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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 STUDIES IN H T L V - I - A S S O C I A T E D MYELOPATHY AND MULTIPLE  SCLEROSIS  by ABDULAZIZ AL-FAHIM M . S c , Microbiology & Immunology, The University of British Columbia, 1994 A T H E S I S 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 O F 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 O F 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 STUDIES 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 A l - F a h i m , 2000  In  presenting  degree freely  at  this  the  thesis  in  partial  fulfilment  University  of  British  Columbia, I agree  available for  copying  of  department publication  this or of  reference  thesis by  this  for  his  and  scholarly  or  thesis  study.  her  for  I further  purposes  financial  <  ^  ^  ^  /  ^  /  ^  DE-6  (2/88)  ,<?ujj  £ i  y  ?,<?<r>  requirements that the  agree that  It  gain shall not  The University of British C o l u m b i a Vancouver, Canada  D a t e  the  may  representatives.  permission.  Department  of  be is  Library  permission  granted  by  understood be  for  allowed  an  advanced  shall make for  the that  without  it  extensive  head  of  my  copying  or  my  written  ABSTRACT  Multiple sclerosis ( M S ) and human T-lymphotropic virus type I ( H T L V - I ) associated myelopathy ( H A M ) are inflammatory demyelinating diseases o f the central nervous system ( C N S ) . Current opinion implicates immune mediated factors, particularly T cells in the pathogenesis o f 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 o f peripheral blood M N C to cerebral endothelial cells that constitute the blood-brain barrier (BBB). The objective o f this thesis was to investigate, first, the phenotype o f lymphocytes of H A M patients.with a focus on T cell activation and adhesion related antigens; second, the adhesion and mechanism of adhesion o f blood M N C of H A M and M S patients to endothelial cells; and third, the effects o f immunomodulating drugs on lymphocyte subsets and function in M S . W e utilized direct two-color flow cytometry to study lymphocyte subsets in a group  o f patients with H A M and compared the results  with those o f 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 o f Cr-labeled 51  M N C s to the monolayers. Adhesion experiments indicated that M N C from H A M and  ii  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 o f 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 o f 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 o f "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 ( A v o n e x ™ , Betaseron®, and Rebif®). We found that A v o n e x ™ had the highest inhibitory effect followed by Rebif® and Betaseron® respectively. In this study, we also examined the effects o f IFN-P on M N C - H U V E C adhesion and demonstrated  that IFN-P pretreatment o f M N C , but not H U V E C  significant reduction in M N C - H U V E C  results in  adhesion. This might partially explain the  beneficial effects o f IFN-p in M S .  111  T A B L E OF CONTENTS  Abstract Table o f Contents List o f Abbreviations List o f Tables List o f Figures Acknowledgements  CHAPTER ONE  ii iv X VI viii xiv t  INTRODUCTION 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8  CHAPTER TWO  H A M and M S : an overview o f their immunopathology Role o f T cells in the immunopathogenesis of H A M and M S Leukocyte-endothelial cell adhesion and its implication in H A M and M S immunopathology Adhesion molecule classification, cascade and regulation Adhesion molecules in H A M and M S Immunotherapy in M S Challenges in studying M S and H A M Rationale, hypothesis and objectives  2 11 12 13 22 23 29 32  MATERIALS AND METHODS 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10  Study subjects Preparation o f peripheral blood M N C Monoclonal antibodies and two-color flow cytometry Culture o f H U V E C Culture o f E C V - 3 0 4 Adhesion assay Monoclonal antibody blocking studies PWM-induced IgG secretion Determination o f IgG content by E L I S A Statistical analysis  iv  45 48 48 49 51 52 53 53 54 55  CHAPTER THREE  RESULTS  3.1 3.2 3.3 3.4 3.5 3.6  CHAPTER FOUR  H T L V - I infection Lymphocyte subsets in H A M , H T L V - I carriers and healthy controls Blood M N C - H U V E C adhesion in H A M Blood M N C - H U V E C adhesion in M S Blood M N C - E C V - 3 0 4 adhesion in M S In vitro effects o f IFN-P on PWM-induced IgG secretion and M N C - H U V E C adhesion  DISCUSSION A N D 4.1 4.2 4.3 4.4 4.5 4.6 4.7  60 60 63 64 66 68  CONCLUSIONS  Lymphocyte subsets in H A M and H T L V - I carriers 96 Blood M N C - H U V E C adhesion in H A M 99 Blood M N C - H U V E C adhesion in M S 104 Comparing adhesion properties of H U V E C and E C V - 3 0 4 for blood M N C 109 The effects o f immunomodulatory drugs on lymphocyte subsets and function in M S Ill Summary and conclusions 117 Future experimental considerations 120  REFERENCES  123  APPENDIX A  150 A. 1  The effects o f immunomodulatory drugs on lymphocyte phenotype in M S  v  151  LIST OF TABLES  Table 1.1  Table 1.2  Adhesion molecules involved in leukocyte-endothelial cells adhesion  37  Similarities and differences between A v o n e x ™ , 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 Cell subsets o f fresh and cultured peripheral blood lymphocytes from controls, H A M , and carriers 72 Percentage o f CD3+, CD4+, and C D 8 + cells bearing putative functional markers in fresh and cultured peripheral blood from controls, H A M and carriers 73  Table 3.2  Table 3.3  Table 3.4  Table 3.5a  Table 3.5b  Table 3.6  Percentage o f CD3+, CD4+, and C D 8 + cells expressing activation-related antigens in fresh and cultured peripheral blood from controls, H A M and carriers  74  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  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  Adhesion o f blood M N C to untreated and IFN-y or L-929 supernatant treated H U V E C (cryopreserved M N C )  76  Adhesion o f s R R - M S , S P - M S , and healthy subjects blood M N C to HUVEC  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 o f I F N - p - l b on M N C - H U V E C adhesion  Table A . 1  Lymphocyte subsets o f a R R - M S patient enrolled in Schering-Plough trial  153  Lymphocyte subsets o f a R R - M S patient enrolled in I C O S trial who received 2 mg/kg o f A n t i - r h u - L F A (Hu23F2G)  154  Lymphocyte subsets of a R R - M S patient enrolled in I C O S trial who received 1 mg/kg o f A n t i - r h u - L F A (Hu23F2G)  155  Lymphocyte subsets of a R R - M S patient enrolled in I C O S trial who received placebo  156  Lymphocyte subsets o f a R R - M S patient enrolled in I C O S trial who received intravenous Methylprednisolone  157  Table A . 2  Table A . 3  Table A . 4  Table A . 5  Viii  80  LIST OF FIGURES  Figure 1.1  Sequential steps in a simplified model o f leukocyte-endothelial cells adhesion  43  Figure 3.1  Adhesion o f 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 o f anti-adhesion molecule antibodies on the adhesion o f M N C o f H A M patients to activated H U V E C  82  Effects o f anti-adhesion molecule antibodies on the adhesion o f M N C o f R R - M S patients to untreated H U V E C  83  Effects o f anti-adhesion molecule antibodies on the adhesion o f M N C o f healthy subjects to untreated H U V E C  84  Effects o f anti-adhesion molecules antibodies on the adhesion o f M N C of R R - M S patients to untreated and IFN-y treated H U V E C  85  Effects o f anti-adhesion molecule antibodies on the adhesion o f M N C o f R R - M S patients to untreated E C V - 3 0 4  86  Figure 3.3  Figure 3.4  Figure 3.5  Figure 3.6  Figure 3.7  Figure 3.8  Figure 3.9  Effects o f anti-adhesion molecules antibodies on the adhesion o f M N C o f healthy subjects to untreated E C V - 3 0 4  87  Adhesion of blood M N C to untreated or IFN-y and L-929 supernatant treated H U V E C and E C V - 3 0 4  88  Effects o f anti-adhesion molecules antibodies on the adhesion o f M N C o f R R - M S patients to untreated and IFN-y treated E C V - 3 0 4  89  Figure 3.10a  Effects o f IFN-p on PWM-induced IgG secretion in healthy subjects  90  Figure 3.1 Ob  Effects o f IFN-P on PWM-induced IgG secretion in R R - M S patients  90  Figure 3.10c  Effects o f A v o n e x ™ on PWM-induced IgG secretion in R R - M S and healthy subj ects  91  Effects o f Rebif® on PWM-induced IgG secretion in R R - M S and healthy subjects  91  Effects of Betaseron® on PWM-induced IgG secretion in R R - M S and healthy subjects  91  Figure 3.1 Od  Figure 3.10e  yiii  Figure 3.11  Figure 3.12  Figure 3.13  Same dose comparison of A v o n e x ™ , Betaseron® and Rebif® on inhibition of PWM-induced IgG secretion  92  Per dose comparison of A v o n e x ™ , Betaseron® and Rebif® on inhibition of PWM-induced IgG secretion  93  Fraction of weekly dose comparison of A v o n e x ™ , Betaseron® and Rebif® on inhibition of PWM-induced IgG secretion  94  IX  LIST OF ABBREVIATIONS  A 405 nm  Absorbance at 405 nm  ADCC  Antibody-dependent cell-mediated cytotoxicity  ANOVA  Analysis o f variance  APC  Antigen-presenting cells  ATCC  American type culture collection  BBB  Blood-brain-barrier  BSA  Bovine serum albumin  CD  Cluster o f differentiation  CHO  Chinese hamster ovary  CNS  Central nervous system  Con. A  Concavalin A  5 !  