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Functional and cellular studies in HTLV-I-associated myelopathy and multiple sclerosis Al-Fahim, Abdulaziz 2000

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F U N C T I O N A L A N D C E L L U L A R S T U D I E S I N H T L V - I - A S S O C I A T E D M Y E L O P A T H Y A N D M U L T I P L E S C L E R O S I S by A B D U L A Z I Z A L - F A H I M M . S c , Microbiology & Immunology, The University of British Columbia, 1994 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF D O C T O R OF P H I L O S O P H Y in T H E F A C U L T Y OF G R A D U A T E S T U D I E S Department of Medicine, Experimental Medicine Program We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A November 2000 © Abdulaziz Al-Fahim, 2000 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 < ^ ^ ^ / ^ / ^ The University of British Columbia Vancouver, Canada D a t e ,<?ujj £ i y ?,<?<r> DE-6 (2/88) ABSTRACT Multiple sclerosis (MS) and human T-lymphotropic virus type I (HTLV-I ) associated myelopathy ( H A M ) are inflammatory demyelinating diseases of the central nervous system (CNS). Current opinion implicates immune mediated factors, particularly T cells in the pathogenesis of both diseases. Histopathological studies in H A M and M S show perivascular mononuclear cell ( M N C ) infiltration into the central nervous system (CNS). The mechanism by which M N C gain access the C N S involves adhesion of peripheral blood M N C to cerebral endothelial cells that constitute the blood-brain barrier ( B B B ) . The objective of this thesis was to investigate, first, the phenotype of lymphocytes of H A M patients.with a focus on T cell activation and adhesion related antigens; second, the adhesion and mechanism of adhesion of blood M N C of H A M and M S patients to endothelial cells; and third, the effects of immunomodulating drugs on lymphocyte subsets and function in M S . We utilized direct two-color flow cytometry to study lymphocyte subsets in a group of patients with H A M and compared the results with those of H T L V - I asymptomatic carriers and seronegative controls. We found that in H T L V - I carriers, lymphocytes are activated and that activation is even more profound in H A M patients. To investigate the factors regulating the entry of blood M N C into the C N S , we used human umbilical vein endothelial cells ( H U V E C ) as a model for endothelial function and, after growing them to confluence, studied the adhesion of 5 1 Cr-labeled M N C s to the monolayers. Adhesion experiments indicated that M N C from H A M and i i from clinically active (secondary progressive) M S patients adhered significantly more to H U V E C monolayers than M N C from controls. This supports the view that infiltration of M N C across the B B B into the C N S in H A M and M S is due to increased interaction between blood M N C and endothelium. Monoclonal antibody blocking studies indicated that the adhesion molecules L F A - l / I C A M - 1 pathway plays a pivotal role in adhesion both under inflammatory and non-inflammatory conditions, while the V L A - 4 / V C A M - 1 pathway contributes to M N C -H U V E C adhesion only when H U V E C are stimulated and therefore, might be important in recruiting immune cells under inflammatory conditions as in H A M and M S . Studies of IgG secretion by peripheral blood M N C in stable relapsing-remitting (sRR) M S and healthy controls after Pokeweed mitogen ( P W M ) stimulation indicated that s R R - M S patients produced more immunoglobulin (lg) G and had a higher percentage of "high responders" compared with controls. This increase in IgG secretion was significantly inhibited by interferon beta (IFN-P). This inhibition was not equivalent among three commercially available preparations of IFN-(3 (Avonex™, Betaseron®, and Rebif®). We found that Avonex™ had the highest inhibitory effect followed by Rebif® and Betaseron® respectively. In this study, we also examined the effects of IFN -P on M N C - H U V E C adhesion and demonstrated that IFN -P pretreatment of M N C , but not H U V E C results in significant reduction in M N C - H U V E C adhesion. This might partially explain the beneficial effects of IFN-p in M S . 111 T A B L E OF CONTENTS Abstract i i Table of Contents iv List of Abbreviations Xt List of Tables VI List o f Figures v i i i Acknowledgements xiv C H A P T E R O N E I N T R O D U C T I O N 1.1 H A M and M S : an overview of their immunopathology 2 1.2 Role of T cells in the immunopathogenesis of H A M and M S 11 1.3 Leukocyte-endothelial cell adhesion and its implication in H A M and M S immunopathology 12 1.4 Adhesion molecule classification, cascade and regulation 13 1.5 Adhesion molecules in H A M and M S 22 1.6 Immunotherapy in M S 23 1.7 Challenges in studying M S and H A M 29 1.8 Rationale, hypothesis and objectives 32 C H A P T E R T W O M A T E R I A L S A N D M E T H O D S 2.1 Study subjects 45 2.2 Preparation of peripheral blood M N C 48 2.3 Monoclonal antibodies and two-color flow cytometry 48 2.4 Culture of H U V E C 49 2.5 Culture of ECV-304 51 2.6 Adhesion assay 52 2.7 Monoclonal antibody blocking studies 53 2.8 PWM-induced IgG secretion 53 2.9 Determination of IgG content by E L I S A 54 2.10 Statistical analysis 55 iv C H A P T E R T H R E E R E S U L T S 3.1 H T L V - I infection 60 3.2 Lymphocyte subsets in H A M , H T L V - I carriers and healthy controls 60 3.3 Blood M N C - H U V E C adhesion in H A M 63 3.4 Blood M N C - H U V E C adhesion in M S 64 3.5 Blood M N C - E C V - 3 0 4 adhesion in M S 66 3.6 In vitro effects of IFN-P on PWM-induced IgG secretion and M N C - H U V E C adhesion 68 C H A P T E R F O U R D I S C U S S I O N A N D C O N C L U S I O N S 4.1 Lymphocyte subsets in H A M and H T L V - I carriers 96 4.2 Blood M N C - H U V E C adhesion in H A M 99 4.3 Blood M N C - H U V E C adhesion in M S 104 4.4 Comparing adhesion properties of H U V E C and ECV-304 for blood M N C 109 4.5 The effects of immunomodulatory drugs on lymphocyte subsets and function in M S I l l 4.6 Summary and conclusions 117 4.7 Future experimental considerations 120 R E F E R E N C E S 123 A P P E N D I X A 150 A . 1 The effects of immunomodulatory drugs on lymphocyte phenotype in M S 151 v L I S T O F T A B L E S Table 1.1 Adhesion molecules involved in leukocyte-endothelial cells adhesion 37 Table 1.2 Similarities and differences between Avonex™, Betaseron®, and Rebif® P-interferon 38 Table 1.3 Principal features of the C D molecules referenced in this thesis 39 Table 2.1 Individual M S patient characteristics and treatment participating in ICOS trial 56 Table 2.2 Monoclonal antibody pairs used for lymphocyte subset analysis 57 Table 3.1 T Cel l subsets of fresh and cultured peripheral blood lymphocytes from controls, H A M , and carriers 72 Table 3.2 Percentage of CD3+, CD4+, and CD8+ cells bearing putative functional markers in fresh and cultured peripheral blood from controls, H A M and carriers 73 Table 3.3 Percentage of CD3+, CD4+, and CD8+ cells expressing activation-related antigens in fresh and cultured peripheral blood from controls, H A M and carriers 74 Table 3.4 Percentage of CD3+, CD4+, and CD8+ cells expressing adhesion-related antigens in fresh and cultured peripheral blood from controls, H A M and carriers 75 Table 3.5a Adhesion of blood M N C to untreated and IFN-y or L-929 supernatant treated H U V E C (freshly isolated M N C ) 76 Table 3.5b Adhesion of blood M N C to untreated and IFN-y or L-929 supernatant treated H U V E C (cryopreserved M N C ) 76 Table 3.6 Adhesion of s R R - M S , S P - M S , and healthy subjects blood M N C to H U V E C 77 Table 3.7 Adhesion of healthy and s R R - M S blood M N C to E C V - 3 0 4 78 Table 3.8 In vitro IgG secretion in s R R - M S and healthy subjects 79 Table 3.9 Effects of IFN-p- lb on M N C - H U V E C adhesion 80 Table A . 1 Lymphocyte subsets of a R R - M S patient enrolled in Schering-Plough trial 153 Table A .2 Lymphocyte subsets of a R R - M S patient enrolled in ICOS trial who received 2 mg/kg of Ant i - rhu-LFA (Hu23F2G) 154 Table A.3 Lymphocyte subsets of a R R - M S patient enrolled in ICOS trial who received 1 mg/kg of Ant i - rhu-LFA (Hu23F2G) 155 Table A .4 Lymphocyte subsets of a R R - M S patient enrolled in ICOS trial who received placebo 156 Table A . 5 Lymphocyte subsets of a R R - M S patient enrolled in ICOS trial who received intravenous Methylprednisolone 157 Viii L I S T O F F I G U R E S Figure 1.1 Sequential steps in a simplified model of leukocyte-endothelial cells adhesion 43 Figure 3.1 Adhesion of H A M and n o n - H A M blood M N C to activated H U V E C 81 Figure 3.2 Effects of anti-adhesion molecule antibodies on the adhesion of M N C of H A M patients to activated H U V E C 82 Figure 3.3 Effects of anti-adhesion molecule antibodies on the adhesion of M N C of R R - M S patients to untreated H U V E C 83 Figure 3.4 Effects of anti-adhesion molecule antibodies on the adhesion of M N C of healthy subjects to untreated H U V E C 84 Figure 3.5 Effects of anti-adhesion molecules antibodies on the adhesion of M N C of R R - M S patients to untreated and IFN-y treated H U V E C 85 Figure 3.6 Effects of anti-adhesion molecule antibodies on the adhesion of M N C of R R - M S patients to untreated ECV-304 86 Figure 3.7 Effects of anti-adhesion molecules antibodies on the adhesion of M N C of healthy subjects to untreated ECV-304 87 Figure 3.8 Adhesion of blood M N C to untreated or IFN-y and L-929 supernatant treated H U V E C and ECV-304 88 Figure 3.9 Effects of anti-adhesion molecules antibodies on the adhesion of M N C of R R - M S patients to untreated and IFN-y treated E C V - 3 0 4 89 Figure 3.10a Effects of IFN-p on PWM-induced IgG secretion in healthy subjects 90 Figure 3.1 Ob Effects of IFN-P on PWM-induced IgG secretion in R R - M S patients 90 Figure 3.10c Effects of Avonex™ on PWM-induced IgG secretion in R R - M S and healthy subj ects 91 Figure 3.1 Od Effects of Rebif® on PWM-induced IgG secretion in R R - M S and healthy subjects 91 Figure 3.10e Effects of Betaseron® on PWM-induced IgG secretion in R R - M S and healthy subjects 91 yiii Figure 3.11 Same dose comparison of Avonex™, Betaseron® and Rebif® on inhibition of PWM-induced IgG secretion 92 Figure 3.12 Per dose comparison of Avonex™, Betaseron® and Rebif® on inhibition of PWM-induced IgG secretion 93 Figure 3.13 Fraction of weekly dose comparison of Avonex™, Betaseron® and Rebif® on inhibition of PWM-induced IgG secretion 94 I X LIST OF ABBREVIAT IONS A 405 nm Absorbance at 405 nm A D C C Antibody-dependent cell-mediated cytotoxicity A N O V A Analysis of variance A P C Antigen-presenting cells A T C C American type culture collection B B B Blood-brain-barrier B S A Bovine serum albumin C D Cluster of differentiation C H O Chinese hamster ovary C N S Central nervous system Con. A Concavalin A 5 ! C r Radio-isotope chromium-51 C S F Cerebrospinal fluid C T L Cytotoxic T-lymphocytes E A E Experimental allergic/autoimmune encephalomyelitis E C Endothelial cells E C M Extracellular matrix E L I S A Enzyme linked immunosorbent assay ESL-1 E-selectin ligand-1 F A C S Fluorescence activated cell sorter F C S Fetal calf serum FITC Fluorescein isothiocyanate FSc Forward scatter G l y C A M - 1 Glycosylation-dependent cell adhesion molecule-1 H A M HTLV-I-associated myelopathy H B S S Hanks' balanced salt solution H L A Human leukocyte antigen H R P Horse radish peroxidase HS Horse serum H T L V - I Human T lymphotropic virus type I H U V E C Human umbilical vein endothelial cell I C A M - 1 Intracellular adhesion molecule-1 IFN-y Human recombinant interferon-gamma IFNs Interferons IFN-B Human recombinant interferon-beta IgG Immunoglobulin G isotype IGSF Immunoglobulin supergene family LL-10 Interleukin-10 IL-2 Interleukin-2 IL-2R Interleukin-2 receptor ILs Interleukins IV Intravenous k D Kilodalton kg Kilogram L A D Leukocyte adhesion deficiency L F A - 1 Leukocyte function-associated antigen-1 mAb Monoclonal antibody M A C Membrane attack complex M a d C A M - 1 Mucosal addressin cell adhesion molecule-1 M A G Myelin-associated glycoprotein M B P Myel in basic protein mg Mil l igram M H C Major histocompatiblity complex M I U M i l l i o n international units M N C Mononuclear cells M O G Myel in oligodendrocyte glycoprotein M R I Magnetic resonance imaging m R N A Messenger ribonucleic acid M S Multiple sclerosis N K Natural killer cells N O D Non-obese diabetic P B S Phosphate buffered saline P E Phycoerthyrin P L P Proteolipid protein P M A Phorbol myristate acetate P P - M S Primary progressive multiple sclerosis PSGL-1 P-selectin glycoprotein ligand-1 Xii P W M Pokeweed mitogen rhu- Recombinant and humananized R R - M S Relapsing-remitting multiple sclerosis s Stable SD Standard deviation S E M Standard error of the mean S P - M S Secondary progressive multiple sclerosis SSc Side scatter T A C T cell activation antigen T C R T cell receptor TGF-B Tumor growth factor-beta T h l T helper-1 subtype Th2 T helpler-2 subtype T N F - a Tumor necrosis factor-alpha TNF-(3 Tumor necrosis factor-beta TSP Tropical spastic paraparesis TSP Tropical spastic paraparesis V C A M - 1 Vascular cell adhesion molecule-1 V L A - 4 Very late antigen-4 X i i i A C K N O W L E G E M E N T I would like to express my thanks and appreciation to my supervisor, Dr. Joel Oger for his guidance, encouragement and patience throughout the course of this project. I also like to acknowledge the support and direction of my thesis committee, Dr. Katerina Dorovini-Zis, Dr. Geoffrey Hoffmann, Dr. Lome Kastrukoff, and Dr. Hermann Ziltener. I am also grateful to M r . Terry A z i z , Dr. Philippe Cabre, Mrs. Rukmini Prameya, and Dr. Lucy Wang for their technical support. I wish to express my personal gratitude to my wife and daughters Fatima and Maha for their compassion, encouragement and understanding during this endeavor. I also like to thank the United Arab Emirates University for their financial support. xiv C H A P T E R ONE INTRODUCTION 1.1 H T L V - I - A S S O C I A T E D M Y E L O P A T H Y A N D M U L T I P L E S C L E R O S I S : A N O V E R V I E W OF T H E I R I M M U N O P A T H O L O G Y 2 1.1.1 HTLV-I-associated myelopathy 2 1.1.2 Multiple sclerosis 5 1.2 R O L E OF T C E L L S IN T H E I M M U N O P A T H O G E N E S I S OF H A M A N D M S 11 1.3 L E U K O C Y T E - E N D O T H E L I A L C E L L A D H E S I O N A N D ITS I M P L I C A T I O N IN H A M A N D M S I M M U N O P A T H O L O G Y 12 1.4 A D H E S I O N M O L E C U L E C L A S S I F I C A T I O N , C A S C A D E A N D R E G U L A T I O N 13 1.4.1 Classification of adhesion molecules 13 1.4.2 Adhesion cascade (leukocyte-endothelial adhesion) 17 1.4.3 Regulation of adhesion 19 1.5 A D H E S I O N M O L E C U L E S IN H A M A N D M S 22 1.6 I M M U N O T H E R A P Y IN M S 23 1.6.1 Interleukin-10 24 1.6.2 Interferon-P 25 1.6.3 A n t i - L F A - 1 monoclonal antibody 27 1.7 C H A L L E N G E S I N S T U D Y I N G M S A N D H A M 29 1.8 R A T I O N A L E , H Y P O T H E S E S A N D O B J E C T I V E S 32 1.8.1 Studies of lymphocyte subsets in H A M 32 1.8.2 Studies of lymphocyte-endothelial adhesion in H A M and M S 33 1.8.3 Studies of immune modulating agents in M S 35 1 INTRODUCTION 1.1 HTLV-I-ASSOCIATED MYELOPATHY AND MULTIPLE SCLEROSIS: AN OVERVIEW OF THEIR IMMUNOPATHOLOGY 1.1.1 HTL V-I-associated myelopathy The human T cell lymphotropic virus type I (HTLV-I ) can be associated with a slowly progressive neurological disease called HTLV-I-associated myelopathy ( H A M ) (Osame et al., 1986) or Tropical spastic paraparesis (TSP) (Gessain et al., 1985). These two conditions have been shown by subsequent comparative studies to be identical to each other, but are endemic in different geographical locations (Roman and Osame, 1988). H A M usually begins in adulthood and affects more women than men. The disease has usually been reported in high H T L V - I endemic areas and occurs mainly in persons of African or Asian origin but can also be observed in whites (Gessain 1996). Despite the fact that H T L V - I has been established as the etiologic agent in H A M , its pathogenic mechanisms remain unknown. The neuropathology of H A M provides evidence that immunological processes in association with H T L V - I infection may play a significant role in the pathogenesis of the disease. Pathologically, H A M is characterized by perivascular cuffing by mononuclear cells and demyelination in the central nervous system (CNS), predominantly in the thoracic region of the spinal cord (Iwasaki et al., 1992, Itoyama et a l , 1988 a,b; Jacobson et al., 1988). Early in the disease, lymphocytes are shown to be abundant and consist of equal numbers of CD4+ and CD8+ T cells as well as some B cells (Moore et al., 1989). However, some reports indicate the 2 preponderance of CD4+ T cells in very early stages of the spinal cord lesions (Iwasaki et al., 1992). In patients with chronic H A M , lymphocytic infiltrates are less abundant and consist mostly of CD8+ T cells (Umehara et al., 1993). Immune abnormalities, including an increase in activated T cells and spontaneous T cell proliferation have also been demonstrated in peripheral blood of H A M patients (Itoyama et al., 1988, Jacobson et al., 1988). Although these abnormalities are believed to result from the active replication of H T L V - 1 genome, the exact mechanism of these activated T cells involvement in the pathogenesis of H A M is still unclear (Oger and Dekaban, 1995; Yoshida et al., 1989). It also remains unclear why only a small percentage of H T L V - I infected individuals develop H A M and what determines the progression from the carrier state to clinical disease (Kaplan et al., 1990). Analysis of cerebrospinal fluid (CSF) in H A M patients shows activated T cells, mild lymphocytic pleocytosis, protein elevation, elevated IgG synthesis, and oligoclonal bands (Jacobson et al., 1990; Ceroni et al., 1988; Link et al., 1989; Ijichi et a l , 1989). Some of the oligoclonal bands are directed to H T L V - I proteins (Levin and Jacobson, 1997). Magnetic resonance imaging (MRI) of the spinal cord may also reveal atrophy and M R I of the brain shows periventricular white matter lesions in large number of H A M patients (Nakagawa et al., 1995). There are two major hypotheses that have been proposed to explain the immunopathogenesis of H A M . In the first hypothesis, H T L V - I infects the glial cells in the C N S , and a subsequent cytotoxic immune response against the infected cells results in demyelination (Moore et al., 1989, Levin and Jacobson, 1997). In the second hypothesis, H T L V - I infection leads to the random activation of autoreactive T cells and the induction 3 of an autoimmune process (Oger et al., 1995). Recent evidence that the frequency of H L A class I restricted and H T L V - I tax-specific C D 8+ cytotoxic T-lymphocytes (CTL) is high in blood mononuclear cells and in C S F of H A M patients but not in carriers or in patients with adult T-cell leukemia favors the first hypothesis (Jacobson et al., 1992; Elovaara et al., 1993). Demyelination mediated by cytotoxic T cells could occur either by direct ki l l ing of proposed HTLV-I-infected glial cells in a manner restricted by M H C class I antigen, or by secretion of cytokines from cytotoxic T cells that could adversely affect the function of uninfected neurons and glial cells within the C N S (Giraudon et al., 1996). Whereas the presence of CD8+ T lymphocytes in H A M lesions is well documented, it is controversial whether H T L V - I infects the resident cells of the C N S . The direct demonstration of H T L V - I antigens in glial cells is difficult, because of the close association with potentially infected T cells. It is possible that CD8+ cytotoxic T cells are recognizing viral products presented by HTLV-I-infected T cells. This might result in the activation of cytotoxic T cells and the subsequent secretion of pro-inflammatory cytokines such as IFN-y and T N F - a . A n autoimmune attack on C N S in H A M could be explained by at least two different mechanisms. In the first scenario, CD4+ T cells displaying cross-reactivity between viral antigens and C N S antigens could mediate autoimmune reactions. This is supported by findings that T cell receptors are much more cross-reactive than had been previously thought (Oldstone 1987, Wucherpfennig and Strominger 1996). Furthermore, Nagai et al., have recently characterized a T cell clone that displays reactivity to both HTLV-I-infected cells and a yet unknown spinal cord antigen (Nagai et al., 1996). A n alternative mechanism for an autoimmune pathogenesis in H A M relies on random 4 infection of CD4+ T cells. It is known that T cells with specificity for self-antigens including myelin basic proteins are part of the normal T cell repertoire. The presence of activated T cells with specificity for myelin basic protein can induce experimental autoimmune encephalomyelitis ( E A E ) in mice, indicating that the control of the activation state of autoreactive T cells is critical in avoiding self-reactivity. According to this scenario, in H A M , autoreactive CD4+ T cells are infected by H T L V - I in the periphery, and become activated and migrate to the C N S where they recognize a C N S autoantigen, resulting in a specific immune response and subsequent demyelination. However, the analysis of H T L V - I in vivo infection of autoreactive T cells is hampered by the HTLV-I-mediated T-cell activation. Therefore, it is impossible to discriminate between antigen reactivity and virally mediated spontaneous proliferation. Thus, it is unknown whether a subset of HTLV-I-infected T cells cross reacts with self-autoantigens. 1.1.2 Multiple sclerosis Multiple sclerosis (MS) is the most common neurological disease of young and middle-aged adults of Northern European decent. M S affects women more commonly than men (Duquette et al., 1992; Sadovnick et al., 1993). M S is a chronic inflammatory disease of the C N S . The clinical course of M S is highly unpredictable. In the majority of cases, M S starts with a relapsing-remitting course (RR-MS) that eventually changes to a secondary progressive (SP-MS) course; less commonly the course is progressive from the onset (primary progressive, PP-MS) (Lublin and Reingold, 1996). While the etiology of M S is still unknown, immunological factors are believed to play an important role. The 5 histopathology of the lesion in M S is characterized by multifocal and periventicular infiltration of the white matter by inflammatory immune cells in the C N S , and a selective destruction of myelin and myelin-forming oligodendrocytes (Raine, 1994; Cannella and Raine, 1995). Immune cells consist mostly of T-cells and macrophages. Both C D 4 positive and C D 8 positive T-cells are present in M S lesions. It is believed that macrophages play a primary role as effector cells in the destruction and removal of C N S myelin, while the lesion progression is driven by the activity of CD4+ T cells (Scholding et al., 1994). The main immune abnormality in M S probably involves T cell-mediated immune function, but one of the hallmarks of M S disease is the presence of immunoglobulin (lg) G of restricted heterogeneity in the cerebrospinal fluid in majority of patients (Johnson and Nelson, 1977) also referred to as "oligoclonal bands". Since oligoclonal bands are only occasionally found in the serum of M S patients, it is conceived that a few clones of plasma cells are activated intrathecally. Recently it was reported that oligoclonal band negative M S patients were significantly less disabled compared with matched oligoclonal band-positive M S patients (Zeman et al., 1996). A dysregulation of IgG synthesis in the peripheral blood has also been described by in vitro studies. Analyzing IgG production in lymphocyte cultures stimulated by a T cell-dependent B cell activator like Pokeweed mitogen ( P W M , Fauci et a l , 1980), it was found that M S patients synthesize larger amount of IgGs (Goust et al., 1982; O'Gorman et al., 1987). Furthermore, B cells isolated from C S F of M S patients have been shown to produce antibodies targeted against myelin components such as: proteolipid protein (PLP), myelin basic protein ( M B P ) , and myelin-associated glycoprotein ( M A G ) (Olsson et al., 1990; Sun et al, 1991; Baig et a l , 1991). 