #123] or hosts were given anti-viral agents [Fohlman, 1996 #122]. Thus, immune responses appear to be protective in CVB3 infection of the myocardium. Furthermore compelling evidence comes from the use of molecular techniques, such as in-situ hybridization, which have allowed the localization of CVB3 genome within the myocardium throughout the course of CVB3 infection. Thus, CVB3 has been demonstrated to be present early within cardiac myocytes following infection (acute phase), and for up to 42 days post-infection (chronic phase) in immune-competent mice [Klingel, 1996 #124]. 6. CV and Diabetes In addition to the association of group B coxsackieviruses with heart disease, C V B infections have been linked to the onset of type 1 diabetes, or insulin-dependent diabetes mellitus (IDDM) [Fohlman, 1993 #152; D'Alessio, 1992 #151; Yoon, 1979 #150]. Type 1 diabetes is a disease characterized by a deficiency of insulin production due to the loss of beta (P) cells within the islets of Langerhans of the endocrine pancreas [Toniolo, 1988 #90]. The loss of P cells can be attributed to numerous factors, such as genetics, immunology, and environment, but the current view is that type 1 diabetes involves an environmental trigger which results in immunopathology and autoimmunity causing P cell damage. The role of viruses as environmental triggers has long been recognized. The link of CVBs, in particular, with type 1 diabetes was initially suggested in 1969 [Gamble, 1969 #135]. Studies have recovered CVB-specific IgM antibodies in newly diagnosed cases of diabetes [D'Alessio, 1992 #151] and virus was isolated from the pancreas of a 7 child with diabetic ketoacidosis [Yoon, 1979 #150]. The strain most often implicated with diabetes is CVB4 compared to the other CVBs in various serological investigations, although all CVBs have the potential of causing diabetes [Toniolo, 1988 #90]. Many strains of CVB4 have been shown to directly infect P cells inducing hypoinsulinemia and hyperglycemia in mice [Yoon, 1978 #153]. The complete nucleotide sequence of two murine p-cell tropic CVB4 strains, CVB4 (E2) [Kang, 1994 #154] and P-CVB4 [Titchener, 1994 #155] have been determined, but the viral genomic sequences that determine the diabetogenic phenotype of CVB4 in mice are presently unknown. Although the mechanism of the pathogenesis of CV-induced diabetes in humans is not clear, pancreatic damage cannot be attributed solely to viral replication [Chapman, 1997 #92]. The widely accepted hypothesis is the occurrence of autoimmunity, thought to occur due to molecular mimicry, with a P cell specific antigen or alternatively, bystander activation of resting autoreactive T cells [Wekerle, 1998 #91]. The activation of autoreactive T cells by presentation of excessive amounts of self antigen released from pancreatic islets during inflammation has also been suggested as another mechanism [Horwitz, 1998 #89]. 7. Factors Affecting the Course of CV Infection Host susceptibility to C V infection varies considerably., and the ultimate outcome of C V challenge to the host is dependent on a number of factors [Gauntt, 1997 #35]: 1) Host factors i) Genetic background 8 ii) Age iii) Immune status prior to infection iv) Immune system function v) Hormones (e.g., pregnancy) vi) Nutritional status 2) Virus factors i) Virus genetics, i.e., virulence properties of the C V serotype ii) Dose of inoculation iii) Tissue tropism Additional conditions affecting the response to C V infection include the route of inoculation, and the temperature of infection conditions. [Loria, 1988 #36]. 7.1 Host Genetics It is well-established that most viruses differ in host range, with some species of animals being susceptible, and others, resistant to infection with a virus. In addition, variation in susceptibility among members of the same animal species is seen, indicating that genetics play an important role [Mogensen, 1979 #37]. Differences in genetic susceptibility to viral disease may be related to many factors, including the ability to produce interferon and activate an efficient N K cell and macrophage response, the nature of peptides presented to T cells and thereby, the specificity of the CTL and antibody responses. In addition, the presence of a suitable cell receptor for virus on the target tissue is clearly critical to enable viral replication in the host. The cellular receptors of 9 poliovirus II, CVB3, and echovirus II have all been mapped to chromosome 19, although these viruses do not share a receptor [Loria, 1988 #36]. Presence of the appropriate receptor is essential but not necessarily sufficient to permit viral replication, as intracellular host factors are often also required. Minor alterations in the receptors are thought to alter the affinity of virus attachment and play a major role in host susceptibility to infection. Associated with the immune response, one study analysing a number of genetically defined mouse strains found wide variation in the extent and duration of CVB3 viremia, as well as in the time of appearance of antibody, and in the prevalence and degree of myocarditis [Wolfgram, 1986 #44]. In conclusion, host-virus interactions are a complex phenomenon; while host susceptibility may be determined by a single dominant gene in certain cases, more commonly it is under polygenic control [Mogensen, 1979 #37]. 7.2 Host Immune Status and Function The majority of coxsackievirus infections in immunocompetent individuals are inconsequential, indicating that the host immune response is adequate to control the infective process. The individual is then protected from reinfection by that particular strain but is susceptible to infection by the large number of other CV serotypes. Moreover, secondary infection by a different strain of virus may be enhanced by prior infection with a different serotype in a phenomenon known as antibody-dependent enhancement (ADE) [Fust, 1997 #167; Porterfield, 1986 #166]. Immunologic determinants have been shown to influence susceptibility to CV in experimental infections in mice. Maturation of the immune system is important in the decline of susceptibility which occurs with aging, and is discussed in detail in section 8. 10 Susceptibility of adult mice to CVB is increased by treatments known to interfere with the immune system including corticosteroids, cyclophosphamide, stress, and undernutrition [Bendinelli, 1988 #38]. Moreover, experimental models have clearly proved that immunological factors not only contribute to recovery and resistance from CV infection, but also contribute to the generation of disease. Antibody has been shown to participate in CV-induced disease, such as in immune complex nephritis [Sun, 1967 #98], and in the generation of myocarditis, through generation of autoantibodies directed to heart tissue antigens [Wolfgram, 1989 #99]. T cells also have been noted to play a role in pathogenesis, as their transfer into SCID mice caused autoimmune myocarditis [Schwimmbeck, 1997 #100]. Neutrophils and macrophages are known to be vital cellular components to limit CV infection, but their secretion of mediators, such as leukotrienes, may mediate inflammatory pathogenesis to the heart [Maisch, 1987 #101]. 7.3 Hormones Susceptibility to CV is dependent on sex-associated hormones, since males are more prone to infection than females. Androgens (testosterone and progesterone) enhance susceptibility to virus, while estrogens decrease susceptibility [Huber, 1993 #136]. The reasons for these hormonal differences are not well-understood but relate to the nature or effectiveness of the immune response generated. 7.4 Virus Factors Different serotypes of CV cause infections of varying severity. Some serotypes are more virulent, causing diseases such as severe paralysis in certain hosts; while other 11 serotypes are less virulent, causing mild, often subclinical infections [Melnick, 1996 #97], The genotype of the virus is one of the variables that influences induction and severity of disease. The great majority of genetic characterization of C V has been performed with laboratory strains, in an attempt to relate a set of characteristics of a given strain to its pathogenicity in cell cultures or in animal models [Gauntt, 1988 #96]. Mutations in different regions of the C V B genome may have a direct result in virulence and tissue tropism [Tracy, 1995 #146]. It has been shown that a mutation in the capsid region within the genome of a CVB3 antibody escape mutant, called H310A1, limits virus binding to receptors [Knowlton, 1996 #147]. Regions outside of the coding region of the C V genome have also been identified to be important determinants of virulence. For example, the 5' non-coding region, which contains many stem-loop structures and the IRES (internal ribosome entry site), is the region to which the host cell translational machinery binds to direct the translation of C V B R N A during an active infection. Mutations in this region may affect viral replication within infected cells and corresponding virulence. 8. Effect of Host Age on Coxsackievirus Infection Hosts infected with Group B coxsackieviruses demonstrate a marked age-related susceptibility. Neonates, of humans and mice, are highly susceptible to severe C V B infection. The high mortality rate seen in the newborn is in contrast to the mild or inapparent infections seen in adults. A progressive resistance to C V B viruses develops as the neonates become older, commencing at about 8 days of age (Fig. 2) [Rager-Zisman, 1973 #10]. 12 Fig. 2. Development of age-dependent resistance to CVB3. BALB /c mice were inoculated i.p. with 105 pfu of virus. Each point represents mortality in a group of 12-20 mice. (Adaptedfrom reference [Rager-Zisman, 1973 #10]). Various explanations have been proposed to account for the increased vulnerability of the newborn to severe coxsackievirus infection. The first hypothesis is that various tissues in the neonate possess an innate susceptibility to C V [Holland, 1961 #30] and that this property is due to the presence of viral receptors on the cell membrane [Hsu, 1988 #31; Shafren, 1995 #32]. Thus, it is possible that the expression of certain cell surface proteins that act as enterovirus receptors is up-regulated early in life, and that this enhances the susceptibility of specific tissues or organs to C V infection [Modlin, 1997 #33]. Another explanation for the extreme susceptibility of neonates is the immaturity of the neonatal host immune response to C V infection [Modlin, 1997 #33]. For example, the 13 reduced functional capability of human neonatal macrophages relative to older macrophages has been suggested to contribute to the susceptibility of newborns to C V infection [Mills, 1983 #34]. This idea is supported by the finding that neonatal mice are protected against severe CVB3 infection when peritoneal exudate cells from adult mice are transferred to the neonates prior to infection (Fig. 3) [Rager-Zisman, 1973 #10]. p.e.c. + CVB3 CVB3 0 2 4 6 8 10 12 14 16 18 20 Time after virus infection (days) Fig. 3. Effects of transfer of peritoneal exudate cells (p.e.c.) on mortality of newborn mice infected with CVB3. BALB/c mice, 48 hrs old, were inoculated i.p. with 103 pfu CVB3 (control), or with 107 p.e.c. i.p. and 103 pfu CVB3 i.p. Groups consisted of 10 mice each. (Adaptedfrom reference [Rager-Zisman, 1973 #10]). 9. Immune Responses to CV Infection C V can appear in target tissues (such as the heart) within hours of inoculation of mice [Lodge, 1987 #107]. Virus replicates to high titres, reaching a peak between 2 and 7 14 days after infection. Thereafter, the virus is eliminated, which can take anywhere from two to four weeks, or perhaps even longer, depending on the host. Different strains of mice exhibit different timecourses of infection with CVB3 with respect to localization and clearance of virus in target tissues [Huber, 1993 #136]. Host resistance to infection involves innate immune responses, which serve to limit virus replication early in infection, and acquired immune responses, which serve to clear the infection. 9.1 Innate Mechanisms of Resistance A variety of innate mechanisms exist to eliminate virus, including the activity of macrophages, N K cells, and interferon. Control of virus early in infection is important as early intervention in the replication cycle would dramatically reduce the viral load, which in turn, would result in fewer new cells becoming infected [Huber, 1993 #136]. Macrophages are of pivotal importance in host defense against a wide range of virus infections [Bendinelli, 1988 #38]. They can eliminate, phagocytize, and degrade virus through the release of a vast array of soluble mediators (See 11). Extensive data from studies in mice indicate that the cells of the monocyte-macrophage lineage provide efficient protection against C V B [Conaldi, 1988 #39]. Susceptibility has been shown to be influenced by the functional state of macrophages, and the impairment of these cells resulted in severe C V B infection [Kabiri, 1978 #40; Rager-Zisman, 1973 #10]. Natural killer (NK) cells contribute to host resistance against viruses through direct cellular cytotoxicity, involving secretion of perforins and enzymes (granzymes), and by the release of cytokines such as IFN-y. In the case of CVB3, activated N K cells have been demonstrated to limit viral replication both in vivo and in vitro [Conaldi, 1988 #39]. 