Cr  Radio-isotope chromium-51  CSF  Cerebrospinal fluid  CTL  Cytotoxic T-lymphocytes  EAE  Experimental allergic/autoimmune encephalomyelitis  EC  Endothelial cells  ECM  Extracellular matrix  ELISA  Enzyme linked immunosorbent assay  ESL-1  E-selectin ligand-1  FACS  Fluorescence activated cell sorter  FCS  Fetal calf serum  FITC  Fluorescein isothiocyanate  FSc  Forward scatter  GlyCAM-1  Glycosylation-dependent cell adhesion molecule-1  HAM  HTLV-I-associated myelopathy  HBSS  Hanks' balanced salt solution  HLA  Human leukocyte antigen  HRP  Horse radish peroxidase  HS  Horse serum  HTLV-I  Human T lymphotropic virus type I  HUVEC  Human umbilical vein endothelial cell  ICAM-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  kD  Kilodalton  kg  Kilogram  LAD  Leukocyte adhesion deficiency  LFA-1  Leukocyte function-associated antigen-1  mAb  Monoclonal antibody  MAC  Membrane attack complex  MadCAM-1  Mucosal addressin cell adhesion molecule-1  MAG  Myelin-associated glycoprotein  MBP  M y e l i n basic protein  mg  Milligram  MHC  Major histocompatiblity complex  MIU  M i l l i o n international units  MNC  Mononuclear cells  MOG  M y e l i n oligodendrocyte glycoprotein  MRI  Magnetic resonance imaging  mRNA  Messenger ribonucleic acid  MS  Multiple sclerosis  NK  Natural killer cells  NOD  Non-obese diabetic  PBS  Phosphate buffered saline  PE  Phycoerthyrin  PLP  Proteolipid protein  PMA  Phorbol myristate acetate  PP-MS  Primary progressive multiple sclerosis  PSGL-1  P-selectin glycoprotein ligand-1  Xii  PWM  Pokeweed mitogen  rhu-  Recombinant and humananized  RR-MS  Relapsing-remitting multiple sclerosis  s  Stable  SD  Standard deviation  SEM  Standard error o f the mean  SP-MS  Secondary progressive multiple sclerosis  SSc  Side scatter  TAC  T cell activation antigen  TCR  T cell receptor  TGF-B  Tumor growth factor-beta  Thl  T helper-1 subtype  Th2  T helpler-2 subtype  TNF-a  Tumor necrosis factor-alpha  TNF-(3  Tumor necrosis factor-beta  TSP  Tropical spastic paraparesis  TSP  Tropical spastic paraparesis  VCAM-1  Vascular cell adhesion molecule-1  VLA-4  Very late antigen-4  Xiii  ACKNOWLEGEMENT  I would like to express my thanks and appreciation to my supervisor, Dr. Joel Oger for his guidance, encouragement and patience throughout the course o f 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, M r s . 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  CHAPTER ONE INTRODUCTION  1.1  HTLV-I-ASSOCIATED M Y E L O P A T H Y A N D M U L T I P L E SCLEROSIS: A N O V E R V I E W OF THEIR I M M U N O P A T H O L O G Y 2  1.1.1 1.1.2  HTLV-I-associated myelopathy Multiple sclerosis  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  1.3  2 5  11  LEUKOCYTE-ENDOTHELIAL CELL ADHESION AND ITS I M P L I C A T I O N I N H A M A N D M S I M M U N O P A T H O L O G Y  12  ADHESION M O L E C U L E CLASSIFICATION, C A S C A D E A N D REGULATION  13  1.4.1 1.4.2 1.4.3  Classification o f adhesion molecules Adhesion cascade (leukocyte-endothelial adhesion) Regulation o f adhesion  13 17 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 IN STUDYING M S A N D H A M  29  1.8  RATIONALE, HYPOTHESES A N D OBJECTIVES  32  1.8.1 1.8.2 1.8.3  Studies o f lymphocyte subsets in H A M Studies o f lymphocyte-endothelial adhesion in H A M and M S Studies o f immune modulating agents in M S  32 33 35  1.4  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 ( H T L V - 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 o f 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 o f 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  o f the disease. Pathologically, H A M is characterized by  perivascular cuffing by mononuclear cells and demyelination in the central nervous system ( C N S ) , predominantly in the thoracic region o f 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 o f C D 4 + and C D 8 + T cells as well as some B cells (Moore et al., 1989). However, some reports  2  indicate the  preponderance o f C D 4 + T cells in very early stages o f the spinal cord lesions (Iwasaki et al., 1992). In patients with chronic H A M , lymphocytic infiltrates are less abundant and consist mostly o f C D 8 + 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 o f H A M patients (Itoyama et al., 1988, Jacobson et al., 1988). Although these abnormalities are believed to result from the active replication o f H T L V - 1 genome, the exact mechanism of these activated T cells involvement in the pathogenesis o f H A M is still unclear (Oger and Dekaban, 1995; Yoshida et al., 1989). It also remains unclear why only a small percentage o f 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 o f 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; L i n k et al., 1989; Ijichi et a l , 1989). Some o f 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 o f H A M patients (Nakagawa et al., 1995). There are two major hypotheses that have been proposed  to explain the  immunopathogenesis o f 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 o f autoreactive T cells and the induction  3  of an autoimmune process (Oger et al., 1995). Recent evidence that the frequency o f H L A class I restricted and H T L V - I tax-specific C D 8+ cytotoxic T-lymphocytes ( C T L ) is high in blood mononuclear cells and in C S F o f H A M patients but not i n 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 killing o f proposed HTLV-I-infected glial cells in a manner restricted by M H C class I antigen, or by secretion o f cytokines from cytotoxic T cells that could adversely affect the function o f uninfected neurons and glial cells within the C N S (Giraudon et al., 1996). Whereas the presence  o f CD8+ T lymphocytes in H A M lesions is well  documented, it is controversial whether H T L V - I infects the resident cells o f the C N S . The direct demonstration of H T L V - I antigens in glial cells is difficult, because o f the close association with potentially infected T cells. It is possible that C D 8 + cytotoxic T cells are recognizing viral products presented by HTLV-I-infected T cells. This might result in the activation o f cytotoxic T cells and the subsequent secretion o f proinflammatory 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, C D 4 + 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  4  in H A M relies on random  infection o f C D 4 + 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 o f activated T cells with specificity for myelin basic protein can induce experimental autoimmune  encephalomyelitis ( E A E ) in mice, indicating that the control o f the  activation state o f 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 o f H T L V - I in vivo infection o f 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 ( M S ) is the most common neurological disease of young and  middle-aged adults o f 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 o f the C N S . The clinical course of M S is highly unpredictable. In the majority o f cases, M S starts with a relapsing-remitting course ( R R - M S ) that eventually changes to a secondary progressive (SP-MS) course; less commonly the course is progressive from the onset (primary progressive, P P - M S ) (Lublin and Reingold, 1996). While the etiology o f M S is still unknown, immunological factors are believed to play an important role. The  5  histopathology o f the lesion in M S is characterized by multifocal and periventicular infiltration o f the white matter by inflammatory immune cells in the C N S , and a selective destruction o f myelin and myelin-forming oligodendrocytes (Raine, 1994; Cannella and Raine, 1995). Immune cells consist mostly o f 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 o f C N S myelin, while the lesion progression is driven by the activity o f C D 4 + T cells (Scholding et al., 1994). The main immune abnormality in M S probably involves T cell-mediated immune function, but one o f the hallmarks o f M S disease is the presence o f immunoglobulin (lg) G o f restricted heterogeneity in the cerebrospinal fluid in majority o f patients (Johnson and Nelson, 1977) also referred to as "oligoclonal bands". Since oligoclonal bands are only occasionally found in the serum o f M S patients, it is conceived that a few clones o f 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 o f 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 o f IgGs (Goust et al., 1982; O'Gorman et al., 1987). Furthermore, B cells isolated from C S F o f 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 myelinassociated glycoprotein ( M A G ) (Olsson et al., 1990; Sun et al, 1991; Baig et a l , 1991).  6  Studies in animal models o f 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 o f M S , enchephalitogenic T cells only  cause full demyelination in the presence o f 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 o f B cells are also an integral component o f 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 o f 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 o f 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 o f 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 o f M S came from the detection o f myelin-reactive T cells in the blood and C S F o f M S patients. Further studies however, revealed that the autoreactive T cells also form part o f 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 o f 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 o f a single antigen. This is because o f the phenomenon o f 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 o f one protein epitope can subsequently lead to activation o f T cells recognizing other epitopes o f the same protein. In time, there might also be activation o f 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 o f 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, C D 4 + T lymphocytes differentiate into two main types o f 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 o f T h l cells and inhibits the development of the Th2 response. Alternatively, IL-4 and IL-10 favor the development o f 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 o f 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 o f IL-10 suppresses the development o f 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 o f cytokine production. Increased levels o f 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 o f 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 o f 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 T h l / T h 2 dichotomy is less clear in the human immune system and represents the extreme o f a range of cytokine production profiles. Many instances exist in humans o f cells that secrete combinations o f 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 inflammatory  properties  may be more  appropriate  o f cytokines with pro- or antiin classifying their  functional  properties. Some scientists do not favor the autoimmune hypothesis o f 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 o f 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 o f 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  progressive form o f M S (Roos, 1983; D a l Canto et al., 1995).  