6 Studies in animal models of M S have shown that autoreactive T cells are not sufficient to provoke E A E , since IgG-deficient rats fail to develop E A E (Willenberg and Prowse, 1983) and, in Callithrix Jacchus primate model of M S , enchephalitogenic T cells only cause full demyelination in the presence of anti-myelin antibodies (Genain et al., 1995). Recently, it was reported that IgG, including IgG directed against peptides of M B P and M O G , was localized within acute M S lesions (Raine et al., 1999). These data indicate that the role and abnormalities of B cells are also an integral component of M S pathogenesis (Levinson et al., 1983; Oger et al., 1981 and 1988; Raine et al., 1999). B cells can be involved either as antigen presenting cells (APCs) or as antibody-secreting cells. They can interfere directly with the mechanism of demyelination or act thereafter (Glynn and Linington, 1989). B cells may also damage the myelin sheath and clear them with aid of complement and/or the microglial cells (Goldenberg et al., 1989; Mosley and Cuzner, 1996; Ulvestad et al., 1994). The combined actions of the cellular and humoral immune components in M S lesions, the association of M S with specific M H C genes and the failure to detect a specific infectious agent support the notion that M S could be an autoimmune disease (Bertram and Kuwert, 1982; Marrosu et al., 1988; Gran et al., 1999). Further evidence to support the possible autoimmune nature of M S has been derived based on the analogy with an experimental animal model, experimental autoimmune encephalomyelitis ( E A E ) (Bernard et al., 1992). Additional evidence to support an autoimmune origin of M S came from the detection of myelin-reactive T cells in the blood and C S F of M S patients. Further studies however, revealed that the autoreactive T cells also form part of the normal T-cell repertoire in healthy donors. Despite an extensive search for an autoantigen 7 that elicits a self-reactive immune response in M S , none of the candidates proved to be causative. Most of the candidates studied are myelin proteins that have been shown to be encephalitogenic in E A E . The antigenic target in the C N S is unlikely to be found in a discovery of a single antigen. This is because of the phenomenon of epitope spreading (Lehmann et al., 1992) that also may operate in M S pathogenesis. According to this scenario, inflammatory process initiated by T cell recognition of one protein epitope can subsequently lead to activation of T cells recognizing other epitopes of the same protein. In time, there might also be activation of T cells that recognize other proteins that presumably get degraded and then presented by local antigen-presenting cells ( A P C ) in association with M H C . Data showing myelin reactive T cells activated against both myelin basic protein and proteolipid protein in the same M S patient support this concept (Zhang etal. , 1994). Imbalances in the cytokine network have also been implicated in M S pathology. It has been suggested that M S pathology is due to a T h l cell-mediated immune response, driven by a specific antigen that triggers the production of proinflammatory cytokines and secondary immune cells recruitment and activation. Cytokines regulate immune responses by modulating lymphocyte and monocyte function, and may directly cause demyelination and gliosis. Studies in murine models have revealed that upon activation, CD4+ T lymphocytes differentiate into two main types of effector cells that can be separated based on their cytokine secretion profile: T h l cells that secrete cytokines interleukin-2 (IL-2), tumor necrosis factor alpha (TNF-oc) and interferon gamma (IFN-y) and Th2 subset that produce IL-4, IL-5, IL-6, IL-10, and IL-13. The T h l subset regulates proinflammatory effector mechanisms involved in cell-mediated immunity such as 8 delayed type hypersensitivity response and macrophage activation, while the Th2 subset regulates humoral immunity and also downregulates local inflammation (Romagnani, 1997). The cytokines that are produced by T h l or Th2 can also affect each other's development. For example IFN-y produced by T h l cells promotes the differentiation of T h l cells and inhibits the development of the Th2 response. Alternatively, IL-4 and IL-10 favor the development of Th2 cells and inhibit the T h l response. The following observations support this view point: susceptibility to E A E correlates with a predominant T h l response to myelin antigens, and resistance to disease induction correlates with a predominant Th2 immune response (Smeltz and Swanborg, 1998), myelin-reactive T cell clones capable of transferring resistance to other animals in E A E models secrete IL-4 and IL-10 (Chen et al., 1994), the recovery from E A E is associated with an increase in Th2 type cytokines in C N S , and administration of IL-10 suppresses the development of E A E (Rott et al., 1994). In M S , cells isolated from the C S F during active disease expressed a T h l pattern of cytokine production. Increased levels of T N F - a are detected only during active disease and not in inactive M S (Drulovic et a l , 1997). M S relapses precede the increased IFN-y and TNF-P secretion by blood mononuclear cells. (Link et al., 1994). Proteolipid protein (PLP)-specific T-cell clones generated from M S patients during relapse secreted mostly I F N - y and T N F - a , but clones isolated during remission secreted high levels of IL-10 (Correale et al., 1995). Studies of M S lesions based on composition of the inflammatory cells expressing adhesion molecules, and histocompatibility antigen also support the role of T h l response in M S pathology (Woodroofe and Cuzner, 1993; Schluesener H , Meyermann, 1993; Traugott et al., 1983). However, it should be noted that no clear bias toward T h l or Th2 profile has been found in M S , as both pro-9 inflammatory as well as regulatory cytokines are present in M S lesions (Canella and Raine, 1995). Furthermore, the Th l /Th2 dichotomy is less clear in the human immune system and represents the extreme of a range of cytokine production profiles. Many instances exist in humans of cells that secrete combinations of T h l and Th2 cytokines. For example, cytokines such as IL-10, can be produced by both T h l and Th2 cell subsets (Romagnani et al, 1997). Thus, the descriptions of cytokines with pro- or anti-inflammatory properties may be more appropriate in classifying their functional properties. Some scientists do not favor the autoimmune hypothesis of M S ; they favor an infectious aetiology. According to this hypothesis a neurotropic virus infects the C N S in M S , and T cells infiltrate the brain to target the foreign viral antigens. The neural tissue is then damaged, either directly or via a bystander effect of the ongoing inflammatory response. However, no causative virus has been reproducibly identified yet (Karpas et al., 1986). There is indirect evidence that viruses may play a role in initiating M S . For example, infections with measles, rubella or mumps virus predisposes an individual to an increased risk of developing M S (Waksman, 1989, Martin et al., 1996; Monteyne, 1998). Furthermore, several cases have been documented of brain virus infection leading to M S (Challoner et al., 1995; Sanders et al., 1996). The experimental viral model best characterized is Theiler's virus-induced encephalomyelitis. Theiler's virus is a naturally occurring pathogen in mice that produces a chronic persistent C N S infection resulting in inflammatory demyelination similar pathologically and clinically to the chronic progressive form of M S (Roos, 1983; Dal Canto et al., 1995). 10 1.2 R O L E O F T C E L L S I N T H E I M M U N O P A T H O G E N E S I S O F H A M A N D M S Although different mechanisms of pathology have been proposed, there is a general agreement that the pathogenesis of both H A M and M S is immune mediated and that T lymphocytes play a central role in both disease processes (Moore et al., 1989; Hafler and Weiner, 1989; Chang et al., 1992; Utz and McFarland, 1994). H T L V - I has a preferential tropism for CD4+CD45+ T lymphocytes in patients with H A M in vivo (Richardson et al., 1990) and T lymphocytes are the predominant cell type in the cerebrospinal fluid (CSF) of H A M patients (Moore et al., 1989). H A M patients have also been shown to have high levels of activated T lymphocytes in their peripheral blood (Itoyama et al., 1988a) and C S F (Mori et al., 1988). This is highlighted by an increase in the number of large CD3+ cells that also express markers of activation, such as H L A - D R and IL-2 receptor molecules. In addition, peripheral blood lymphocytes of H A M patients show spontaneous proliferation in the absence of any exogenous antigen/mitogen in vitro (Itoyama et al., 1988b). Furthermore, H A M patients have a high frequency of H T L V - I specific CD8+ C T L in their circulating blood (Jacobson et al, 1990; Elovaara et al., 1993) and C S F (Elovaara et al., 1993). T lymphocytes are also the predominant cell type in the cerebrospinal fluid (CSF) of M S (Hafler and Weiner, 1989), and activated T cells have been localized in the M S plaques (Hofman et al., 1986; Bellamy et al., 1985), C S F (Noronha et al, 1980; Hafler et al., 1985) and circulating blood (Hartung et al., 1990). Patients with acute relapsing M S have also been found to have increase T cell adherence to their brain endothelial cells 11 during exacerbation (Tsukada et al., 1993a). Furthermore, experimental autoimmune encephalomyelitis (EAE) , an animal model for M S , is T-cell dependent and can be induced through the transfer of myelin basic protein (MBP)-sensitized T lymphocytes (Mokhtarian et al., 1984). 1.3 L E U K O C Y T E - E N D O T H E L I A L C E L L ADHESION AND ITS IMPLICATION IN H A M AND MS I M M U N O P A T H O L O G Y The recruitment of circulating leukocytes into inflammatory lesions requires adhesion to vascular endothelium, followed by migration between endothelial cells into the underlying tissue. The recruitment of leukocytes into the central nervous system is complicated by the existence of a specialized microvasculature, characterized by the presence of a continuous network of high resistance and complex tight junctions. Under the control of surrounding astrocytes, this microvasculature constitutes the blood-brain barrier ( B B B ) that limits the exchanges between the blood and brain of soluble substances such as growth factors, cytokines and immunoglobulins as well as immune cells (Goldstein and Betz, 1983; Pardridge, 1988; Joo, 1993). On the basis of the existence of the B B B and of low levels of major histocompatibility complex ( M H C ) molecules on brain cells, the C N S has often been considered an "immunologically privileged" site and not normally accessible to leukocyte traffic (Baker and Bill ingham, 1977). However, this viewpoint has been challenged. Recent evidence indicates that leukocytes can invade the brain parenchyma at very low levels under normal conditions and at much higher levels when T cells are activated (Wekerle et al, 1986; Raine et al., 12 1990; Hickey et al., 1991). Furthermore, there is an emerging view that there is a definite connection between the C N S and the peripheral blood through draining lymphatic channels to the cervical lymph nodes (Cserr and Knopf, 1992; Weller et al., 1996). Histopathological and M R I studies of the C N S in H A M and in M S indicate that the breakdown of the B B B and infiltration of leukocytes are early events in the formation of H A M and M S lesions (Kermode et al., 1990, Umehara et al., 1993). A prerequisite for the passage of lymphocyte across the B B B into the C N S parenchyma is binding to endothelial cells. The molecular mechanisms that governs leukocytes infiltration into the C N S involves selective and sequential adherence between cell surface molecules on both leukocytes and endothelium. Adhesion molecules also mediate the subsequent migration of leukocytes into the surrounding tissue. While adhesion molecules also participate in T cell costimulation (Davignon et al., 1981), helper function for B cell immunoglobulin production (Miedema et al., 1985), antibody dependent cell mediated cytotoxicity (Capron et al., 1987), and cytotoxic T cell mediated cytolysis (Krensky et al., 1983), in the following sections the general role of these cell surface molecules in adhesion of leukocyte to endothelial cells are emphasized and discussed. 1.4 A D H E S I O N M O L E C U L E C L A S S I F I C A T I O N , C A S C A D E A N D R E G U L A T I O N 1.4.1 Classification of adhesion molecules Based on their structure adhesion molecules have been classified into three major groups: selectin, integrin, and immunoglobulin supergene family (IGSF) members 13 (Osborn, 1990S; Springer, 1990, 1994) (Table 1.1). Selectins are expressed on leukocytes, platelets, and endothelial cells, and their common structural component is a N-terminal lectin-binding domain. Selectins have been subclassified according to the cell type on which they were first identified: L-selectin (lymphocyte), P-selectin (platelets/endothelium), and E-selectin (endothelium) (Bevilacqua and Nelson, 1993; Tedder et al., 1995). L-selectin is expressed constitutively on all leukocytes and has a critical role in the adhesion of lymphocytes to peripheral lymph node cells and activated endothelium. (Tedder et al., 1995) Upon activation, L-selectin is lost rapidly from the surface of leukocytes (Tedder et a l , 1990). In fact, L-selectin and B2 integrin Mac-1 (CD1 l b / C D 18) expression appear to be regulated inversely (Kishimoto et al, 1989). P-selectin, found on platelets and in Weibel-Palade bodies of endothelial cells (EC) synergizes with cytokines to upregulate leukocyte integrin expression (Bevilacqua and Nelson, 1993). E-selectin, expressed on endothelium, is upregulated after exposure to tumor necrosis factor alpha (TNF-a) , and shed rapidly after loss of cytokine stimulation (Doukas and Pober; 1990). E-selectin binding of leukocyte triggers more stable adherence by integrin receptors (Lawrence and Springer, 1991). A l l selectins bind in a Ca+ +-dependent manner to sialylated carbohydrate structures. The main counter-receptors for L-selectin that have been characterized so far are: glycosylation-dependent cell adhesion molecule-1 ( G l y C A M - 1 ) , the glycosylated and sulphated form of CD34 expressed by endothelial cells, and mucosal addressin cell adhesion molecule-1 (MadCAM-1) , and P-selectin. P-selectin binds to P-selectin glycoprotein ligand-1 (PSGL-1) and E-selectin. E-selectin interacts with E-selectin ligand-1 (ESL-1) (Varki, 1997). Binding of E-selectin to CD66 14 (Kuijpers et a l , 1992), CD11/CD18 (Kotovuori et al., 1993), and L-selectin (Picker et al., 1991) has also been reported. Despite their short intracellular regions, selectins are able to generate costimulatory signals that contribute to leukocyte activation after interaction with their counter-receptors. The physiological importance of selectins in inflammatory responses is seen in leukocyte adhesion deficiency ( L A D ) type II, in which the congentical absence of selectin ligands produces significant adhesion defects and recurrent life-threatening infections (Etzioni et al., 1992). Integrin adhesion molecules are heterodimeric structures composed of noncovalently linked a heavy chain and B light subunits. Subunit combinations form functionally different receptors (Larson and Springer, 1990). The name integrin was based on the function of these transmembrane molecules to "integrate" extracellular information into the cytoskeleton. Integrins are arranged in subfamilies according to the B subunits and each B subunit may have from one to eight different a subunits associated with it. It is also possible for individual a subunits to be associated with different B chain. Within the integrin family of adhesion receptors so far only five members have been shown to be involved in leukocyte adhesion to endothelium: The B2 leukocyte integrins ( C D l l a / C D 1 8 , C D l l b / C D 1 8 , and C D l l c / C D 1 8 ) , the Bi integrin V L A - 4 (a 4 B,, CD49d/CD29), and OC4B7. The B2 integrins share a common B chain (CD 18) and can be noncovalently associated with any of the three C D l l a (LFA-1) , C D l l b (Mac-1), and C D l l c (pl50, 96) a subunits. Peripheral blood lymphocytes mainly express C D l l a / C D 1 8 (LFA-1) whereas neutrophil, monocytes, and N K cells express all three B2 integrins. Ligands for B2 integrins include immunoglobulin supergene family (IGSF) protein family, which are I C A M - 1 for C D l l a / C D 1 8 , C D l l b / C D 1 8 and I C A M - 2 , -3 for 15 C D l l a / C D 1 8 . B 2 integrins also bind soluble proteins such as fibrinogen, and factor X (Larson and Springer, 1990). The Bi integrins share CD29 as their common B subunit. OC4B1 ( V L A - 4 , CD49d/CD29) a prototypical Bi integrin is mostly prominent on cells of the hematopoietic system. Ligands for OC4B1 include V C A M - 1 and extracellular matrix ( E C M ) proteins fibronectin, vitronectin, laminin, collagen, von Willebrand factor, and fibrinogen (Larson and Springer, 1990) and the ligands for OC4B7 are V C A M - 1 and M A d C A M - 1 (Springer, 1994). Integrins exhibit important functional features such as their ability to increase the avidity for their counter-receptors (Clark and Brugge, 1995; A p l i n et al., 1998). The enhancement of integrin avidity is due to intracellular signals that are generated through other cell surface receptors. Integrins are also linked through the cytoskeleton, to molecules involved in the generation of intracellular signals such as focal adhesion kinase or the PI 3-kinase. Thus, the interaction of integrins with their ligands induces costimulatory signals that contribute to cell activation and differentiation (Clark and Brugge, 1995). The cell adhesion molecules that belong to IGSF have one or more domains homologous to those found in immunoglobulin and therefore are named and classified together (Springer, 1990, 1994; Carlos and Harlan, 1994). Members of this superfamily are expressed by endothelial cells (e.g. M a d C A M - 1 and vascular cell adhesion molecule-1 ( V C A M - 1 ) , or by both endothelial cells and leukocytes [e.g. intracellular adhesion molecule-1 and - 2 ( ICAM-1 and -2)]. I C A M - 1 and V C A M - 1 are detected on activated endothelial cells, whereas I C A M - 2 is expressed by both resting and activated endothelial cells. A n additional member of this family, P E C A M - 1 (CD31) plays a role in homotypic 16 adhesion of leukocytes and promotes adhesion between the endothelial cells and leukocytes. ISGF cell adhesion molecules may interact among themselves in a heterotypic or homotypic fashion or with cell adhesion molecules from the integrin families. Additional receptors in this family that function as adhesion molecules include L F A - 2 (CD2) and L F A - 3 (CD58) (Dustin and Springer, 1991). There are additional intercellular adhesion molecules that also participate in inflammatory phenomenon. Cadherins are calcium-dependent adhesion proteins that mainly interact homotypically (Takeichi, 1995; Yap et al., 1997). Members of this superfamily are expressed by, and are responsible for the integrity of endothelial and epithelial cells. Cadherins found at intercellular endothelial junctions seem to play a key role in the extravasation of inflammatory cells. Lastly, other molecules mainly involved in signal transduction such as the chemokine/chemokine receptor may also function as cell adhesion receptors (Imai et al., 1997). 1.4.2 Adhesion cascade (leukocyte-endothelial adhesion) Leukocyte adhesion and migration is a complex phenomenon regulated by a cascade of molecular events that take place in an ordered series of steps involving close interactions between adhesion receptors expressed by migrating leukocytes and endothelial cells (EC). A consensus model of leukocyte migration in four sequential steps is now generally accepted (figure 1.1) (Butcher, 1991; Shimizu et al., 1992; Springer, 1994). In the first step (tethering/rolling), some of the flowing leukocytes come into brief contact with the vessel wall , slow their movement, and roll on the endothelium. This step is transient, reversible and mediated by constitutively expressed selectin molecules and 17 their cognate oligosaccharide ligands (Bevilacqua and Nelson, 1993). In addition to selectins, it has been found that V L A - 4 integrin is also able to sustain the rolling of leukocytes both in vivo (Johnson et al., 1996) and in vitro (Berlin et al., 1995). In the second step (triggering/activation), rolling leukocytes are exposed to the local endothelial microenvironment in the presence of inflammatory mediators such as chemoattractant/cytokines that further deliver activating signals to the leukocytes resulting in upregulation of adhesion molecules and leading to adhesion arrest (Schall and Bacon, 1994, Campbell et al., 1998). The third step (firm adhesion) is primarily mediated by activated Bi ( V L A - 4 ) and B2 (LFA-1 and Mac-1) integrins, which bind to their counter receptors V C A M - 1 and I C A M s , respectively (Hogg and Landis; 1993). The activation of leukocytes induces a rapid shedding of L-selectin caused by cleavage of the extracellular portion of L-selectin by an unidentified endogenous protease (Kansas, 1996). The activation of leukocytes also results in an increase in avidity of integrins for their ligands due to the conformational changes in integrin heterodimer. The increased avidity of the leukocytes integrins results in the firm adhesion of leukocytes to E C . During this phase, leukocytes change shape and acquire a flattened morphology. Then leukocytes transmigrate between E C (diapedesis), or through them following the chemotactic gradient generated by inflammatory foci (fourth step or extravasation). The molecular interactions that are involved in the extravasation of leukocytes are those that mediate the firm adhesion ( L F A - l / I C A M - 1 , -2 and V L A - 4 / V C A M - 1 ) , but adhesion receptors located at the E C junctions such as CD31 and VE-cadherin also have an important role in leukocyte extravasation (Piali et al., 1995; Bianchi et al., 1997). The migration of leukocytes from the blood vessel wall toward the inflammatory foci also involves 18 interactions of the leukocyte receptors mainly R\ integrins with the components of the extracellular matrix such as collagen, fibronectin, and laminin. 