15 NK cell-mediated cytotoxicity is activated during the early stages of CVB 3 infection, peaking at day 3 post-infection, and then declining [Bendinelli, 1988 #38]. Interferons participate in controlling many virus infections [Vilcek, 1996 #138]. Interferons have been demonstrated to inhibit all phases of viral interactions within cells, including viral entry, viral uncoating , and synthesis of mRNA and proteins [Peters, 1996 #139]. Three types of interferon are known. These are EFN-a and IFN-P (known as type I interferon) and IFN-y (type II interferon). All three interferons are produced after viral infection and display anti-viral effects, however IFN-y functions predominantly as an immunomodulator. Macrophages represent the major cellular source for the early production of IFN-a and IFN-P in response to infection [Belardelli, 1995 #140], while IFN-y is produced by NK cells (important during early phases of infection) and by T lymphocytes. IFN-y, in turn stimulates the activation of macrophages to release a number of non-specific mediators such as nitric oxide; IFN-y also stimulates NK cell cytotoxicity. The importance of interferons in CVB infections have been highlighted in studies in which IFN-a and IFN-P administered to CVB3 -infected mice have been shown to reduce virus titres in heart by at least 100-fold [Kandolf, 1987 #141]. In addition, administration of antibody against these cytokines to infected mice caused enhanced replication [Kandolf, 1987 #141]. Additional innate mechanisms of defense which contribute to CV resistance include restricted expression of cell receptors on tissue, and barrier and clearance mechanisms operative in the gut [Loria, 1974 #42; Bendinelli, 1988 #38]. The host employs many defense tactics to reduce viral titres early in infection until acquired immunity is generated some days later. The collaborative effects of innate 16 immune mediators with acquired immune constituents provides for most efficient host resistance against CV. 9.2 Acquired Mechanisms of Resistance Resistance to picornaviruses, including the CVBs is largely mediated by antibodies [Conaldi, 1988 #39]. Virus-specific antibody binds to the elevated regions of the virus capsid (coat) and interferes with virus-receptor binding (virus neutralization). IgM neutralizing antibody has been detected as early as 2 days after infection and IgG antibody is observed by day 6 [Lodge, 1987 #107]. In humans, the pattern of humoral immunity against CVB is varied, but characterized by the generation of both type- and group-specific antibodies of the IgA, IgM, and IgG classes [Conaldi, 1988 #39]. As in mice, differences in antibody production are thought to determine the type and severity of CVB infection [Schernthaner, 1985 #45; Tilzey, 1986 #46]. Later studies correlated the incidence of CVB-induced disease to the host MHC genes, which control timing and extent of neutralizing antibody production [Wolfgram, 1986 #44], as well as CTL induction. Antibody functions coordinately with innate immune cells, such as macrophages and NK cells by neutralizing free extracellular virus while the cells prevent new virus production. Antibody can clump viruses together producing antigen-antibody aggregates, which then enhances phagocytosis by macrophages by the binding of these aggregates to Fc receptors. CD4+ and CD8+ T cells begin to appear at around day 5 after infection in a ratio of approximately 2:1, but later the proportion of CD8+ cells increases [Huber, 1993 17 #136]. Cell-mediated immunity does not appear to be as critical as antibody induction in host resistance against CVB, since depletion of T lymphocytes in mice did not affect their ability to clear CVB infections [Huber, 1983 #49]. These results conform with clinical observations, which demonstrate that CVB-induced diseases occur with higher frequency in patients with antibody deficiencies, but not among individuals with T cell deficiencies [Moore, 1984 #50]. CMI, however, does play a role in resistance to CVB, since CVB has been shown to persist for longer periods in the heart of T cell deficient mice [Schurr, 1984 #51]. In addition, mice infected with immunosuppressive murine retroviruses, which cause the suppression of CMI but leave antibody responses intact, demonstrated an increase of tissue invasion and lethality by CVB3 [Specter, 1987 #52]. 10. The Role of Macrophages in CV Infection Macrophages are involved at all stages of the immune response during infections. As mentioned above, they act as a rapid protective mechanism that can respond to microbes before the amplification and development of aquired immunity. Before a look at macrophage interactions with CV specifically, a more general view on macrophage-virus interactions will first be discussed. 10.1 Macrophage-Virus Interactions: General Aspects The importance of mononuclear phagocytes (MP) in innate resistance to viruses was first recognized in 1964 [Mims, 1964 #54]. Since then, their crucial role in the innate resistance, and in the induction, regulation, and amplification of acquired immune 18 responses through antigen-presentation and secretion of cytokines, as well as their role in inflammation and tissue repair has been well characterized (Fig. 4). Selection of Mechanism To Be Activated IL-10 (Th2) IL-12 (Thl) Lymphocyte Activation antigen processing, antigen presentation, IL-1 production Inflammation and Fever IL-6, TNF-oc, IL-1 prostaglandins complement factors clotting factors t Tissue Damage H 2 0 2 , acid hydrolases, C3a, TNF-cx Tumoricidal Activity cytotoxic action, toxic factors, H 2 0 2 , C3a, proteases, arginase, NO, TNF-ct Tissue Reorganization secreted factors, elastase, collagenase, hyaluronidase, fibroblast stimulating factors, angiogenesis factors Microbicidal Activity Oxygen-dependent H 2 0 2 , 0 2 , NO, OH Oxygen-independent lysozyme, acid hydrolases, cationic proteins Fig. 4. The various roles of macrophages in immunity and inflammation. (Adapted from reference [Roitt, 1998 #164]). M P comprise a widely distributed cell system that includes immature cells in the bone marrow, circulating monocytes and tissue macrophages. Macrophages are situated 19 strategically throughout the body to meet foreign particles, particularly in sub-epithelial tissues of the nasopharynx and gut, where they are in a position to encounter virus early in the infectious process [Mogensen, 1988 #57]. Macrophages play various roles in viral infections in vivo. As scavenger cells, mononuclear phagocytes employ phagocytosis to eliminate virus from circulation following a blood-borne infection [Gendelman, 1992 #59], constituting a first line of defense that reduces virus load until acquired immune responses have been induced. A second line of defense arises when macrophages restrict virus infection in other cells by various antimicrobial effector mechanisms, such as the lysis of virus-infected cells by antibody-dependent cellular cytotoxicity (ADCC), or by the initiation of a series of immune reactions through the cytokine network [Gendelman, 1992 #59]. Macrophages themselves may become activated by cytokines (such as IFN-y) released by virus-specific T cells, which cause expression of various macrophage inducible genes, such as nitric oxide synthase, which generates nitric oxide. A further line of defense arises when MP act as antigen-presenting cells, which take up and process virus, and deliver viral antigens to T cells in regional lymph nodes for the initiation of aquired immune responses, which then clear the virus infection [Gordon, 1986 #61]. In certain instances, a host's antiviral defense system may not be sufficient to clear a virus infection, leading to viral persistence or latency within the macrophage and late-onset disease [Morahan, 1988 #64; Semenov, 1981 #63; Oldstone, 1989 #62]. For example, human immunodeficiency virus (HIV) is well known to be tropic for macrophages, establishing persistent infections within these cells. 20 The interaction of macrophages and viruses has been studied in detail using in vitro systems, and it has been demonstrated that the outcome of the interaction is dependent on a number of factors (Table 1) that vary depending on the virus or the macrophage itself: Virus Factors Strains Passage history Mutation Multiplicity of infection Defective interfering particles MP Factors Organ source of MP (peritoneal, blood, bone marrow, lung, spleen, liver) Animal species Animal age: neonatal, adult Genetic background Culture conditions in vitro (medium, duration, adherent or suspension cultures) Treatment of animals in vivo (elicitation with thioglycollate broth, mineral oil, proteose-peptone; activation with C. parvum, BCG; immunization with live or killed viruses) Table 1. Experimental factors that affect mononuclear phagocyte-virus interactions. (Adaptedfrom reference [Morahan, 1985 #53]). There are two kinds of interactions in vitro between viruses and macrophages: intrinsic and extrinsic [Morahan, 1979 #58]. Intrinsic interaction is defined as the ability of macrophages to either permit or restrict virus replication within the macrophage itself, while extrinsic interaction relates to the ability of macrophages to influence (restrict) extracellular virus, by interfering with virus replication in surrounding permissive cells [Morahan, 1985 #53]. 21 Why macrophages are more restrictive (non-permissive) for many virus infections compared to most other cell types is not known. Attempts have been made to correlate interferon production by macrophages to permissiveness for viruses [Ellermann-Eriksen, 1986 #144; Mogensen, 1987 #143], since it was suggested that low, undetectable levels of endogenous interferon maintain an antiviral state in macrophages, since anti-interferon treatment of normal mice before macrophage harvest rendered these cells permissive to subsequent viral challenge [Mogensen, 1988 #57]. But most studies on interferon as a mechanism of intrinsic resistance have been inconclusive. Recent explanations for macrophage permissiveness take into account the state of macrophage differentiation and activation. It is known that initial virus-macrophage interactions are dependent on specific and non-specific viral receptors on the macrophage surface, and that after binding and uptake, the ability of the virus to complete a full replication cycle depends upon the differentiation and activation state of the macrophage, as well as the nature of the infecting virus [Gendelman, 1992 #59]. Macrophages are clearly heterogenous in their permissiveness for viral infection, since only a small percentage become infected at a given time. Thus, a transient differentiation state (or activation state) of the macrophage may lead to the acquisition of specific virus receptors resulting in viral replication, along with intracellular factors [Gendelman, 1992 #59]. Although macrophages restrict many viruses, a number of viruses are known to be capable of replicating in monocytes or macrophages (Table 2) and a proportion of these persist for prolonged periods in this cell population. 22 Table 2. Some viruses known to be permissive for macrophages. HIV CMV Rubella Influenza A Hepatits B Herpes Simplex When a virus infects a macrophage, the result is either cytolysis or persistent infection of the macrophage. Cytolytic infections of macrophages may eliminate the macrophages, either locally or systemically and the subsequent decrease in resistance allows for a more severe viral infection. The more subtle outcomes of intrinsic interactions, i.e., abortive or persistent infections, which do not kill macrophages, alter their functions instead (Table 3). In these experiments, depressed function of macrophages is usually observed, although enhanced activity or no activity has also been noted [Mogensen, 1982 #65]. Table 3. MP functions that may be altered by virus infection. (Adaptedfrom reference [Morahan, 1985 #53]). Chemotaxis Attachment and phagocytosis of particles through nonspecific, Fc or complement receptors Intracellular oxidative response Lysosome-phagosome fusion Intracellular microbicidal activity Synthesis and/or secretion of biologically active molecules (prostaglandins, neutral proteases, interferon, and complement) Antigen presentation Regulation of immune responses, i.e., accessory and suppressor activity Macrophage activation process for microbicidal and tumoricidal activity Antibody-dependent cytotoxicity (ADCC) Wound healing DNA synthesis and macrophage proliferation Macrophage differentiation 23 Extrinsic resistance on the other hand (i.e., the ability of macrophages to suppress virus replication in another cell) is independent of the ability of the macrophage to support virus replication itself [Stohlman, 1982 #60]. Many mechanisms are involved in limiting viruses extrinsically (Table 4), and the specific mechanism utilized by a given virus-macrophage system depends on the particular macrophage and target cell [Morahan, 1985 #53]. Table 4. Anti-viral mediators produced by macrophages. Complement components Arachidonic acid metabolites Neutral proteases Cytokines: Interferons (IFN-a, IFN-P) Tumour necrosis factor (TNF-a) Reactive oxygen intermediates Reactive nitrogen intermediates 10.2 Macrophages and CV In murine models of C V infection, macrophages have been shown to be important components of host defenses at the portal of entry (the gut), limiting virus spread to target organs. They have also been shown to contribute to viral clearance from infected tissues [Mogensen, 1979 #37; Conaldi, 1988 #39]. Early studies correlated age of susceptibility to CVB3 with the functional maturation of peritoneal cells (macrophages). Thus, neonatal mice with immature peritoneal cells, displayed extreme susceptibility and mortality to CVB3 infection as 24 compared to adult mice [Rager-Zisman, 1973 #10], and the transfer of peritoneal cells from uninfected syngeneic adult mice to neonatal mice protected them against the lethal effects of virus [Rager-Zisman, 1973 #10]. Moreover, pretreatment of mice with macrophage-activating substances increased protection, whereas blocking substances showed the opposite effects [Bendinelli, 1988 #38]. Similarity, the inhibition of the synthesis of prostaglandins (which are produced predominantly by macrophages) resulted in more severe myocardial inflammation and necrosis during infection with CVB3 [Costanzo-Nordin, 1985 #66], implying that the cells responsible for production of these mediators are major factors in the clearance of virus. Although macrophages play a major role in resistance to CV, the control of CV is most efficient with combined effects of mononuclear phagocytes and aspects of adaptive immunity [Bendinelli, 1988 #38]. This was demonstrated in experiments in which passive protection of CVB3-infected neonates containing transferred adult peritoneal cells was increased by concurrent administration of anti-CVB3 antibody [Rager-Zisman, 1973 #10]. Macrophages, however, have been shown to exert independent antiviral activity in vitro in which peritoneal macrophages from nonimmune mice were shown to take up and inactivate extracellular CVB3 [Rager-Zisman, 1973 #10]. 