10  clinically  to  the  chronic  1.2  R O L E OF T C E L L S IN T H E IMMUNOPATHOGENESIS OF H A MA N D MS  Although different mechanisms o f pathology have been proposed, there is a general agreement that the pathogenesis o f 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; U t z 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) o f H A M patients (Moore et al., 1989). H A M patients have also been shown to have high levels o f 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 o f large C D 3 + cells that also express markers of activation, such as H L A - D R and IL-2 receptor molecules. In addition, peripheral blood lymphocytes o f H A M patients show spontaneous proliferation in the absence o f any exogenous antigen/mitogen in vitro (Itoyama et al., 1988b). Furthermore, H A M patients have a high frequency o f H T L V - I specific C D 8 + 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 ( C S F ) 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 ( E A E ) , an animal model for M S , is T-cell dependent and can be induced through the transfer o f 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 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  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 o f leukocytes into the central nervous system is complicated by the existence of a specialized microvasculature, characterized by the presence o f a continuous network o f high resistance and complex tight junctions. Under the control o f surrounding astrocytes, this microvasculature constitutes the blood-brain barrier ( B B B ) that limits the exchanges between the blood and brain o f soluble substances such as growth factors, cytokines and immunoglobulins as well as immune cells (Goldstein and Betz, 1983; Pardridge, 1988; Joo, 1993). O n the basis o f the existence o f the B B B and of low levels o f 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 Billingham, 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 o f the C N S in H A M and i n M S indicate that the breakdown o f 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 o f 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 o f these cell surface molecules in adhesion o f leukocyte to endothelial cells are emphasized and discussed.  1.4  ADHESION  MOLECULE  CLASSIFICATION,  CASCADE  AND  REGULATION  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)  13  members  (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  (platelets/endothelium),  were  first  identified:  L-selectin (lymphocyte),  P-selectin  and E-selectin (endothelium) (Bevilacqua and Nelson, 1993;  T e d d e r et al., 1995).  L-selectin is expressed constitutively on all leukocytes and has a critical role i n the adhesion o f 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 o f 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 ( T N F - a ) , and shed rapidly after loss of cytokine stimulation (Doukas and Pober; 1990). E-selectin binding o f 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 o f C D 3 4 expressed by endothelial cells, and mucosal addressin cell adhesion molecule-1 ( M a d C A M - 1 ) , and P-selectin. Pselectin 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 o f E-selectin to C D 6 6  14  (Kuijpers et a l , 1992), C D 1 1 / C D 1 8 (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 o f selectins in inflammatory responses is seen in leukocyte adhesion deficiency ( L A D ) type II, in which the congentical absence o f 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 (CDlla/CD18,  CDllb/CD18,  C D 4 9 d / C D 2 9 ) , and  OC4B7.  and  CDllc/CD18),  the  Bi integrin V L A - 4  (a B,, 4  The B2 integrins share a common B chain ( C D 18) and can be  noncovalently associated with any of the three C D l l a ( L F A - 1 ) , C D l l b (Mac-1), and CDllc  (pl50,  96)  a  subunits.  Peripheral  blood  lymphocytes  mainly  express  C D l l a / C D 1 8 ( L F A - 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 integrins also bind soluble proteins such as fibrinogen, and factor X 2  (Larson and Springer, 1990). The Bi integrins share C D 2 9 as their common B subunit. OC4B1 ( V L A - 4 , CD49d/CD29) a prototypical Bi integrin is mostly prominent on cells o f 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 o f 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 o f intracellular signals such as focal adhesion kinase or the PI 3-kinase. Thus, the interaction o f 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 o f this superfamily are expressed by endothelial cells (e.g. M a d C A M - 1 and vascular cell adhesion molecule1 ( V C A M - 1 ) , or by both endothelial cells and leukocytes [e.g. intracellular adhesion molecule-1 and - 2 ( I C A M - 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 o f leukocytes and promotes adhesion between the endothelial cells and leukocytes. I S G F  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; Y a p et al., 1997). Members o f this superfamily are expressed by, and are responsible for the integrity o f endothelial and epithelial cells. Cadherins found at intercellular endothelial junctions seem to play a key role in the extravasation o f 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 o f molecular events that take place in an ordered series o f steps involving close interactions  between  adhesion  receptors  expressed  by migrating leukocytes  and  endothelial cells (EC). A consensus model o f 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 o f 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 o f 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  chemoattractant/cytokines  the  presence  that further  of  inflammatory  mediators  such  as  deliver activating signals to the leukocytes  resulting i n upregulation o f 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 ( L F A - 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 o f leukocytes induces a rapid shedding o f L-selectin caused by cleavage o f the extracellular portion o f L-selectin by an unidentified endogenous protease (Kansas, 1996). The activation o f leukocytes also results in an increase in avidity o f integrins for their ligands due to the conformational changes i n integrin heterodimer. The increased avidity o f the leukocytes integrins results in the firm adhesion o f leukocytes to E C . During this phase, leukocytes transmigrate  change  shape and acquire  a flattened  morphology. Then  leukocytes  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 o f 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 i n leukocyte extravasation (Piali et al., 1995; Bianchi et al., 1997). The migration o f leukocytes from the blood vessel wall toward the inflammatory foci also involves  18  interactions o f the leukocyte receptors mainly R\ integrins with the components o f the extracellular matrix such as collagen, fibronectin, and laminin.  1.4.3  Regulation of adhesion Regulation o f adhesion occurs through increased avidity o f existing adhesion  molecules or increased expression of molecules on the cell surface. For example, activation o f T cells with the phorbol myristate acetate ( P M A ) has been demonstrated to increase the affinity o f 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 o f integrins cell surface expression (Wilkins et a l , 1991; Shimizu et al., 1991; Dustin and Springer 1989). Cross-linking o f 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 o f T lymphocytes, because spontaneous adhesion is low or absent in resting T lymphocytes (Dustin and Springer, 1989). Furthermore, binding o f cell adhesion molecules with their respective ligands causes alteration in the expression and affinity of adhesion molecules (Frelinger etal., 1988; L o u et al., 1996). Activation o f 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  o f 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 o f adhesion molecule expression determines homing o f lymphocytes to organs  and sites o f 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 o f E-selectin, but expression is typically short lived. Concomitant  stimulation with  interferon  gamma  (IFN-y)  enhances and  expression o f E-selectin (Doukas and Pober, 1990). If chemoattractant  prolongs  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 o f leukocytes into the E C M and tissue (Springer, 1994; Tedder, 1995). Besides changes in avidity and cell surface expression, differential distribution and expression o f adhesion molecules on T lymphocyte subsets may  modulate  immune/autoimmune response patterns. O n the one hand, C D 8 + T