1.4.3 Regulation of adhesion Regulation of adhesion occurs through increased avidity of existing adhesion molecules or increased expression of molecules on the cell surface. For example, activation of T cells with the phorbol myristate acetate ( P M A ) has been demonstrated to increase the affinity of L F A - 1 and V L A - 4 for their counter receptor I C A M - 1 and V C A M - 1 , respectively, without changing the levels of integrins cell surface expression (Wilkins et a l , 1991; Shimizu et al., 1991; Dustin and Springer 1989). Cross-linking of T-cell receptors also increases L F A - 1 avidity; however, T cell receptor-induced changes in L F A - 1 avidity are transient (Dustin and Springer, 1989). Cell-cell adhesion is generally more efficient when an appropriate antigen: M H C complex is recognized (Martz, 1987). Antigen-independent adhesion requires prior activation of T lymphocytes, because spontaneous adhesion is low or absent in resting T lymphocytes (Dustin and Springer, 1989). Furthermore, binding of cell adhesion molecules with their respective ligands causes alteration in the expression and affinity of adhesion molecules (Frelinger etal., 1988; Lou et al., 1996). Activation of cells not only induces changes in avidity but also can upregulate adhesion receptor expression (Springer, 1994). For example, cytokines such IL-4, IL-1B, T N F - a , and IFN-y have been shown to promote the adhesiveness of T cells for endothelial cells by increasing the expression of V C A M - 1 , I C A M - 1 and E L A M - 1 on endothelial cells (Hughes et al, 1988; Minovsky et al., 1990; Thornhill et al., 1991; 19 Shimizu et al., 1991). Differential regulation of adhesion molecule expression determines homing of lymphocytes to organs and sites of inflammation. Adhesion receptor expression on leukocytes, vascular endothelium, and other cell types may be constitutive or regulated (Springer, 1994, Imhof and Dunon, 1995). For example, selectin adhesion is mediated by the presence or absence of receptor expression on the cell surface. Selectin adhesion is transient and unstable under intravascular conditions but slows leukocyte circulations dramatically (Springer, 1994; Bevilacqua and Nelson, 1993). TNF-ct or IL-1 induces endothelial expression of E-selectin, but expression is typically short lived. Concomitant stimulation with interferon gamma (IFN-y) enhances and prolongs expression of E-selectin (Doukas and Pober, 1990). If chemoattractant or additional adhesion mechanisms are not present at a microvascular site, transiently adherent leukocytes are released back into circulation. If secondary adhesion molecules (integrins, IGSF receptors) are expressed, leukocyte adherence to endothelium becomes more stable, leading to infiltration of leukocytes into the E C M and tissue (Springer, 1994; Tedder, 1995). Besides changes in avidity and cell surface expression, differential distribution and expression of adhesion molecules on T lymphocyte subsets may modulate immune/autoimmune response patterns. On the one hand, CD8+ T cells have been reported to have higher L F A - 1 expression compared with CD4+ T cells (Pardia et al., 1989). On the other hand, it has been shown that m A b against V L A - 4 inhibited CD4+ but not CD8+ T cell infiltration of the pancreas in the non-obese, diabetic (NOD) mouse model of diabetes (Baron et al., 1994). This finding implies that differential adhesion 20 molecule expression occurs on lymphocyte subsets and determines transendothelial migration patterns for CD4+ and CD8+ T cells. In addition to the broadly expressed proinflammatory cytokines such as T N F - a , IFN-y, IL-1, IL-4, and IL-6, chemokines are also important soluble mediators of inflammation (Bacon, 1994; Baggiolini, 1998). Once released by immune cells, they strongly modulate adhesion molecules expression and affinity on both endothelial cells and lymphocytes (Wong and Dorovini-Zis, 1996; Merr i l l and Benveniste, 1996). Important chemokines include R A N T E S , IL-8, MIP-1B, M l P - l a M C P - 1 , colony stimulating factors (G-CSF, G M - C S F ) , chemoatractant peptides (C5a, F M L P ) and neuropeptides (Luster, 1998; Baggiolini, 1998). Binding of chemokines, cytokines or chemoattractants to leukocyte expressing complimentary receptors transduces signals that can augment B l or B2 integrin-dependent adhesion. Some chemoattractant factors influence narrow population of cells. For example, R A N T E S acts primarily on memory T cells (Schall et al., 1990). M I P - a attracts monocytes and CD8+ T cells, whereas the closely related MIP-1B acts on CD4+ T cells (Ming Wang et al., 1993; Taub et al., 1993). Furthermore, M I P - a but not MIP-B increases the adherence of T cells to endothelial cell (Tanaka et al., 1993). Most "classic" cytokines (e.g. IL-1, IL-4, TGF-B) exert their adhesion modulating effects on a variety of cells, but they may show different effect on different cells. For example, TGF-B induces migration of T cells and monocyte but not granulocytes and IL-4 induces adhesion molecules on H U V E C but inhibits monocyte adhesion. 21 1.5 A D H E S I O N M O L E C U L E S I N H A M A N D M S Analysis of the spinal cord lesions of H A M patients has shown enhanced expression of V C A M - 1 and E-selectin on endothelium, and high levels of V L A - 4 , L F A - 1 and Mac-1 expression on infiltrating mononuclear cells (Umehara et al., 1996). Increased levels of soluble I C A M - 1 and V C A M - 1 have been detected in the serum and C S F of H A M patients (Mainnolfi and Rothlein, 1992; Tsukada et al., 1993b; Matsuda, 1995a). Significant elevation of soluble L-selectin has also recently been reported in the sera of H A M patients (Tsujino et al., 1998). Expression of adhesion molecules on freshly isolated lymphocytes from H A M patients have not been studied. However, enhanced expression of V L A - 4 and V L A - 5 integrins has been shown in peripheral blood lymphocytes of healthy controls that were infected with H T L V - I in vitro (Dhawan et al., 1993). Enhanced expression of I C A M - 1 and L F A - 3 has also been reported in T cell lines carrying H T L V - I (Fukodome et al., 1992; Imai et al., 1993) Most of the studies of the role of adhesion molecules during M S have relied on immunochemical analyses of the expression of adhesion molecules during the different stages of disease on autopsy C N S material or on blood or CSF-derived lymphocytes from patients with M S . In typical M S lesions, upregulation of I C A M - 1 , V C A M - 1 and E -selectin on the endothelium, and V L A - 4 , and L F A - 1 on the infiltrating mononuclear cells have been described (Washington et a l , 1994; Brosnan et al., 1995; Cannella and Raine, 1995). In addition, in M S lesions some resident cells of the C N S , such as astrocytes or microglia, show increased cell surface expression of I C A M - 1 and L F A - 1 (Bo et al., 1996; Cannella and Raine, 1995). Lymphocytes from either C S F or the blood of M S patients 22 also express increased levels of adhesion molecules such as L F A - 1 , L F A - 3 , C D 2 , and CD44 on their surface (Svenningsson et al., 1993). Cultured brain microvascular endothelial cells derived from M S patients were shown also to constitutively express high levels of I C A M - 1 and were demonstrated to have a high capacity in adhering to isolated leukocytes (Lou et al., 1997). A further clue that lends support to the importance of adhesion molecules in M S is derived from measurement of circulating soluble adhesion molecules during M S . Investigators found that circulating forms of I C A M - 1 , I C A M - 3 , V C A M - 1 , and L-selectin were increased in serum and C S F from most M S patients (Rieckmann et al., 1994b, Mobner et al., 1996, Droogan et a l , 1996). Circulating levels of I C A M - 1 , V C A M - 1 , E - , and L-selectin are also correlated with clinical relapse (Sharief et al., 1993; Rieckmann et al., 1994b, Hartung et al, 1995; Dore-Duffy et al., 1995) and the appearance of new gadolinium-enhancing lesions in M R I (Mobner et al., 1996) (this is a parameter of B B B breakdown and disease activity). 1.6 I M M U N O T H E R A P Y I N M S Proinflammatory cytokines, such as IFN-y and T N F - a , have been shown to be associated with exacerbation in patients with M S (Panitch et al, 1987). Furthermore, increase in T N F - a m R N A expression and decrease in IL-10 m R N A expression positively correlates to exacerbation of M S (Rieckmann et al, 1994a). Thus, cytokine-based strategies for the treatment of M S have focused on anti-inflammatory cytokines such as IL-10 and IFN-B. 23 The adhesion and subsequent migration of circulating leukocytes across the B B B into the C N S in inflammatory conditions, such as M S , involve a complex series of adhesion molecules expressed on leukocytes and endothelium. A n approach to immunotherapy in M S has also been to inhibit this interaction using anti-adhesion molecules monoclonal antibodies. A n important and central adhesion molecule in leukocyte-endothelium interaction is L F A - 1 integrin. 1.6.1 Interleukin-10 Interleukin-10 (IL-10) is believed to be produced by Th-2 cells and to inhibit the function of T h l cells (Mosmann and Moore, 1991). IL-10 is also thought to participate in recovery from the inflammatory events by down-regulating the activated state of endothelial cells and macrophages (Olsson, 1995). In M S , levels of IL-10 m R N A were reported to be higher in stable M S compared with relapsing M S and the levels of IL-10 were shown to decline prior to relapse in M S (Rieckmann et al., 1994a). These observations prompted Schering-Plough Corporation to develop recombinant human interleukin-10 (rhuIL-10) for potential therapeutic use in multiple sclerosis. RhuIL-10 was produced in a strain of Escherichia coli bearing a genetically engineered plasmid that contains a rhuIL-10 gene. With the exception of methionine residue at the amino-terminus, rhuIL-10 is identical to endogenous human IL-10 protein. The effect of rhuIL-10 has been examined in several rodent models of E A E . In one study where T N F - a was used to induce relapses of E A E in SJL mice that had recovered from acute E A E , rhuIL-10 given with T N F - a provided complete protection against relapses (Crissi et al., 1995). In another study where acute E A E in the Lewis rat was induced by M B P , administration 24 of rhuIL-10 during the initial phase of the disease suppressed the subsequent induction of E A E (Rott et a., 1994). In a different study in which an acute E A E was induced in mice, conflicting results were obtained. A single dose of rhuIL-10 given immediately after M B P injection showed no effect (Smith, 1994). However, a similar dose of rhuIL-10 given at the onset of E A E symptoms showed a trend toward improvement (Schering-Plough, Data on file as D-27219). However, repeated doses of rhu-IL-10 at 7 and 14 days during the initial phase of the disease resulted in an exacerbation of the disease (Schering-Plough, Data on file as P-5806). The safety studies in multiple dose pilots clinical trials with healthy volunteers, patients with Crohn's disease, Ulcerative Colitis, and Rheumatoid Arthritis indicated that the rhuIL-10 is safe with minimal side affects up to 25 ug/kg dose levels. In a multi-center, randomized, double blind, placebo-controlled study, the subcutaneous injection of single dose of rhuIL-10 was tested in clinical trial o f relapsing-remitting form of M S with M R I evidence of disease activity. The results of study indicated that there was no significant benefit for the use of rhuIL-10 in R R - M S . 1.6.2 Interferon Interferons are a family of proteins that inhibit viral activities and cellular proliferation and modify the immune response (Becker et al., 1995). One member of the IFN family, IFN-B has been shown to be of benefit in relapsing-remitting M S . The mechanisms underlying the efficacy of IFN-P in M S patients are still not completely known. However, there are several proposed mechanisms that may play a role in the efficacy of IFN-p in M S (Yong et al., 1998). These include: down-regulation of the IFN-y activity; induction of T suppressor cell function; augmentation of IL-10 production; 25 inhibition of T cells migration into C N S and antiviral effect. Interferon p has received regulatory approval in the United States, Canada, Europe, and Australia for the treatment of M S . Currently there are three preparation of interferon P in use. These are, Avonex™ (IFN-p-la, Biogen), Betaseron® (IFN-p-lb, Berlex), and Rebif® (IFN-p-la, Serono). The similarities and differences between the three preparations of IFN-P are summarized in Table 1.2. Betaseron® (IFN-P-lb) was initially tested in a multicentre trial involving 372 patients with relapsing-remitting M S and mild to moderate disability. Treatment consisted of either 8 M I U (250 ug) or 1.6 M I U (50 ug) or placebo given by subcutaneous injection on alternate days. The high dose was set on the basis of patient tolerance to a single injection. Compared with placebo, treatment with the higher dose reduced the relapse rate by 31 %, increased the time to first relapse and the proportion of patients who were relapse free (The IFN-P Multiple Sclerosis Study Group, 1993, 1995). In addition, there was a significant reduction in disease activity as measured by the analysis of new or enlarging lesions on serial M R I (Paty and L i and the U B C M S / M R I Study group, 1993). A second multicentre trial of Betaseron® was recently completed in Europe, comprising 718 patients with secondary progressive M S who had been clinically active in the 2 years preceding the study (European Study Group on interferon P-lb, 1998). Treatment consisted of either 8 M I U or placebo subcutaneously on alternate days over 3 years. Treatment with Betaseron® resulted in the significant delay of disease progression and reduced the disease activity. Furthermore, it increased the time to first relapse and the proportion of patients who were relapse free. 26 Avonex™ (IFN-B-la) was tested in a trial involving 301 patients with relapsing form of M S and mild to moderate neurological impairment. Treatment consisted of weekly intramuscular injections with 6 M I U (30 jug) Avonex or placebo. A n 18% reduction in exacerbation rate was seen for the treated group (Jacobs et al., 1996). The treatment was also accompanied by a reduction of gadolinium enhancement and of new or enlarging lesions on annual M R I (Simon et al., 1998). Rebif® (IFN-P-la) was investigated in a large study involving 560 patients with active relapsing-remitting M S and mild to moderate disability who were randomized to treatment with I F N - p - l a at 6 M I U (22 pg) or 12 M I U (44 pg) or placebo, given subcutaneously three times a week for 2 years. The result showed that, compared with placebo, Rebif® significantly decreased the number and severity of exacerbation by 27% and 33% in the 22 pg and 44 pg groups, respectively, (PRISMS study group, 1998). Rebif® also increased the time to first and second relapse, and increased the percentage of patients who were relapse free during the study. Furthermore, there was a significant reduction in the disease activity on M R I , as defined by new or enlarging lesions (PRISMS study group, 1998). 1.6.3 Anti-LFA-1 monoclonal antibody The importance and beneficial effects of anti-LFA-1 monoclonal antibody (mAb) in inflammation has been demonstrated in animal models in bacterial meningitis (Tuomanen et al., 1989) and acute lung injury (Mulligan et al., 1992). In rodent E A E , m A b against L F A - 1 had either no effect (Canella et al., 1992) or a deleterious effect (Welsh et al., 1993). However, mAbs against Mac-1 suppressed E A E , but only i f given 27 prior to the disease onset (Huitanga et al., 1993). Thus, the role of L F A - 1 in the pathogenesis of E A E remains not well defined. The presence of brain inflammation in E A E and the anti-inflammatory effects of antibodies against L F A - 1 adhesion molecule led ICOS Corporation to develop and test a humanized anti-LFA-1 named Hu23F2G. Hu23F2G is produced by Chinese hamster ovary (CHO) as y4 immunoglobulin. This mAb recognizes both L F A - 1 and Mac-1. A s expected, in vitro Hu23F2G has the following properties: inhibits L F A - 1 dependent cell aggregation; blocks binding of L F A - 1 bearing cells to purified I C A M - 1 and blocks L F A -1-dependent leukocyte transmigration through endothelial cells monolayers. Furthermore, ICOS tested the effects of Hu23F2G in nonhuman primate Macaca fascicularis E A E . In this E A E model animals were treated with intravenous injection of either Hu23F2G (2 mg/kg for 7 days) or dexamethasone (4 mg/kg for 3 days). The result indicated less severity of E A E , more resolution of brain M R I abnormalities, and longer survival than animals given dexamethasone alone. These in vivo and vitro observations indicate that anti-LFA-1 might also have a favorable effect on M S . This led ICOS to determine the safety and efficacy of Hu23F2G in the treatment of acute exacerbations of M S . A multi-center, randomized, double-blind, placebo-controlled trial was conducted. A total of 169 patients were enrolled within seven days of the onset of symptoms recurrence to one of four treatment groups: Placebo (n=43), methylprednisolone at 1 gram intravenously (IV) for 3 days (n=41), Hu23F2G at 1 mg/kg IV (n=44) or Hu23F2G at 2 mg/kg IV (n=41). The result indicated that while Hu23F2G was safe and well tolerated at the doses used, it was ineffective at either dose as compared to placebo (Lublin, 1999). 28 1.7 CHALLENGES IN STUDYING MS AND HAM Variability in disease progression is a prominent clinical feature of M S . Some patients have initial attacks, complete recovery, and no further symptoms, while others progress rapidly within months of initial involvement. Most patients have exacerbation and remission that follow an unpredictable course. Immunopathological studies of lesions in a single M S patient reveal a fairly uniform pattern of inflammation, demyelination, axonal loss, and remyelination. However, profound heterogeneity exists in lesional structure between different patients (Lassmann et al., 1998). To further dissect which specific mechanisms are involved in M S pathogenesis, Lucchinetti et al. (1996) have analyzed the M S lesions in a large number of early biopsy and autopsy of lesions. Based on this study, the M S lesions were categorized to five different types; demyelination with minimal oligodendrocyte damage; demyelination associated with extensive oligodendrocyte loss; primary demyelination with a gradient of oligodendrocyte loss toward the inactive plaque center; demyelination paralleled with nonselective destruction of oligodendrocytes, axons and astrocytes; and primary oligodendrocyte damage in the periplaque white matter with secondary demyelination. In addition to structural differences in M S lesions, the predominant immunopathological contribution to lesions formation was also seen. The cases were segregated into those with antibody and complement involvement, those with a dominant T cells/macrophage reaction and those with primary oligodendrocyte involvement. The diverse clinical course and pathological findings might imply the diversity in M S pathogenesis (Lucchinetti et al., 1996; Lassmann et al., 1998). Based on these studies it was suggested that M S might represent 29 a neurological syndrome with several different immunopathological mechanisms that lead to a final common trail of C N S injury rather than a single disease with a single cause (Lucchinetti et a l , 1996; Lassmann et al., 1998). These diverse clinical and pathogenic findings in M S have contributed to ambiguity and uncertainty in understanding M S pathology and have slowed the course of development of effective therapy in M S . To elucidate the mechanism of pathogenesis of M S , investigators have relied on studies of experimental autoimmune encephalomyelitis (EAE) , considered to be an animal model of M S . Acute and chronic variants of this T-cell driven autoimmune disorder of the C N S have been established in rodents (Swanborg, 1995) and non-human primates (Massacaesi et al., 1995). Generally E A E is induced by sensitization with myelin antigens such as myelin basic protein (MBP) , proteolipid protein (PLP), myelin oligodendrocyte glycoprotein ( M O G ) or S-100 (an intracellular protein present in astrocytes). A n additional approach to inducing E A E in an animal is by injection of myelin-specific T cells that have been activated in vitro. Frequently used animals include mice (strains SJL/J , P L / J , Biozzi) , rats (strain Lewis and D A ) , guinea pigs (strain 13, Hartley) and more recently marmosets. Depending on the species and the agents used to elicit E A E , distinct histological and clinical features result, each resembling M S to a variable degree (Brochet and Dousset, 1999). Many animal models of M S feature mostly acute attacks and full recovery often not associated with destruction of myelin sheath. M S , in contrast to E A E is chronic, lasting a lifetime, and commonly manifests episodes of inflammation in the white matter, leading to permanent disabilities. Furthermore, M S is a spontaneous chronic disorder of the C N S within a heterogeneous population in contrast to E A E , which is an experimentally induced acute inflammatory reaction, mostly 30 in inbred rodents. These observations indicate that, while helpful in the study of the basic mechanisms involved in the immune attack on the C N S , E A E does not adequately represent M S pathogenesis and lacks the clinical complexity of M S . Studies of M S are further complicated by unidentified environmental influences and their interactions with individual immuno-genetic backgrounds. One approach to advance our understanding of the M S pathophysiology is to study immune-mediated diseases of the C N S associated with well-known etiology. A n example of such a disease is H A M . The presence of activated T cells associated with proinflammatory cytokines as well as elevated levels of IgG and the presence of oligoclonal bands in the C S F of both H A M and M S patients indicate that demyelinating factors in both diseases might be similar. While there are clear clinical and histopathological differences between H A M and M S , there are subsets of M S patients such as those with progressive M S that are clinically similar to H A M (Kira et al., 1993, Godoy et al., 1995). Magnetic resonance imaging studies of the brain and spinal cord may also be indistinguishable. In general, H A M and M S share the common findings of demyelination and axonal damage associated with inflammatory cell infiltrates in the C N S (Moore et al., 1989). On the one hand, multiple sclerosis is a common neurological disease among people of European descent in British Columbia. On the other hand, the number of H A M patients is very few and confined mostly to West Coast Amerindian populations. In fact, the total number of known H A M patients in British Columbia, Canada is believed to be 15 cases (Oger, personal communication). Furthermore, unlike M S patients who live mostly in urban areas, the H A M patients mostly live in remote regions. Therefore, the low number of patients combined with inaccessibility makes phenotypic and functional 31 immunological studies that require fresh blood in H A M patients a particularly challenging task. 1.8 R A T I O N A L E , H Y P O T H E S E S A N D O B J E C T I V E S 1.8.1 Studies of lymphocyte subsets in HAM The technique of flow cytometry allows for large number of cells to be analyzed and divided into subsets based on expression of surface antigens (Tragnos, 1984) and is particularly suitable for quantification of immune cells (Landay, 1988). The development of fluorochrome-conjugated monoclonal antibodies with emission wavelengths, which can be separated spectrally, has contributed a great deal in assessing diseases. Fluorescein isothiocyanate (FITC) was the first fluorochrome to be developed for use in biology (Coon et al., 1950) and continues in common use today. The development of antibodies conjugated to the phycoerthyrin (PE) dye allowed simultaneous analysis of two different molecules with a single laser line (Oi et al., 1982), thus providing more information than single color analyses. F low cytometry has also been used to study lymphocyte subsets in peripheral blood of H A M patients. However, to date, most lymphocyte subset studies in H A M have relied on single color analysis of lymphocyte expressing markers of activations confined to H L A - D R , or CD25. In the last few years due to increased development and availability of monoclonal antibodies against lymphocyte surface antigen, much has been learned regarding the immunoregulatory roles that various lymphocyte subsets play in both health and diseases. Some of the lymphocyte subsets characterized by monoclonal antibodies 32 have been assigned to participate in a defined immunological function(s). T lymphocytes are believed to play a critical role in the immunopathogenesis of H A M . Therefore to further distinguish and characterize immunoregulatory lymphocytes subsets, we investigated lymphocyte subsets employing two-color flow cytometry using a panel of well defined monoclonal antibodies against T lymphocyte markers of activation and adhesion related antigens (CD molecules) in a group of patients with H A M , and compared the results with those of H T L V - I asymptomatic carriers, and seronegative controls. The Principal features of the C D molecules referenced in this thesis are indicated in Table 1.3. One of the immunological hallmarks of H T L V - I infection is spontaneous T cell proliferation in culture and this phenomenon has been reported to be more intense in H A M than in carriers (Itoyama et al., 1988). To investigate whether this difference in T cell spontaneous mitogenesis is accompanied by changes in cell surface phenotypes, we also studied lymphocyte subsets after 2 days in culture without mitogen. This timing selection was based on our laboratory observations that spontaneous M N C proliferation initiates after 2 days in culture. 