11. Nitric Oxide Nitric oxide (NO) is a free radical gas with diverse physiologic functions. It acts as a messenger between cells, as well as a cytotoxic agent, playing a role in immune defense. Nitric oxide was initially discovered to be produced by macrophages [Stuehr, 1985 #157], and was shortly identified to be synthesized by endothelial cells [Palmer, 1988 25 #158] and neurons [Martin, 1988 #159]. To date, many cell-types are known to produce nitric oxide, including epithelial cells, hepatocytes, and neutrophils, to name a few. NO is produced by an enzyme within cells called nitric oxide synthase (NOS), which is present in one of three isoforms [Forstermann, 1994 #68]. Isoform I was the first to be purified and originally isolated from the brain (neurons), and later identified in other cells types; it is constitutively expressed, and its activity is regulated by Ca 2 + and calmodulin [Forstermann, 1994 #68]. Isoform II was purified next and is expressed by macrophages, as well as other cell types; its activity is not regulated by Ca 2 + and it is not constitutively expressed, rather it is inducible by bacterial endotoxin and/or cytokines (see below) [Forstermann, 1994 #68]. Isoform III is very similar to isoform I in that it is constitutively expressed, and its activity is regulated by Ca 2 + and calmodulin; this form has been identified primarily in endothelial cells [Forstermann, 1994 #68]. The function of NO varies depending on the cell isoform from which it was produced. NO produced by isoform I within neurons functions as an atypical neurotransmitter and is responsible for synaptic plasticity [Forstermann, 1994 #68]. NO from isoform II within macrophages is produced in large amounts and functions as an immune defense molecule [Forstermann, 1994 #68]. Endothelial isoform III produces NO in relatively small amounts (similar to isoform I NO) to promote vasodilation [Forstermann, 1994 #68]. Thus, it can be said that NO functions at high concentrations as a toxic mediator against microbes (as produced by isoform II), and at low concentrations as a signal (as produced by isoforms I and III) [Crane, 1997 #156]. 26 11.1 Biosynthesis of NO All NOS isoforms are heme proteins which utilize L-arginine as the substrate and require several cofactors (Fig. 5). The reaction of L-arginine, in the presence of molecular oxygen, is catalyzed by NOS to generate NO, along with a co-product, L-citrulline. Al l isoforms of NOS are inhibited by L-arginine analogs such as NG-monomethyl-L-arginine ( L - N M M A ) [Forstermann, 1994 #68]. NO, an extremely unstable molecule with a half-life of seconds, reacts with several different molecular targets. In the presence of oxygen and water, N O interacts with itself to generate other reactive nitrogen oxide intermediates, and ultimately decomposes to form nitrite (N02") and nitrate (N0 3 '), (more stable end products) [Coligan, 1994 #69]. Fig. 5. Biosynthesis of NO from L-arginine and 02. 27 11.2 Macrophage NO Only activated macrophages produce NO [Forstermann, 1994 #68] and its synthesis is believed to contribute significantly to their antimicrobial activity [Nathan, 1991 #70]. The NO produced has been shown to exert potent inhibitory effects on the replication of many different types of microbes (intracellular bacteria, fungi, protozoa), tumor cells, and more recently, viruses. Numerous cytokines and microbial products, often acting synergistically and sequentially, induce NOSH enzymatic activity within macrophages [MacMicking, 1997 #71]. Lipopolysaccharide (LPS), a constituent of the cell wall of Gram-negative bacteria, alone or in synergy with interferon-gamma (IFN-y) induces maximum levels of enzyme activity. Tumour necrosis factor-alpha or -beta (TNF-a or -0), are inactive on their own, but synergize with IFN-y to induce type II NOS. In contrast to LPS, TNF and IFN-y play prominent roles in viral infections. Other cytokines, including IL-ip, IL-2, and IL-12 also activate induction of type II NOS [Lincoln, 1997 #160]. The actions of NO depend on the intracellular environment of both the generating cell and the target [Lincoln, 1997 #160]. Since NO can undergo a vast range of reactions in biological conditions (it is a highly reactive species possessing an unpaired electron), the exact mechanism of NO interactions with targets are not known, but may depend upon the redox environment within cells. Several potential ways that NO may render toxicity to cells, however, have been identified. NO has been demonstrated to affect the DNA of a cell by inhibiting the activity of ribonucleotide reductase, the rate-limiting enzyme in DNA synthesis, and can also induce mutagenesis by causing direct damage to DNA from deamination by NO [Lincoln, 1997 #160]. In addition, NO can destroy a cell's (and 28 mitochondrial) membrane potential by interacting with transport proteins and ion channels [Kroncke, 1995 #161]. NO can induce energy depletion within cells by inhibiting enzymes involved in metabolism, such as iron-sulfur cluster enzymes (complex I and II) of the mitochondrial electron transport chain and cis-aconitase (enzyme converting citrate to isocitrate) in the Krebs cycle [Nathan, 1991 #70]. NO can also interact with iron-sulphur clusters in proteins, zinc-finger domains in proteins, and free thiol groups near the active sites of enzymes [Lincoln, 1997 #160]. Thus, the main targets of NO within cells are transition metal- (e.g. iron) and thiol-containing proteins. Induction of isoform II NOS in macrophages is associated with production of large amounts of NO for prolonged periods of time, and thus can contribute to pathology. The high levels of NO produced by activated macrophages (and probably neutrophils and other cells) is not only be toxic to undesired microbes, or tumor cells, but can also harm healthy surrounding tissue [Forstermann, 1994 #78]. Most inflammatory and autoimmune lesions are characterized by an infiltrate of activated macrophages, as well as neutrophils, which secrete high amounts of NO. It is unclear, however, if the tissue damage is related to the NO radical itself (NO), or an interaction of NO with superoxide anion ( O2), which forms peroxynitrite (ONOO") and eventually hydroxyl radical (HO) [Forstermann, 1994 #78]. Induced isoform II NOS has also been noted for a role in septic shock [Forstermann, 1994 #78]. 11.3 Antiviral Role of NO The involvement of NO as an antiviral mediator of macrophages has only recently received attention. Experiments providing strong evidence for the antiviral role of NO 29 was carried out with viruses such as herpes simplex type 1 (HSV-1), extromelia virus, and vaccinia virus. Replication of all of these viruses was significantly inhibited upon NO synthase induction in macrophages by IFN-y [Karupiah, 1993 #73]. Other studies reproduced the effect of endogenous NO on HSV-1 replication upon addition of a NO donor compound, called S-nitroso-N-acetyl penicillamine (SNAP), which also inhibited HSV-1 replication, and showed that the diminished replication of HSV-1 could be partially reversed by using NOS inhibitors [Croen, 1993 #74], The mechanism of NO inhibition on virus replication is not known. A wide range of viruses have been shown to be inhibited by NO, including DNA viruses, RNA viruses, and retroviruses [Mannick, 1995 #67]. These viruses have varying modes of replication, therefore, NO may work at a variety of levels depending on the virus [Mannick, 1995 #67]. It is likely that NO, being highly reactive, exerts its antiviral effect by reacting with both viral and cellular targets; this would be advantageous for host defence since viruses would be limited in their ability to develop resistance [Mannick, 1995 #67]. Several mechanisms to explain the antiviral effects of NO have been proposed for different viruses. As mentioned earlier, the known cellular targets for NO have been identified as transition metal- and thiol-containing enzymes; haem-containing enzymes have also been identified [Mannick, 1995 #67]. Ribonucleotide reductase, an iron-containing enzyme in DNA synthesis, is possessed not only by cells, but by vaccinia viruses as well, and may be a target for NO action [Mannick, 1995 #67]. Another iron-containing enzyme, guanylate cyclase, which is found within cells, may mediate NO antiviral effects of Epstein Barr virus, in which cGMP interactions are regulated by redox mechanisms which utilize thiol-containing proteins [Stamler, 1992 #77; Stamler, 1994 30 #76]. Other potential targets of NO include viral and cellular transcription factors, since they are redox-regulated; examples include Zta, which is an E B V transcriptional transactivator, or N F - K B , a cellular transcription factor [Mannick, 1995 #67]. Finally, NO may inhibit virus replication indirectly by interfering with cellular functions and metabolism, such that the cell host can no longer support virus infection (mechanisms are mentioned in the section of "Macrophage NO"). 11.4 Nitric Oxide and CV The role of nitric oxide in C V infections has only recently been examined. In one study, it was shown that CVB3 infection of BIO. A mice was associated with the induction of iNOS expression in macrophages found within myocarditic infiltrates [Lowenstein, 1996 #17]. Moreover, inhibition of NOS increased both mortality and the survival time of infected mice [Lowenstein, 1996 #17]. Subsequent studies explored the mechanisms by which NO exerted its antiviral effects on CVB3 in vitro by examining NO effects at the specific stages of the life cycle CVB3 by using the NO donor, S-nitroso-amino-penicillamine (SNAP) [Zaragoza, 1997 #16]. While NO did not affect CVB3 attachment to cultured cells, viral replication, specifically RNA synthesis, was inhibited, which led to a subsequent inhibition of protein synthesis [Zaragoza, 1997 #16]. The mechanism of this RNA synthesis inhibition by NO is currently not known. In contrast to its protective role, NO has been shown to be involved in the pathogenesis of CVB3-induced myocarditis as well, since excess production of NO can lead to inflammation and tissue injury. One study investigated the timecourse of induction of iNOS in the hearts of C3H/He mice during CVB3 infection [Mikami, 1996 #20]. The 31 induction of iNOS was correlated with inflammatory cell infiltration in the heart, suggesting that NO production by inflammatory cells might be a major mechanism of heart injury in CVB3-induced myocarditis. In another study, the detrimental role of NO in CVB3 myocarditis was shown by using a low-dose NOS inhibitor treatment in C3H/He mice, which improved their survival rate and decreased myocardial injury [Mikami, 1997 #102]. 32 RATIONALE AND OBJECTIVES Clinically, coxsackieviruses (CV) manifest severe infections in the pediatric population. The extreme susceptibility seen in the young is in contrast to the mild infections that occur in adults. Understanding the differences that exist in the young host relative to the adults is imperative in learning how these differences may play a role in disease development. In mouse models, it has been demonstrated that macrophages are one of the key participants in relaying host resistance against viruses. Being an important component of the immune system, macrophages possess diverse functions that limit infections (see Introduction). The overall objective of this project was to compare the functional differences of macrophages between young and adult mice and to correlate these differences to CVB3 susceptibility. The importance of NO as a determinant of tissue damage in the pathogenesis of C V infections has recently been shown (Mikami et al., 1997). In addition, a direct antiviral role of NO has been suggested by Zaragoza et al., 1997. Thus, the first aim was to compare the ability of macrophages from young and adult CD-I mice to produce NO during CVB3 infection. Other macrophage functions to be compared included virus inactivation, and production of anti-viral cytokines, such as TNF-a. In addition to host age, genetics are also important determinants of the outcome of virus infection since differences in C V susceptibility among individuals of the same species are seen. Thus, another objective of this research project was to characterize macrophage functional differences between young and adults in another mouse strain, B A L B / c . 33 MATERIALS 1. Cell line 1.1 HeLa (human cervical epithelioid carcinoma) cell line was a gift from Charles Gauntt. 2. Cell culture reagents 2.1 DMEM/F12 media (Gibco/BRL) was obtained as powder and prepared according to the manufacturer's instructions. 2.2 Fetal bovine serum and fetal lamb serum (Gibco/BRL). 2.3 Heat-inactivated fetal bovine serum was prepared by heating the fetal bovine serum at 60 °C for 30 minutes. 2.4 Gentamicin antibiotic (lmg/ml) (Gibco/BRL) and used at a concentration of 1% (v/v). 2.5 Trypsin (0.25%): 2.5 g of 1:250 Trypsin (Gibco/BRL) "T* 2"|_ Dissolved in 1 L of Hank's balanced salt solution without Ca or M g Trypsin solution was sterilized using a 0.22 um pore diameter filter. 2.6 Hank's balanced salt solution without Ca + or M g 2 + 0 . 