cells have been reported to have higher L F A - 1 expression compared with C D 4 + T cells (Pardia et al., 1989). O n the other hand, it has been shown that m A b against V L A - 4 inhibited C D 4 + but not C D 8 + T cell infiltration o f the pancreas in the non-obese, diabetic ( N O D ) mouse model o f 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 C D 8 + T cells. In addition to the broadly expressed proinflammatory cytokines such as T N F - a , IFN-y, I L - 1 , IL-4, and IL-6, chemokines are also important soluble mediators o f 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, Important  chemokines  stimulating factors  include R A N T E S ,  1996; Merrill and Benveniste,  IL-8, MIP-1B,  MlP-la  ( G - C S F , G M - C S F ) , chemoatractant peptides  MCP-1,  1996). colony  (C5a, F M L P )  and  neuropeptides (Luster, 1998; Baggiolini, 1998). Binding o f 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 o f 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 C D 8 + T cells, whereas the closely related MIP-1B acts on CD4+ T cells ( M i n g Wang et al., 1993; Taub et al., 1993). Furthermore, M I P - a but not MIP-B increases the adherence o f T cells to endothelial cell (Tanaka et al., 1993). Most "classic" cytokines (e.g. I L - 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, T G F - B induces migration o f 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  ADHESION M O L E C U L E S IN H A M AND MS  Analysis o f the spinal cord lesions of H A M patients has shown  enhanced  expression o f V C A M - 1 and E-selectin on endothelium, and high levels o f V L A - 4 , L F A - 1 and Mac-1 expression on infiltrating mononuclear cells (Umehara et al., 1996). Increased levels o f soluble I C A M - 1 and V C A M - 1 have been detected in the serum and C S F o f H A M patients (Mainnolfi and Rothlein, 1992; Tsukada et al., 1993b; Matsuda, 1995a). Significant elevation o f soluble L-selectin has also recently been reported in the sera o f H A M patients (Tsujino et al., 1998). Expression o f adhesion molecules on freshly isolated lymphocytes from H A M patients have not been studied. However, enhanced expression o f V L A - 4 and V L A - 5 integrins has been shown in peripheral blood lymphocytes o f healthy controls that were infected with H T L V - I in vitro (Dhawan et al., 1993). Enhanced expression o f 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 o f the studies of the role o f adhesion molecules during M S have relied on immunochemical analyses of the expression o f adhesion molecules during the different stages o f 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 o f 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, i n M S lesions some resident cells o f the C N S , such as astrocytes or microglia, show increased cell surface expression o f 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 o f M S patients  22  also express increased levels o f adhesion molecules such as L F A - 1 , L F A - 3 , C D 2 , and C D 4 4 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 o f 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 o f adhesion molecules in M S is derived from measurement o f circulating soluble adhesion molecules during M S . Investigators found that circulating forms o f 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 o f new gadolinium-enhancing lesions in M R I (Mobner et al., 1996) (this is a parameter o f B B B breakdown and disease activity).  1.6  I M M U N O T H E R A P Y IN 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 o f 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 o f circulating leukocytes across the B B B into the C N S i n inflammatory conditions, such as M S , involve a complex series o f 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 i n 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 o f T h l cells (Mosmann and Moore, 1991). IL-10 is also thought to participate i n recovery from the inflammatory events by down-regulating the activated state o f endothelial cells and macrophages (Olsson, 1995). In M S , levels o f IL-10 m R N A were reported to be higher i n stable M S compared with relapsing M S and the levels o f IL-10 were shown to decline prior to relapse i n M S (Rieckmann et al., 1994a).  These  observations prompted Schering-Plough Corporation to develop recombinant  human  interleukin-10 (rhuIL-10) for potential therapeutic use i n multiple sclerosis. RhuIL-10 was produced in a strain o f Escherichia coli bearing a genetically engineered plasmid that contains a rhuIL-10 gene. With the exception o f methionine residue at the aminoterminus, rhuIL-10 is identical to endogenous human IL-10 protein. The effect o f rhuIL10 has been examined i n several rodent models o f E A E . In one study where T N F - a was used to induce relapses o f E A E i n S J L mice that had recovered from acute E A E , rhuIL10 given with T N F - a provided complete protection against relapses (Crissi et al., 1995). In another study where acute E A E i n the Lewis rat was induced by M B P , administration  24  of rhuIL-10 during the initial phase o f the disease suppressed the subsequent induction o f 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 o f rhuIL-10 given immediately after M B P injection showed no effect (Smith, 1994). However, a similar dose o f rhuIL-10 given at the onset o f E A E symptoms showed a trend toward improvement (ScheringPlough, Data on file as D-27219). However, repeated doses of rhu-IL-10 at 7 and 14 days during the initial phase o f the disease resulted in an exacerbation o f 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 o f single dose o f rhuIL-10 was tested in clinical trial o f relapsing-remitting form o f 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 o f proteins that inhibit viral activities and cellular  proliferation and modify the immune response (Becker et al., 1995). One member o f the I F N family, IFN-B has been shown to be o f benefit in relapsing-remitting M S . The mechanisms underlying the efficacy o f IFN-P in M S patients are still not completely known. However, there are several proposed mechanisms that may play a role i n the efficacy o f IFN-p in M S (Yong et al., 1998). These include: down-regulation o f the IFN-y activity; induction o f T suppressor cell function; augmentation o f IL-10 production;  25  inhibition o f 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 o f interferon P in use. These are, A v o n e x ™ (IFN-p-la, Biogen), Betaseron® (IFN-p-lb, Berlex), and Rebif® (IFN-p-la, Serono). The similarities and differences between the three preparations o f 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 o f 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 o f 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 o f 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 o f 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 o f 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 o f either 8 M I U or placebo subcutaneously on alternate days over 3 years. Treatment with Betaseron® resulted in the significant delay o f disease progression and reduced the disease activity. Furthermore, it increased the time to first relapse and the proportion o f patients who were relapse free.  26  A v o n e x ™ (IFN-B-la) was tested in a trial involving 301 patients with relapsing form o f M S and mild to moderate neurological impairment. Treatment consisted o f 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 o f 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 o f exacerbation by 27% and 33% in the 22 pg and 44 pg groups, respectively, ( P R I S M S 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 ( P R I S M S study group, 1998).  1.6.3  Anti-LFA-1 monoclonal antibody The importance and beneficial effects o f 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, m A b s 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 o f L F A - 1 i n the pathogenesis o f E A E remains not well defined. The presence o f brain inflammation in E A E and the anti-inflammatory effects o f antibodies against L F A - 1 adhesion molecule led I C O S Corporation to develop and test a humanized anti-LFA-1 named H u 2 3 F 2 G . H u 2 3 F 2 G is produced by Chinese hamster ovary ( C H O ) as y4 immunoglobulin. This m A b recognizes both L F A - 1 and M a c - 1 . A s expected, in vitro H u 2 3 F 2 G has the following properties: inhibits L F A - 1 dependent cell aggregation; blocks binding o f 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, I C O S tested the effects o f H u 2 3 F 2 G i n nonhuman primate Macaca fascicularis E A E . In this E A E model animals were treated with intravenous injection o f either H u 2 3 F 2 G (2 mg/kg for 7 days) or dexamethasone (4 mg/kg for 3 days). The result indicated less severity o f E A E , more resolution o f 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 I C O S to determine the safety and efficacy o f H u 2 3 F 2 G i n the treatment o f acute exacerbations o f M S . A multicenter, randomized, double-blind, placebo-controlled trial was conducted. A total o f 169 patients were enrolled within seven days o f the onset o f symptoms recurrence to one o f four treatment groups: Placebo (n=43), methylprednisolone at 1 gram intravenously (IV) for 3 days (n=41), H u 2 3 F 2 G at 1 mg/kg I V (n=44) or H u 2 3 F 2 G at 2 mg/kg I V (n=41). The result indicated that while H u 2 3 F 2 G 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 H A M  Variability in disease progression is a prominent clinical feature o f M S . Some patients have initial attacks, complete recovery, and no further symptoms, while others progress rapidly within months o f initial