1.8.2 Studies of Lymphocyte-endothelial cell adhesion in HAM and MS To understand the pathology of H A M and M S it is also important to define the influences that permit the circulating immune cells to enter the C N S , and also the factors that downregulate the inflammatory cell invasion of the C N S . A prerequisite to the passage of lymphocyte across blood vessel walls is binding to endothelial cells. In vitro studies have indicated that adhesion of lymphocytes to endothelium is increased by 33 activation of lymphocytes (Brown et al., 1993) and it is these cells rather than quiescent lymphocytes that are more likely to penetrate the blood-brain barrier (Wekerle et al., 1986). A s circulating lymphocytes are activated in patients with H A M and M S (Hafler et al., 1985; Jacobson et al., 1988), this current study was undertaken to determine whether adhesion of H A M and M S lymphocyte to endothelium in vitro is augmented as well . We studied this interaction in an in vitro model that measures the binding of 5 1 Cr-labeled human lymphocyte to endothelial cell monolayers (Brown et al., 1993). Using this system, the contribution of different well-known adhesion molecules in lymphocyte binding to endothelium was also investigated by monoclonal antibody blocking. Due to difficulties in obtaining and growing endothelial cells from human sources, several laboratories have relied extensively on the recent availability of endothelial cell lines to investigate adhesion, transmigration and the responsiveness of the endothelium to pro- and anti-inflammatory agents. ECV-304 was reported to be a spontaneously transformed and immortalized human umbilical vein endothelial cell line. ECV-304 cells do not require special growth factors, can be maintained in culture indefinitely, and have been described to display and retain most of the characteristic markers of endothelial cells such as Ulex europaeus agglutunin-1 binding and secretion of von Willebrand's factor (Takahashi et al, 1990; Bowie et al., 1995; Hughes, 1996; Dolman et al., 1997). ECV-304 has also been previously used in adhesion as well as transmigration assays (Romanic and Madri , 1994; Bowie et al., 1995; Romanic et al., 1997; Kuchler-Bopp et al., 1999). In a part of this project, we also utilized this cell line as a source of endothelial cells for adhesion and inhibition of adhesion assays. However, once this project was initiated, we were informed ( A T C C official letter) that E C V - 3 0 4 is 34 a subclone of the epithelial bladder cancer cell line T-24 (Dirks et al., 1999), rather than an endothelial cell line. Since ECV-304 has been used both by us and other investigators, we further wanted to compare it to H U V E C and assess its relevance in adhesion assays. Thus experiments were undertaken in which the adherent properties of E C V - 3 0 4 cell line for lymphocytes were compared with those of H U V E C . 1.8.3 Studies of immune modulating agents in MS Three different preparations of IFN-P, namely Avonex™, Betaseron® and Rebif®, are currently in use for the treatment of relapsing and remitting M S patients. These drugs were shown in controled trials to be effective in reducing the frequency of relapses, the number of demeylinating plaques and suppression toward disability. The beneficial effect of IFN-B in M S is likely due to immuno-modulating properties of IFN-B and not due to suppression of viral infections (Panitch, 1994). Nevertheless, the relative units of these IFN-P are all measured by a viral plaque assay and represent the antiviral potency of the drugs, which may not correlate with their beneficial effect in M S . In trials, evaluating the safety and efficacy of the three IFN-P, different doses, dosing regimen, and application routes were used. Due to differences in study design and populations, no direct comparison can be made on efficacy of these three IFN-P in use. Therefore, the optimal dose for treatment of M S with each of the IFN-P in use is still under debate. In an approach to resolving this issue, we directly compared the effects of each of the three preparations of IFN-P in use in an in vitro assay representing an immunomodulating model that relies on inhibition of T-cell dependent B cell activation. 35 More specifically, the potency of the three IFN-P was calculated from their ability to suppress P W M induced IgG secretion in the culture supernatant. The potential roles of L F A - 1 adhesion molecules and IL-10 in M S are addressed in the above paragraphs. During the course of this thesis, two separate double blind, placebo controlled, multicenter trials were initiated by Schering-Plough and ICOS Corporation to respectively, test the safety and efficacy of IL-10 and anti-LFA-1 in relapsing-remitting M S . The University of British Columbia multiple sclerosis clinic was one of the trial sites. Therefore, we had an opportunity to study the potential in vivo effects of IL-10 and Ant i -LFA-1 on lymphocytes surface activation and adhesion related antigens using flow cytometry. IFN-B has been shown to favorably alter the disease course of relapsing-remitting multiple sclerosis patients. This clinical efficacy is accompanied by a more profound reduction in the number and size of lesions as measured by gadolinium enhanced M R I (Paty et al., 1993). Thus, it is possible that IFN-P may mediate its effect in M S at least in part by affecting the interaction of blood M N C with the B B B and inhibiting the migration of M N C into the C N S . However, the mechanisms by which IFN-P works in M S remain unclear. Since an important pathologic feature of M S is the transmigration of leukocytes across the B B B into the C N S , we proposed that a potential mechanism of action of IFN-P could be due to the ability of the IFN-P to inhibit adhesion of circulating lymphocytes to endothelium. 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A fl .3 S 2 kH -fl -fl s ' c CD > CD • - f l S - f l " _ H 60 Id fl fl 2 - f l ° ^ oo +-» (D t i 60 2 60 t50 "HS g * fl CD « O -f l fl ^ - f l 00 O AH VH CD OH O ro C H A P T E R T W O M A T E R I A L S A N D M E T H O D S 2.1 S T U D Y S U B J E C T S 45 2.2 P R E P A R A T I O N OF P E R I P H E R A L B L O O D M O N O N U C L E A R C E L L S 48 2.3 M O N O C L O N A L A N T I B O D I E S A N D T W O - C O L O R F L O W C Y T O M E T R Y 48 2.4 C U L T U R E OF H U M A N U M B I L I C A L V E I N E N D O T H E L I A L C E L L S 49 2.5 C U L T U R E OF ECV-304 C E L L L I N E 51 2.6 A D H E S I O N A S S A Y 52 2.7 M O N O C L O N A L A N T I B O D Y B L O C K I N G STUDIES 53 2.8 P O K E W E E D M I T O G E N - I N D U C E D IgG S E C R E T I O N 53 2.9 D E T E R M I N A T I O N OF IgG C O N T E N T B Y E L I S A 54 2.10 S T A T I S T I C A L A N A L Y S I S 55 44 M A T E R I A L S A N D M E T H O D S 2.1 S T U D Y S U B J E C T S The human T cell lymphotropic virus type I (HTLV-I ) associated myelopathy ( H A M ) patients and H T L V - I carriers involved in this study were seen at the Vancouver Hospital and Healthy Sciences Center, University of British Columbia Site ( V H & H S C / U B C ) outpatient medical clinic. The diagnosis of H A M was based on clinical criteria (Osame et al., 1987; Oger et al., 1993). This included patients with gradual and progressive spasticity that had the following characteristics: 1) antibody titers to H T L V - I in serum and C S F ; 2) predominantly upper motor neuron disorder, symmetrical, sensory and bladder disturbance; and 3) presence of adult T-cell leukemia-like cells (cells with lobulated nuclei) in both peripheral blood and C S F . The presence of H T L V - I in the investigated H A M and H T L V - I carriers was previously confirmed by P C R on blood mononuclear cells (Dekaban et al., 1993). For comparing lymphocyte subsets in H A M , H T L V - I carriers and healthy controls by flow cytometry, blood samples were obtained from 7 H A M patients, 3 males and 4 females whose age ranged from 37 to 79 years (mean ± S.D., 56.4 ± 15.9 years), 9 H T L V - I carriers, 3 males and 6 females (age range 33-88, 52.2 ± 17.4 years) and 10 healthy subjects, 5 males and 5 females (age range 23-67, 41.1 ± 13.8 years). For flow cytometric analysis of lymphocytes after 2 days in culture, blood samples of 7 of the 7 H A M patients, 7 of the 9 H T L V - I carriers, 3 males and 4 females (age range 34-69, 48.5 ± 1 1 . 2 years) and 8 of the 10 healthy subjects, 4 males and 4 females (age range 25-67, 43.7 ± 13.7 years) were used. For the adhesion assays, comparing H A M and n o n - H A M , blood was obtained from 45 8 patients with H A M (4 males, and 4 females) whose ages ranged from 43 to 79 years (56.8 ± 1 3 . 8 years). Blood was also obtained from 8 n o n - H A M (4 H T L V - I seropositive carriers, 46.8 ± 10.2 years and 4 healthy control subjects, 40.5 ± 9.7). These were 3 males and 5 females (age range 33-57, 43.6 ± 9.8 years). Seven of the H A M patients, all the H T L V - I carriers and 4 of the healthy controls were Coastal Amerindians from British Columbia, Canada. The multiple sclerosis (MS) patients who participated in this study were seen at the V H & H S C / U B C M S clinic. A l l M S patients had clinically definite M S as were diagnosed according to Poser's criteria (Poser et al., 1983). This means patients qualified i f they had two attacks and clinical evidence of two separate lesions or two attacks and clinical evidence of one lesion and paraclinical evidence of another separate lesion. Furthermore, the attacks should involve different parts of the central nervous system (CNS) and be separated by a period of at least one month. A n attack in M S is defined as the occurrence of one or more symptoms of neurological dysfunction that lasts more than 24 hours and which is not due to temporary factors such as fevers. None of the M S patients had other chronic diseases and they had not received anti-inflammatory or immunoregulatory drugs for at least 2 months preceding the tests. The relapsing-remitting (RR) M S patients who were free of clinical attacks during the 2 months prior to the study were classified as stable (s) R R - M S . Clinically active secondary progressive (SP) M S patients were those who had lost at least 1 point on Extended disability Status Scale (EDSS) (Kurzke, 1984) during the last 6 months preceding the assays. For the adhesion assays using human umbilical vein endothelial cells ( H U V E C ) as a source of endothelial cells in comparing multiple sclerosis (MS) and healthy controls, subjects 46 included 12 S P - M S , 4 males and 8 females (age range 32-69, 54 ± 11.3 years), 14 sRR-M S disease, 3 male and 11 female (age range 18-58, 40 ± 9.7 years), and 18 healthy controls, 11 male and 7 female.(age range 27-65, 44.9 ± 12.2 years). The adhesion assays were also done using ECV-304 cell line as substrate in comparing M S and healthy controls, subjects were 12 s R R - M S , 2 male and 10 female (age range 32-56, 43.2 ± 8.8 years) and 12 healthy controls, 2 male and 10 female (age range 21-54, 38.7 ± 9.4 years). For comparing pokeweed mitogen-induced IgG secretion, subjects were 39 sRR-M S , 9 male and 30 female (age range 22-57, 41.8 ± 9.6) and 23 healthy controls, 6 male and 17 female (age range 21-60, 38.6 ± 8.9). Healthy controls included in this study were patients' relatives/companions and V H & H S C personnel who were free of chronic infection and inflammation. A l l blood samples from patients and healthy controls were obtained with written informed consent from the donors. The characteristics of individual M S patients participating in the trial with recombinant anti-LFA-1 (Hu23F2G, ICOS C O R P O R A T I O N , Bothel, W A ) are stated in Table 2.1. Two-color flow cytometric analysis of lymphocyte subsets were performed at screening and 5 days post treatment. F low cytometric analysis of lymphocyte subsets were also done at screening and at 2 and 7 days post treatment for 1 R R - M S patient participating in a separate trial with recombinant human interleukin-10 (rhuIL-10, S C H 5200, Schering-Plough). However, this trial was still blinded. 47 2.2 PREPARATION OF PERIPHERAL BLOOD MONONUCLEAR CELLS Peripheral blood was collected in heparinized (50 U/ml) Vacutainer tubes (Beckton Dickinson, Mountain View, C A ) . Blood mononuclear cells ( M N C ) were isolated by density-gradient centrifugation on Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) at 400g at room temperature for 30 min. Interface cells were washed twice in C a + 2 - and Mg + 2-free Hanks' balanced salt solution (Gibco, Grand Island, N Y ) at 300g at 4°C for 10 min. The viability of M N C was greater than 95% as measured by Trypan blue exclusion. In some experiments blood M N C were cultured as follows: After suspending at 1 X 10^ cells/ml in medium consisting of RPMI-1640 (Gibco) supplemented with 10% heat-inactivated fetal calf serum (Gibco), 25 m M H E P E S , 100 pg/ml streptomycin, 100 U / m l penicillin (Gibco) (complete RPMI-1640), cells were cultured for 2 days at 37°C in a 5% C02-humidified atmosphere and 95% air in a 25-cm^ tissue culture flask (Corning, Cambridge, M A ) without mitogen. 2.3 MONOCLONAL ANTIBODIES AND TWO-COLOR FLOW CYTOMETRY Fluorochrome-labelled monoclonal antibodies (mAbs) to the following cell surface antigens were used as outlined in Table 2:2: CD45, CD14, CD19, CD16, CD56, C D 3 , C D 4 , C D 8 , H L A - D R , CD25, CD57, CD26, CD27, CD69, CD49d, C D 6 2 L , CD54 (Beckton Dickinson, San Jose, C A ) and C D 4 5 R A , CD29, CD38, CD28, CD30 (Coulter 48 Immunology, Hialeah, FL) . Controls included nonspecific IgG2a and nonspecific I g G l (Beckton Dickinson). After washing, M N C were divided into aliquots each containing 3-5 X 10^ cells and stained with specific mAbs at dilutions recommended by the manufacturer. After incubation for 30 min at 4°C, the cells were washed twice with phosphate-buffered saline (PBS) supplemented with 1% F C S and 0.1% NaN3 and fixed in 500wl of 1% paraformaldehyde in P B S , 1% F C S , 0.1% NaN3. Two color immunofluorescence was recorded using a FACStar® plus (Becton Dickinson, Mountain View, Ca). Lymphocytes were gated on their forward scatter (FSc) and side scatter (SSc) characteristics resulting in more than 95% of them being C D 4 5 b r i g h t C D 1 4 - . The FITC and P E gains were optimized by using FITC-nonspecific IgG2a and PE-nonspecific I g G l . Data analysis was performed using CELLQuest™ software. The operation of the FACStar® equipments and analysis of data was carried out in the Department of Medicine, Divis ion of Neurology at Vancouver Hospital and Health Sciences Center by Abdulaziz A l -Fahim. 2.4 C U L T U R E O F H U M A N U M B I L I C A L V E I N E N D O T H E L I A L C E L L S Primary and secondary cultures of human umbilical vein endothelial cells ( H U V E C ) were obtained from Dr. Doronini-Zis laboratory and established as described by slightly modified methods of Jaffe et. al. (Jaffe et al., 1973). In brief, umbilical cords obtained at normal deliveries were perfused with H B S S . The umbilical veins were then treated with 0.1 % collagenase (Sigma Chemical Co., St. Louis, M O ) in M l 9 9 medium 49 for 15 min at 37°C water-bath. Subsequently, the collagenase suspension was harvested and H U V E C were obtained by centrifugation. The pelleted cells were suspended and maintained in Medium 199 (StemCell Technologies Inc., Vancouver, B C ) supplemented with 10% heat-inactivated horse serum (Gibco), 25 m M H E P E S (Gibco), 20 ug/ml endothelial cell growth supplement (Sigma Chemical Co.), 100 ng/ml heparin (Sigma Chemical Co.), 100 ng/ml penicillin, 100 ng/ml streptomycin and 2.5 [ig/m\ amphotericin B (Gibco). The endothelial nature of isolated cells was previously confirmed by Dr. Dorovini-Zis laboratory personnel according to their binding for factor VHI-related antigen and of Ulex Europaeus Agglutinin I (UEA-I) lectin as described (Dorovini-Zis et al., 1991). For demonstration of factor VIII related antigen, cultured monolayers were incubated with a 1:100 dilution of polyclonal antibodies to rabbit anti-factor VIII antigen (Dakopatts). To demonstrate the binding of U E A - I lectin by endothelial-specific receptors, the cultured monolayers were incubated with 1:400 dilution of U E A - I lectin (Vector, Mississauga, Ontario). After washing, they were incubated with a 1:100 dilution of rabbit antiserum to U E A - I . Subsequently the monolayers were incubated with a 1:400 dilution of HRP-conjugated goat anti-rabbit IgG (Jackson Immunoresearch Laboratories). After further washing, cultured monolayers were incubated with amino-ethyl-carbazol, and counterstained with hematoxylin. Stained monolayers were then examined under a light microscope (Nikon, Labphot). H U V E C were grown to confluence on fibronectin (Sigma Chemical Co. , 100 ng/ml) coated 96-well flat bottom microtitre plates (Falcon, Beckton Dickinson, Franklin Lakes, N J . Culture media were changed every 2-3 days. 50 To examine the binding of M N C to cytokine-treated H U V E C , monolayers were treated for 48 h with 100 U / m l of human recombinant interferon-gamma (IFN-y) (Chemicon International Inc., Temecula, C A ) or 50% filtered (0.2 pm, Gelman Sciences, A n n Arbor, MI) murine L-929 fibrosarcoma supernatant prior to adhesion assay. Confluent monolayers of H U V E C were obtained after 7-11 days of culture at 37°C in a 5% C02-humidified atmosphere and 95% air. 2.5 C U L T U R E O F E C V - 3 0 4 C E L L L I N E E C V - 3 0 4 cells (American Type Culture Collection, Rockvil le, M D ) were cultured in M-199 medium containing 100 pg/ml penicillin, 100 pg/ml streptomycin, 2.5 pg/ml amphotericin B (Gibco) and 10% heat-inactivated fetal bovine serum (Gibco) (culture medium). ECV-304 were grown to confluence on 0.5% (w/v) gelatin (Sigma) coated 25mm tissue culture flasks (Corning) at 37°C in a 5% C02-humidified atmosphere and 95% air. When confluent monolayers of ECV-304 had formed, the cells were detached from the culture flasks by brief (1-2 min) treatment with 0.025% trypsin (Sigma) in P B S . The enzyme digestion was arrested by addition of 10 ml E C V - 3 0 4 culture medium. ECV-304 was pelleted at 400g for 10 min, resuspended in culture medium to 1 X 10 5 cells/ml and 100 pi added to each well of fibronectin coated 96-well flat bottom microtitre plates (Falcon). Culture media were changed every 2-3 days and confluent monolayers were obtained after 4-6 days of culture. 51 2.6 A D H E S I O N A S S A Y Cultured M N C were pelleted by centrifugation and 200 u C i of N a 2 5 1 C r 0 4 ( ICN Biomedicals Inc, Costa Mesa, C A ) were added to 5 X I 0 6 cells in a total volume of 200 ul of complete RPMI-1640 medium. After incubation for 90 min at 37 °C, cells were washed three times and resuspended at 1 X 10^ cells/ml. Confluent endothelial cells or ECV-304 monolayers in 96-well flat-bottom microtitre plates were washed twice with pre-warmed R P M I 1640 supplemented with 10% heat inactivated fetal calf serum medium, and 100 ul of fresh medium was added to each well . Then 1 X 10 5 5 1 Cr- labe l l ed M N C were added in a further 100 ul volume of medium. After a 1 h incubation at 37°C, non-adherent cells were removed by gently washing the monolayers five times with 200 ul of pre-warmed medium. Wells were examined by phase-contrast microscopy before and after washing to determine the evenness of cell settling and potential damage to the endothelial/ECV-304 cells monolayers during washing. The H U V E C or ECV-304 monolayers and remaining adherent cells were lysed by the addition of 200 ul of 0.1 M HC1. The lysate was collected and counted in a y counter (Beckman gamma 5500). Each assay was performed using four to six replicate wells. The percentage of adhesion to the H U V E C or E C V - 3 0 4 monolayer was calculated as follows: C P M in 100 nl lysate % adherence = — X 100 C P M in 100 ul original cell suspension 52 2.7 MONOCLONAL ANTIBODY BLOCKING STUDIES Antibodies directed against V L A - 4 (CD49d), I C A M - 1 (CD54), and L-selectin (CD62L) (all from Beckton Dickinson) and L F A - 1 (CD11/CD18, ICOS, Bothel, W A ) , were diluted in medium to a final concentration of either 2.5 pg/ml or 0.5 pg/ml and were added to H U V E C or ECV-304 monolayers. Then, 1 X 10 5 5 1 Cr- l abe l l ed cells were added. Each assay was performed using four to six replicate wells. Control m A b included was mouse IgG2b isotype anti-dansyl, (monoclonal antibody specific for hapten dansyl (5-[dimethyl amino naphthalene-1 sulfonyl) (PharMinogen Canada, Mississauga, ON). Percent inhibition was calculated as: C P M in 100 pi lysate with inhibitor % inhibition = [1- ( )] X 100 C P M in 100 pi lysate without inhibitor 2.8 POKEWEED MITOGEN-INDUCED IgG SECRETION Pokeweed-mitogen-induced IgG secretion was carried out in vitro as previously described (O'Gorman et al., 1988). Briefly, mononuclear cells ( M N C ) isolated by Ficol l -hypaque (Pharmacia) density gradient centrifugation from peripheral blood were washed four times in C a + 2 and M g + 2 free Hanks' balanced salt solution (Gibco) and one time with the culture medium consisting of R P M I 1640 with 10% fetal calf serum, 2 m M L -glutamine and 2 mg of genitmycine per 100 ml . Afterward, the blood M N C were 53 suspended at 10 6 M N C in 1 ml of the culture medium with pokeweed mitogen ( P W M , Gibco) at the optimal final dilution of 1:300 in 12 x 75 mm plastic capped tubes (Falcon). Control cultures, consisting of unstimulated blood M N C , were set up in parallel for each sample. The effects of different preparations of IFN-B on PWM-induced IgG secretion was tested by adding varying doses (ranging from 0.000384 to 312 ng/ml) of human recombinant interferon fi to the culture. The three interferons currently available for use in R R - M S that were also used in this assay are Betaseron® (IFNB-lb , Berlex Laboratories), Avonex™ (IFNfi-la, Biogen Inc.), and Rebif® (IFNB-la , Serono). After 7 days at 37°C and 5% CO2 in air, the cultures were centrifuged at 400 X g for 10 min and the cell free supernatants were harvested for IgG content. 2.9 D E T E R M I N A T I O N O F IgG C O N T E N T B Y E L I S A IgG content of the . supernatant was measured by an enzyme-linked immunosorbent assay (ELISA) . Microtitre plates (Maxisorb, Nunc, Rosklide, Denmark) were coated with 100 ul of goat anti-human IgG (Cappel, West Chester, P A ) at 10 ng/ml diluted in 0.05 M carbonate-bicarbonate buffer (pH 9.6), incubated overnight at 4°C. A l l washes were done with P B S containing 0.05% (v/v) Tween-20. The plates were washed 3 times then blocked with P B S containing 0.1% bovine serum albumin (BSA) for 1 h at room temperature. Serial dilution of known concentration of human IgG (NIH, Betheda, M D ) were used as standard in parallel with samples to be tested and added to the wells for 1 h at room temperature. The plates were washed as above and 1:1000 dilution of alkaline phosphatase conjugated goat anti-human IgG (Biosource, Camarillo, C A ) was 54 added. After 1 h at room temperature, the plates were washed and then developed by adding 100 pi of the phosphatase substrate ;?