4 g K C l 0 .06gKH 2 PO 4 8.0gNaCl 0.35gNaHCO 3 0.12gNa 2 HPO 4 1.00 gD-glucose Diluted to 1 L with ddH 2 0 2.7 Phosphate buffered saline (PBS): 34.0 g NaCl 4 .28gNa 2 HP0 4 1 . 3 8 g N a H 2 P 0 4 H 2 0 Diluted in 4 L of ddH 2 0 and autoclaved. 2.8 Agarose (SeaKem/Mandel Scientific) was used at 1% for plaque assays and 0.7% of agarose (Gibco/BRL) was used for gel electrophoresis 3. Stains and fixatives 3.1 Coomassie blue stain 100 ml glacial acetic acid (10%) 250 ml isopropanol (25%) 2.5 g Coomassie blue powder (0.25%) Diluted to 1 L with ddH 2 0 3.2 Haematoxylin 0.2 g Haematoxylin (0.2%) 0.7 g A1 2(S0 4) 3-15H 20 (12.6 mM) 0.008 gNaI0 3 (0.4 mM) 25 ml 1-2-ethanediol (25%) 2 ml glacial acetic acid (2%) Diluted to 100 ml with ddH20 35 3.3 Eosin 70 ml ethanol (70%) 25 ml glacial acetic acid (25%) 0.5 g eosin (0.05%) 3.4 Masson's Trichrome Bouin's Sodium bicarbonate (2%) Weigert's iron hematoxylin (a mixture of 2 solutions in equal parts): Solution A: 1% (w/v) hematoxylin in absolute ethanol Solution B: 40% (w/v) FeCl 3 and 10 ml HC1 per IL Ponceau 2R (1% w/v) / acid tuchsin (1%> w/v) mixed (70/30) in acetic acid (1%) Acetic acid (1%) Phosphomolybdic acid (1%) Aniline Blue (2.5%) in acetic acid (2.5%) 3.5 Carnoy's Fixative 3:1 mixture of 95% ethanol:glacial acetic acid: 750 ml 95% ethanol 250 ml glacial acetic acid 3.6 Paraformaldehyde 4% 40.5mlNa 2 HPO 4 (0.1M) 9.5 m l N a H 2 P 0 4 H 2 0 (0.1M) The phosphate buffer was heated to 80 °C and 2 g of paraformaldehyde 36 (Mallinckrodt) was added. Five to ten drops of 5N NaOH was added to pHto 7.4. 4. Solutions for large scale pCVB3-Rl plasmid preparation using PEG precipitation 4.1 2 X YT medium 20 g Bacto-tryptone 10 g Bacto-yeast extract 20 g NaCl Diluted to 1L with ddH 2 0 4.2 Solution I 0.45 g glucose (50 mM) 0.15 g Tris-HCl pH 8.0 (25 mM) 1.25 ml 0.4 M E D T A (10 mM) Diluted to 50 mL with ddH 2 0 4.3 Solution II 0.4 g NaOH (0.2 M) 0.5 mL 10%SDS (0.1%) Diluted to 50 mL with ddH 2 0 4.4 Solution III 30 mL potassium acetate (5M) 5.75 mL glacial acetic acid Diluted to 50 mL with ddH20 4.5 TEpH8 .0 37 0.12 g Tris-HCl pH 8.0 (10 mM) 0.25 ml 0.4 M EDTA (1 mM) Diluted to 100 ml with ddH20 5. Solutions for quantification of DIG-labeled transcript (dot blot) All solutions came in a DIG Nucleic Acid Detection Kit (Boehringer Mannheim) 5.1 Buffer 1 5.8 g maleic acid (100 mM) 4.3.8 gNaCl (150 mM) pHto7.5 Diluted to 500 mL using DEPC-ddH20 5.2 Buffer 2 1.0 g blocking reagent (proteolytic fragments of casein in powdered form) (1%) Diluted to 100 mL with Buffer 1 5.3 Buffer 3 3.03 g Tris-HCl pH 9.5 (100 mM) 1.46 gNaCl (100 mM) 2.54gMgC12(50mM) Diluted to 250 mL using DEPC-ddH20 5.4 Anti-digoxygenin-alkaline phosphatase (anti-DIG-AP), Fab fragments (from sheep) (Boehringer Mannheim) 5.5 Colour substrates (NBT and BCIP) 0.045 mL nitro blue tetrazolium salt (NBT) (75 mg/ml in 70% 38 dimethylformamide) 0.035 mL 5-bromo-4-chloro-3-indolyl phosphate toluidinium salt (BCIP) (50 mg/ml in 100% dimethylformamide) Diluted to 10 mL using Buffer 3 6. Solutions for in-situ hybridization 6.1 DEPC-ddH20 0.1% diethylpyrocarbonate (v/v) 6.2 10%SDS 10 g SDS in 100 ml DEPC-ddH20 6.3 4MNaCl 467.4 g NaCl Diluted to 2 L with DEPC-ddH20 6.4 20XSSC 350.6 g NaCl 176.4 g Na citrate Diluted to 2 L with DEPC-ddH20 6.5 1M Tris pH 7.4 242.2 gTris Dissolved in 1L with DEPC-ddH20, pH to 7.4 with HC1, and diluted up to 2L with DEPC-ddH20 6.6 1M Tris/0.1M EDTA pH 7.4 60.55 g Tris 18.61 g g E D T A Dissolved in 250 ml DEPC-ddH20, pH to 7.4 and diluted up to 500 ml with DEPC-ddH 2 0 6.7 0 .2MCaCl 2 H . l g C a C l 2 Diluted to 500 ml DEPC-ddH 2 0 6.8 0 . 5 M M g C l 2 101.7 g M g C l 2 Diluted to IL with DEPC-ddH 2 0 6.9 10 mg/ml Proteinase K 10 mg proteinase K (Gibco/BRL) Diluted to 1 ml DEPC-ddH 2 0 6.10 100% Formamide (Gibco/BRL) 6.11 1.8MNaCl(10%DexS) 10.52 gNaCl 10 g Dextran Sulfate Diluted to 100 ml DEPC-ddH 2 0 6.12 1 M D T T 0.25 g D T T (Gibco/BRL) I. 62 mlDEPC-ddH 2 0 6.13 100X Poly vinylpyrrolidone, Ficoll, B S A (PFB) (2%) 2 g poly vinylpyrrolidone (Sigma) 2 g Ficoll (Pharmacin) 5 g B S A (Gibco/BRL) Diluted to 100 mL with DEPC-ddH 2 0 6.14 Salmon sperm D N A solution (1 mg/ml) (Gibco/BRL) 6.15 Wash Buffer 500 ml 100% Formamide (50%) 10 ml 1M Tris/0.1 mM E D T A (pH 7.4) (lOmM) 150 ml 4 M N a C l (600 mM) Diluted to 1L with DEPC-ddH 2 0 6.16 Buffer 1 37.5 ml 4 M NaCl (0.15 M) 100 ml 1M Tris (pH 7.5) (0.1 M) Diluted to 1L with DEPC-ddH 2 0 6.17 Buffer 2 37.5 ml 4 M NaCl (0.15 M) 100 ml 1M Tris (pH 7.5) (0.1 M) 20 ml lamb serum (Gibco/BRL) Diluted to 1L with DEPC-ddH 2 0 6.18 Buffer 3 25 ml 4 M NaCl (0.1M) 100 ml l M T r i s (0.1 M) 100 ml 0.5M M g C l 2 (0.05 M) Dissolved in 500 ml ddH20, pH to 9.5 with NaOH, and diluted up to lLwi thDEPC-ddH 2 0 6.19 BCIP/NBT Sigma Fast tablets (Sigma) One and a half tablets were dissolved in 10 mL DEPC-ddH 20 and made up to 200 mL DEPC-ddH 2 0 6.20 0.2NHC1: 13.9ml32%HCl Diluted to 600 ml with DEPC-ddH 2 0 6.21 20 mM Tris/2 mM CaCl 2 12 ml l M T r i s 6 ml 0.2 M CaCl 2 Diluted to 600 ml with DEPC-ddH 2 0 7. Solutions for nitric oxide experiments 7.1 Murine recombinant interferon gamma (IFN-Y) (Genzyme) used at 100 U/ml 7.2 Murine recombinant tumor necrosis factor alpha (TNF-a) (Genzyme) used at 500 U/ml 7.3 Sodium nitrite (NaN0 2) (Sigma) 0.014gNaNO 2 (2mM) 100 mL medium (DMEM/F12 supplemented with 5% FUFBS and 1% gentamicin) 7.4 Greiss reagents (2 solutions): Reagent#1 1 g sulfanilamide (1%) 2.5mlH 3 P0 4 (2 .5%) 42 Diluted to 100 mL in ddH20 Reagent#2 0.1 g naphthylethylenediamine dihydrochloride (0.1%) 2.5 ml H3PO4 (2.5%) Diluted to 100 mL in ddH20 7.5 N^monomethyl-L-arginine acetate salt (L-NMMA) (Sigma) used at 10 mM 8. TNF-a Assay 8.1 Murine recombinant interferon gamma (IFN-y) (Genzyme) used at 100 U/ml 8.2 Lipopolysaccharide (LPS) from E. coli serotype 0111:B4 (Sigma) used at 1 ug/ml 9. TNF-a ELISA 9.1 Anti-mouse/rat TNF-a antibody, purified (PharMingen) 9.2 Wash Buffer: PBS with 0.05% Tween 9.3 Blocking Buffer: PBS with 2% BSA 9.4 TNF-a standard, mouse (Genzyme) 9.5 Biotin rat anti-mouse TNF-a detection antibody (PharMingen) 9.6 HRP-streptavidin (Genzyme) 9.7 TMB (3,3',5,5'-Tetramethylbenzidine) (Sigma) 43 10. Mice Three week old and 9 week old CD-I (Swiss outbred strain) and BALB/c (inbred strain) male mice were obtained from Charles River Laboratories (St.-Constant, Quebec, Canada), housed in isolation upon their arrival, and regularly monitored by personnel from the Animal Unit at the Women and Children's Research Institute. CD-I mice and BALB/c mice are established models of CVB3-induced myocarditis. 11. Virus The CG (Charles Gauntt) strain of coxsackievirus B3 was obtained from Charles Gauntt (The University of Texas Health Science Centre at San Antonio, San Antonio, Texas, U.S.A.). The RK (Reinhardt-Kandolf) strain of coxsackievirus B3 was obtained from Reinhardt-Kandolf (Max Planck Institut fuer Biochemie, Martinsried, Germany). 12. Silica Depletion 12.1 Silica powder (Sigma) was suspended in DMEM/F12 medium with 10% HIFBS, sonicated, and 0.2 mL was injected i.p. at 100-200 mg/kg per mice. 13. Solutions For Cytospin 13.1 Hemacolor™ stain set (Diagnostic Systems, Inc.): Hemacolor™ Solution 1 (Fixative): Methyl alcohol (assay 99.8%) min.) Hemacolor™ Solution 2: Phosphate buffered eosin solution (1.25 g/L) Sodium azide (0.1 g/L) Hemacolor™ Solution 3: Phosphate buffered thiazine solution (1.2 g/L) 45 METHODS 1. Cell Maintenance HeLa cells were incubated in a 5% C0 2 humidified incubator at 37°C. To sub-culture, the intact monolayer was washed with PBS and incubated with 0.25% (w/v) trypsin at 37°C for approximately 5-10 mins until the cell monolayer detached from the bottom surface of the flasks. The cells were resuspended in cell medium (DMEM/F12 supplemented with 10% FBS, and 1% gentamicin) and distributed to new tissue culture flasks (for cell maintenance) or petri dishes (for plaque assay). 2. Coxsackievirus B3(CG) Stock Preparation HeLa cell monolayers were infected at 95% confluence (24 hrs following sub-culture) with 0.1-1 pfu CVB3(CG) per cell. First, CVB3(CG) was diluted into 1 ml of medium (DMEM/F12 supplemented with 5% HJTBS and 1% gentamicin) and added to a HeLa cell monolayer in a flask that was washed once with PBS. The flask was incubated for 1 hr at 37°C in a 5% CO2 humidified incubator to allow for adsorption. Additional fresh medium was then added and the flask was incubated further until widespread cytopathology was affecting over 95% of the cell sheet, as evident by light microscopy (usually 24 hrs later). The flask was harvested by freezing at -70 °C. The flask was frozen/thawed for three cycles to lyse infected cells and allow release of virus. The cell debris was separated from virus-containing supernatant by centrifugation at 3,000 x g for 10 mins at R/T. The supernatant was removed, divided into aliquots, and stored at -70°C. A plaque assay was performed on an aliquot to determine the virus titre. 46 3. Coxsackievirus B3(CG) Titration Virus titration was performed by a standard plaque assay method (Van Houten et al., 1991). Virus was serially diluted with medium (DMEM/F12 without additional supplements) and plated in duplicate on HeLa cell monolayers at 95% confluence (24 hrs after sub-culture). The petri dishes were then incubated at 37°C in 5% CO2 humidified incubator for 1 hr to allow viral adsorption to occur. The unadsorbed (cell-free) virus was removed from the petri dishes by aspiration and a 1:1 mixture of medium (2XDMEM/F12 supplemented with 10% FflFBS and 2% gentamicin) combined with an equal volume of 1% agarose was added to each petri dish. The dishes were incubated for an additional 48 hrs at 37°C. Carnoy's Fixative was added to each dish and the dishes were incubated at R/T for 30 mins. The agarose gel above the monolayer was removed, and the bottom of the dishes was stained with Coomassie blue stain to aid in the visualization of plaques, or areas of clearing of the monolayer where cell death had occurred. The plaques were counted and the concentration of virus in the original preparation was determined. 4. CVB3 Infection of CD-I Mice Three week old and 9 week old CD-I mice (6/group) were infected with 0.2 ml containing 105 pfu CVB3 (CG) per mouse. Control mice were injected with an equal volume of PBS. Mice were sacrificed at day 3 and day 7 post-infection, and tissues (blood, heart, and pancreas) were removed. One-half of each tissue (except blood) was snap-frozen in liquid nitrogen and stored at -70°C for virus titration, and the other half was fixed in 4% paraformaldehyde (PF) for subsequent sectioning and mounting onto 47 slides for histopathological examination or in-situ hybridization. The apices of each heart were used for plaque assay, while the centre portion of the heart was fixed for slide preparation. 5. In-Situ Hybridization 5.1 Large scale pCVB3-Rl plasmid preparation using PEG precipitation A culture of JM109 bacteria carrying pGVB3-Rl was prepared by inoculating a flask containing 200 ml of 2 X YT medium and 50 mg/ml ampicillin with an aliquot of cells and incubated GVN at 37°C in a shaker-waterbath. Cells were collected by centrifugation for 10 mins at 10,000 x g using a GSA rotor in a Sorvall centrifuge at 20°C. The cell pellet was loosened and Solution I was added to stabilize the sample. Lysozyme was added to digest bacterial cell walls, and the resuspended pellet was incubated at R/T for 10 mins. Solution II was added to allow alkaline lysis and the sample was incubated on ice for 10 mins. Neutralization of the solution was brought about by addition of Solution III and the sample was incubated on ice for a further 20 mins. The sample was then centrifuged at 25,000 x g for 20 mins and the supernatant was collected and filtered with cheesecloth. Isopropanol (50%) was added to precipitate the nucleic acid and then washed with 70% ethanol. The pellet was dissolved in TE pH (8.0) and 5M LiCl (1:1) was added to precipitate large bacterial RNA strands. The mixture was centrifuged at 20,000 x g for 10 mins. The supernatant was collected and isopropanol was added to precipitate the plasmid. The pellet was washed with 70% ethanol to remove excess salt and dried. The pellet was dissolved in TE (pH 8.0), mixed with NaCl, and precipitated with 13% PEG (polyethylene glycol 8000) to remove small-48 size nucleic acids. The pellet was dissolved in TE (pH 8.0) and the DNA was extracted with phenol/chloroform followed by chloroform alone. The DNA was then precipitated with 3M NaOAc and 100% ethanol. The DNA pellet was washed twice with 70% ethanol, dried, and dissolved in 100 ul TE (pH 8.0). The plasmid concentration was determined by electrophoresis of a 1 ul sample on a 0.7% agarose gel using a known quantity of lambda DNA digested with Hind III as the standard. 5.2 RNA probe labelling by in vitro transcription Plus (+) sense and minus (-) sense RNA probes were synthesized by in vitro transcription of pCVB3-Rl, a plasmid which contains CVB3(RK) DNA, cloned into the EcoRl site of plasmid pSPT18 and flanked by SP6 and T7 RNA promoters. The SP6 promoter transcribes minus strands and the T7 promoter transcribes plus strands. The transcribed RNA was labeled with digoxygenin (DIG)-labeled UTP's in the NTP mixture. Purified plasmid (1 ug) was linearized using restriction enzymes Sal 1 (for T7 transcript) and Sma I (for SP6 transcript) at 37°C and 25°C, respectively. The labeling reaction (in vitro transcription) was carried out in a final volume of 20 ul, which contained 1 u.1 of 0.05 ug/ul linearized plasmid, 1 ul of 20 ug/ul RNase inhibitor (obtained from human placenta (Boehringer Mannheim)), 2 ul of 10X DIG-labeled NTP's (10 mmol/L ATP, CTP, GTP, and 3.5 mmol/L of DIG-labeled UTP), 2 ul of 10X transcription buffer, 2 ul of T7 or SP6 polymerase, 1 u.1 of 0.1 M DTT, and 11 u.1 of DEPC-dH20. The reaction was incubated at 37 °C for 2 hrs. The labeled RNA transcript 49 was precipitated with 3M NaOAc, washed with 70% ethanol, dried, and suspended in 20 u.1 of TE pH 8.0 for storage. 