involvement. Most patients have exacerbation and remission that follow an unpredictable course. Immunopathological studies o f lesions in a single M S patient reveal a fairly uniform pattern o f 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 o f early biopsy and autopsy o f 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 o f 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 o f development of effective therapy in M S . To elucidate the mechanism of pathogenesis o f M S , investigators have relied on studies o f experimental autoimmune encephalomyelitis ( E A E ) , considered to be an animal model o f M S . Acute and chronic variants o f this T-cell driven autoimmune disorder o f 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 ( M B P ) , 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 o f 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 o f M S feature mostly acute attacks and full recovery often not associated with destruction o f 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 o f 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 o f M S . Studies o f M S are further complicated by unidentified environmental influences and their interactions with individual immuno-genetic backgrounds. One approach to advance our understanding o f the M S pathophysiology is to study immune-mediated diseases o f the C N S associated with well-known etiology. A n example o f such a disease is H A M . The presence o f activated T cells associated with proinflammatory cytokines as well as elevated levels o f IgG and the presence o f oligoclonal bands in the C S F o f 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 o f 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 o f the brain and spinal cord may also be indistinguishable. In general, HAM  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). O n the one hand, multiple sclerosis is a common neurological disease among people o f European descent in British Columbia. O n the other hand, the number o f H A M patients is very few and confined mostly to West Coast Amerindian populations. In fact, the total number o f 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 o f 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  RATIONALE, HYPOTHESES AND OBJECTIVES  1.8.1  Studies of lymphocyte subsets in HAM The technique o f flow cytometry allows for large number o f cells to be analyzed  and divided into subsets based on expression o f surface antigens (Tragnos, 1984) and is particularly suitable for quantification o f 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 o f antibodies conjugated to the phycoerthyrin (PE) dye allowed simultaneous analysis o f two different molecules with a single laser line (Oi et al., 1982), thus providing more information than single color analyses. F l o w cytometry has also been used to study lymphocyte subsets in peripheral blood o f H A M patients. However, to date, most lymphocyte subset studies in H A M have relied on single color analysis o f lymphocyte expressing markers o f activations confined to H L A - D R , or C D 2 5 . 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 i n both health and diseases. Some o f 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 o f H A M . Therefore to further  distinguish and  characterize  immunoregulatory  lymphocytes  subsets,  we  investigated lymphocyte subsets employing two-color flow cytometry using a panel o f well defined monoclonal antibodies against T lymphocyte markers o f activation and adhesion related antigens ( C D 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 o f the C D molecules referenced in this thesis are indicated in Table 1.3. One o f the immunological hallmarks o f 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 o f the C N S . A prerequisite to the passage o f lymphocyte across blood vessel walls is binding to endothelial cells. In vitro studies have indicated that adhesion o f lymphocytes to endothelium is increased by  33  activation o f 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 o f H A M and M S lymphocyte to endothelium in vitro is augmented as well. W e studied this interaction in an in vitro model that measures the binding o f Cr-labeled 51  human lymphocyte to endothelial cell monolayers (Brown et al., 1993). Using this system, the contribution o f 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 o f endothelial cell lines to investigate adhesion, transmigration and the responsiveness o f the endothelium to pro- and anti-inflammatory agents. E C V - 3 0 4 was reported to be a spontaneously transformed and immortalized human umbilical vein endothelial cell line. E C V - 3 0 4 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 o f 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).  E C V - 3 0 4 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 o f this project, we also utilized this cell line as a source o f endothelial cells for adhesion and inhibition o f 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 o f the epithelial bladder cancer cell line T-24 (Dirks et al., 1999), rather than an endothelial cell line. Since E C V - 3 0 4 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 o f 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  o f IFN-P, namely A v o n e x ™ , 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 o f relapses, the number of demeylinating plaques and suppression toward disability. The beneficial effect o f IFN-B in M S is likely due to immuno-modulating properties o f IFN-B and not due to suppression o f viral infections (Panitch, 1994). Nevertheless, the relative units o f these IFN-P are all measured by a viral plaque assay and represent the antiviral potency o f the drugs, which may not correlate with their beneficial effect in M S . In trials, evaluating the safety and efficacy o f 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 o f these three IFN-P i n use. Therefore, the optimal dose for treatment o f M S with each o f the IFN-P in use is still under debate. In an approach to resolving this issue, we directly compared the effects o f each o f the three preparations o f IFN-P in use in an in vitro assay representing an immunomodulating model that relies on inhibition o f T-cell dependent B cell activation.  35  More specifically, the potency o f the three IFN-P was calculated from their ability to suppress P W M induced IgG secretion in the culture supernatant. The potential roles o f L F A - 1 adhesion molecules and IL-10 in M S are addressed in the above paragraphs. During the course o f this thesis, two separate double blind, placebo controlled, multicenter trials were initiated by Schering-Plough and I C O S Corporation to respectively, test the safety and efficacy o f IL-10 and anti-LFA-1 i n relapsing-remitting M S . The University o f British Columbia multiple sclerosis clinic was one o f the trial sites. Therefore, we had an opportunity to study the potential in vivo effects o f IL-10 and A n t i - L F A - 1 on lymphocytes surface activation and adhesion related antigens using flow cytometry. IFN-B has been shown to favorably alter the disease course o f relapsing-remitting multiple sclerosis patients. This clinical efficacy is accompanied by a more profound reduction i n the number and size o f lesions as measured by gadolinium enhanced M R I (Paty et al., 1993). Thus, it is possible that IFN-P may mediate its effect i n M S at least i n part by affecting the interaction o f 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 i n M S remain unclear. Since an important pathologic feature o f M S is the transmigration o f leukocytes across the B B B into the C N S , we proposed that a potential mechanism o f action o f IFN-P could be due to the ability o f the IFN-P to inhibit adhesion o f circulating lymphocytes to endothelium. 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Q U  1  —i >  1  "Q  H—»  s- fl  4 3  1  > 2 <  CL)  cd  - -H  <D  -A s  3 <D - J  60 c  60  u  >,  o o  O  o  o c Ui T3  -fl  <D  -tn  >  «  00  Is  CD O CD  !-H CL) O  JS  00  I  CH  — ' -t—'  S o  CD  fl  •fl cd  g  H  J D  ft  3 fl cd cu  I >  !i  o o 3  D  H  CCJ  CD  CT CD  H  4 J  fl  u  ^ A ,  CD  td  -a CL)  fl  chemcik  60  o Ji <% 8? oo  Endot helial eel  1  o  l) - O fl -fl  O  <u c  H> -  fl  - f l GO fl  ICAM-•1 VCAM-•1  Chemokine i eceptdr  H  LFA-1 VLA-4  Leukoc;ytes  i  00  fl  CL)  H—»  CN  CH  C  CD  IJ  H  S-H  O -iH -fl 5 •  cd  i  CD  'oo cd cd  o •a c u  —1  60  d.9" fl .2 —i  >  <•  CL)  o y  <-| cd H S3  2  H  -fl  JJ  fl .s  -fl  O  fl  '  fl  ^  ~ c ^  tl  60  fl fl -fl H o u  S to  O  .fl  .A  T3 +->  <u cd  -fl  o  CD  & s  fl .3 2  S  kH -fl  fl -fl .fl oo  -fl s  'c  fl  CD  1  V J  - .2  •  CD CD -fl  Id  fl  >  S-fl"  60  _ H  -T3  CD CD O  "cd  t -H *CD  H-»  o C  w  ^ •1  «  fl 2  GO  C  -fl°^ oo +-»  i-J  oo  SH CT CD  •D:  1  CD  O  (D  ti  2  60 60  t50  "HS g  -! ° * fl CD « -fl  fl ^ O  -fl  00  O AH VH  OH  CD O  ro  CHAPTER TWO MATERIALS AND METHODS  2.1  STUDY SUBJECTS  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 ANTIBODIES A N D TWO-COLOR FLOW CYTOMETRY  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  ADHESION ASSAY  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  STATISTICAL ANALYSIS  55  44  MATERIALS AND METHODS  2.1  STUDY SUBJECTS  The human T cell lymphotropic virus type I ( H T L V - 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 o f 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 o f adult T-cell leukemialike cells (cells with lobulated nuclei) in both peripheral blood and C S F . The presence o f 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 o f  lymphocytes after 2 days in culture, blood