-nitrophenyl phosphate (Sigma) at 1 mg/ml in diethanolamine buffer. After 30 min, the absorbance at 405 nm was read on a specrophotometer ( M R X microplate reader, Dynex Technologies) and the results were expressed as ng/ml. In each experiment, a 10-point standard curve was generated from a standard human serum containing known concentration of IgG. The level of IgG in the unknown samples was then derived from the standard curve using Revelat ion™ software. A l l samples were run in triplicates. 2.10 S T A T I S T I C A L A N A L Y S I S Data are reported as mean ± S E M or mean ± S.D where indicated. Statistical analysis utilized paired and unpaired Student Mest as indicated with p<0.05 accepted as statistically significant. The differences between the inhibitory effects of Avonex™, Betaseron®, and Rebif® (3-interferon on PWM-induced IgG secretion were assessed using two-way analysis of variance ( A N O V A ) . If the differences between the three P-interferon treatments were significant, Tukey multiple comparison test was used to rule out the probability of type I error (erroneously declaring something by chance). Student t- tests were performed using Stat Works™ software. Two-way A N O V A and Tukey analyses were done in S-Plus™ software. 55 Table 2 .1 Individual M S Patient Characteristics and Treatment Part icipat ing in I C O S T r i a l Patient No . Age (years) Sex Treatment D E S #0801 34 Male Hu23F2G (2.0 mg/kg) M C E #0802 36 Female Hu23F2G (1.0 mg/kg) M O S #0803 36 Female Placebo TES #0804 48 Female Methy lpredni so lone 56 Table 2.2 Monoclona l Ant ibody Pairs Used for Lymphocyte Subset Analysis Phycoerythrin-conj ugated Fluorescein-conjugated Nonspecific IgGl Nonspecific IgG2a A n t i - C D 14 (Leu M3) Ant i -CD45 ( L C A ) A n t i - C D 19 (Leu 12) Ant i -CD3 (Leu 4) Ant i -CD4 (Leu 3 a) Ant i -CD3 Ant i -CD8 (Leu 2a) Ant i -CD3 Ant i -CD56 (NKH-1) A n t i - C D 16 (FC YRIII) Ant i -CD4 A n t i - C D 4 9 d ( V L A - 4 a ) Ant i -CD8 Ant i -CD49d Ant i -CD4 A n t i - C D 6 2 L ( L E C A M - 1 ) Ant i -CD8 A n t i - C D 6 2 L Ant i -CD54 ( ICAM-1) Ant i -CD3 Ant i -CD4 A n t i - C D 4 5 R A Ant i -CD4 Ant i -CD29 Ant i -CD8 Ant i -CD28 Ant i -CD8 Ant i -CD57 (Leu 7) Ant i -CD27 Ant i -CD3 Ant i -CD4 A n t i - H L A - D R (HLA-II) Ant i -CD8 A n t i - H L A - D R Ant i -CD4 Ant i -CD25 (Tac) Ant i -CD8 Ant i -CD25 Ant i -CD4 Ant i -CD38 Ant i -CD8 Ant i -CD38 Ant i -CD3 Ant i -CD26 Ant i -CD3 Ant i -CD30 Ant i -CD3 Ant i -CD69 (AIM) 57 C H A P T E R T H R E E R E S U L T S 3.1 H T L V - I I N F E C T I O N 60 3.2 L Y M P H O C Y T E S U B S E T S I N H A M , H T L V - I C A R R I E R S A N D H E A L T H Y C O N T R O L S 60 3.2.1 Double staining for CD3+, CD4+, and CD8+ T cells 61 3.2.2 Double staining for putative markers of function 61 3.2.3 Double staining for markers of activation 61 3.2.4 Double staining for markers of adhesion 62 3.3 B L O O D M N C - H U V E C A D H E S I O N IN H A M 63 3.3.1 Adhesion of blood M N C to IFN-y and L-929 supernatant treated H U V E C 63 3.3.2 Adhesion of H A M and n o n - H A M blood M N C to L-929 supernatant treated H U V E C 63 3.3.3 Effects of anti-adhesion molecule antibodies on the adhesion of H A M patients blood M N C to L-929 supernatant treated H U V E C 63 3.4 B L O O D M N C - H U V E C A D H E S I O N IN M S 64 3.4.1 Adhesion of healthy, s R R - M S and S P - M S blood M N C to H U V E C 64 3.4.2 Effects of anti-adhesion molecule antibodies on the adhesion of R R - M S and healthy blood M N C to H U V E C 64 3.4.3 Effects of anti-adhesion molecule antibodies on the adhesion of R R - M S blood M N C to untreated and IFN-y treated H U V E C 65 3.5 B L O O D M N C - E C V - 3 0 4 A D H E S I O N IN M S 66 3.5.1 Adhesion of healthy and s R R - M S blood M N C to ECV-304 ..66 3.5.2 Effects of anti-adhesion molecule antibodies on the adhesion of s R R - M S and healthy blood M N C to ECV-304 66 3.5.3 Adhesion of blood M N C to IFN-y and L-929 supernatant treated H U V E C and ECV-304 67 3.5.4 Effects of anti-adhesion molecule antibodies on the adhesion of R R - M S blood M N C to untreated and IFN-y treated E C V - 3 0 4 68 3.6 IN VITRO E F F E C T S OF IFN -P O N P W M - I N D U C E D IgG S E C R E T I O N A N D M N C - H U V E C A D H E S I O N 68 3.6.1 In vitro IgG secretion in healthy and s R R - M S 68 3.6.2 In vitro effects of IFN-B on PWM-induced IgG secretion 69 58 3.6.3 Comparing the in vitro effects of Avonex™, Betaseron® and Rebif® on PWM-induced IgG secretion 70 3.6.4 Effects of IFN-P on M N C - H U V E C adhesion 71 59 RESULTS 3.1 HTLV-I INFECTION The human T lymphotropic virus type I (HTLV-I ) has been identified as the etiological agent of the inflammatory and chronic progressive demyelinating disease called HTLV-I-associated myelopathy ( H A M ) . The major clinical features of H A M consist of spasticity and hyper-reflexia of the lower extremities, bladder disturbance, lower extremity muscle weakness, and sensory disturbance (Osame et al., 1986). H A M appears one or more decades following infection with H T L V - I . However, the onset of H A M is substantially shorter in patients infected by transfusion of HTLV-I-contaminated blood than in patients who acquire the infection by breast-feeding or by the venereal route (Osame et al., 1990). The mean age of onset is 43 years and the male: female ratio of occurrence is 1:2.9. The lifetime risk of development of H A M among H T L V - I carriers is estimated to be less than 5% and most (-95%) individuals chronically infected with H T L V - I remain clinically asymptomatic (Kaplan et al., 1990; Maloney et al., 1998). 3.2 LYMPHOCYTE SUBSETS IN HAM, HTLV-I CARRIERS AND HEALTHY CONTROLS Lymphocytes were analyzed at isolation and after 2 days in culture without mitogen. The mean percentage of each lymphocyte subset for the patients with H A M , the H T L V - 1 carriers and controls are presented in Tables 3.1 to 3.4. Statistically 60 significant differences (p<0.05) between H A M , carriers, and controls are identified in Tables 3.1 to 3.4. 3.2.1 Double staining for CD3+, CD4+, and CD8+ T cells The mean percentage of CD3+, CD4+ and CD8+ T cells were not significantly different between H A M patients, H T L V - 1 carriers and seronegative controls (Table 3.1). 3.2.2 Double staining for putative markers offunction The mean percentage of CD4+CD29+ (memory/helper inducer) cells was higher in both H A M and carriers compared with controls (p<0.05) at isolation (Table 3.2). Furthermore, H A M patients had a significantly higher percentage of CD8+CD57+ (cytotoxic) cells compared with H T L V - I carriers and seronegative controls. A t isolation, the percentage of CD3+CD27- (primed T cells) was significantly higher in H A M patients compared with controls. No significant difference was observed between H T L V - I carriers and controls in the percentage of CD3+CD27-. 3.2.3 Double staining for markers of activation Table 3.3 shows markers of activation on CD3+, CD4+, and CD8+ subsets. The percentage of CD4+ cells coexpressing activation markers H L A - D R or CD25, and of CD8+ cells expressing H L A - D R were significantly higher in H A M patients and H T L V - I carriers than controls (p<0.05). The percentage of CD8+ cells coexpressing CD25 was low; however, after 2 days in culture without any mitogenic stimulation, a significant increase in the percentage of CD8+CD25+ population was observed in H A M patients but 61 not in carriers or controls. We also assessed CD4+ and CD8+ cells for the expression of the "activation" antigen CD38. There was no significant difference between H A M patients, carriers, and controls in percentage of CD4+CD38+ or CD8+CD38+ subpopulations at isolation. However, after 2 days in culture, the mean percentage of CD8+CD38+ cells increased significantly in H A M patients and carriers compared with controls (p<0.05). Interestingly, a significantly higher percentage of CD8+CD38+ cells were observed in patients with H A M compared with carriers. With respect to T cells coexpressing the early activation marker, CD69, we only observed significant increases in the percentage CD3+CD69+ in H A M and carriers after 2 days in culture. 3.2.4 Double staining for markers of adhesion The percentage of CD4+ and CD8+ cells co-expressing CD49d (a-chain of V L A - 4 ) was generally higher in patients with H A M and in H T L V - I carriers (Table 3.4) than in controls, but this difference reached significance only in carriers. There were significantly lower numbers of CD4+ and CD8+ cells co-expressing C D 6 2 L (L-Selectin) in H A M patients compared with healthy controls. There were also a significantly fewer double staining CD4+CD62L+ cells in H A M patients compared with H T L V - I carriers. Table 3.4 also reveals that, compared with controls, the percentage of mature T cells expressing adhesion molecule CD54 ( ICAM-1) was higher in patients with H A M and H T L V - I carriers than in controls, but this difference reached significance only in H A M ; however, after 2 days in culture this difference reached to a statistically significant level for both H A M patients and H T L V - I carriers. 62 3.3 B L O O D M N C - H U V E C A D H E S I O N I N H A M 3.3.1 Adhesion of blood MNC to IFN-y and L-929 supernatant treated HUVEC Table 3.5a shows that treatment of H U V E C with IFN-y (100 U/ml) or L-929 supernatant (50%) for 48 h resulted in significant increase in adhesion of M N C from two controls, two R R - M S and two H A M patients (p<.02). Similar results were obtained using cryopreserved M N C from two controls, two H T L V - 1 carriers and two H A M patients (Table 3.5b). Thus, it is likely that M N C maintain their binding characteristics to endothelial cells after cryopreservation. 3.3.2 Adhesion of HAM and non-HAM blood MNC to L-929 supernatant treated HUVEC Figure 3.1 summarizes the results of 8 paired assays comparing H A M and non-H A M (4- H T L V - I carriers and 4 healthy) controls blood M N C to L-929 supernatant treated H U V E C . The H A M patients' M N C adhered significantly more to activated H U V E C (mean 30%) than n o n - H A M cells (mean 19.1%) in 7 out of 8 assays. 3.3.3 Effects of anti-adhesion molecule antibodies on the adhesion of HAM patients blood MNC to activated HUVEC To assess the contribution of I C A M - 1 , V L A - 4 and L-selectin in promoting the binding of H A M derived M N C to endothelium, M N C where co-incubated with H U V E C in the continued presence of mAbs against each of theses adhesion molecules. Figure 3.2 show that the binding of M N C from H A M patients to L-929 supernatant activated 63 H U V E C was reduced by antibodies directed against V L A - 4 (mean 31% inhibition; p<0.001), I C A M - 1 (mean 43% inhibition; pO.OOl) , and L-selectin (mean 38% inhibition; p<0.001) at 2.5 ug/ml. A t 0.5 ug/ml of antibodies, significant inhibition only occurred with anti-ICAM-1 (mean 16% inhibition), and anti-L-selectin (mean 9% inhibition). 3.4 BLOOD MNC-HUVEC ADHESION IN MS 3.4.1 Adhesion of healthy, sRR-MS and SP-MS blood MNC to HUVEC Table 3.6 shows the results of adhesion comparing s R R - M S , S P - M S and healthy controls blood M N C to untreated H U V E C . The S P - M S patients' M N C adhered significantly more to H U V E C than healthy controls M N C (p<0.02). The adhesion of s R R - M S blood M N C were generally higher than that of healthy subjects. However, with the number of patients that we tested, this difference was not statistically significant. 3.4.2 Effects of anti-adhesion molecule antibodies on the adhesion of RR-MS and health blood MNC to HUVEC Several specific antibodies directed against adhesion molecules were used in order to determine the contribution of adhesion molecules in the adherence of R R - M S and healthy blood M N C to H U V E C . M N C where co-incubated with H U V E C in the continued presence of mAbs against each of L F A - 1 , I C A M - 1 , V L A - 4 and L-selectin or combinations of all of theses adhesion molecules. Irrelevant mAb against dansyl hapten was also used. Anti-dansyl mAb was selected because it does not react to any know 64 human leukocyte antigen. Results from six experiments using M N C from R R - M S are shown in Figure 3.3. A t 2.5 pg/ml, the anti-LFA-1 and anti-ICAM-1 produced a mean 54% and 28% inhibition of adhesion, respectively (p<0.01). A t 0.5 pg/ml of antibodies, inhibition with anti-LFA-1 (mean 47% inhibition) and anti-ICAM-1 (mean 16% inhibition) were still significant (p<0.02). Antibodies against V L A - 4 and L-selectin did not significantly inhibit M S M N C binding to H U V E C . Inclusion of a combination of m A b to L F A - 1 , I C A M - 1 , V L A - 4 , and L-selectin in the adhesion assay significantly increased the inhibition. However, the level of inhibition was not much higher than when m A b to L F A - 1 was used alone. Irrelevant m A b against dansyl hapten also did not influence the adhesion of blood M N C to H U V E C . Figure 3.4 shows results from 3 experiments measuring the inhibition of adhesion of healthy blood M N C to H U V E C . Similar results were obtained when M N C of healthy control subjects were used. These results indicate that the binding pathways of both R R - M S and control subjects M N C to resting H U V E C are similar and mainly utilize L F A - 1 / I C A M - 1 . 3.4.3 Effects of anti-adhesion molecule antibodies on the adhesion of RR-MS blood MNC to untreated and IFN-y treated HUVEC To compare the contribution of different adhesion molecules under both stimulatory and unstimulatory conditions in M N C - H U V E C adhesion, H U V E C was left untreated or treated with 100 U / m l of IFN-y for 48 h and the monoclonal antibody-blocking assays were subsequently performed. The results from three separate paired experiments using R R - M S blood M N C are shown in Figure 3.5. When H U V E C were untreated, antibodies directed against L F A - 1 and to a lesser degree I C A M - 1 produced 65 significant inhibition of adhesion, whereas ant i -VLA-4 or anti-L-selectin m A b had no significant effect. When H U V E C were stimulated with I F N - y , an t i -VLA-4 also significantly inhibited M N C - H U V E C interactions. These data indicate that adhesion of M N C to H U V E C mainly involve L F A - 1 / I C A M - 1 when H U V E C are untreated. However, when H U V E C are treated with IFN-y, in addition to L F A - 1 / I C A M - 1 , V L A -4 / V C A M - l pathway also mediates adhesion. 3.5 B L O O D M N C - E C V - 3 0 4 A D H E S I O N I N M S 3.5.1 Adhesion of healthy and sRR-MS blood MNC to ECV-304 Table 3.7 shows the results comparing adhesion of s R R - M S and controls blood M N C to untreated ECV-304 . In 12 paired assays, no significant differences were found between s R R - M S and healthy controls blood M N C in binding to ECV-304 . 3.5.2 Effects of anti-adhesion molecule antibodies on the adhesion of sRR-MS and healthy blood MNC to ECV-304 Contributions of different adhesion molecules to the adherence of s R R - M S and healthy blood M N C to ECV-304 was also determined using monoclonal antibody-blocking functional assays. M N C where co-incubated with ECV-304 in the continued presence of mAbs against each of L F A - 1 , I C A M - 1 , V L A - 4 and L-selectin or combinations of all monoclonal antibodies to theses adhesion molecules. Results from eight experiments using M N C from s R R - M S and eight experiments using M N C from healthy subjects are shown in Figures 3.6 and 3.7, respectively. At both 2.5 and 0.5 66 pg/ml, the anti-LFA-1 and anti-ICAM-1 significantly reduced adhesion of blood M N C in sPvR-MS and healthy subjects to untreated ECV-304 . Antibodies against V L A - 4 and L-selectin did not significantly inhibit binding of s R R - M S or healthy blood M N C to ECV-304 . Using a combination of m A b to L F A - 1 , I C A M - 1 , V L A - 4 , and L-selectin also significantly increased the inhibition; however, the level of inhibition was not much higher than when m A b to L F A - 1 was used alone. These results indicate that the binding pathways of both s R R - M S and healthy control subjects M N C to uninduced E C V - 3 0 4 are similar and mainly involve L F A - 1 / I C A M - 1 pathway. 3.5.3 Adhesion of blood MNC to IFN-y and L-929 supernatant treated HUVEC and ECV-304 In the next series of experiments, we directly compared adhesion properties of H U V E C and ECV-304 for M N C in parallel assays. Figure 3.8 shows the results from two separate experiments using blood M N C from two healthy, two s R R - M S and two H A M patients. In these experiments, H U V E C and ECV-304 were left untreated or treated with IFN-y (100 U/ml) or L-929 (50%) supernatant for 48 h prior to the adhesion assay. The results indicate that treatment of H U V E C with either IFN-y or L-929 supernatant significantly increases their adherence for blood M N C . However, similar treatment in ECV-304 fails to increase their adherence for M N C . 67 3.5.4 Effects of anti-adhesion molecule antibodies on the adhesion of RR-MS blood MNC to untreated and IFN-y treated ECV-304 To further compare the adhesion properties of ECV-304 to those of H U V E C for M N C , we determined the contribution of different adhesion molecules in binding to untreated or IFN-y treated ECV-304 . ECV-304 monolayers were untreated or treated with 100 U / m l of IFN-y for 48 h prior to the monoclonal antibody-blocking assays. For direct comparison, the M N C used in these experiments (Figure 3.9) are the same as those used in experiments of figure 3.5. Furthermore, the assays were also performed in parallel to that of the experiments of Figure 3.5. When ECV-304 monolayers were untreated, antibodies directed against L F A - 1 and to a lesser degree I C A M - 1 produced significant inhibition, whereas an t i -VLA-4 or anti-L-selectin m A b had no significant effect on M N C - E C V - 3 0 4 interaction (Figure 3.9). This is similar to what was seen when H U V E C was used as substrate in binding M N C (figure 3.5). However, unlike the results obtained using H U V E C (Figure 3.5), when ECV-304 monolayers were activated with IFN-y, anti-V L A - 4 did not inhibit M N C adhesion (figure 3.9). These data indicate that adhesion of M N C to ECV-304 involve L F A - 1 / I C A M - 1 whether or not ECV-304 are stimulated with IFN-y. 3 . 6 IN VITRO EFFECTS OF IFN-B ON PWM-INDUCED IgG SECRETION AND MNC-HUVEC ADHESION 3.6.1 In vitro IgG secretion in healthy and sRR-MS Table 3.8 shows that IgG concentration in unstimulated cultures was comparable in 39 stable relapse and remitting multiple sclerosis (sRR-MS) and in 24 healthy control 68 subjects (95 ± 52 ng/ml vs 116 ± 80 ng/ml, respectively). It increased significantly after pokeweed (PWM) stimulation in both sRR-MS and healthy controls. However, after PWM stimulation, the amount of IgG concentration was significantly higher in sRR-MS (2173 ± 1432 ng/ml) compared with healthy controls (1159 ± 913 ng/ml, p<0.02). Based on their response to PWM stimulation, s R R - M S and healthy subjects were divided to two distinct populations: one population of " low responder" subjects (producing <900 ng/ml IgG) and one population of "high responder" subjects (producing >900 ng/ml IgG). Table 3.8 further shows that the percentage of high responders was larger in sRPv-MS patients (66%) than in healthy controls (38%). 3.6.2 In vitro effects of IFN-P on PWM-induced IgG secretion The in vitro effects of different preparations of IFN-P were studied on blood MNC of six sRR-MS and five healthy subjects whom were categorized as "high responders" in response to PWM stimulation (producing >900 ng/ml IgG). In the absence of IFN-P, the concentration of IgG was similar for sRR-MS (2519 ± 629 ng/ml) and healthy (2476 ± 936 ng/ml). Figure 3.10a, and 3.10b show that Avonex™, Betaseron® and Rebif® used at concentration ranging from 30 ng/ml to 0.194 pg/ml all inhibited PWM-induced IgG secretion in a dose-related manner in both healthy (3.10a, 69% to 20%o inhibition) and sRR-MS (3.10b 76% to 21% inhibition) subjects. Comparing Figure 3.10a and b also indicates that IFN-P at different dilutions, tended to suppress PWM-induced IgG secretion more in sRR-MS than in healthy subjects. However, this difference did not reach a statistically significant level. The comparative inhibition of PWM-induced 69 IgG secretion in s R R - M S and healthy controls by Avonex™, Rebif® and Betaseron® are shown in Figures 3.10c, 3.1 Od, and 3.10e. 3.6.3 Comparing the in vitro effects of Avonex™, Betaseron® and Rebif® on PWM-induced IgG secretion Although all three preparations of IFN-p are available for clinical use in R R - M S , few data have been reported to directly compare their immunomodulatory effects. Therefore, we expanded this study to include an additional 16 "high responding" sRR-M S patients and compared the in vitro effects of Avonex™, Betaseron® and Rebif® in parallel for their ability to inhibit PWM-induced IgG secretion. This high responding group of 16 s R R - M S patients produced 91 ± 5 6 ng/ml of IgG (mean ± S E M ) spontaneously. However, when M N C were stimulated with P W M and in the absence of IFN-P, they produced 3070 ± 1507 ng/ml of IgG (mean ± sem). Figure 3.11 shows inhibition of PWM-induced IgG secretion using the same dose (ng/ml) of different IFN-P preparation. A t concentration of 312 ng/ml, no significant differences were found between Avonex™, Betaseron® and Rebif® in inhibiting PWM-induced IgG secretion, perhaps reaching a saturation point. However, at 31.2 ng/ml and 3.12 ng/ml, Avonex™ inhibited IgG secretion significantly more than Betaseron® (p<0.05). Moreover at 31.2 and 3.12 ng/ml, Rebif® also inhibited IgG secretion significantly more than Betaseron® (p<0.05). When we took into consideration the amount of IFN-B used in vitro as a approximate proportion of the daily recommended dose of each of these IFN-Bs (this is 300 pg, 44 pg, and 30 pg for Betaseron®, Rebif® and Avonex, respectively), Avonex™ 70 still had generally higher in vitro inhibitory effects on IgG synthesis compared with Betaseron® and Rebif® (Figure 3.12). However, these differences did not reach a statistically significant level. Furthermore, when we calculated the amount of IFN-B used in vitro as a fraction of weekly administration using their antiviral activity in Units (Log) Betaseron®, Avonex™ and Rebif® had equivalent activity with respect to their influence on PWM-induced IgG secretion (Figure 3.13). 3.6.4 Effects oflFN-pon MNC-HUVEC adhesion To determine the role of IFN-p in M N C - H U V E C adhesion, H U V E C and/or M N C were cultured in the absence or continued presence of IFN-P-lb (Betaseron®, 1000 U/ml) for 48 h. The IFN-P-lb was then washed and adhesion assays were carried out as in Materials and Methods. Table 3.9 shows that treatment of M N C with 1000 U / m l of IFN-p- lb for 48 h significantly reduced M N C adhesion to H U V E C in both R R - M S and healthy controls (p<0.001). In contrast, treatment of H U V E C with the same dose of IFN-P-lb had no significant effect on M N C adhesion. The combined treatment of H U V E C and M N C of R R - M S patients with IFN-P-lb did not result in further reduction of adhesion beyond what was seen with M N C treatment alone. 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S H © PH CU ^ CO fi o cu > CO O P H CU cu o H-H O C o ' „ o P H O OJ) C H ^ CO CU I H f i + f - H CU • * i-H <a ."fi PN 0 0 < CH CO CO 3 1-i-CU H-» fi „ o cS o o CO CO fi fi CO co ) H S H cu CU > > in © © © © V V C H & * Table 3.5a Adhesion of Blood M N C to Untreated and IFN-y or L-929 Treated H U V E C % Adhesion Subject N o treatment mean ± S E M I F N - y (100 U/ml) mean ± S E M L-929 (50%) mean ± S E M Healthy 12.7 ± 2 . 3 15.6 ± 2 . 5 * 16.1 ± 2 . 1 * M S 12.9 ± 0 . 7 14.7 ± 0 . 9 * 15.2 ± 1.1* H A M 15.3 ± 1.7 18.0 ± 1.7* 18.7 ± 1.4* Table 3.5b % Adhesion Subject N o treatment mean ± S E M IFN-y (100 U/ml) mean ± S E M L-929 (50%) mean ± S E M Healthy 11.7 ± 2 . 2 14.4 ± 2 . 2 * 15.5 ± 1.8* Carrier 13.0 ± 0.5 15.2 ± 1.3* 16.3 ± 1.2* H A M 2 1 . 7 ± 2 . 2 28.0 ± 2 . 8 * 25.9 ± 2 . 