5.3 Quantification of DIG-labeled transcript Ten-fold serial dilutions of DIG-labeled control RNA (of known concentration) and the RNA transcript to be quantified were prepared. One microliter of each dilution was spotted on a Hybond nitrocellulose membrane and UV-crosslinked by placement of the membrane on a UV-transilluminator for 2 mins. The membrane was moistened by incubation with Buffer 1 in a petri dish. Buffer 2 was then added and the membrane was incubated at R/T for 10 mins on a shaker. Anti-DIG-AP conjugate (diluted 1:5000) was applied and the dish was incubated on a shaker for 10 mins. The membrane was washed in Buffer 1 and then equilibrated to pH 9.5 with Buffer 3 for 2 mins. NBT and BCIP, colour substrates were added to the membrane and colour development was allowed to proceed for 15 mins in the dark. The spot intensities (insoluble blue precipitate) were compared between the probe and control, and the concentrations of the RNA probe were deduced. 5.4 In-situ hybridization using a DIG-labelled RNA probe Tissues removed from mice were fixed in 4% PF overnight, washed in 70% ethanol, embedded in paraffin blocks, cut in 4 u.m sections and placed onto silanated (charged) glass slides. The sections were incubated O/N at 60°C to adhere tissue sections onto the slide, deparaffinized using xylene, and hydrated in graded alcohols (90%, 70%, 40% ethanol). The tissues were then incubated in 0.2 N HC1, 2 X SSC, and then 50 permeabilized by incubation in 20 mM Tris/2mM CaCk containing proteinase K (1 u,g/ml). The reaction was quenched in 0.25% acetic anhydride with 0.1M triethanolamine. The tissues then underwent dehydration in graded alcohols (70%, 90%, 100% ethanol) and were incubated at 30°C to dry the sections further. The sections were then hybridized with DIG-labeled RNA probe (sense and anti-sense), overlayed with siliconized coverslips, placed in a large, sealed humidified petri dish and incubated O/N at 42°C. The coverslips were removed and the tissues underwent post-hybridization washing by incubating overnight in a Buffer 1 containing 50% formamide, lOmM Tris/lmM EDTA, and 600 mM NaCl and then washing with 2 X SSC. The sections were equilibrated to pH 7.5, blocked with lamb serum, and then re-equilibrated to pH 7.5. Anti-DIG antibody linked to alkaline phosphatase (AP) (100u.l) was applied over the tissue sections and incubated for 1 hr at R/T while being covered with petri-dish lids to prevent drying. The tissues were washed with Buffer 1 and equilibrated to pH 9.5 to activate the AP enzyme. The tissues were incubated with NBT/BCL? colour substrate, covered in foil, and incubated O/N at R/T with gentle rotation. The slides were counterstained with eosin and examined by light microscopy for a blue-black precipitate on a pale pink background indicating a positive reaction. 6. Histology and Histopathological Interpretation Tissues removed from mice were fixed in 4% PF, washed with 70% ethanol and sent to the histology laboratory in the Department of Pathology and Laboratory Medicine of the University of British Columbia. Tissues were cut into 3 urn sections using a Leica microtome. The sections were stained with Masson's trichrome for pathological 51 assessment. Necrosis of myocytes within the heart tissue section is evidenced by areas stained with a pale pink colour, containing cells with degenerated (or lack of any) nuclei. Damage is also evidenced by myocardial scarring which is represented by areas of fibrosis, or connective tissue deposition within the myocardial tissue, in which collagen, cartilage, and basic granules stain purple-blue. 7. Virus Titration of Mice Tissue Tissues (except blood) from mice were snap-frozen by placement in liquid nitirogen immediately following removal and stored at -70°C. Tissues were homogenized in 1 ml of medium (DMEM/F12 without supplements) using a pellet pestle homogenizer (KONTES). The homogenate was centrifuged at 12,000 x g to remove tissue debris, and the supernatant was removed and stored at -70°C for virus titration. Serum was obtained from clotted blood samples and stored at -70°C for virus titration. 8. In- Vitro Inactivation of CVB3 by Peritoneal Cells Peritoneal cells were removed from mice by peritoneal lavage using ice-cold medium (DMEM/F12 supplemented with HEPES buffer, 10 % HUBS, 1% gentamicin). Cells were pooled and distributed into tissue culture tubes at 1 X 106 per tube. These were incubated for 2 hrs at 37°C in a 5% C0 2 humidified chamber to allow cells to adhere to bottom of tube. The tubes were washed once with medium to remove non-adhered cells (which were counted and found to comprise approximately 5-10% of the total number of cells), and the remaining cells were infected with CVB3(CG) at a M.O.I, of 5 and incubated further for a period of 6 days at 37°C. Triplicate samples were 52 removed every 48 hrs and stored at -70°C. Tubes were frozen/thawed and assayed for total virus infectivity by plaque assay on HeLa cell monolayers. 9. Quantification of Nitric Oxide Production To determine levels of nitric oxide (NO), a microassay was used, which measured levels of nitrite (NO2), a stable oxidative product of N O . Mouse peritoneal cells were collected by peritoneal lavage using ice-cold medium (DMEM/F12 supplemented with HEPES buffer, 10% HTJFBS, and 1% gentamicin), pooled together, and adjusted to 106 cells/ml. Aliquots of 0.3 ml cell suspension were added to wells of a 96-well flat-bottom microtiter plate (Costar) and the plate was incubated for 2 hrs at 37°C in a 5% CO2 humidified chamber to allow macrophage cells to adhere. The wells were washed with medium, and IFN-Y (100 U/ml) was added to appropriate wells to serve as a priming agent, and the plate was incubated for an additional 2 hrs. T N F - a (500 U/ml) was then added to appropriate wells to serve as the triggering agent; in other wells, CVB3(CG) was added at 5 pfu/cell without prior priming with IFN-y, and the plate was incubated for up to 24 hrs. All samples were done in triplicate. Supernatants from wells (50 u.1) were transferred to wells of a new 96-well flat-bottom microtiter plate. Serial dilutions of NaN02 solutions (125 uM to 1 uM) were added to wells to serve as standards. To the samples and standards, 50 u.1 of each Greiss reagent solution was added (sulfanamide solution was added before naphthylethylenediamine dihydrochloride solution for optimal results). The plate was incubated at R/T for 10 mins, and the absorbance of the samples and standards was measured at 550 nm using a microtiter plate reader (BioRad Model 3550). The dilution absorbance values were plotted against NaN02 concentrations and 53 the resulting standard curve was used to determine the amount of NOV (in uM) in the samples. 10. CVB3 Infection of CD-I Mice In the Presence of a NOS Inhibitor N^monomethyl-L-arginine acetate salt (L-NMMA), a NOS inhibitor, was supplied in the drinking water of 3 week old and 9 week old CD-I mice at 10 mM (30 ml) supplied each day for 3 days: 24 hours prior to virus infection, on the day of infection, and one day after infection. CVB3(CG) was injected at 105 pfu in 0.2 ml per mouse. Control mice were injected with virus only and did not receive L-NMMA. Three mice/group were used. All mice were sacrificed at day 2 post-infection, and tissues (blood, heart, and pancreas) were removed for virus titration. 11. TNF-a Production In Vitro To induce the in vitro production of TNF-a by peritoneal cells, the technique was virtually identical as for the induction of NO (see 9). Mouse peritoneal cells were isolated from the mouse peritoneum by lavage using ice-cold medium (DMEM/F12 supplemented with FfJEPES buffer, 10% FBS, and 1% gentamicin) and pooled. Aliquots of 5 x 105 to 1 x 106 cells were added to each well of a 96-well flat-bottom microtiter plate and macrophages were allowed to adhere to the well-bottom by incubating the plate for 2 hrs at 37 °C in a 5% CO2 humidified chamber. The wells were washed with medium to remove non-adherent cell populations, then IFN-y (100 U/ml) was added to prime the macrophages, and the plate was incubated for an additional 2 hrs. Lipopolysaccharide (LPS) was then added at 1 ug/ml to wells, and the plate was 54 incubated for up to 24 hrs. Supernatants were removed and stored at 4 °C, until analysis using a TNF-a ELISA. 12. TNF-a ELISA Supernatant TNF-a concentrations were measured using a sandwich ELISA. The wells of a 96-well microtiter plate were coated overnight with 8 ug/ml of monoclonal anti-mouse/rat TNF-a antibody (capture antibody) and incubated at 4 °C. The plate wells were washed three times with Wash Buffer, blocked with Blocking Solution and incubated for 1 hr at 37 °C. The plate was washed again four times with Wash Buffer. Dilutions of mouse TNF-a standards, ranging from 0 to 2000 pg/ml were added to wells, along with supernatant samples diluted 1:5 and 1:20, and the plate was incubated for 1 hr at 37 °C. The plate was washed five times with Wash Buffer, and biotin rat anti-mouse TNF-a detection antibody was added at 1 u,g/ml and incubated for 1 hr at 37 °C. The plate was washed four times with Wash Buffer, and 100 ul of horseradish peroxidase (FTJRP)-streptavidin was added to each well and incubated for 15 mins at 37 °C. The plate was washed four times with Wash Buffer and 100 ul of the substrate, TMB (3,3',5,5-tetramethylbenzidine), was warmed to R/T before it was added to individual wells. The plate was read immediately at 370 nm using a microtiter plate reader (SpectraMax 190). 13. Silica Depletion of Peritoneal Cells in BALB/c Mice A silica solution was made using DMEM/F12 medium containing 10% FflFBS, sonicated for 2 mins, and stored OVN at 4°C. Just prior to use, the silica solution was re-55 sonicated for 1 min and 0.2 ml was injected i.p. into BALB/c mice at 100-200 mg/kg. Silica was injected twice per week for 2 weeks, prior to infection (i.p.) with 105 pfu of CVB3(RK) in 0.2 ml. In total, twenty-four 5 week old male BALB/c mice were used in the experiment, and treated according to the following protocol: 6 mice received silica and virus, 6 mice received silica and PBS, 6 mice received no silica and virus, 6 mice received no silica and PBS. Mice were sacrificed on day 3 and day 7 post-infection (3 mice from each group per day), and tissues (heart and pancreas) were removed for virus titration. 14. Calculation of Macrophage Content of Peritoneal Cell Populations Peritoneal cells were analysed for purity of the macrophage cell population by microscopic examination of cytospin slides. A protein cushion was prepared by adding 100 u.1 of medium (DMEM/F12 supplemented with HEPES buffer, 10% HLFBS, and 1% gentamicin) and spun at 1000 rpm for 5 mins at high acceleration in a centrifuge (CytoSpin 2/Shandon). Freshly isolated peritoneal cells were adjusted to a concentration of 5 X 105 cells/ml and an aliquot of 50-100 u.1 was added on top of the cushion and centrifuged at 650 RPM for 3 minutes at low acceleration. The resulting cell smear on the microscope slide was allowed to air dry for 30 mins to 1 hr. The slide was then stained using a rapid differential Hemacolor™ stain set, by dipping 5 times for a second each in Solution 1 (Fixative), then 3-5 times for a second each in two buffered staining solutions, Solution 2 and Solution 3. The slide was rinsed with deionized or distilled water, blotted, and examined by oil immersion microscopy. Adherent macrophage populations were counted as being >95% pure. 56 15. Statistical Analysis Results are expressed as mean ± SEM for the indicated number of determinations. Comparison between groups was made employing a Student's 7-test. Statistical significance was assessed at the 95% confidence level (p<0.05). 57 RESULTS AND DISCUSSION 1. AGE-DEPENDENT RESISTANCE TO CVB3 Classical studies on the development of age-dependent resistance of mice to infection by CVB3 showed that it follows a course similar to that found for other virus-host interactions (Johnson, 1964; Hirsch, Zisman et al., 1970; Zisman, Wheelock et al., 1971). Specifically, while CVB3 infections in newborn mice are lethal, mice a few weeks of age survive, with susceptibility further decreasing as the mice age, until immune senescence is reached, when susceptibility to infection is re-established. 1.1. CVB3 Infection of Young and Adult CD-I Mice To confirm these previous findings on the susceptibility of young mice to CVB3 infection, the course of infection was compared between 3 week old and 9 week old CD-1 mice. Instead of examining viral LDso's, as was carried out in the original experiments, viral titres in specific target tissue were determined, and the amount of viral genome present was assessed by in-situ hybridization. This was correlated with the degree of tissue damage seen by histological examination of the tissue. Four groups of CD-I male mice (6 mice/group) were used in the experiment and treated according to the following protocols: Group 1: 3 week old CD-I mice injected i.p. with 105 CVB3(CG) pfu/mouse. Group 2: 3 week old CD-I mice injected i.p. with PBS (control). Group 3: 9 week old CD-I mice injected i.p. with 105 CVB3(CG) pfu/mouse. Group 4: 9 week old CD-I mice injected i.p. with PBS (control). 