samples o f 7 o f the 7 H A M patients, 7 o f 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 o f 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 o f the H A M patients, all the H T L V - I carriers and 4 o f the healthy controls were Coastal Amerindians from British Columbia, Canada. The multiple sclerosis ( M S ) 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 o f two separate lesions or two attacks and clinical evidence o f one lesion and paraclinical evidence of another separate lesion. Furthermore, the attacks should involve different parts o f the central nervous system ( C N S ) and be separated by a period o f at least one month. A n attack in M S is defined as the occurrence o f 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 o f 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 relapsingremitting ( R R ) M S patients who were free o f 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 ( E D S S ) (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 o f endothelial cells in comparing multiple sclerosis ( M S ) 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 s R R 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 E C V - 3 0 4 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 s R R 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, I C O S 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 l o w cytometric analysis o f 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 V i e w , 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 M g 2 - f r e e Hanks' balanced salt solution (Gibco, Grand Island, N Y ) at 300g +  +  at 4 ° C for 10 min. The viability o f 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 i n medium  consisting  of R P M I - 1 6 4 0 (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 i n a 25cm^ 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: C D 4 5 , C D 1 4 , C D 1 9 , C D 1 6 , C D 5 6 , CD3, CD4, C D 8 , H L A - D R , CD25, CD57, CD26, CD27, CD69, CD49d, CD62L, CD54 (Beckton Dickinson, San Jose, C A ) and C D 4 5 R A , C D 2 9 , C D 3 8 , C D 2 8 , C D 3 0 (Coulter  48  Immunology, Hialeah, F L ) . 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 m i n at 4°C, the cells were washed twice with  phosphate-buffered  saline ( P B S ) supplemented with 1% F C S and 0.1% NaN3 and fixed in 500wl o f 1% paraformaldehyde in P B S , 1% F C S , 0.1% N a N 3 . Two color immunofluorescence was recorded using a F A C S t a r ® plus (Becton Dickinson, Mountain V i e w , 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 C D 1 4 - . The F I T C and P E gains were h t  optimized by using FITC-nonspecific IgG2a and PE-nonspecific I g G l . Data analysis was  performed  using CELLQuest™  software.  The operation  o f the  FACStar®  equipments and analysis o f data was carried out in the Department of Medicine, Division of Neurology at Vancouver Hospital and Health Sciences Center by Abdulaziz A l Fahim.  2.4  CULTURE OF HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS  Primary and secondary cultures o f 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 o f 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 m i n 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 M e d i u m 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 Chemical  Co.), 100  ng/ml heparin (Sigma  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 o f Ulex Europaeus Agglutinin I ( U E A - 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 o f polyclonal antibodies to rabbit antifactor VIII antigen (Dakopatts). To demonstrate the binding o f U E A - I lectin by endothelial-specific receptors, the cultured monolayers were incubated with  1:400  dilution o f U E A - I lectin (Vector, Mississauga, Ontario). After washing, they were incubated with a 1:100 dilution o f 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  monolayers  Immunoresearch  were  incubated  with  Laboratories).  After  further  amino-ethyl-carbazol, and  washing,  cultured  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 C o . , 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 o f M N C to cytokine-treated H U V E C , monolayers were treated for 48 h with 100 U / m l o f human recombinant interferon-gamma  (IFN-y)  (Chemicon International Inc., Temecula, C A ) or 50% filtered (0.2 pm, Gelman Sciences, Ann  Arbor, M I ) murine L-929 fibrosarcoma supernatant prior to adhesion  assay.  Confluent monolayers o f 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 ECV-304 C E L L L I N E  ECV-304  cells (American Type Culture Collection, Rockville, M D ) were  cultured in M - 1 9 9 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). E C V - 3 0 4 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 o f E C V - 3 0 4 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 o f 10 m l E C V - 3 0 4 culture medium. E C V - 3 0 4 was pelleted at 400g for 10 min, resuspended in culture medium to 1 X 10 cells/ml and 100 p i added to each well o f fibronectin coated 96-well 5  flat bottom microtitre plates (Falcon). Culture media were changed every 2-3 days and confluent monolayers were obtained after 4-6 days o f culture.  51  2.6  ADHESION ASSAY  Cultured M N C were pelleted by centrifugation and 200 u C i o f N a 2 C r 0 4 ( I C N 5 1  Biomedicals Inc, Costa Mesa, C A ) were added to 5 X I 0  6  cells in a total volume o f 200  ul o f 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 E C V - 3 0 4 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 1 0  5  51  C r - l a b e l l e d M N C were added in a further 100 ul volume o f  medium. After a 1 h incubation at 37°C, non-adherent cells were removed by gently washing the monolayers five times with 200 ul o f pre-warmed medium. Wells were examined by phase-contrast  microscopy before and after washing to determine the  evenness o f cell settling and potential damage  to the endothelial/ECV-304 cells  monolayers during washing. The H U V E C or E C V - 3 0 4 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 o f adhesion to the H U V E C or E C V - 3 0 4 monolayer was calculated as follows:  % adherence =  C P M in 100 nl lysate — C P M in 100 ul original cell suspension  52  X 100  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 ( C D 1 1 / C D 1 8 , I C O S , 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 E C V - 3 0 4 monolayers. Then, 1 X 1 0  5  51  C r - l a b e l l e d 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, O N ) . Percent inhibition was calculated as:  C P M in 100 p i lysate with inhibitor % inhibition = [1- (  )] X 100 C P M in 100 p i 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 F i c o l 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 o f R P M I 1640 with 10% fetal calf serum, 2 m M L glutamine and 2 mg o f genitmycine per 100 m l . Afterward, the blood M N C were  53  suspended at 10 M N C in 1 m l of the culture medium with pokeweed mitogen ( P W M , 6  Gibco) at the optimal final dilution of 1:300 in 12 x 75 m m plastic capped tubes (Falcon). Control cultures, consisting of unstimulated blood M N C , were set up in parallel for each sample. The effects o f different preparations of IFN-B on P W M - i n d u c e d 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® ( I F N B - l b , Berlex Laboratories), A v o n e x ™ (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 ( E L I S A ) . 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 ( B S A ) for 1 h at room temperature. Serial dilution o f known concentration o f 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 o f 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 o f IgG. The level of IgG in the unknown samples was then derived from the standard curve using Revelation™ software. A l l samples were run in triplicates.  2.10  STATISTICAL  ANALYSIS  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 o f A v o n e x ™ , Betaseron®, and Rebif® (3-interferon on PWM-induced IgG secretion were assessed using two-way analysis o f variance ( A N O V A ) . If the differences between the three Pinterferon treatments were significant, Tukey multiple comparison test was used to rule out the probability o f type I error (erroneously declaring something by chance). Student t- tests were performed using Stat W o r k s ™ software. Two-way A N O V A and Tukey analyses were done in S-Plus™ software.  55  Table 2 . 1 I n d i v i d u a l M S Patient Characteristics and Treatment P a r t i c i p a t i n g i n I C O S T r i a l  Patient N o .  