6 * Note. In experiments of Table 5a, M N C were from two healthy controls, two multiple sclerosis (MS) and two H A M patients. In experiment of Table 5b, M N C were from two healthy controls, two H T L V - 1 carriers and two H A M patients that were previously cyopreserved at -70 °C. In each experiment, M N C were added to H U V E C monolayers that were untreated or pretreated with I F N - y (100 U/ml) or conditioned medium containing L-929 (50%) supernatant for 48 h. Each number represents the mean ± S E M percentage of quadruplicates in two separate experiments. *p<0.02 compared with untreated monolayers. 76 Table 3.6 Adhesion of s R R - M S , S P - M S , and Healthy Subjects Blood M N C to H U V E C % Adhesion (mean ± S E M ) Healthy s R R - M S S P - M S (n=18) (n=14) (n=12) 12.4 ± 3 . 4 14.5 ± 3 . 0 16.1 ± 2 . 9 * Blood M N C s from healthy, stable relapsing-remitting multiple sclerosis (sRPv-M S ) , and secondary progressive multiple sclerosis (SP-MS) were incubated with untreated H U V E C for 1 hr. In these experiments the binding of M N C to H U V E C was expressed as the percentage of total binding. The M N C from S P - M S adhered significantly more to H U V E C than M N C from healthy subjects. Each assay was performed in four replicates. The numbers of subjects studied in each category are indicated in the parentheses. p<.02 versus healthy controls 77 Table 3.7 Comparat ive Adhesion of Healthy and R R - M S Blood M N C to E C V - 3 0 4 % Adhesion (mean ± S E M ) Healthy (n= 12) 17.1 ± 5 . 9 sRR-MS(n=12) 19.6 ± 5 . 1 Blood M N C s from stable relapsing-remitting (RR-MS) and healthy subjects were incubated with untreated ECV-304 . Each assay consisted of adding M N C from one sRR-M S and one healthy subject to ECV-304 monolayers. The number indicates the mean ± S E M from 12 paired assays. Each assay was performed using four replicate wells. N o significance differences were seen between s R R - M S and healthy subjects in binding to ECV-304 . 78 Table 3.8 In V i t r o I g G Secretion in s R R - M S and Healthy Subjects s R R - M S (n=39) Healthy (n=24) P W M - (ng/ml IgG) 95 ± 5 2 116 ± 8 0 P W M + (ng/ml IgG) 2173 ± 1 4 3 2 * 1159 ± 913 High responders 66% 38% (>900 ng/ml) The number of subjects for each category is indicated in parentheses. In these experiments blood M N C s were incubated without (PWM-) or with pokeweed mitogen (PWM+) for 7 days at 37°C. The IgG content of supernatant was measured by an E L I S A and expressed as mean ± S E M in ng/ml. The results indicate that stable relapsing and remitting M S (sRR-MS) patients produce significantly (p<0.02) higher amount of IgG in response to P W M compared with healthy controls. Furthermore, the percentage of individuals producing greater than 900 ng/ml of IgG in response to P W M ("high responders") are higher in s R R - M S than in healthy controls. *p<0.02 versus healthy controls 79 Table 3.9 Effects of I F N - p - l b on M N C - H U V E C Adhesion IFN-P-lb treatment of: Untreated M N C H U V E C M N C & H U V E C R R - M S (n=8) 13.2 ± 1 . 3 8.4 ± 1 . 3 * 14.6 ± 2 . 9 9.5 ± 1 . 9 * Healthy (n=7) 12.6 ± 1 . 8 7.8 ± 0.9* nd nd Blood M N C and/or H U V E C were pretreated with 1000 U / m l of IFN-P- lb (Betaseron®) for 48 hr prior to doing the adhesion assay. The results indicate that pretreatment of M N C with I F N - p - l b results in a significant reduction of their bindings to H U V E C in both healthy and R R - M S subjects. However, treatment of H U V E C with IFN-P does not influence M N C - H U V E C adhesion. Each assay was performed in four replicates and the number of subjects studied is indicated in parentheses. Results are expressed as mean ± S E M . *p<0.03 versus untreated H U V E C monolayers 80 Adhesion of H A M and N o n - H A M Blood M N C to Activated H U V E C Pair Number Figure 3.1. M N C from eight H A M patients and eight n o n - H A M controls (four H T L V - I carriers and four healthy) were incubated with L -929 supernatant activated H U V E C . Each assay consisted of adding M N C from one H A M and one n o n - H A M subject to H U V E C derived from the same umbilical cord vein. Each assay was performed using six replicate wells. The adhesion of H A M patients' M N C to H U V E C was significantly greater than that observed with n o n - H A M controls in seven out of eight paired assays. In panel (a) controls were H T L V - I carriers, while in panel (b) controls included were healthy subjects. Vertical bars represent S E M . *p<0.01 compared with n o n - H A M . 81 Figure 3.2. M N C from four different H A M patients were coincubated with anti-ICAM-1 (CD54), an t i -VLA-4 (CD49d), and anti-L-selectin (CD62L) antibodies at concentration of 2.5, 0.5 ug/ml or medium alone, during the assay. In these experiments, H U V E C monolayers were previously treated with conditioned medium containing L-929 (50%) supernatant. Each assay was performed using six replicate wells. Results are representative of four separate experiments and are expressed as mean ± S E M percentage inhibition of adhesion. *p< 0.05 compared with M N C incubated with medium. 82 Effect of Ant i -Adhes ion Molecule Antibodies on the Adhesion of M N C of R R - M S patients to Untreated H U V E C mAbs used Figure 3.3. M N C from six different R R - M S patients were co-incubated with recombinant and humanized (rhu) anti-LFA-1 (CD11/CD18), anti-ICAM-1 (CD54), an t i -VLA-4 (CD49d), anti-L-selectin (CD62L) , or combination of all anti-adhesion antibodies during the assay. Control included was anti-dansyl mAb. Each assay was performed using four replicate wells. Results are from six separate experiments and are expressed as mean ± S E M percentage inhibition of adhesion. *p<0.03 compared with M N C incubated with medium. 83 Effect of Anti-Adhesion Molecule Antibodies on the Adhesion of M N C of Healthy Subjects to Untreated H U V E C mAbs used Figure 3.4. M N C from three different healthy subjects were co-incubated with recombinant and humanized (rhu) anti-LFA-1 (CD11/CD18), anti-ICAM-1 (CD54), an t i -VLA-4 (CD49d), anti-L-selectin (CD62L) or combination of all antibodies during the assay. Control included was anti-dansyl mAb. Each assay was performed using four replicate wells. Results are from three separate experiments and are expressed as mean ± S E M percentage inhibition of adhesion. *p<0.02 compared with M N C incubated with medium 84 Effects of Anti-Adhesion Molecules antibodies on the Adhesion of M N C of R R - M S Patients to Untreated and IFN-y Treated H U V E C < < mAbs used Figure 3.5. M N C from three different R R - M S patients were coincubated with recombinant and humanized (rhu) anti-LFA-1 (CD 11 a/CD 18), anti-ICAM-1 (CD54), V L A - 4 (CD49d), anti-L-selectin (CD62L) or combination of all antibodies during the assay. H U V E C monolayers were untreated (a) or treated with 100 U / m l of IFN-y for 48 h prior to doing the assay (b). Each assay was performed using four replicate wells. Results are from three separate experiments and are expressed as mean ± S E M percentage inhibition of adhesion. *p< 0.001 compared with M N C incubated with medium. 85 Effect of Anti-adhesion Molecule Antibodies on the Adhesion of MNC of RR-MS Patients to untreated ECV-304 • 2.5 ug/ml M 0.5 ng/ml mAbs used Figure 3.6 M N C from eight different R R - M S patients were coincubated with recombinant and humanized (rhu) anti-LFA-1 (CD11/CD18), an t i -VLA-4 (CD49d), anti-ICAM-1 (CD54), anti-L-selectin (CD62L) or combination of all antibodies during the assay. Each assay was performed using four replicate wells. Results are from eight separate experiments and are expressed as mean ± S E M percentage inhibition of adhesion. *p< 0.01 compared with M N C incubated with medium. 86 Effects of anti-adhesion molecules antibodies on the adhesion of M N C of healthy subjects to untreated E C V - 3 0 4 • 2.5 pg/ml ^0 .5 pg/ml mAbs used Figure 3.7 M N C from eight different healthy subjects were coincubated with recombinant and humanized (rhu) anti-LFA-1 (CD11/CD18), an t i -VLA-4 (CD49d), anti-ICAM-1 (CD54), anti-L-selectin (CD62L) or combination of all antibodies during the assay. Each assay was performed using four replicate wells. Results are from eight separate experiments and are expressed as mean ± S E M percentage inhibition of adhesion. *p< 0.01 compared with M N C incubated with medium. 87 Figure 3.8. In these experiments, M N C were from two healthy controls, two multiple sclerosis (MS) and two H A M patients. In each experiment, M N C were added to H U V E C (a) or E C V - 3 0 4 (b) monolayers that were untreated or pretreated with IFN-y (100 U/ml) or conditioned medium containing L-929 (50%) supernatant for 48 h. Each column represents the mean ± SD percentage of quadruplicates of two separate experiments. *p<0.01 compared with untreated H U V E C monolayers. 88 Effects of Anti-adhesion Molecules Antibodies on the Adhesion of M N C of R R - M S Patients to Untreated and IFN-y Treated E C V - 3 0 4 70 -60 -50 --3 40 -1 H H 30 -20 -10 -0 -mAbs used Figure 3.9. M N C from three different R R - M S patients were coincubated with anti-LFA-1 (CD11/CD18), an t i -VLA-4 (CD49d), anti-ICAM-1 (CD54), anti-L-selectin (CD62L) or combination of all antibodies during the assay. E C V - 3 0 4 monolayers were untreated (a) or treated with 100 U / m l of IFN-y for 48-h prior to the assays (b). Each assay was performed using four replicate wells. Results are from three separate experiments and are expressed as mean ± S E M percentage inhibition of adhesion. *p< 0.005 compared with M N C incubated with medium. 89 Effects of IFN-p on P W M - i n d u c e d IgG Secretion in Healthy Controls (a) and in s R R - M S (b) • Avonex • Betaseron Rebif Concentration of IFN-P ( ng/ml) used Figure 3.10a and b. Suppression of PWM-induced IgG secretion by IFN-p. Blood M N C were stimulated with P W M and co-incubated without and with indicated concentrations (ng/ml) of IFN-p from Avonex™, Betaseron® and Rebif®. After 7 days at 37°C, the IgG content of the supernatants were measured by an E L I S A . These figures show a dose-dependent inhibition (mean ± S E M ) of PWM-induced IgG secretion by different preparations of IFN-P in 5 high responding healthy individuals (a) and 6 high responding s R R - M S patients (b). 90 Inhibition of PWM-induced IgG Secretion by Avonex™ (c), Betaseron® (d), and Rebif® (e) in Multiple sclerosis and Healthy controls 90 0 -I 1 1 1 1 1 1 1 1 : i 1 3 0 6 1.2 0 .24 0 .048 0 .0096 Concentration of IFN-B (ng/ml) used Figure 3.10 c, d, and e. Inhibition (mean + S E M ) of PWM-induced IgG secretion by Avonex™ (c), Betaseron® (d), and Rebif® (e) in 5 high responding healthy individuals and 6 high responding stable R R - M S patients. 91 Same Dose Compar ison of Avonex™, Be ta se ron® and Rebi f® on Inhibit ion of P W M - i n d u c e d IgG Secretion 312 31.2 3.12 Concentration of IFN-P ( ng/ml) used Figure 3.11. In these experiments blood M N C from 16 high responding s R R - M S were stimulated with P W M and co-incubated without and with indicated doses (ng/ml) of IFN-P from Avonex™, Betaseron® and Rebif®. After 7 days at 37°C, the IgG content of the supernatants were measured by an E L I S A . Each column indicates mean ± S E M inhibition of PWM-induced IgG secretion by different preparation of IFN-p. At 312 ng/ml, no significant differences were seen between different IFN-p in inhibition of PWM-induced IgG secretion. At 31.2 and 3.12 ng/ml, Avonex™ or Rebif® inhibited significantly more IgG secretion than that of Betaseron® (p<0.05). Significance p<0.05 * Avonex™ versus Betaseron® + Rebif® versus Betaseron® 92 Per Dose Compar ison of A v o n e x ™ , Be ta se ron® and Rebi f® on Inhibit ion of P W M - i n d u c e d IgG Secretion Concentration of IFN-B ( ng/ml) used Figure 3.12. Effects of Avonex™, Betaseron® and Rebif® on inhibition of PWM-induced IgG secretion were compared according to their proportion of daily recommended doses. Each column indicates mean ± S E M inhibition of PWM-induced IgG secretion by different preparation of IFN-B. No significant differences was found between the three treatment groups. 93 a o • ta •PM JS c c o _ Q V I * -B C « C ta s- o I £ W C/3 s H V C o O r—I -a u w 3 a o e S o 5 c3 * a S ° o U VI O Q o CU o a ca fe ro T O O iooo T0000 £0-31 90-HI I0-HI 80-31 60-31 01-31 8 8 8 4? uopaioas O^I JO uoniqnruT o o q CN CN 60 _o CD c/3 O Q (D (D C+H O fl o • r H fe CD cd "2 2 fl > "fl "+3 •S s •v fl <fe O •<3 60 -fl fe ~ n-60 O O .s rr! 'oo 3 O O O T3 60 C CN O fl CD •S fl « -2 - f l CD 60 fl fl fl fl cij fl @> - f l ~o ^ CD U C r * co 0 + 3 ; 3 CD O CO. CO fe 1 3 60 2; ^ 5 m ^ s" o 0 i= 8 A * & 0 CD ' fl T3 fl > Us < 11 ^ i § CD CD O (U « CD S - i - f l C+H > C c CO ro o ^ fe S ro 2H e £ 8 £ CO OXJO fe fe ^ £ r f ON C H A P T E R F O U R D I S C U S S I O N A N D C O N C L U S I O N 4.1 L Y M P H O C Y T E S U B S E T S IN H A M , H T L V - I C A R R I E R A N D H E A L T H Y C O N T R O L S 96 4.2 B L O O D M N C - H U V E C A D H E S I O N IN H A M 99 4.2.1 Mechanism of adhesion of H A M blood M N C to activated H U V E C 101 4.2.2 Expression of adhesion molecules on H A M and n o n - H A M lymphocytes 103 4.3 B L O O D M N C - H U V E C A D H E S I O N IN M S 104 4.3.1 Mechanism of adhesion of R R - M S and healthy blood M N C to H U V E C 106 4.3.2 Mechanism of adhesion of R R - M S blood M N C to unstimulated and IFN-y-stimulated H U V E C 108 4.4 C O M P A R I N G A D H E S I O N P R O P E R T I E S OF H U V E C A N D E C V - 3 0 4 F O R B L O O D M N C 109 4.5 T H E E F F E C T S OF I M M U N O M O D U L A T O R Y D R U G S O N L Y M P H O C Y T E S U B S E T S A N D F U N C T I O N IN M S I l l 4.5.1 Lymphocyte subsets in a R R - M S patient participating in Schering-Plough trial I l l 4.5.2 Lymphocyte subsets in R R - M S patients participating in ICOS trial I l l 4.5.3 PWM-induced IgG secretion in s R R - M S patients and healthy controls and the effects of IFN-P on this function 113 4.5.4 Comparing the effects of different preparations of IFN-P on PWM-induced IgG secretion 114 4.5.5 Effects of IFN-P on M N C - H U V E C adhesion 115 4.6 S U M M A R Y A N D C O N C L U S I O N S 117 4.7 F U T U R E E X P E R I M E N T A L C O N S I D E R A T I O N S 120 95 DISCUSSION AND CONCLUSION 4.1 LYMPHOCYTE SUBSETS IN HAM, HTLV-I CARRIER AND HEALTHY CONTROLS In this study, we demonstrated that lymphocyte subsets are altered in patients with H A M and in asymptomatic H T L V - I carriers. We did not find significant differences among major lymphocyte subsets including CD3+, CD4+, and CD8+ T cells between H A M , H T L V - I carriers and controls. This is in contrast with some (Itoyama et al., 1988) but also in agreement with other (Yasuda et al., 1986; Prince, 1990; Mukae et al., 1994) previous published reports on major lymphocyte subsets in H A M and H T L V - I carriers. We furthermore found that the percentage of CD4+CD29+ was significantly higher in H A M patients and carriers compared with controls. CD4+CD29+ lymphocytes represent "memory " cells (Sanders et al., 1988; Akbar et al., 1988). After encounter with an antigen, activated T cells acquire CD29 expression, which is paralleled by a downregulation of C D 4 5 R A expression (Akbar et al., 1988). CD4+CD29+ cells have also been called helper-inducer since they produce a variety of cytokines including IL-2, IL-4, IL-5, and IFN-y and provide help for immunoglobulin production (Morimoto et al., 1989). Therefore, the increase in CD4+CD29+ cells may account for high H T L V - I antibody titers and polyclonal B cell activation observed in the serum and cerebrospinal fluid of H A M and H T L V - 1 carriers (Itoyama et al., 1988; Yasuda et al., 1986; Geroni et al., 1988; Link et al., 1989; Osame et al., 1987; Mor i et al., 1988). We observed significantly higher levels of CD8+CD57+ cells in H A M compared with both carriers and controls. Increased number of CD8+CD57+ cells have been 96 reported to be associated with a number of clinical disorders including human immunodeficiency virus (HIV) infection (Lewis et a l , 1985; Borthwick et al., 1994), rheumatoid arthritis (Burns et al., 1992), Crohn's disease (James et a l , 1984), and in recipients of cardiac (Maher et al., 1985) and bone morrow (Leroy et al., 1986) transplants. It is difficult to propose a specific role for CD8+CD57+ subset and its role in H A M pathogenesis, since a broad range of functions has been proposed for CD8+CD57+ cells. Some of the proposed functions are: lectin-dependent and antibody-directed cytotoxicity (Phillips and Lanier, 1986), cytotoxic responses (Joly et al., 1989; Autran et al., 1991), and suppression of the generation of cytotoxic T lymphocytes (Wang et al., 1994). If we assume a cytotoxic role for CD8+CD57+ cells, increased CD8+CD57+ could explain the high levels of the virus-specific cytotoxic T lymphocytes observed in H A M but not in carriers (Jacobson et al., 1990; Elovaara et al., 1993). We found high levels of CD3+CD27- cells only in H A M patients and not in carriers. The CD27 molecule is a member of the tumor necrosis factor superfamily (Goodwin et al., 1993). T cell activation studies in vitro have shown that CD27- cells arise from C D 4 5 R A - , CD45RO+, CD27+ T cells after prolonged restimulation (De Jong et al., 1992; Hintzen et al., 1993). A n increased percentage of CD4+CD27- cells have also been reported in peripheral blood, synovial fluid, and synovial tissue of patients with rheumatoid arthritis, and these cells exhibit an enhanced capacity for transendothelial migration (Kohem et al., 1996). This could also explain the enhanced binding of lymphocytes to endothelial cells seen in H A M patients (Ichinose et al., 1992). This is also consistent with recent findings of high levels of the soluble form of CD27 in the cerebrospinal fluid of patients with H A M and multiple sclerosis (Hintzen et al., 1999). 97 Our study revealed that the percentage of both CD4+ and CD8+ cells which expressed H L A - D R was significantly higher in H A M and carriers. H L A - D R is the class II M H C antigen and acts as a restricting element required to mediate T cell activation (Corley et al., 1985). The activation molecule CD25, is an a-chain of the receptor for interleukin-2 (Uchiyama et al., 1981) and here we showed that it is upregulated on CD4+ cells of both H A M and carriers. These findings are consistent with previous observations that showed H A M patients and H T L V - I carriers have high levels of CD25+ and H L A -DR+ cells in their P B L (Itoyama et al., 1988). It is known that lymphocytes in peripheral blood from H A M and H T L V - I carriers show an enhanced spontaneous proliferation in vitro (Itoyama et al., 1988). A n IL-2 autocrine mechanism may operate in this phenomenon because a unique transregulatory protein Tax which is encoded by the p X region of the H T L V - I proviral genome has been shown to induce the expression of host cellular genes IL-2 and IL-2 receptor a chain (Tender et al., 1990). Therefore, increase of CD4+CD25+ cells in peripheral blood in H A M and H T L V - I carriers may result from the activation of IL-2 and its receptor gene by this transregulatory protein. However, the enhanced expression of H L A - D R on CD4+ and CD8+ cells and upregulation of CD25 molecules on CD4+ cells have not been observed in American asymptomatic H T L V - I carriers (Prince, 1990). We also found a high percentage of activation markers CD38, H L A - D R , and CD25 on CD8+ cells of H A M patients after 2 days in culture. This is of particular interest and potentially relevant to the pathological findings of H A M patients in whom lymphocytes infiltrating the central nervous system were predominantly CD8+ (Jacobson et al., 1992). The levels of CD8+CD38+ cells have been reported to have prognostic value for H I V disease progression (Ho et al., 1993). High levels of 98 CD8+CD25+, CD8+CD38+, and C D 8 + H L A - D R + cells in H A M patients may be due to chronic antigenic stimulation in response to H T L V - I infection and may be indicative of progression of the infectious process or disease development. The levels of early activation antigen CD69 expressed on CD3+ (Hara et al., 1986) cells was low, but after 2 days in culture increased significantly in both H A M and in H T L V - I carriers. This may indicate that the circulating T lymphocytes of H T L V - I infected individuals have not been activated recently. It is possible, however, that recently activated lymphocytes expressing CD69 may be sequestered in lymph node or other organs. 4.2 B L O O D M N C - H U V E C A D H E S I O N I N H A M In this study, we used primary and secondary culture of human umbilical vein endothelial cells ( H U V E C ) monolayers as a source of endothelial cells, which are more readily available than cerebral endothelial cells. Cerebral endothelium that constitutes the B B B is different in many ways from extracerebral endothelium particularly in the structural features. For example cerebral endothelium is further supported by astrocytic processes and contain tight junctions. However, endothelial cells from H U V E C also share many similarities with cerebral endothelial cells (Pober, 1988). For example stimulation of both umbilical and cerebral endothelial cells with cytokines increases their adherence for leukocytes (Pober, 1988; Tsukada et al., 1994). Furthermore, T N F - a increases the expression of I C A M - 1 and V C A M - 1 on both cerebral endothelial cells and H U V E C (Stins et al., 1997). A s lymphocytes were subjected to extensive manipulation, we performed our adhesion assays after 48 h in culture. We also speculate that 99 lymphocytes in culture could partially mimic the C N S microenvironment: secreted lymphokines and cytokines accumulate in culture, a situation that probably occurs at the blood brain barrier in C N S inflammation. In the past oligoclonal bands were generated in vitro using this strategy (Oger et al., 1981). The adhesion of lymphocytes to the brain microvascular endothelium, which form the blood-brain barrier, is a critical step in the initiation of the inflammatory response in the C N S and therefore probably plays an essential role in the pathogenesis of many neurological diseases (Raine et al., 1990; Martin et a l , 1992). Adhesion is mediated by multiple receptor-ligand systems including cell adhesion molecules selectins, integrins and immunoglobulins expressed on lymphocytes and endothelial cells (Bevilacqua, 1993). The expression of adhesion molecules on endothelial cells is regulated by several cytokines (Yu et a l , 1985; Carley et al., 1988; Pober, 1988). Indeed, we have verified here that treatment with IFN-y enhanced the M N C - H U V E C adhesion. We have also used conditioned medium containing L-929 supernatant to stimulate H U V E C in some of our experiments as it contains many different cytokines (Oger et al., 1974; Tonetti et al., 1997), thus mimicking more closely the situation in inflammatory conditions in C N S . Unknown soluble factors in L-929 supernatant were previously shown to induce the release of T N F - a from a macrophage cell line (Tonetti et al., 1997). L-929 supernatant could have similar effects on H U V E C , as H U V E C is also capable of producing T N F - a (Nilsen et al., 1998), and thus enhancing M N C - H U V E C adhesion. We have confirmed earlier observations (Ichinose et al., 1992) that M N C obtained from H A M patients adhere more readily to H U V E C than those of H T L V - I carriers or healthy controls, and this is highly relevant to the pathogenesis of H A M . The 100 greater binding of H A M patients' blood M N C to activated H U V E C could be due to their greater recognition of ligands on activated H U V E C . The activation of M N C is important in this regard, and it is the highly activated, rather than the quiescent M N C , that are more likely to bind endothelial cells (Brown et al., 1993; Oen et al., 1994; Vora et al., 1995). It has been shown that the activation of T cells with P M A increases the affinity of L F A - 1 and V L A - 4 integrins for their counter ligands I C A M - 1 and V C A M - 1 on endothelial cells without changing the levels of cell surface expression (Dustin and Springer, 1989; Wilkins et al., 1991). Pro-inflammatory cytokines such as T N F - a , IL-1 and IFN-y are secreted by activated Thl-lymphocytes and these cytokines have been demonstrated to increase both the affinity and the induction of adhesion molecules on lymphocytes ( Y u et al., 1985; Pober and Cotran, 1990). In H A M , blood M N C produce large amounts of I F N -y in culture (Nishiura et al., 1994) and therefore, could function in an autocrine fashion in promoting their binding to endothelial monolayers. 