58 The time of infection constituted day 0 of infection. Three mice per group were sacrificed on day 3 p.i. (timepoint when virus titres peak in pancreas and heart, and pancreatic histopathology is apparent) and day 7 p.i. (timepoint when cardiac histopathology is apparent). Upon sacrifice, a number of tissues including heart, pancreas, and serum were removed for analysis. A plaque assay was performed on tissue homogenates to determine the titre of infectious virus present. The degree of histopathology was examined microscopically on sections of tissue stained with Masson's trichrome and in-situ hybridization was performed to determine the levels of virus genome present within the tissues. The results of the plaque assay (Fig. 6) indicated the presence of higher virus titres in the young CD-I mice as compared to the adult mice. In the heart tissue (Fig. 6a), significantly more virus was recovered in the young mice than in the adult mice (approximately one log-fold more) at both days of analysis (day 3 and day 7 p.i.). In the pancreas (Fig. 6b), however, a significant difference could be seen only later in the infection, at day 7 p.i., when adult mice had substantially cleared virus, while the younger mice still had high titres of virus present. At day 3, although the virus titres appear similar in both age groups, it is likely that the levels of virus at this time of infection were too high for significant differences to be noted due to maximum virus replication in the extremely susceptible pancreatic tissue. In serum, CVB3(CG) concentrations peaked at different days (Fig. 6c) in the two age groups of mice. In the young animals, virus titres were shown to peak early in infection, ie., at day 2 p.i., while in the older animals, maximum virus was recovered later, at day 3 p.i. A significant difference between the young and adult mice is seen at 59 day 2 and day 4 p.i. At day 3, although there is not a significant difference between the young and adult mice, it is at this point that the levels of virus are declining in the younger animals and peaking in the older animals. Heart H Q . U l I -> X Day 3 Day 7 Fig. 6. CVB3(CG) titres in heart, pancreas, and serum of young and adult CD-I mice. A., Heart tissue at day 3 and day 7 p.i., B., Pancreas tissue at day 3 and day 7 p.i., C , Serum at day 1, day 2, day 3, and day. 4 p.i. (Differences between groups were considered significant where *p<0.05). Serum 60 The differences in the timecourse of viremia between the young and adult mice, such that the young mice have higher levels of virus in circulation early in infection relative to the older mice, demonstrates an inability of the younger animals to control CVB3 during the early stage of infection compared to the older animals. During the early phases of infection, innate immune responses play a prominent role in limiting most infections; adaptive immunity, i.e., antibodies and CTL's, do not become significant until a few days later and are responsible for clearing the infection. The plaque assay results, which demonstrated a relatively greater susceptibility of young CD-I mice to CVB3(CG) infection, were corroborated qualitatively by microscopic examination of tissue histopathology (Fig. 7). It can be seen that young CD-1 mice (Fig. 7B) had relatively more damage to the myocardium than older animals (Fig. 7C), as indicated by the presence of more diffuse pale-staining, fibrotic areas within the myocardium, which becomes apparent by day 7 p.i. Damage to the pancreas, on the other hand, is apparent as early as day 3 p.i., and is seen to be greater in young mice (Fig. 7E) relative to adult mice (Fig. 7F). Damage to the pancreas is represented by widespread necrosis of the acinar cells of the exocrine tissue, while the endocrine pancreas, comprising the islets of Langerhans, is left intact. Localization of virus genome within tissues by in-situ hybridization allowed for further assessment of the relative susceptibility of CD-I mice (Fig. 8 and 9). As can be seen in Fig. 8, relatively greater levels of virus was present within the myocardium of young CD-I mice (Fig. 8B) than adult mice (Fig. 8C) at day 3. By day 7, little virus genome could be recovered from either young (Fig. 8D) or adult (Fig. 8E) mice, although somewhat more virus was still detected in the younger animals, as seen in the periphery 61 Fig. 7. Histopathological analysis of CVB3(CG) infection of young and adult CD-I mice. Three week old and 9 week old CD-I mice were infected with 105 pfu CVB3(CG)/mouse and tissues including heart and pancreas were removed at day 3 and day 7 p.i., fixed in 4% PF, and stained with Masson's trichrome for microscopic examination. Damage to the heart, evident at day 7 p.i, was diffuse and more prominent in younger mice, B, relative to the older mice, C, in comparison to the non-infected control heart, A. In the pancreas at day 3 p.i., damage in the young mice, E, was somewhat greater relative to adult mice, F, when compared to the control pancreas, D. 62 Fig. 8. In-situ hybridization of CVB3(CG) RNA in heart tissue from young and adult CD-I mice. Three week old and 9 week old CD-I mice were infected with 105 pfu CVB3(CG)/mouse and heart tissue was removed at day 3 and day 7 p.i., fixed in 4 % PF, and analysed for the presence of CVB3 genome by the technique of in-situ hybridization. Control, non-infected heart is indicated in A. At day 3, significantly greater levels of virus were found to be localized in hearts of young animals, B, compared to adult animals, C. By day 7, most of the virus was cleared from the hearts of both age groups, but young mice had somewhat more virus left (in the periphery) of the tissue, D, compared to adult mice, E, from which, virus was not detectable. 63 Fig. 9. In-situ hybridization of CVB3(CG) RNA in pancreas from young and adult CD-I mice. As described in the legend of Fig. 8, 3 week old and 9 week old CD-I mice were infected with 105 pfu CVB3(CG)/mouse and pancreatic tissue was removed at day 3 and day 7 p.i., fixed in 4% PF, and analysed for the presence of CVB3 genome by the technique of in-situ hybridization. Control, non-infected pancreas is indicated in A. At day 3, similar (high) levels of virus were found to be localized in the pancreas from both young, B, and adult mice, C. By day 7, most of the virus had been cleared from the tissue in both age groups, with somewhat greater levels of virus still present in the young mice, D, compared to adult mice, E. 64 of the tissue (Fig. 8D). In the pancreas (Fig. 9), however, similar quantities of virus were detected in both the young (Fig. 9B) and adult (Fig. 9C) mice at day 3. By day 7, the levels of virus within the pancreas decreased in both the young (Fig. 9D) and adult (Fig. 9E) mice, but the younger animals still had somewhat more virus within the tissue. It is evident from both Fig. 8 and 9 that virus starts to become cleared from tissues by day 7, concurrent with the time of the development of acquired immune responses. 65 2. ROLE OF MACROPHAGES IN CVB3 INFECTION The goal of this research project was to focus on the early responses to CVB3 infection and to identify the roles played by the various factors involved in innate immunity. Although a single mechanism is unlikely to account for age-dependent resistance, the role of macrophages, was examined for its contribution, since these cells are believed to play a critical role in limiting many viruses early in the infectious process (see Introduction). Therefore, the maturation of macrophage function during development from very young animals to adult animals may constitute an important contribution to age-dependent resistance to CVB3 and other infections (Stevens and Cook, 1971; Zisman, Wheelock et al., 1971). 2.1 Depletion of Peritoneal Cells With Subsequent CVB3 Infection Several previous observations have suggested that macrophages play an important role in limiting the spread of systemic virus infections. For example, injection of silica particles, which are selectively taken up by macrophages and impair their function (Allison, Harington et al., 1966) was found many years ago to increase the mortality of adult mice infected with viruses such as herpes simplex virus (Zisman, Hirsch et al., 1970). Later studies have employed many other substances to deplete macrophage populations in vivo, including anti-Mac-2 monoclonal antibodies (Baek and Yoon, 1990; Hirasawa, Tsutsui et al., 1996), and the administration of liposomes (Van Rooijen, 1989). Preliminary work in our laboratory examined the effect of the impairment of macrophage function on the course of CVB3 infection. In this experiment, 5 week old BALB/c mice were injected with silica intraperitoneally twice per week for two weeks 66 prior to injection with CVB3(RK). Heart and pancreas were removed at day 3 and day 7 p.i. for analysis by plaque assay to determine viral titres (Fig. 10). While impairment of macrophage function did not result in increased mortality of the mice, the CVB3 titres were significantly increased in both the pancreas (Fig. 10A) and heart tissue (Fig. 10B) by approximately 10-fold. While a different mouse strain and virus strain were used, this experiment exemplifies the importance of macrophages in controlling CVB3 infections in general. Pancreas PBS (-silica) H CVB3(RK) (-silica) PBS (+silica) • CVB3(RK) (+silica) Fig. 10. Effects of silica on CVB3(RK) infection in BALB/c mice. BALB/c mice at 5 weeks age were pretreated with silica or PBS (control) prior to injection with CVB3(RK) or PBS (control). A., Virus titre in pancreas tissue at day 3 p.i., B., Virus titre in heart tissue at day 7 p.i. (Differences between groups containing CVB3(RK) with and without silica were considered significant, p<0.05). (D U> I - Q. M u> 3 O » - T -> X 4 0 3 0 a> n n 3 © 20 1 0 0 4) O) •b .3 in *> 3 O I - T -> >< D a y 3 Heart Ml D a y 7 B 67 3. COMPARISON OF SPECIFIC MACROPHAGE FUNCTIONS IN YOUNG AND ADULT MICE It is apparent from the previous results that macrophages play a role in limiting CVB3 infection. Next, we wanted to examine specific macrophage functions that may be involved in resistance to CVB3 infection as the mice mature. A number of macrophage functions have been described that may potentially counteract viruses. Both intrinsic and extrinsic antiviral mechanisms of macrophages have been described (see Introduction) (Baskin, Ellermann-Eriksen et al., 1997). Although several mechanisms have been indicated to be involved in macrophage antiviral functions, their relative importance in different virus infections, such as coxsackievirus infections, is not well understood. 3.1 Inactivation of CVB3 By Peritoneal Cells Isolated from Young and Adult CD-I Mice The ability of peritoneal cells to inactivate CVB3(CG) infection was compared between young and adult CD-I mice. Briefly, cultures of peritoneal cells from 3 week old and 9 week old animals were infected with CVB3(CG) at a M.O.I, of 5, and incubated at 37°C. At 48 hour intervals, up to a period of 6 days, samples were harvested by freezing at -70°C. The samples were assayed for total virus infectivity (intracellular and extracellular virus) by plaque assay in HeLa cell monolayers. As illustrated in Fig. 11, infectivity decreased more rapidly in cultures of adult peritoneal macrophages than cultures of young peritoneal macrophages, indicating a greater capability of the macrophages from the adult mice to contain CVB3. During the first 2 days following 68 infection, peritoneal cells from the adult mice reduced the virus titre somewhat more rapidly than cells from the young mice, but this was not considered significant. However, at day 4 and day 6 p.i., the cells from adult mice showed a significantly greater capacity to eliminate virus. These results indicate that adult peritoneal cells are more adept in containing CVB3 infection than young cells, which may partially account for the increased resistance of adult hosts to CVB3 infections. - ^ 3 w k old - * - 9 w k old 0 2 4 6 Time After Virus Infection (days) Fig. 11. Timecourse of inactivation of CVB3(CG) by peritoneal cell cultures from young and adult CD - I mice. The rate of inactivation of CVB3 was compared between 3 week old and 9 week old CD-I mice by incubating virus at a M.O.I, of 5 with lxlO 6 peritoneal cells/tube. Culture tubes were incubated at 37°C and samples were harvested at day 0, day 2, day 4, and day 6 p.i. Graph represents results from one of two similar experiments. (Differences between 3 wk old and 9 wk old mice were significant at day 4 and day 6, p<0.05). 3.2 The Role of Macrophage Nitric Oxide in Controlling CVB3 Infection The contribution of nitric oxide as a mediator of macrophage-induced resistance against various pathogens, such as intracellular bacteria, protozoa, fungi, is well 69 documented. The role of NO in viral infections, however, has only recently received attention. Initial studies demonstrating the inhibition of herpes virus type 1, ectromelia virus, and vaccinia virus by IFN-y-induced NO in mouse macrophages have paved the way for research into other viruses, such as HIV, flaviviruses, influenza viruses, Epstein-Barr virus, and more recently the coxsackieviruses. Recent studies on NO inhibition of CVB3 have attempted to define the mechanism of antiviral activity in vitro (Zaragoza, Ocampo et al., 1997), or have suggested a detrimental effect of NO on CVB3-induced myocarditis (Mikami, Kawashima et al., 1996). No previous studies have been directed towards understanding the role of NO in age-dependent susceptibility to CVB3 infection. 3.2.1 Inhibition of Nitric Oxide Synthase In Vivo With Subsequent CVB3 Infection To analyze the age-dependent antiviral role of nitric oxide in protection against CVB3 in a CD1 mouse model, a substrate analogue (N -^monomethyl-L-arginine, L-NMMA) for the nitric oxide synthase (NOS) enzyme was administered in vivo to diminish the activity of NOS induced by viral infection. Briefly, CD1 mice (young and adult) containing 3 mice/group were given the following treatment protocols: Group 1: 3 week old CD-I mice orally administered L-NMMA and injected i.p.with 105 CVB3(CG) pfu/mouse. Group 2: 3 week old CD-I mice injected i.p. with 105 CVB3(CG) pfu/mouse. Group 3: 9 week old CD-I mice orally administered L-NMMA and injected i.p 70 with 105 CVB3(CG) pfu/mouse. Group 4: 9 week old CD-I mice injected i.p. with 105 CVB3(CG) pfu/mouse. At day 2 p.i., mice were sacrificed and whole tissue specimens (heart and pancreas), and serum, were removed for analysis by plaque assay (Fig. 12). Fig. 12. Effect of inhibition of NOS on CVB3(CG) infection in young and adult CD-I mice. Analysis of tissue at day 2 p.i. A., Virus titres in heart tissue, B., Virus titres in pancreas tissue, C , Virus titres in serum. (Differences between groups were considered significant where *p<0.05). Graphs represent results from one of two similar experiments. 