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  T E S #0804  48  Female  Methy lpredni so lone  56  Table 2.2 M o n o c l o n a l A n t i b o d y Pairs Used for L y m p h o c y t e Subset Analysis Phycoerythrin-conj ugated  Fluorescein-conjugated  Nonspecific I g G l  Nonspecific IgG2a  A n t i - C D 14 (Leu M 3 )  Anti-CD45 ( L C A )  A n t i - C D 19 (Leu 12)  A n t i - C D 3 (Leu 4)  A n t i - C D 4 (Leu 3 a)  Anti-CD3  A n t i - C D 8 (Leu 2a)  Anti-CD3  Anti-CD56 (NKH-1)  A n t i - C D 16 (F RIII)  Anti-CD4  Anti-CD49d(VLA-4a)  Anti-CD8  Anti-CD49d  Anti-CD4  Anti-CD62L (LECAM-1)  Anti-CD8  Anti-CD62L  Anti-CD54 (ICAM-1)  Anti-CD3  Anti-CD4  Anti-CD45RA  Anti-CD4  Anti-CD29  Anti-CD8  Anti-CD28  Anti-CD8  A n t i - C D 5 7 (Leu 7)  Anti-CD27  Anti-CD3  Anti-CD4  A n t i - H L A - D R (HLA-II)  Anti-CD8  Anti-HLA-DR  Anti-CD4  A n t i - C D 2 5 (Tac)  Anti-CD8  Anti-CD25  Anti-CD4  Anti-CD38  Anti-CD8  Anti-CD38  Anti-CD3  Anti-CD26  Anti-CD3  Anti-CD30  Anti-CD3  Anti-CD69 (AIM)  CY  57  CHAPTER THREE RESULTS  3.1  HTLV-I INFECTION  60  3.2  L Y M P H O C Y T E SUBSETS IN H A M , HTLV-I CARRIERS A N D HEALTHY CONTROLS  60  3.2.1 3.2.2 3.2.3 3.2.4  Double staining Double staining Double staining Double staining  61 61 61 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 3.3.2  Adhesion o f blood M N C to IFN-y and L-929 supernatant treated H U V E C Adhesion o f H A M and n o n - H A M blood M N C to L-929 supernatant treated H U V E C Effects o f 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 3.4.2  Adhesion o f healthy, s R R - M S and S P - M S blood M N C to H U V E C Effects o f anti-adhesion molecule antibodies on the adhesion of R R - M S and healthy blood M N C to H U V E C Effects o f anti-adhesion molecule antibodies on the  64  3.3.3  3.4.3  for for for for  CD3+, CD4+, and CD8+ T cells putative markers o f function markers o f activation markers o f adhesion  63 63  64  adhesion o f 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 MNC-ECV-304 ADHESION IN M S  66  3.5.1 3.5.2  Adhesion o f healthy and s R R - M S blood M N C to E C V - 3 0 4 Effects o f anti-adhesion molecule antibodies on the adhesion o f s R R - M S and healthy blood M N C to E C V - 3 0 4 Adhesion o f blood M N C to IFN-y and L-929 supernatant treated H U V E C and E C V - 3 0 4 Effects o f anti-adhesion molecule antibodies on the adhesion o f R R - M S blood M N C to untreated and IFN-y treated E C V - 3 0 4  3.5.3 3.5.4  3.6  3.6.1 3.6.2  ..66 66 67 68  IN VITRO E F F E C T S O F IFN-P O N P W M - I N D U C E D IgG SECRETION A N D M N C - H U V E C ADHESION  68  In vitro IgG secretion in healthy and s R R - M S In vitro effects o f IFN-B on PWM-induced IgG secretion  68 69  58  3.6.3 3.6.4  Comparing the in vitro effects of A v o n e x ™ , Betaseron® and Rebif® on P W M - i n d u c e d IgG secretion Effects of IFN-P on M N C - H U V E C adhesion  59  70 71  RESULTS  3.1  HTLV-I INFECTION The human T lymphotropic virus type I ( H T L V - I ) has been identified as the  etiological agent o f the inflammatory and chronic progressive demyelinating disease called HTLV-I-associated myelopathy ( H A M ) . The major clinical features o f H A M consist o f 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 o f H A M is substantially shorter in patients infected by transfusion o f 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 o f onset is 43 years and the male: female ratio of occurrence is 1:2.9. The lifetime risk o f development o f 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 i n culture without mitogen.  The mean percentage o f 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+, C D 4 + and C D 8 + 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 o f 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 o f C D 8 + C D 5 7 + (cytotoxic) cells compared with H T L V - I carriers and seronegative controls. A t isolation, the percentage o f C D 3 + C D 2 7 - (primed T cells) was significantly higher in H A M patients compared with controls. N o significant difference was observed between  HTLV-I  carriers and controls in the percentage o f CD3+CD27-.  3.2.3  Double staining for markers of activation Table 3.3 shows markers o f activation on CD3+, CD4+, and C D 8 + subsets. The  percentage o f C D 4 + cells coexpressing activation markers H L A - D R or C D 2 5 , and o f C D 8 + 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 o f CD8+ cells coexpressing C D 2 5 was low; however, after 2 days in culture without any mitogenic stimulation, a significant increase in the percentage o f CD8+CD25+ population was observed in H A M patients but  61  not in carriers or controls. We also assessed C D 4 + and CD8+ cells for the expression o f the "activation" antigen C D 3 8 . There was no significant difference between H A M patients,  carriers,  and  controls  in percentage  of CD4+CD38+ or C D 8 + C D 3 8 +  subpopulations at isolation. However, after 2 days in culture, the mean percentage o f CD8+CD38+ cells increased significantly in H A M patients and carriers compared with controls (p<0.05). Interestingly, a significantly higher percentage o f C D 8 + C D 3 8 + cells were observed in patients with H A M compared with carriers. W i t h respect to T cells coexpressing the early activation marker, C D 6 9 , 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 o f C D 4 + and C D 8 + cells co-expressing C D 4 9 d (a-chain o f 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 o f 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 C D 4 + C D 6 2 L + 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 o f mature T cells expressing adhesion molecule C D 5 4 ( I C A M - 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  BLOOD MNC-HUVEC ADHESION IN 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 o f H U V E C with IFN-y (100 U / m l ) or L-929  supernatant (50%) for 48 h resulted in significant increase in adhesion o f 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 o f 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 o f 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 o f 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 o f theses adhesion molecules. Figure 3.2 show that the binding o f 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 3 1 % inhibition; p<0.001), I C A M - 1  (mean 43% inhibition; p O . O O l ) ,  and L-selectin (mean 38%  inhibition; p<0.001) at 2.5 ug/ml. A t 0.5 ug/ml o f 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  HUVEC.  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 o f s R R - M S blood M N C were generally higher than that o f healthy subjects. However, with the number o f 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 o f 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 o f mAbs against each o f L F A - 1 , I C A M - 1 , V L A - 4 and L-selectin or combinations o f all o f theses adhesion molecules. Irrelevant m A b against dansyl hapten was also used. Anti-dansyl m A b 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 o f adhesion, respectively (p<0.01). A t 0.5 pg/ml o f 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 o f a combination o f 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 o f 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 o f blood M N C to H U V E C . Figure 3.4 shows results from 3 experiments measuring the inhibition o f adhesion o f healthy blood M N C to H U V E C . Similar results were obtained when M N C o f 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 o f IFN-y for 48 h and the monoclonal antibodyblocking 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 o f adhesion, whereas a n t i - V L A - 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 ,  anti-VLA-4  also  significantly inhibited M N C - H U V E C interactions. These data indicate that adhesion o f MNC  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 MNC-ECV-304 ADHESION IN 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 E C V - 3 0 4 . 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 E C V - 3 0 4 .  3.5.2  Effects of anti-adhesion molecule antibodies on the adhesion of sRR-MS and healthy blood MNC to ECV-304 Contributions o f different adhesion molecules to the adherence o f s R R - M S and  healthy blood M N C to E C V - 3 0 4 was also determined using monoclonal antibodyblocking functional assays. M N C where co-incubated with E C V - 3 0 4 in the continued presence o f m A b s against  each of L F A - 1 ,  ICAM-1,  VLA-4  and L-selectin or  combinations o f 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. A t both 2.5 and 0.5  66  pg/ml, the anti-LFA-1 and anti-ICAM-1 significantly reduced adhesion o f blood M N C in sPvR-MS and healthy subjects to untreated E C V - 3 0 4 . 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 E C V - 3 0 4 . 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 o f 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 o f 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 o f H U V E C and E C V - 3 0 4 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 E C V - 3 0 4 were left untreated or treated with IFN-y (100 U / m l ) or L-929 (50%) supernatant for 48 h prior to the adhesion assay. The results indicate that treatment o f 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 E C V - 3 0 4 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 o f E C V - 3 0 4 to those o f H U V E C for M N C , we determined the contribution o f different adhesion molecules in binding to untreated or I F N - y treated E C V - 3 0 4 . E C V - 3 0 4 monolayers were untreated or treated with 100 U / m l o f 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 o f figure 3.5. Furthermore, the assays were also performed in parallel to that o f the experiments o f Figure 3.5. When E C V - 3 0 4 monolayers were untreated, antibodies directed against L F A - 1 and to a lesser degree I C A M - 1 produced significant inhibition, whereas a n t i - V L A - 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 E C V - 3 0 4 monolayers were activated with IFN-y, antiV L A - 4 did not inhibit M N C adhesion (figure 3.9). These data indicate that adhesion o f M N C to E C V - 3 0 4 involve L F A - 1 / I C A M - 1 whether or not E C V - 3 0 4 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 ( s R R - M S ) 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 P W M stimulation, the amount o f 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 P W M stimulation, s R R - M S and healthy subjects were divided to two distinct populations: one population o f " l o w 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 o f different preparations o f IFN-P were studied on blood  M N C o f six sRR-MS and five healthy subjects whom were categorized as "high responders" in response to P W M stimulation (producing >900 ng/ml IgG). In the absence of IFN-P, the concentration o f IgG was similar