4,2.1 Mechanism of adhesion of HAM blood MNC to activated HUVEC Using monoclonal antibody blocking studies, we have shown the respective role of V L A - 4 a , I C A M - 1 , and L-selectin molecules in H A M blood M N C adhesion to activated H U V E C . These adhesion molecules may also play an important role in directing T cells to inflammatory sites in patients with H A M . In our studies, antibody to I C A M - 1 was the most effective in inhibiting the binding of H A M cells to H U V E C . This is in agreement with previous findings that effective blocking of lymphocyte-endothelial cell interactions could be achieved both in vivo and in vitro by antibody to I C A M - 1 (Whitcup et al., 1993; Greenwood et a l , 1995; Wong et al., 1999). Antibody to V L A - 4 101 was the least effective in inhibiting the binding of cultured H A M lymphocytes to activated H U V E C ; it is probably because T cells are fully activated and activated cells preferentially use L F A - 1 / I C A M - 1 interactions rather than V L A - 4 / V C A M - 1 (Van Kooyk et al., 1993). We also have shown that antibodies to L-selectin block the binding of H A M patients' lymphocytes to H U V E C . This inhibition was unexpected, since an inhibitory effect with anti L-selectin antibody has been demonstrated only under flow but not static conditions (Spertini et al., 1991). This difference could be because our experimental system resulted in activation of both lymphocytes and H U V E C . This inhibition could be attributed to L-selectin recognition of its ligand on E C with higher affinity or binding to an alternative ligand on activated E C . A n inhibitory role by E -selectin has been reported when both H U V E C and CD4+ T cells are activated (Shimizu et al., 1991). A more recent study (Wong et al., 1999), indirectly supporting the role of L-selectin other than rolling, has shown that E-selectin, a ligand for L-selectin inhibits transmigration of T cells across T N F - a activated human brain microvessel endothelial cells. Furthermore, its has been shown that E-selectin mediates a major role for adhesion of Adult T-cell Leukemia (also caused by infection with H T L V - 1 ) cells to H U V E C (Ishikawa et al., 1993). It is therefore possible that L-selectin plays a role not only in rolling but also in binding when lymphocyte and endothelial cells are activated. In our antibody blocking experiments, control mouse serum also slightly inhibited M N C attachment to E C when used at 2.5 ug/ml of immunoglobulin concentration (data not shown). We also used mouse IgG2b m A b isotype control anti-dansyl, (monoclonal specific for hapten dansyl (5-[dimethylamino] naphthalene- 1-sulonyl), normal human serum, and normal mouse serum to inhibit adhesion of M N C of healthy controls and 102 multiple sclerosis patients to EC and found that both normal mouse as well as pooled human serum occasionally inhibit M N C - E C adhesion, whereas anti-dansyl mAb does not. Therefore, anti-dansyl mAb serves as a more appropriate control. 4.2.2 Expression of adhesion molecules on HAM and non-HAM lymphocytes To investigate whether the increase in binding of lymphocytes from H A M patients was mediated by specific receptor-ligand interactions, we analyzed the expression of adhesion molecules on lymphocytes after two days in culture. We found that the expression of L-selectin on CD4+ and CD8+ cells decreased in HTLV-I carriers and even more in H A M . L-selectin is an adhesion molecule of the selectin family that mediates the initial step of lymphocyte attachment to vascular endothelium ("rolling") (Lawrence and Springer, 1991). Upon extensive and prolonged cellular activation, this molecule is shed (Jung and Dailey, 1990). Elevated serum levels of soluble L-selectin have been reported during the period of active disease in patients with multiple sclerosis (Hartung et al., 1995) and adult T-cell leukemia (Tatewaki et al., 1995). In addition, significant elevation of soluble L-selectin has recently been reported in the sera of H A M patients (Tsujino et al., 1998). The reduced expression of L-selectin on lymphocytes thus probably best represents chronic cell activation. Furthermore, as activation of lymphocytes following receptor engagement results in lymphocyte shedding of L-selectin, this may allow the leukocyte to break its tight bonds with the vascular endothelium and proceed with emigration into the underlying tissue into the CNS. We further showed high levels of expression of ICAM-1 in T cells from HTLV-I carriers and at even higher levels in patients with H A M following two days in culture. This fits well 103 with the fact that ICAM-1 is upregulated in T cell lines carrying HTLV- I and in lymphocytes of patients with adult T cell leukemia (ATL) (Yamamoto et al., 1982; Yamamoto and Hinuma, 1985). It is known that cell-free HTLV- I exhibits very low infectivity and cell-to-cell infection is regarded as the major route of HTLV- I transmission both in vitro and in vivo (Weiss et al., 1985; Yamamoto and Hinuma, 1985). Therefore, constitutive expression of ICAM-1 in HTLV-1-infected T cells might be important for cell-mediated transmission by prompting cell adhesion between H T L V -I-positive T cells and uninfected T cells. Increased levels of soluble ICAM-1 have also been reported in the sera of multiple sclerosis and H A M patients (Sharief et al., 1993; Tsukada et al., 1993b). Analysis of adhesion molecules on lymphocytes at isolation showed that the percentage of CD4+ and CD8+ cells expressing V L A - 4 was generally higher in both patients with H A M and carriers than in controls. Therefore, it is likely that V L A - 4 does not contribute much to the increased binding of lymphocytes to H U V E C seen in H A M patients. Antibody blocking experiments further demonstrated that V L A - 4 was the molecule least involved in this adhesion. 4.3 BLOOD MNC-HUVEC ADHESION IN MS In this study we also investigated the adhesion properties of peripheral blood M N C of sRR-MS and SP-MS to H U V E C . Blood M N C from SP-MS patients exhibited significantly higher adhesion capacity than M N C from normal donors. This is consistent with the previous published report indicating increased adhesion of chronic progressive 104 M S blood M N C to cultured cerebral endothelium (Lou et a l , 1997; Tsukada et al., 1993a). This increase in adhesion was attributed in part to higher expression of L F A - 1 on circulating blood M N C than on those of normal controls (Lou et a l , 1997). Increased adhesion of S P - M S blood M N C to H U V E C is also probably due to increased levels of activated lymphocytes seen in circulating peripheral blood of S P - M S patients (Hafler et al., 1985) and it is activated lymphocytes that are highly adherent to endothelial monolayers (Brown et al., 1993). Our data are also in agreement with a recent report indicating enhanced transmigration of secondary progressive M S lymphocytes across fibronectin-coated membranes (Prat et al., 1999). In previous studies, increased IFN-y and T N F - a production capacity were found in cultured M N C from patients with S P - M S (Beck et al., 1988; Chofflon et al., 1992). Since we performed the adhesion assay after two days in culture, IFN-y and T N F - a , the well known inducers of adhesion molecules (Yu et al., 1985; Pober and Cotran, 1990) also might have contributed in an autocrine fashion to the enhanced S P - M S blood M N C adhesion observed in this study. The adhesion of clinically stable R R - M S blood M N C was generally higher than that of healthy subjects (although not statistically significant). This is probably because a certain proportion of stable R R - M S in this study had biological activity that could have been recognized by M R I . M R I was not done for this study. Our data might also explain the reason for significant impairment of blood brain barrier from R R - M S to secondary progressive M S (McLean et a l , 1993). Increased binding of R R - M S blood M N C to endothelial monolayers have been previously reported, but only during the clinically active phase of the disease (Tsukada et al., 1993a). This might also imply that the expression of some adhesion molecules on active R R - M S and S P - M S differ from that on 105 stable R R - M S . In another study of heterogeneous populations of M S it was found that M S blood M N C adhered to H U V E C more than did healthy, but only after IFN-y, T N F - a , and IL-1 cytokine activation of H U V E C (Vora et al., 1996). Such differences were not seen with resting H U V E C . However, in studies of Tsukada et al. (1993a), no significant differences were found between adhesion of M N C from clinically active R R - M S and healthy control after T N F - d treatment of cerebral endothelium. 4.3.1 Mechanism of adhesion of RR-MS and healthy blood MNC to HUVEC We also compared the relative contribution of major adhesion molecules in binding blood M N C of R R - M S and of normal subjects to H U V E C by monoclonal antibody blocking studies. The adhesion of M N C from both R R - M S and healthy subjects to untreated H U V E C was significantly reduced by mAbs. to L F A - 1 and I C A M - 1 . This is consistent with previous observation that inhibition of M N C adhesion to untreated H U V E C can be achieved with monoclonal antibodies to L F A - 1 and I C A M - 1 (Shimizu et al., 1991; Oppenheimer-Marks et al., 1991; Watson et al., 1996). The m A b to I C A M - 1 was not as effective as L F A - 1 in inhibition of adhesion. This is perhaps due to L F A - 1 binding to I C A M - 2 in addition to I C A M - 1 . I C A M - 2 is constitutively expressed on both resting and activated H U V E C (Staunton et al., 1989). In fact, I C A M - 2 is expressed at greater levels than I C A M - 1 on untreated H U V E C (Staunton et al., 1989). Antibody directed against V L A - 4 or L-selectin had no inhibitory effects. This is probably because untreated H U V E C do not express V C A M - 1 or E-selectin, the main ligands for V L A - 4 and L-selectin, respectively (Shimizu et al., 1991; van Kooyk et al., 1993), although there is some dispute (Hughes, 1996). Previous studies has shown that adhesion of 106 lymphocytes to resting cerebral endothelium cells also involve V L A - 4 adhesion molecule (Matsuda et al., 1995b). Perhaps, this is because resting cerebral endothelium expresses low levels of V C A M - 1 , the main ligand for V L A - 4 (Wong and Dorovini-Zis, 1995). In a study using retinal endothelial cells, it was found that antibody to V L A - 4 does not inhibit resting lymphocyte adhesion to untreated retinal endothelial cells. However, when lymphocytes were activated by ConA, antibody to V L A - 4 significantly inhibited adhesion to untreated retinal endothelial cells (Greenwood et al., 1995). A s retinal endothelial cells were shown not to express V C A M - 1 , this inhibition was attributed to the ability of the antibody to V L A - 4 to induce aggregation of C o n A -activated lymphocytes in vitro. Lymphocyte aggregates are then easier to remove during the washing stage of the assay, thus producing an apparent reduction in binding. Combinations of L F A - 1 , I C A M - 1 , V L A - 4 and L-selectin mAbs failed to show greater inhibition than seen with the L F A - 1 m A b alone, indicating that the L F A - 1 pathway predominates in mediating adhesion of R R - M S and healthy subjects to untreated H U V E C . We further have shown that the major adhesion molecules investigated in this study are equivalently involved in adhesion of both R R - M S and healthy subjects to resting H U V E C with L F A - 1 / I C A M - 1 pathway mediating a predominant role. Our data do not support the previous report that indicates the involvement of L F A - 1 adhesion pathway in binding of R R - M S , and not in binding of healthy subjects blood M N C to cerebral endothelium (Tsukada et al., 1993a). 107 4.3.2 Mechanism of adhesion of RR-MS blood MNC to unstimulated and IFN-y-stimulated HUVEC To characterize further the adhesion molecule-ligand pairs involved in inflammation, we extended this study to test antibodies directed against various adhesion molecules to block the adhesion of R R - M S blood M N C to H U V E C stimulated with I F N -y. Monoclonal antibodies directed against L F A - 1 and I C A M - 1 strongly inhibited R R - M S blood M N C - H U V E C interaction. Our results are in agreement with a previous study showing significant role for L F A - 1 and I C A M - 1 in adhesion of M N C to both resting and IL-6 activated H U V E C (Watson et al., 1996). Contrary to our finding is a report indicating significant involvement of L F A - 1 and I C A M - 1 on binding of M N C to resting but not to IL-1 activated H U V E C (Oppenheimer-Marks et al., 1991). The reasons for this disparity are not clear. In addition to L F A - 1 and I C A M - 1 , the adhesion of R R - M S blood M N C to IFN-y activated H U V E C was shown to also significantly involve V L A - 4 adhesion molecule. These results are in agreement with previous published observation on the inhibitory role of antibody to V L A - 4 on M N C adhesion to activated endothelial cells (Oppenheimer-Marks et al., 1991; Watson et al., 1996). In our study, it is likely that IFN-y might have induced the expression of V C A M - 1 , the main ligand for V L A - 4 on H U V E C . Indeed IFN-y has been previously shown to enhance the expression of V C A M -1 on H U V E C (Lindington et a l , 1999). A n increase in levels of expression of V L A - 4 on lymphocytes (Svenningsson et al., 1993) and V C A M - 1 on brain microvessel endothelial cells (Washington et al., 1994) have been found in M S . Therefore, V L A - 4 might play an important role in mediating M N C adhesion and subsequently transmigration in M S . Our result could also partly explain the previous observation that administration of an t i -VLA-108 4 antibody alleviated the clinical and pathological symptoms of E A E (Yednock et al., 1992). In the present study antibody to V L A - 4 inhibited M N C adhesion to activated H U V E C but to a lesser extend than did ant i -LFA-1. This may be explained by differences in the distribution of these two adhesion molecules. Unlike L F A - 1 , which is present on all lymphocytes, V L A - 4 expression is confined to a subpopulation of the cells (Shimizu et al., 1990). We were not able to completely inhibit M N C adhesion to H U V E C using a combination of the studied mAbs. This indicates the potential involvement of other receptor/ligand interactions. Some other candidate that might be involved in adhesion include CD44, which has been implicated in the adhesion of activated T cell to activated H U V E C (Oppenheimer-Marks et al., 1990) and C D 2 which mediates T cell adhesion to other cell types by binding to its ligand, L F A - 3 (Makgoba et a l , 1989). 4.4 COMPARING ADHESION PROPERTIES OF HUVEC AND ECV-304 FOR BLOOD MNC In a part of this project, we also assessed the suitability of E C V - 3 0 4 cell lines as a substitute for H U V E C for the adhesion of blood M N C . The adhesion of s R R - M S and healthy controls blood M N C to ECV-304 monolayers were not significantly different. This is consistent with our observations using H U V E C to compare s R R - M S and healthy control M N C . However, we cannot conclude with absolute certainty that the adhesion pathways for s R R - M S and healthy controls M N C to untreated H U V E C and to E C V - 3 0 4 monolayers are similar as we did not use M N C from the same individuals and the assays 109 were not done in parallel. We furthermore found significant differences between E C V -304 and H U V E C in their ability to respond to IFN-y and L-929 supernatant. Treatment of H U V E C with IFN-y and L-929 supernatant resulted in significantly enhancing their adhesion for M N C . However, similar treatment of ECV-304 failed to enhance their adhesion for M N C . It is possible that unlike in H U V E C , IFN-y and L-929 supernatant does not result in upregulation of adhesion molecules expression and subsequent functional enhancement for M N C adhesion to ECV-304 . Monoclonal antibody blocking studies demonstrated that adhesion pathways of R R - M S and healthy controls to untreated H U V E C and ECV-304 are similar and predominantly involve L F A - 1 / I C A M - 1 pathway. In a parallel study, we found that when both monolayers were treated with proinflammatory IFN-y cytokine, V L A - 4 was involved in M N C adhesion to H U V E C but not to ECV-304 . Our results agree with recent observations that indicate ECV-304 constitutively express I C A M - 1 but not V C A M - 1 (Dobbie et al., 1999). Moreover, our results are also in agreement with the findings that unlike in H U V E C , the treatment of ECV-304 with T N F - a does not result in the induction of V C A M - 1 expression (Lindington et al., 1999). Our data however, conflicts with a report that demonstrated constitutive expression of V C A M - 1 on E C V -304 which was upregulated following activation with L P S (Hughes, 1996). The exact reason (s) for the discrepancy between these studies is unclear but could be due to variations in adhesion molecule expression in response to different activators, differences in antibodies utilized and in the sensitivity of the detection methods employed. 110 4.5 T H E EFFECTS OF IMMUNOMODULATORY DRUGS ON L Y M P H O C Y T E SUBSET AND FUNCTION IN MS 4.5.1 Lymphocyte subsets in a RR-MS patient participating in Schering-Plough trial Analyzing lymphocytes subset of a R R - M S patient who participated in a double-blind and placebo controlled trial of Schering-Plough, we observed a major shift in lymphocyte subsets at both 2 and 7 days post treatment. However, at present we do not know whether the patient received placebo or rIL-10. Therefore, we cannot conclude whether the observed shifts in lymphocyte subsets are due to rIL-10 treatment or spontaneous. 4.5.2 Lymphocyte subsets in RR-MS patients participating in ICOS trial We also were interested in verifying whether a short-term treatment with Hu23F2G (recombinant and humanized anti-LFA-1 antibody) is able to modify peripheral blood lymphocyte subsets in R R - M S . We observed a major shift in some lymphocyte subset in 4 patients who participated in this study at the V H & H S C / U B C site Multiple Sclerosis Cl inic . Due to the limited number of participants, statistical analysis was not possible and all changes in lymphocyte subsets cannot be discussed rationally. Hu23F2G treatment had no major effect on the number of circulating CD3+, CD4+ or CD8+ T cells. Focusing only on few lymphocyte subsets, we observed a major reduction of CD3+CD26+ (activated T) cell and CD4+ and CD8+ cells expressing adhesion molecules V L A - 4 (CD49d) and L-selectin (CD62L) following in vivo treatment with high dose (2 mg/kg) Hu23F2G. At lower dose (1 mg/kg), H U 2 3 F 2 G effects were less 111 profound, and resulted only in a large reduction in the percentage of CD4+ and CD8+ cells expressing V L A - 4 adhesion molecules. On the contrary, in a placebo treated patient the percentage of CD4+ and CD8+ cells expressing V L A - 4 and L-selectin increased while that of CD3+CD26+ remained unchanged. Furthermore, in a RPv-MS patient treated with intravenous methylprednisolone no major reduction of the discussed adhesion molecules was observed. In contrast to high dose Hu23F2G treated patient, the percentage of CD4+ and CD8+ cells expressing L-selectin increased. These results, with some degree of confidence, indicated that the observed shifts with respect to the discussed lymphocyte subsets are likely due to immunotherapy with Hu23F2G rather than spontaneous. Moreover, our data particularly in the R R - M S patient treated with high dose Hu23F2G indicates that the immunotherpay with Hu23F2G achieved its desired objectives of reducing activation and adhesion related antigens on peripheral blood lymphocytes. The reduction of activation and adhesion related antigen on peripheral blood lymphocytes is relevant in C N S inflammatory cell infiltration in M S since it is these cells that are likely to be involved in the process of endothelial adhesion and extravasation to sites of inflammation (Wekerle et al., 1986; Estess et al., 1999). The significance of adhesion molecules in M S pathology has already been addressed in the Introduction. Furthermore, in peripheral blood of active M S patients' lymphocytes were found to have higher expression of the activation marker CD26 compared to patients with inactive M S , patients with other neurological diseases, or healthy controls (Hafler et al., 1985). Therefore, it is reasonable to expect a beneficial therapeutic effect in Hu23F2G treated M S patients. The reason for clinical inefficacy of Hu23F2G in R R - M S patients is not clear (Lublin 1999). However, we have to bear in mind that the treatment 112 schedule with Hu23F2G is for relapses of M S and this might be too late once the inflammation is irretrievably established in the C N S . Therefore, Hu23F2G might achieve its desired effects i f given at very early stage of the M S relapse. A note of caution should also be raised and stressed here in the interpretation of fluctuations of lymphocyte subsets in the studied patients, as the number of participant in this study was highly limited. 4.5.3 PWM-induced IgG secretion in sRR-MS and healthy controls and the effects of IFN-P on this function We found that PWM-induced IgG secretion by blood M N C was significantly increased in s R R - M S patients compared to the normal healthy populations, as has already been reported (Levitt et al., 1980; Oger et al., 1988, Antel et al., 1984). In agreement with other reports (Antel et al., 1984; Rosenkoetter et al., 1984), we also found that the proportion of high responders to P W M stimulation is higher in R R - M S than in healthy subjects. The main reason for the high in vitro IgG secretion in M S is believed to be defective function of T-suppressor lymphocytes (Antel et al., 1984, 1986). It should also be noted that elevated PWM-induced IgG secretion has not always been found in M S patients (Kelley et al., 1981; Hauser et al., 1985). In our study, all different preparation of IFN-p caused a dose-related inhibition of IgG secretion induced by P W M in peripheral blood M N C of high responders in both s R R - M S and healthy controls. Similar inhibitory effects with IFN-P and closely related I F N - a have been previously reported in both in vivo and in vitro studies (Siegel et al., 1986; O'Gorman et al., 1987; Bratt et al., 1996). It has also been shown that in contrast to 113 their suppressive action on IgG production in unseparated M N C , IFN-B enhanced IgG production in purified B cells (Siegel et al., 1986). The inhibitory action of IFN-P on PWM-induced IgG production in unseparated M N C is believed to be mediated by IFN-P effect on a non-B cell population and in part related to the inhibitory effect of IFN-p on PWM-induced M N C proliferation (Siegel et al., 1986). 