71 As can be seen in Fig. 12 (A, B, C), a more severe viral infection occurred when NOS was inhibited in the CD-I mice. The effects of NOS inhibition were most notable in the heart (Fig. 12A), as approximately 50- and 100-fold more virus was recovered from young and adult heart tissue, respectively, relative to mice injected with virus only. Thus, within each age group, the mice demonstrated a significantly worse infection upon elimination of NOS, as both 3 week old and 9 week old mice were affected. Between the two age groups, moreover, the young mice suffered greater than the adults, as approximately 5-fold more virus was recovered in the heart upon L-NMMA treatment compared to L-NMMA treated adults. In the pancreas (Fig. 12B), high titres of virus were recovered from both young and adult mice upon NOS inhibition, but the difference was not found to be statistically significant with the sample size used. Similarly, between the age groups, more virus was detected in the pancreas from young mice compared to adult pancreas when both were inhibited of NOS, but this was not significant. Overall, the failure to see any statistically significant increase in virus titres either, within each age group, or between the age groups is likely due to the extreme susceptibility of pancreatic tissue for CVB3 infection. In essence, virus titres are already extremely high in the absence of NOS inhibition, and if NOS does become inhibited, then there can only be so much more infection of available pancreatic cells. There was no significant effect on virus concentration within the serum (Fig. 12C) of young mice when NOS was inhibited, but there was an effect seen in the adults, where more than 5-fold more virus was present. But, it is important to note that these serum samples were removed at day 2 p.i., and according to the trend of CVB3 viremia (see Fig. 72 6C), young mice follow a different timecourse of infection in serum compared to adult mice. According to Fig. 6C, at day 2 p.i., virus titres peak in serum of young mice, while the titres peak at day 3 p.i. in adult mice. Therefore, in this experiment a significant difference in virus titres in serum of young mice may not have been detectable when NOS was inhibited at day 2 because the virus levels had already peaked by this time, whereas in the older mice, a significant difference was seen when NOS was inhibited because the virus titres were still relatively low. It is important to note that although L-NMMA was initially identified as an inhibitor of the arginine-dependent cytotoxic response of murine macrophages (Hibbs, Vavrin et al., 1987), it is known that its activity in vivo affects not only NOSH, but the other two isoform types as well (NOSI and NOSIII) (Griffith and Gross, 1996). Thus, when using L-NMMA in vivo, it is impossible to know the contribution of the inhibition of NOSH specifically on CVB3 susceptibility. Recently however, the protective role of NOSH during CVB3 infection was confirmed through the use of transgenic mice which were genetically deficient in the type II NOS locus (Zaragoza, Ocampo et al., 1998). Overall, both young and adult CD-I mice suffered a more serious CVB3 infection upon NOS inhibition, but the younger mice suffered to a greater extent, as seen significantly in the heart tissue. The dramatic effects of NOS inhibition seen in the heart compared to the other tissues analysed may be likely due to the fact that heart tissue contains relatively more NO-producing cell types, such as endothelial cells, smooth muscle cells, and cardiomyocytes than the other tissues. The presence of other cells types, such as macrophages and neutrophils during inflammation (triggered by viral infections), which also generate NO, may also contribute to the effects seen in the tissue, 73 especially i f high numbers of these cells are present. Thus, the presence of a high number of NO-producing cells inherent to the tissue, as well as non-inherent inflammatory cells (the number of which depends on accessibility to the tissue) would greatly affect the tissue of NOS inhibition. 3.2.2 Intrinsic Capability of Peritoneal Cells From Young and Adult CD-I Mice To Produce NO In Vitro Concurrent with the previous experiment examining the importance of NO production in protecting young and adult mice from CVB3 infection, the intrinsic ability of peritoneal cells from each age group of mice to produce NO in vitro was examined. In addition, the direct effect of CVB3 on NO generation in vitro was examined. It is known that macrophage infection with different viruses can result in either increased or decreased N O production, in response to a trigger, depending on the virus in question and the triggering signal (Lyon and Hinshaw, 1993; Adler, Freeh et al., 1994; Kreil and Eibl, 1995) . Studies with CVB3 have demonstrated an increase in iNOS protein expression in macrophage infiltrates from hearts of CVB3-infected mice (Lowenstein, Hi l l et al., 1996) , but in an in vivo situation, various factors (or signals) may be at play in the generation of NO, and the direct effect of virus is not known. The production of NO by peritoneal cells was measured indirectly, by the production of nitrite (NO2"), a stable end product generated by the rapid oxidation of NO. Briefly, peritoneal cells, isolated from young and adult CD-I mice were seeded into 96-well plates, adhered for 2 hours, and incubated with the following substances (triplicate samples): 74 1) IFN-y 2) IFN-y + TNF-a 3) CVB3(CG) After 24 hours, the supernatants from the wells were collected and analysed for levels of nitrite by the Greiss assay (see Methods). Fig. 13. NO production by peritoneal cells from young and adult CD-I mice. Peritoneal cells from 3 week old and 9 week old CD-I mice were incubated for 2 hrs to allow for cell adherence, then incubated with either CVB3(CG) at a M.O.I, of 5, or with IFN-y for 2 hrs plus TNF-a. Control samples contained cells only. Supernatants were collected at 24 hrs and assayed for nitrite levels (Differences between 3 wk old and 9 wk old mice were significant when NO was induced by IFN-y or IFN-y plus TNF-a, p<0.05). Graph represents results from one of three similar experiments. 75 As can be seen from Fig. 13, CVB3 did not induce any significant levels of NO in vitro, from either young or adult CD-I mice peritoneal cells. This suggests that any generation of NO during a CVB3 infection occurs indirectly by intermediary signals, i.e., cytokines such as IFN-y and TNF-a. Other viruses, such as herpes simplex, which are known to be permissive for macrophages, have demonstrated the ability to directly generate NO production in vitro (Baskin, Ellermann-Eriksen et al., 1997). The striking and unexpected result was that the younger CD-I mice produced significantly greater concentrations of NO than the adult mice (Fig. 13). Induction by IFN-y plus TNF-a, synergistically produced greater levels of NO than LFN-y alone, which is well-documented in the literature. Studies on age-related NO production have compared macrophages from adult and senescent hosts (Alvarez, Machado et al., 1996; Chen, Pace et al., 1996); no studies (to our knowledge) had made any comparisons between very young and adult hosts when we initiated this project. The reduced ability of adult mice to produce NO may be due to several reasons. It is possible that adult macrophages possess less receptors on their surface for NO-inducing cytokines (IFN-y or TNF-a) than young mice. Another possibility is that macrophages from older animals may have downregulated signalling pathways downstream from ligand-receptor interaction. For example, adult mice may produce factors that affect transcriptional control at the iNOS promoter, which contains binding sites for N F - K B and interferon-regulatory factor-1 (IRF-1) and other additional IFN-y response sites (Xie, Whisnant et al., 1993; Martin, Nathan et al., 1994). In aged human macrophages stimulated by LPS, it was shown that an increase in intracellular levels of cAMP and loss of protein kinase C translocation from the cytoplasm to plasma 76 membrane resulted in inhibition of IL-1 production (which also induces iNOS) (McLachlan, Serkin et al., 1995). In murine systems, however, it is not known how aging might affect macrophage signal transduction mechanisms. Whatever the reason, it is evident that the ability to generate NO decreases as the mice become older. It is possible that as the mice mature and become more immunocompetent, the reliance on such a crude (simple) molecule as NO (which is known to be the simplest bioactive molecule produced by mammalian cells (Xie, Cho et al., 1992)) to eliminate pathogens is diminished as the animals can instead, rely on more sophisticated mechanisms of immunity. In addition, despite attempts of many investigators, NO has not been shown to be produced by human macrophages, at least in levels generated by murine macrophages. This may serve an evolutionary purpose as well: the availability of more efficient and elaborate mechanisms for immune defense in humans may supercede the requirement for NO by macrophages. Peritoneal macrophages from young animals are therefore intrinsically more capable of producing NO when treated with exogenous IFN-y and TNF-a. Thus, it is possible that younger mice may rely on NO as a major defense molecule to a greater extent than older mice. If this were true, then debilitating the NO system in younger mice would be more detrimental than in older animals. This effect was generally seen in the in vivo experiment using L-NMMA, especially in the heart tissue (Fig. 12A). Of course, the contribution of (macrophage) NOSH may not be solely be responsible for the observed CVB3 age-dependent resistance in the in vivo NOS inhibition experiment, as other NOS isoforms were also inhibited. The inhibition of the other isoforms aside from NOSH probaby explains why adult mice were also quite affected by NOS inhibition, as seen by 77 20-fold more virus in heart of L-NMMA-treated mice compared to control mice (Fig. 12 A). 3.3 Intrinsic Capability of Peritoneal Cells From Young and Adult CD-I Mice To Produce TNF-a In Vitro TNF-a is another major component of the innate immune response and is produced by macrophages and T cells. In addition to being an inducer of NO synthesis, TNF-a is involved in a number of processes, such as activating NK cells, neutrophils, macrophage- and T cell-dependent effector function, induction of class I and class II expression, and upregulation of IL-1, IL-6, and IFN-y expression (Peters, 1996). Intrinsic differences in the amounts of TNF-a produced by macrophages from young and adult mice might, therefore, significantly affect resistance to viral replication. Possible differences in TNF-a production by peritoneal macrophages from young and adult mice was compared using IFN-y and LPS as inducing agents. Briefly, peritoneal cells were removed from 3 week old and 9 week old CD-I mice, seeded into a 96-well plate and incubated with IFN-y and LPS over 24 hours. Cell supernatants were collected and assayed for TNF-a by a sandwich ELIS A. As can be seen in Fig. 14, the adult CD-I mice peritoneal macrophages produced greater levels of TNF-a than the young mice. Thus, the reduced ability of the young CD-1 mice to produce TNF-a may be partially responsible for the increased susceptibility to CVB3 infection. As mentioned earlier, TNF-a is an important inducer of NO synthesis within macrophages. In Fig. 13, it was found that younger CD-I mice produced greater levels of NO than the adult mice (Fig. 13). This higher NO productive capability in the young mice cannot be explained by the production of higher levels of endogenous TNF-a by macrophages, as seen from the results of this experiment. Thus, other (intracellular) events are probably involved, ie. signal transduction. 3 wk old 9 wk old Fig. 14. TNF-a production by peritoneal cells from young and adult CD-I mice. Adherent peritoneal cells (1 x 106 per well) from 3 wk old and 9 wk old CD-I mice were incubated with IFN-y plus LPS for 24 hrs and the TNF-a levels in the cell culture fluid was determined by an ELISA (p<0.05). Graph represents results from one of two similar experiments. 4. STRAIN VARIATION: A COMPARISON WITH BALB/C MICE Prior to analysing the age-dependence of CVB3 susceptibility in the CD-I strain of mice (Fig. 6-9), we had initially performed several experiments in the B A L B / c mouse strain. On inoculation of CVB3(CG) into young and adult B A L B / c mice, we observed extreme susceptibility, not only in the young mice, but in the adult mice as well, such that all of the mice had died by day 6 post-infection. This inability to study CVB3(CG) 79 infection beyond day 6 p.i. in these animals, did not allow for the analysis of heart pathology (which becomes apparent at day 7 p.i.). Therefore, the more resistant CD-I mouse strain was chosen for the majority of experiments carried out. As susceptibility to virus infections depends on a number of host factors, referred to generally as 'genetic susceptibility', we were interested in determining whether the extreme susceptibility of the BALB/c strain was reflected in the functions and capabilities of their macrophages in vitro. Several experiments conducted in the CD-I mice were therefore repeated with the BALB/c strain. 