4.5.4 Comparing the effects of different preparations of IFN-fi on PWM-induced IgG secretion We also directly compared the in vitro biological activity of IFN-p from Avonex™, Betaseron®, and Rebif® utilizing inhibitory effects of IFN-p on P W M -induced IgG secretion in high responding s R R - M S patients. Our results demonstrated that Avonex™ and Rebif® had higher biologic activity compared with Betaseron® when used at a similar concentration. The difference was particularly evident when we compared Avonex™ (IFNp-la) with Betaseron® ( I F N p - l b ) . Avonex™ and Rebif® amino-acid sequence and glycosylation pattern are identical to those of endogenous human IFN-p. B y contrast, in Betaseron® serine is substituted for cysteine at position 17, the N-terminal methionine is missing and the glycosylation of the natural product is lacking. There is evidence that carbohydrate plays a vital role in stabilizing the IFN-P molecules, and its absence from Betaseron® may explain why this molecule in our assay as well as in standard antiviral assay has much less biological activity per milligram of protein compared with Avonex™ and Rebif® (Runkel et al., 1998). We did not expect generally a higher inhibitory effects for Avonex™ compared with Rebif® on P W M -induced IgG secretion as these two preparations of IFN-P are similar and are produced by 114 inserting the natural human gene for IFN-B into Chinese hamster ovary cells. Thus, it is unlikely that there is a structural difference between Avonex™ and Rebif®. We do not have a clear explanation to our observation. The differences in formulation might explain this phenomenon. Avonex™ is formulated in a higher concentration of albumin (15 mg/ml after reconstitution versus 9 mg/ml for Rebif®), at a different p H (7.2 versus 3.8) and in a different buffer (phosphate versus acetate). It is possible that Avonex™ is more stable in its formulation or is better absorbed by IFN-P receptor on M N C than Rebif®. This higher in vitro activity of Avonex™ compared to Rebif® is also consistent with their in vivo pharmacodynamic activity (Alam et al., 1997). It has been shown that when an equal dose (6 M I U ) of Avonex™ and Rebif® was administrated intramuscularly to healthy volunteers, the serum neoptrin concentration was higher in Avonex™ treated individuals ((Alam et al., 1997). When we corrected for specific activity (MIU) and weekly dose and expressed concentration as a fraction of the M I U of IFN-P activity injected per week, Betaseron®, Avonex™ and Rebif® had similar activity with respect to their influence on inhibition of PWM-induced IgG secretion. 4.5.5 Effects of IFN-p on MNC-HUVEC adhesion In this study we demonstrated that I F N p - l b treatment of resting H U V E C has no significant effect on M N C adhesion. This is in agreement with a previous report (Dhib-Jalbut et a l , 1996). We did not study the effects of I F N p - l b on activated H U V E C in binding to M N C ; however, others have shown that the effects of I F N p - l b on I C A M - 1 , V C A M - 1 and E-selectin adhesion molecules expression induced by IFN-y, IL-1 P, or T N F - a on H U V E C is slightly additive, and is associated with significant augmentation of 115 M N C - H U V E C adhesion (Dhib-Jalbut et al., 1996). Contrary to their effects on H U V E C , we demonstrated that the pretreatment of M N C with IFNp- lb results in significant reduction of M N C - H U V E C adhesion. This effect was not a result of a cytotoxic effect because I F N - p - l b had no effect on cell viability as was determined by trypan blue exclusion dye. It is therefore possible that the effects of I F N P - l b on adhesion may be cell-specific. This explanation is supported by the finding that IFN-P is capable of downregulating IFN-y-induced expression of H L A - D R on cerebral endothelial cells (Huynh et al., 1995), but not on monocytes (Soilu-Hanninen et al, 1995). Our findings are also consistent with a previous report showing significant reduction of M N C - H U V E C adhesion after in vivo I F N - p - l b treatment in R R - M S patients (Corsini et al., 1997, Gelati et al., 1999). In addition, it has been shown that pretreatment of epidermal carcinoma cell line with closely related I F N - a results in a significant reduction of their binding to H U V E C (Dao et al., 1995). Our results showing the effects of I F N p - l b on M N C adhesion to H U V E C is also in agreement with a recent report showing that pretreatment of M N C with IFN-p results in significant reduction in migration of M N C through cultured cerebral endothelial cells (Lou et al., 1999). In vitro, I F N - p - l b treatment has also been shown to inhibit the transmigration of activated T cells through fibronectin, by acting on a matrix metalloproteinase (MMP-9) produced by the lymphocytes (Stuve et al., 1996). Recently it has also been shown that the migration across fibronectin-coated membranes of lymphocytes from R R - M S patients receiving I F N p - l b were significantly reduced compared with untreated R R - M S patients (Prat et al., 1999). One of the possible mechanisms for the inhibitory effects of I F N p - l b on M N C - H U V E C binding is that I F N P - l b could alter the level of adhesion molecule expression on M N C . We did not 116 address this question in our study; however, it has been previously shown that pretreatment of M N C with IFN-P results in lower basal and IFN-y-induced expression of V L A - 4 (Soilu-Hanninen et a l , 1995). Downregulated expression of V L A - 4 on lymphocytes has also been shown in R R - M S patients after treatment with IFNp (Calabresi et al., 1997). This does not satisfactorily explain our observation, as we did not activate H U V E C and demonstrated that V L A - 4 is not significantly involved in M N C adhesion to untreated H U V E C . However, it is possible that I F N p - l b might also alter the level of expression or function of other adhesion molecules involved in M N C - H U V E C binding. In fact, a decrease in expression of C D 18 (P chain of L F A - 1 ) on M N C of IFNp-l b treated M S patients has been previously reported (Corsini et al., 1997, Gelati et al., 1999). The effects of I F N p - l b on the functional activity of adhesion molecules might also be responsible for the decreased adhesiveness of M N C to H U V E C since quantitative changes in the expression of cell surface adhesion molecules are not always consistent with the level of cell adhesion (Piela and Korn, 1990; Gamble and Vdas, 1991). In vitro treatment with I F N p - l b has also been shown to downregulate the expression of IL-2 receptor a chain on T cells (Leppert et al., 1996). Thus, it is possible for I F N p - l b to lower the T cell state of activation and thereby inhibit their adhesion to H U V E C . 4.6 SUMMARY AND CONCLUSIONS We have shown that patients with H A M exhibit highly activated and differentiated lymphocyte subsets. It is likely that highly activated and differentiated lymphocyte subsets play a critical role in the pathogenesis of H A M and therefore the 117 assessment of these lymphocyte subsets may be of value in detecting or evaluating inflammatory diseases and monitoring treatment. We have also demonstrated increased adhesion of H A M and clinically active M S blood M N C to H U V E C . Our data and that of others lend support to the view that infiltration of M N C across the B B B into the C N S in H A M and M S is due to increased interaction between blood M N C and endothelium. We also speculate that in both H A M and M S chronic and systemic activation of immune cells result in increased adhesion and initiate events, which lead to central nervous system inflammation. We have shown that pretreatment of M N C with IFN-B significantly inhibits blood M N C - H U V E C adhesion. Therefore, our data supports the postulate that IFN-B might influence the evolution of the M S lesions at the level of B B B by influencing the circulating M N C and inhibiting MNC-endothelial adhesion and the subsequent migration of inflammatory M N C into the C N S . We also investigated mechanism of adhesion of blood M N C in H A M , R R - M S , and healthy controls to H U V E C under different experimental conditions. Our results demonstrate that adhesion of H A M blood M N C to activated H U V E C in addition to I C A M - 1 and V L A - 4 is also mediated by L-selectin. Adhesion pathways in R R - M S and healthy controls to untreated H U V E C were similar and were mainly mediated by L F A -1/ICAM-1. In addition to L F A - 1 / I C A M - 1 pathways, V L A - 4 was also involved in adhesion only after stimulation of H U V E C . These findings are relevant to better understanding the mechanisms of adhesion under both inflammatory and non-inflammatory conditions. 118 We have also compared adhesion properties of H U V E C and E C V - 3 0 4 for adhesion to M N C and demonstrated that when untreated, H U V E C and E C V - 3 0 4 utilize shared adhesion pathways for binding to M N C s that predominantly involve L F A -1 / I C A M - l ; however, when treated with IFN-y, ECV-304 unlike H U V E C does not utilize V L A - 4 / V C A M - 1 pathway. Furthermore, unlike H U V E C , ECV-304 does not respond to INF-y and L-929 supernatant for enhancing their adhesion to M N C adhesion. Therefore, ECV-304 may not be useful for some adhesion assays studies. We demonstrated increased IgG secretion in stable R R - M S in response to P W M . This increase in T cell dependent B cell activity further adds to a spectrum of immune abnormality that has already been reported in this disease. This also supports the view that clinical stability in M S does not necessarily translate into normal functional immune responses. We further demonstrated that IFN-P is capable of significantly inhibiting mitogen-induced IgG secretion. This indicates that another beneficial mechanism of actions of IFN-P in M S might be due to the capacity of IFN-P to downregulate B cell activity and IgG secretion. We also compared the biological activity of different preparations of IFN-P according to their capacity in inhibiting PWM-induced IgG secretion. The results indicated that Avonex™ had the highest in vitro activity followed by Rebif® and Betaseron®, respectively, when used at the same mass dose or proportion of their daily recommended dose. However, when we calculated the amount of different IFN-P used in vitro as a fraction of their weekly injection using their antiviral activity in units, these differences disappeared. These in vitro effects of Avonex™ Rebif® and Betaseron® were more consistent with their antiviral activity and therefore may be used as an 119 alternative assay in evaluating the biological activities of IFN-B. Direct comparative clinical trials have yet to be conducted, but i f in vitro biologic activity of each of these preparations of IFN-B is representative of their physiologic activity in vivo, there might not be significant differences on the clinical outcome when they are used at the recommended dosage and frequency of administration. 4.7 FUTURE EXPERIMENTAL CONSIDERATIONS In the current project, we found that a number of lymphocyte subsets were significantly altered in H A M patients. In order to further elucidate the role of the altered lymphocyte subsets in immunopathology of H A M and examine a potential association between the lymphocyte subsets and disease progression, it would be of significant interest to perform a long follow-up study of early H A M patients. Our understanding of the pathomechanisms of H A M could be further enhanced by defining the functional properties of the altered lymphocyte subsets in terms of determining specificity to H T L V -I antigens, ability to release cytokines, and cytotoxic activity. Immunological studies on C S F cells maybe more relevant to pathogenesis of H A M than cells of the peripheral blood. Therefore, a comprehensive analysis of lymphocyte subsets in the C S F of H A M patients is another avenue that needs to be explored. Longitudinal analysis of lymphocyte subsets in both the peripheral blood and C S F with a focus on T cell adhesion and activation related antigens could also be expanded to M S patients comparing different stages of the disease. 120 In this project endothelial cells from umbilical veins were used to support the binding of M N C . Ideally more appropriate endothelial monolayers would be from human cerebral microvessels, which are more difficult to obtain. To better understand the phenomenon of increased M N C adhesion to endothelial monolayers in H A M and secondary progressive M S patients, flow cytometric techniques could be utilized to characterize the surface phenotype markers of the adhesion and activation related antigens of the M N C that are adherent to the endothelial monolayers. We observed that the adhesion of blood M N C in clinically active M S (SP-MS) was generally higher compared to that of clinically stable M S (sRR-MS) . This might indicate that the increased adhesion of blood M N C to endothelial cells in M S is correlated with disease activity. Longitudinal adhesion assays done in parallel with M R I evaluation of the disease activity in R R - M S patients during both relapse and remission be better in delineating the precise sequence of events. We have also shown that in vitro treatment of M N C with IFN-B results in significant reduction in MNC-endothelial adhesion. To examine whether this phenomenon can also be achieved following in vivo treatment, longitudinal analysis of M N C adhesion to endothelial monolayers could be carried out in R R - M S patients before and during treatment with IFN-B. Furthermore, the mechanism by which IFN-B may influence M N C to alter their adhesion to endothelial monolayers is not fully known. It is possible that that IFN-B may act on M N C by decreasing their activation state. Studies could be conducted to address this possibility by direct in vitro treatment of M N C with IFN-B and subsequent analysis of the expression of markers of activation. Another area of study would be to examine the effects of IFN-P on H A M blood M N C adhesion to the 121 endothelial monolayers. We have shown that H A M blood M N C are highly activated. Therefore, the potential action of IFN-P in downregulation of activated M N C might be more easily observed in H A M . We have evaluated the in vitro biological activity of the three preparations of IFN-p, namely Avonex™, Betaseron®, and Rebif® and found that Avonex™ could suppress PWM-induced IgG secretion the most when used at the same mass concentration. This study could be extended to evaluate the in vivo biological activity of the different preparations of IFN-P. For example, longitudinal measurement of P W M -induced IgG secretion by blood M N C could be performed in R R - M S patients before and after treatment with Avonex™, Betaseron® or Rebif® at the current recommended frequencies, doses, and routes of administration. Using the same assay, in vivo biological activity of the different IFN-P preparations could be further compared in M S patients or healthy volunteers when used at the similar specific activity, frequency and route of administration. 122 R E F E R E N C E S : Akbar A , Terry L , Timms A , Beverley P, Janossy G . 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Med. 179: 973-984; 1994. 149 APPENDIX A A . 1 T H E E F F E C T S OF I M M U N O M O D U L A T O R Y D R U G S O N L Y M P H O C Y T E P H E N O T Y P E IN M S 151 A . 1.1 Lymphocyte subsets in a R R - M S patient participating in Schering-Plough® trial 151 A . l .2 Lymphocyte subsets in R R - M S patients participating in ICOS trial 151 150 A . l T H E E F F E C T S O F I M M U N O M O D U L A T O R Y D R U G S O N L Y M P H O C Y T E P H E N O T Y P E I N M S A.l.l Lymphocyte subsets in a RR-MS patient participating in Schering-Plough® trial Table A . l shows lymphocyte subsets of a R R - M S patient who participated in Schering-Plough® trial at pretreatment, and at 2 and 7 days post therapy. The nature of treatment is still unclear as to whether the patient received placebo or rhuIL-10. Major shifts in lymphocyte subsets are observed. However, we do not know i f the observed shirts in lymphocyte subsets are due to immunotherapy with rhuIL-10 or spontaneous. Following are some of the highlighted change in lymphocyte subsets that occurred either at 2 and/or 7 days post-treatment. Major reduction is seen in the percentage of C D 19+ (B cells), CD4+CD29+ (helper induced), CD3+CD26 (activated T cells), CD4+CD62L+ (selectin expression on CD4+), CD8+CD62L+ (L-selectin expression on CD8+), and CD4+CD49d+ ( V L A - 4 expression on CD4+) cells. O f the lymphocyte subsets that were highly increased after treatment CD8+C57+ (cytotoxic cells) can be pointed out. A.l.2 Lymphocyte subsets in RR-MS patients participating in ICOS trial The results in Table A .2 through A.5 indicate lymphocyte subsets of R R - M S patients participating in ICOS Corporation trial. Lymphocyte subsets were analyzed before treatment and 5 days post treatment. This double blind study on the effect of rhu-anti-LFA-1 on the clinical course of R R - M S is now unblinded, therefore, the nature of treatment is known. 151 Table A . 2 shows lymphocyte subsets in a R R - M S patient who received a single injection of 2 mg/kg of recombinant and humanized anti-LFA-1 mAb. Some of the major highlighted shifts in lymphocyte subsets are as following. Table A .2 shows a major reduction in the percentage of CD3+CD26+, CD4+CD49d+, CD8+CD49d+, CD4+CD62L+, and CD8+CD62L+ cells five days after treatment. Table A . 3 shows lymphocyte subsets for a R R - M S patient who received 1 mg/kg of anti-LFA-1 mAb. After a single injection of 1 mg/kg of ant i -LFA-1, there was a major reduction in the percentage of CD4+CD49d+ and CD8+CD49+ cells. However, unlike the patient who was treated with 2 mg/kg (Table A.2) , this patient showed no major reduction in her percentage of CD3+CD26+, or CD4+CD62L+ and CD8+CD62L+ cells after the treatment. Table A . 4 shows the lymphocyte subsets of a R R - M S patient in ICOS trial that received placebo. Unlike the R R - M S patients who were treated with anti-LFA-1 (Table A.2 and A.3) , this patient showed an increase in the percentage of CD4+CD49d+ and CD8+CD49+ after placebo treatment. The percentage of CD4+CD62L+ and CD8+CD62L+ cells were also moderately increased. Moreover, the percentage of CD3+CD26+ cells remained unchanged. Table A .5 shows the lymphocyte subsets of a R R - M S patient in ICOS Corp. trial that received intravenous methylprenisolone. Similar to the patient who received placebo (Table A.4) , the percentages of CD4+CD62L+ and CD8+CD62L+ cells were moderately increased while the percentage of CD3+CD26+ remained almost unchanged after treatment. The percentage of CD4+CD49d+ was also unchanged. 152 Table A. l Lymphocyte Subsets of a RR-MS Patient Enrolled in Schering Plough Trial Subsets pre- 2 days 7 days treatment Post treatment Post treatment CD3+ 76.4 83.0 72.3 C D 19+ 16.7 5.0 i 18.8 CD16+CD56+ 4.6 5.0 6.0 CD3+CD4+ 55.3 63.4 50 CD3+CD8+ 19.4 18.1 19.4 CD4+CD45RA+ 48.6 51.5 46.2 CD4+CD29+ 39.6 46.7 28.2 CD8+CD28+ 4.2 2.8 i 0.4 CD8+CD57+ 24.6 42.9| 32.8 CD3+CD27- 86.4 82.3 82.8 C D 4 + H L A - D R + 2.4 2.8 1.7 C D 8 + H L A - D R + 3.7 4.3 2.2 CD4+CD25+ 0.6 0.8 0.4 CD8+CD25+ 0 0 0.1 CD4+CD38+ 6.0 10.3| 3.7 CD8+CD38+ 2.8 5.0T 2.4 CD3+CD26+ 40.8 17.0^ 55.2 CD3+CD30+ 0.1 0 0 CD3+CD69+ 0.5 0 1 CD4+CD49d+ 20.4 23.5 10.8| CD8+CD49d+ 42.4 54.6 31.4 CD4+CD62L+ 86.4 90.1 58.4| CD8+CD62L+ 57.5 44.1 29.4| C D 8 + C D l l b + 14.3 14.5 10.8 CD3+CD54+ 2.4 1.1 3.1 Note. CD3+, C D 19+, CD16+CD56+, CD3+CD4+, and CD3+CD8+ lymphocyte subsets are given as percentage of the total number of lymphocyte (CD45 " s h t C D 1 4 - ) . Other lymphocyte subsets were calculated as the proportion of cells expressing a given second marker by using the equation: [% dual positive/(% dual positive + single positive only)] x 100. f and [ indicate major (>30%) increase and decrease, respectively, versus pre-treatment. 153 Table A .2 Lymphocyte Subsets of a R R - M S Patient (DES #0801) Enro l led in I C O S T r i a l W h o Received 2 mg/kg of A n t i - r h u - L F A (Hu23F2G) Subsets Pre-treatment 5 days post-treatment CD3+ 74.8 73.6 CD19+ 8.9 5.81 CD16+CD56+ 8.3 11.3? CD3+CD4+ 56.3 51.5 CD3+CD8+ 15.7 15.9 CD4+CD45RA+ 53.2 34.81 CD4+CD29+ 39 24.51 CD8+CD28+ 11.9 3.81 CD8+CD57+ 18.8 14.4 CD3+CD27- 7.1 3.51 C D 4 + H L A - D R + 5 2.71 C D 8 + H L A - D R + 7.3 4.2 J, CD4+CD25+ 3.2 1.71 CD8+CD25+ 0.1 0 CD4+CD38+ 17 5.4J, CD8+CD38+ 19.4 14.4 CD3+CD26+ 71.1 29.41 CD3+CD30+ 0.4 0 CD3+CD69+ 0.6 1.5 CD4+CD49d+ 15.1 5.41 CD8+CD49d+ 34.1 121 CD4+CD62L+ 84.1 42.81 CD8+CD62L+ 54.1 31.51 C D 8 + C D l l b + 25.8 30.9 CD3+CD54+ 3.6 2.5 Note. CD3+, C D 19+, CD16+CD56+, CD3+CD4+, and CD3+CD8+ lymphocyte subsets are given as percentage of the total number of lymphocyte (CD45 n g h t C D 1 4 - ) . Other lymphocyte subsets were calculated as the proportion of cells expressing a given second marker by using the equation: [% dual positive/(% dual positive + single positive only)] x 100. | and 1 indicate major (>30%) increase and decrease, respectively, versus pre-treatment. 154 Table A.3 Lymphocyte Subsets of a R R - M S Patient ( M C E #0802) Enro l led in I C O S T r i a l W h o Received 1 mg/kg of A n t i - r h u - L F A (Hu23F2G) Subsets Pre-treatment 5 days post-treatment CD3+ 79 78.6 CD19+ 13.2 10.9 CD16+CD56+ 4.5 8.2T CD3+CD4+ 49.8 49.7 CD3+CD8+ 27.8 26.8 CD4+CD45RA+ 23.6 57.7f CD4+CD29+ 39.7 40.5 CD8+CD28+ 6.4 12.9 | CD8+CD57+ 12.8 15.6 CD3+CD27- 5.4 4.3 C D 4 + H L A - D R + 5.6 3 .4 | C D 8 + H L A - D R + 9.2 61 CD4+CD25+ 2.5 2.6 CD8+CD25+ 0.1 0.2 CD4+CD38+ 19.2 14.3 CD8+CD38+ 10.3 14.3 CD3+CD26+ 49.6 45.3 CD3+CD30+ 0.2 0.2 CD3+CD69+ 0.9 1.1 CD4+CD49d+ 25.8 l l . l j , CD8+CD49d+ 26.1 1 3 | CD4+CD62L+ 65.9 64.2 CD8+CD62L+ 50.6 49.3 C D 8 + C D l l b + 13.9 20.9T CD3+CD54+ 1.8 l l . l t Note. CD3+, C D 19+, CD16+CD56+, CD3+CD4+, and CD3+CD8+ lymphocyte subsets are given as percentage of the total number of lymphocyte (CD45 g h t C D 1 4 - ) . Other lymphocyte subsets were calculated as the proportion of cells expressing a given second marker by using the equation: [% dual positive/(% dual positive + single positive only)] x 100. | and J, indicate major (>30%) increase and decrease, respectively, versus pre-treatment. 155 Table A.4 Lymphocyte Subsets of a R R - M S Patient ( M O S #0803) Enro l led in I C O S T r i a l W h o Received Placebo Subsets Pre-treatment 5 days post-treatment CD3+ 78.4 75.4 C D 19+ 4.9 8.4| CD16+CD56+ 7 14.9T CD3+CD4+ 37.4 32.7 CD3+CD8+ 33 33.9 CD4+CD45RA+ 53.3 62.2 CD4+CD29+ 44.8 57.9 CD8+CD28+ 5.4 nd CD8+CD57+ 23.9 29.4 CD3+CD27- 27.8 24.5 C D 4 + H L A - D R + 4.1 3.2 C D 8 + H L A - D R + 3.1 2.9 CD4+CD25+ 1.4 3.3f CD8+CD25+ 0.2 0.1 CD4+CD38+ 24.8 47.2| CD8+CD38+ 12.7 21| CD3+CD26+ 35.9 35.9 CD3+CD30+ 0.1 0.1 CD3+CD69+ 1.5 0.7 CD4+CD49d+ 18.3 29.1 CD8+CD49d+ 27.5 59.9f CD4+CD62L+ 75.2 84.6 CD8+CD62L+ 44.1 56.9 C D 8 + C D l l b + 22.7 29.5 CD3+CD54+ 1.3 1.1 Note. CD3+, C D 19+, CD16+CD56+, CD3+CD4+, and CD3+CD8+ lymphocyte subsets are given as percentage of the total number of lymphocyte (CD45 n g h t C D 1 4 - ) . Other lymphocyte subsets were calculated as the proportion of cells expressing a given second marker by using the equation: [% dual positive/(% dual positive + single positive only)] x 100. | and [ indicate major (>30%) increase and decrease, respectively, versus pre-treatment. 156 Table A.5 Lymphocyte Subsets of a R R - M S Patient ( T E S #0804) Enro l led in I C O S T r i a l W h o Received Intravenous Methylprednisolone Subsets Pre-treatment 5 days post-treatment CD3+ 79.1 76.1 C D 19+ 14.6 20.lt CD16+CD56+ 3.7 3.8 CD3+CD4+ 56 59.2 CD3+CD8+ 21.2 17.4 CD4+CD45RA+ 24.8 50t CD4+CD29+ 36 36.4 CD8+CD28+ 6.5 3.31 CD8+CD57+ 10.8 10.1 CD3+CD27- 7.3 3.9t C D 4 + H L A - D R + 1.7 1.3 C D 8 + H L A - D R + 5.8 5.4 CD4+CD25+ 1.1 0.1 CD8+CD25+ 0.2 0.3 CD4+CD38+ 8.9 7.7 CD8+CD38+ 8.1 10.4 CD3+CD26+ 57.2 62.3 CD3+CD30+ 0.1 0 CD3+CD69+ 0.5 1 CD4+CD49d+ 14.5 12 CD8+CD49d+ nd 33 CD4+CD62L+ 77.8 90.7 CD8+CD62L+ 59.7 74.7 C D 8 + C D l l b + 16.8 12.2 CD3+CD54+ 3.1 2.7 Note. CD3+, CD19+, CD16+CD56+, CD3+CD4+, and CD3+CD8+ lymphocyte subsets are given as percentage of the total number of lymphocyte (CD45 r ' 8 h t CD14- ) . Other lymphocyte subsets were calculated as the proportion of cells expressing a given second marker by using the equation: [% dual positive/(% dual positive + single positive only)] x 100. t and I indicate major (>30%) increase and decrease, respectively, versus pre-treatment 157 

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