4.1 Inactivation of CVB3(CG) By Peritoneal Cells Isolated From Young and Adult BALB/c Mice In contrast to the results in the CD-I mice (Fig. 11), a much more notable difference was found in the ability of peritoneal cells from 3 week old BALB/c mice to inactivate CVB3 than cells from 9 week old animals (Fig. 15). In addition, the young peritoneal cells appeared to be permissive to viral replication, indicated by the increase of CVB3 titres at around day 4 p.i. Adult BALB/c mice peritoneal macrophages, on the other hand, demonstrated a steady inactivation throughout the course of the experiment. By day 2, this was reflected in 5-fold higher titres remaining in the cultures of peritoneal macrophages from younger animals and by day 6, the difference had increased to 100-fold. This sharp difference in macrophage inactivation of virus may play a factor in the severe susceptibility seen in the young BALB/c mice relative to that found for the CD-I mouse strain, but does not explain the high mortality found in the adult BALB/c mice. 80 - • - 3 wk old - • - 9 wk old Time After Virus Infection (days) Fig. 15. Timecourse of inactivation of CVB3(CG) by peritoneal cell cultures from young and adult BALB/c mice. The rate of inactivation of CVB3 was compared between 3 week old and 9 week old BALB/c mice by incubating virus at a M.O.I, of 5 with lxlO 6 peritoneal cells/tube. Culture tubes were incubated at 37°C and samples were harvested at day 0, day 2, day 4, and day 6 p.i. Graph represents results from one of two similar experiments. (Differences between 3 wk old and 9 wk old mice were significant at day 2, day 4, and day 6, p<0.05). 4.2 Intrinsic Capability of Peritoneal Cells From Young and Adult BALB/c Mice To Produce NO In Vitro The trend in NO generation in vitro by BALB/c mice peritoneal cells paralleled that seen in the CD-I mice (Fig. 16). The young mice produced significantly greater levels of NO than the older mice, upon induction by IFN-y or IFN-y plus TNF-a. The ability of young animals to produce greater concentrations of NO than adult animals under optimal conditions of induction is therefore seen in both mouse strains examined. Moreover, a marked difference in NO induction, whether a decrease (which may be associated with less viral inhibition) or an increase (which may be associated with 81 'shock'), therefore does not appear to explain the increased susceptibility of B A L B / c mice to CVB3 infection. The absolute amounts of NO produced by B A L B / c mice was much higher (see Fig. 16 and compare with Fig. 13). Even if inter-experiment variation is considered, the NO induced by IFN-y alone in young B A L B / c mice is significantly higher than in CD-I mice, and is comparable to the NO levels induced by IFN-y plus TNF-a. + TNF-a 3 wk old 9 wk old Fig. 16. NO production by peritoneal cells from young and adult BALB/c mice. Cells from 3 week old and 9 week old BALB/c mice were incubated for 2 hrs to allow for cell adherence, then primed by incubation with IFN-y for 2 hrs, with or without subsequent incubation with TNF-a. Control samples contained cells only. Supernatants were collected at 24 hrs and assayed for nitrite levels (Differences between 3 wk old and 9 wk old mice were significant, p<0.05). Graph represents results from one of three similar experiments. 4.3 Intrinsic Capability of Peritoneal Cells From Young and Adult BALB/c Mice To Produce TNF-a In Vitro Measurement of TNF-a production in young and adult B A L B / c mice gave an unexpected result counter to that obtained with the CD-I strain (Fig. 17). If significance 82 is considered to be at p<0.10, then the results show that the younger B A L B / c mice yielded higher concentrations of the TNF-a compared to the adult mice. The p value for the difference in TNF-a levels produced between young and adult B A L B / c mice was calculated as p=0.09, which makes the result barely insignificant at p<0.05. However, in separate experiments, it was consistently seen that the younger animals produced greater levels of TNF-a than the adult mice, thus, this difference is probably a significant finding. O) Q. 4 i 3.5 3 2.5 2 1.5 1 0.5j oJ -2*3" 3 wkold 9 wkold • IFN-g + LPS m IFN-g • medium Fig. 17. TNF-a production by peritoneal cells from young and adult BALB/c mice. Adherent peritoneal cells (1 x 106 per well) from 3 wk old and 9 wk old BALB/c mice were incubated with IFN-y plus LPS for 24 hrs and the TNF-a levels in the cell culture fluid was determined by an ELISA (p<0.10). Graph represents results from one of two similar experiments. As mentioned previously, B A L B / c mice demonstrated extreme susceptibility with mortality to CVB3(CG) infection. Although the older mice also displayed a high degree of mortality (mean survival time: day 5 p.i.), the younger animals died somewhat earlier 83 (mean survival time: day 4 p.i.). The reasons for mortality in CVB3-infected mice in general are not known. As CVB3 causes widespread, systemic infections in the host, acute damage to any of the vital organs may be a cause of death in the animal, e.g. damage to the liver (Crowell and Landau, 1997). With the BALB/c mouse strain in particular, TNF-a and NO production may provide a clue into the mechanism of the mortality seen. Since NOSH is known to be induced as part of the immune response, and NO is a potent vasodilator, it has been proposed as a major player for causing the hypotension associated with septic shock, which in turn leads ultimately to multiple organ failure and death (Lincoln, Hoyle et al., 1997). While the pathogenesis of shock is not fully understood, it is known that cytokines and their interactions with other inflammatory mediators play a role in its generation (Billiau and Vandekerckhove, 1991). For example, it has been demonstrated that excessive NO production through induction by cytokines, such as TNF, has been linked as a mediator in the generation of toxic shock (Kilbourn and Billiar, 1996; Payen, 1996). The increased TNF-a levels and also high levels of NO induced in the young animals may create a toxic-shock-like syndrome leading to death. The role of NO-mediated death by shock, induced by TNF-a cannot be extended to adult mice, since they produced very low levels of NO. Thus, the reasons for the exquisite sensitivity of the adult BALB/c mice cannot be explained by this mechanism, although it may be a contributing factor in the young animals. 84 CONCLUSIONS Variation in host susceptibility to CVB3 (and many viruses) is multifactorial, with both genetic factors and the age of the host clearly affecting disease severity. The age-dependence of CVB3(CG) was demonstrated in the first set of experiments, which showed that young CD-I mice (3 weeks old) were clearly more susceptible to infection than mice several weeks older (9 weeks old). Susceptibility was measured by comparing infected tissues from the young and adult mice by plaque assays, histopathological examination, and in-situ hybridization. The results of the plaque assay showed that the young mice generally had greater concentrations of virus present within tissues (heart, pancreas, serum) compared to adult mice. In addition, the younger animals were less efficient in clearing virus, as evident from the virus titres in the pancreas and serum at late times post-infection. For instance, in the pancreas, the young mice still retained very high levels of virus on day 7, while the adult mice had very little detectable virus present at this time. In the serum, it was apparent that the younger mice harboured greater amounts of virus, and at earlier timepoints than adults, while the adults cleared virus sooner. The results of the plaque assays were corroborated by visual examination of the infected tissues for damage and by assessing amounts of CVB3 genome by in-situ hybridization. Thus, the first set of experiments clearly showed that young mice were more susceptible to CVB3 than adult mice, as they not only had greater levels of virus present within tissues, but were less adept in clearing infection, especially early in infection. To help understand this age-dependent susceptibility to CVB3, especially early in infection, the innate immune system at the level of macrophage function was compared in 85 the two age groups of mice. First, the role of macrophages in CVB3 infection was established by their depletion in vivo by administering silica to mice. Plaque assays of the infected tissues (heart and pancreas) demonstrated that elimination of macrophages caused more severe infection, as greater concentrations of virus were recovered from the tissues. Thus, macrophages help protect against CVB3 infection. This is counter to that seen in virus systems such as HIV, in which macrophages play a pathogenic role, since these cells are highly tropic for HIV and help disseminate infection. In the next sets of experiments, individual functions of macrophages were compared in vitro between young and adult CD-I mice, such as virus inactivation, NO production, and TNF-a production. Virus inactivation was measured by infecting peritoneal cultures with CVB3 across 6 days, and measuring a drop in virus titres over time. The results demonstrated that virus titres dropped more rapidly in macrophage cultures of adult mice than cultures of young mice, and this difference was seen significantly by day 4 p.i. Thus, young mice are less efficient in containing virus than adult CD-I mice, and this deficiency may contribute to their increased susceptibility to CVB3 in vivo. Various macrophage activities may be responsible for inactivating the virus (see Introduction). Although macrophages from young CD-I mice were less efficient in inactivating virus than adult mice, they were surprisingly more potent producers of NO in vitro. Upon incubation of macrophages with IFN-y or IFN-y plus TNF-a, greater levels of nitrite were detected in the culture fluid from young CD-I mice compared to adult mice. In addition, incubation of macrophages with CVB3 did not result in any production of 86 NO from either young and adult mice. Thus, the increased susceptibility of young mice to CVB3 does not correlate with their ability to produce NO. When NOS was inhibited in vivo by the administration of L-NMMA, a more aggravated infection of CVB3 in tissues was seen in both the young and adult CD1 mice, with the younger mice being generally more affected. The observation that the adults were also severely affected is probably due to the fact that L-NMMA caused the cumulative inhibition of all three isoforms of NOS. Overall from this experiment, it can be concluded that both the young and adult CD-I mice rely on the NOS system for providing protection against CVB3. In addition, the observation that the younger mice generated greater NO levels may explain why NOS inhibition affected this age group greater than the older mice, through inhibition of NOSII. The mechanism for enhanced NO production by macrophages from young mice are not known, but intracellular events may be involved, such as signal transduction pathways and factors involved in NOS gene upregulation. In addition, the density of cell-surface receptors on macrophages involved in NO induction may be greater in the younger mice. Comparison of the TNF-a producing capability of macrophages in vitro showed that young CD-I mice were more limited in its generation. The reduced ability of the young CD-I mice to produce TNF-a may contribute to their increased susceptibility to CVB3, as TNF-a is an important molecule involved in immunity, especially in virus infections. As susceptibility to virus infections depends on many host factors besides age, it was of interest to analyse the effect of host genetics to CVBS susceptibility by examining macrophage functions in another mouse strain, BALB/c. BALB/c mice are more 87 susceptible to CVB3(CG) infection in vivo than CD-I mice, resulting in mortality of both age groups of mice, with the younger mice dying earlier than the adults. A comparison between young and adult B A L B / c mice macrophages in virus inactivation, NO production, and TNF-a production yielded somewhat different results than with CD-I mice. Differences in virus inactivation by macrophages between young and adult B A L B / c mice were more prominent, such that the younger animals were significantly impaired in their ability to contain virus as indicated by a slower drop in virus titres over time. In addition, the young macrophage cells appeared to be permissive to virus infection, as seen by an increase in virus titres by day 4. This limitation in virus restriction of macrophages in young B A L B / c mice may partly explain the enhanced susceptibility to CVB3 infection in comparison to the more resistant CD-I strain. The NO productive capability of B A L B / c mice, on the other hand, followed a similar trend to the CD-I mice, with the younger B A L B / c mice producing more than the adults. An interesting observation, however, was that the younger B A L B / c mice produced greater levels of TNF-a than the adult mice. In the CD-I mice, lower levels of TNF-a were seen to be produced by macrophages from the younger mice, and this was acknowledged to possibly partially explain the increased susceptibility to CVB3. In the case of the B A L B / c strain, the higher levels of TNF-a seen to be produced by the young mice could also explain the increased susceptibility (especially mortality) to virus in terms of a shock-like phenomenon, where excessive levels of TNF-a may be detrimental to the host. The mechanism of a shock-like death in the B A L B / c mice is speculative, and would need further experimentation for substantiation. 88 In conclusion, the age-dependent susceptibility to CVB3 may be partially accounted for by certain functions of macrophages. In the CD-I mice, macrophages from young mice were impaired in their ability to inactivate virus, and possessed a lower intrinsic ability to produce the anti-viral cytokine, TNF-a. 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