for sRR-MS (2519 ± 629 ng/ml) and healthy (2476 ± 936 ng/ml). Figure 3.10a, and 3.10b show that A v o n e x ™ , 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 2 1 % inhibition) subjects. Comparing Figure 3.10a and b also indicates that IFN-P at different dilutions, tended to suppress P W M induced IgG secretion more in sRR-MS than in healthy subjects. However, this difference did not reach a statistically significant level. The comparative inhibition o f PWM-induced  69  IgG secretion in s R R - M S and healthy controls by A v o n e x ™ , 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 PWMinduced 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" s R R M S patients and compared the in vitro effects of A v o n e x ™ , Betaseron® and Rebif® in parallel for their ability to inhibit PWM-induced IgG secretion. This high responding group o f 16 s R R - M S patients produced 91 ± 5 6 ng/ml o f IgG (mean ± S E M ) spontaneously. However, when M N C were stimulated with P W M and i n the absence o f  IFN-P, they produced 3070 ± 1507 ng/ml of IgG (mean ± sem). Figure 3.11 shows inhibition o f P W M - i n d u c e d IgG secretion using the same dose (ng/ml) o f different IFN-P preparation. A t concentration o f 312 ng/ml, no significant differences were found between A v o n e x ™ , Betaseron® and Rebif® in inhibiting P W M - i n d u c e d IgG secretion, perhaps reaching a saturation point. However, at 31.2 ng/ml and 3.12 ng/ml, A v o n e x ™ 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 o f IFN-B used in vitro as a approximate proportion o f the daily recommended dose of each o f these IFN-Bs (this is 300 pg, 44 pg, and 30 pg for Betaseron®, Rebif® and Avonex, respectively), A v o n e x ™  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 o f IFN-B used in vitro as a fraction o f weekly administration using their antiviral activity in Units (Log) Betaseron®, A v o n e x ™ and Rebif® had equivalent activity with respect to their influence on P W M - i n d u c e d IgG secretion (Figure 3.13).  3.6.4 Effects oflFN-pon MNC-HUVEC adhesion To determine the role o f 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 o f I F N - P - l b (Betaseron®, 1000 U/ml) for 48 h. The I F N - P - l b was then washed and adhesion assays were carried out as in Materials and Methods. Table 3.9 shows that treatment o f M N C with 1000 U / m l o f I F N - p - l b 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 o f H U V E C with the same dose o f I F N P-lb had no significant effect on M N C adhesion. The combined treatment o f H U V E C and M N C o f R R - M S patients with I F N - P - l b did not result in further reduction o f adhesion beyond what was seen with M N C treatment alone. O n the contrary, it resulted in slight augmentation o f this adhesion.  71  CD  00 fl fe an  S-H (D U  ii u u  CN  O  -H  -H  -H  CD  fl o  00  -H  tn  < It, 3,  o  ON  vd CN  © od  t--'  vq vd  HH  -H  CO  c o  U  u  o u fe  C  ^  'Ea CD  ON  co CN  VO  ON  u -fc-  E  OH  an fl CD >  ©  <  \-  l/~>  u  at  e  T-H  vd  cd  • PN  U  ON  p^  "E  an  fl <D  co CN  CD  -9 fl  GO  fe -fl ^  CN  CN  -H  fe  HH  -H  IT)  ON  l/"l  fe  CN  ON  CN  HH -H  OO  an CD co CD  GO  W  «"  a  CD  OH  .S C  g  oo  fl  CD  an  <a -<->  -o  o  fl  CD  .fl  an  ^  CD  c3 fe T3  p^  fe •o o o  S  CQ  H  2  a IL.  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S*H  o  '„  3  o  +  +  ON  ON  Q  Q  CO  HO  fi  00  U  +  Q  U  U  + 00 Q  U  + CN  + CN  VO  VO  Q  Q  U  +  Q  a  O  + oo rt u  in Q  U  + CO Q  O  > CO  O  PH  CO  o  o  CO  CO  fi co fi  CO  O  OJ)  cu  CH ^ CU  CO  H f i  I  +  f-H  CU  •  * i-H  <a ."fi PN  <  0  0  CH  CO  3 iH-» CU 1„ fi o cS  CU  PH  +  ^  CO  O  cu  cu  00  cu  -a  ^  cu co  -1 vq  J3 PH  &0  '5b cd  -H  ++  u  cu  +H  ts ^ 3 .g 3  Da  c  CU H f i  CU  u o J3  ©  cr  •o  cu  CU  c  o  ^  CO  co  t3  -3  fa  o  3 fi  cu  -H  o fc.  fc. fa  O  ©  U  J3 CO CU  CO  13 fi  CO  c «  CU >  * i-H  3  1«3  s  Tf  in -H  CN  C «  ro cu  -a 6*  CH  co  _CU  1—1  -x  )H  SH  CU  > > © © V  CH  *  in  © © V  &  Table 3.5a A d h e s i o n of B l o o d M N C to Untreated and I F N - 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 / m l ) mean ± S E M  L-929 (50%) mean ± S E M  Healthy  12.7 ± 2 . 3  15.6 ± 2 . 5 *  16.1 ± 2 . 1 *  MS  12.9 ± 0 . 7  14.7 ± 0 . 9 *  15.2 ± 1.1*  HAM  15.3 ± 1.7  18.0 ± 1.7*  18.7 ± 1.4*  T a b l e 3.5b % Adhesion  Subject  N o treatment mean ± S E M  IFN-y (100 U / m l ) 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*  HAM  21.7±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 ( M S ) and two H A M patients. In experiment o f 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 - 7 0 °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 / m l ) or conditioned medium containing L-929 (50%) supernatant for 48 h. Each number represents the mean ± S E M percentage o f quadruplicates in two separate experiments. *p<0.02 compared with untreated monolayers.  76  Table 3.6 A d h e s i o n of s R R - M S , S P - M S , and Healthy Subjects B l o o d M N C to H U V E C % Adhesion (mean ± S E M ) Healthy (n=18)  sRR-MS (n=14)  SP-MS (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 (sRPvM 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 o f 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 o f subjects studied in each category are indicated i n the parentheses. p<.02 versus healthy controls  77  T a b l e 3.7 C o m p a r a t i v e A d h e s i o n of Healthy and R R - M S B l o o d 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 ( R R - M S ) and healthy subjects were incubated with untreated E C V - 3 0 4 . Each assay consisted o f adding M N C from one s R R M S and one healthy subject to E C V - 3 0 4 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  T a b l e 3.8 In V i t r o I g G Secretion i n 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 (>900 ng/ml)  66%  38%  The number o f subjects for each category is indicated in parentheses. In these experiments blood M N C s were incubated without ( P W M - ) or with pokeweed mitogen ( P W M + ) for 7 days at 37°C. The IgG content o f 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 ( s R R - M S ) patients produce significantly (p<0.02) higher amount o f IgG in response to P W M compared with healthy controls. Furthermore, the percentage o f individuals producing greater than 900 ng/ml o f IgG in response to P W M ("high responders") are higher i n s R R - M S than i n healthy controls. *p<0.02 versus healthy controls  79  T a b l e 3.9 Effects of I F N - p - l b on M N C - H U V E C A d h e s i o n  IFN-P-lb treatment of: Untreated  MNC  HUVEC  MNC& HUVEC  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 o f IFN-P-lb (Betaseron®) for 48 hr prior to doing the adhesion assay. The results indicate that pretreatment o f M N C with I F N - p - l b results i n a significant reduction o f 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 o f subjects studied is indicated in parentheses. Results are expressed as mean ± SEM. *p<0.03 versus untreated H U V E C monolayers  80  A d h e s i o n of H A M and N o n - H A M B l o o d M N C to A c t i v a t e d HUVEC  Pair Number F i g u r e 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 o f 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 o f 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 o f eight paired assays. In panel (a) controls were H T L V - I carriers, while i n 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  F i g u r e 3.2. M N C from four different H A M patients were coincubated with anti-ICAM-1 (CD54), a n t i - V L A - 4 (CD49d), and anti-L-selectin (CD62L) antibodies at concentration o f 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 o f 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 A n t i - A d h e s i o n M o l e c u l e Antibodies on the A d h e s i o n of M N C of R R - M S patients to Untreated H U V E C  m A b s used  F i g u r e 3.3. M N C from six different R R - M S patients were coincubated with recombinant and humanized (rhu) anti-LFA-1 ( C D 1 1 / C D 1 8 ) , anti-ICAM-1 (CD54), a n t i - V L A - 4 (CD49d), antiL-selectin ( C D 6 2 L ) , or combination o f all anti-adhesion antibodies during the assay. Control included was anti-dansyl m A b . Each assay was performed using four replicate wells. Results are from six separate experiments and are expressed as mean ± S E M percentage inhibition o f 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 coincubated with recombinant and humanized (rhu) anti-LFA-1 (CD11/CD18), anti-ICAM-1 (CD54), a n t i - V L A - 4 (CD49d), anti-Lselectin ( C D 6 2 L ) or combination of all antibodies during the assay. Control included was anti-dansyl m A 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<0.02 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 I F N - y Treated H U V E C  <  < mAbs used  F i g u r e 3.5. M N C from three different R R - M S patients were coincubated with recombinant and humanized (rhu) anti-LFA-1 ( C D 11 a/CD 18), anti-ICAM-1 (CD54), V L A - 4 (CD49d), anti-Lselectin ( C D 6 2 L ) or combination o f all antibodies during the assay. H U V E C monolayers were untreated (a) or treated with 100 U / m l o f 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 o f 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), a n t i - V L A - 4 (CD49d), anti-ICAM-1 (CD54), antiL-selectin ( C D 6 2 L ) or combination o f 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 o f 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  F i g u r e 3.7 M N C from eight different healthy subjects were coincubated with recombinant and humanized (rhu) anti-LFA-1 (CD11/CD18), a n t i - V L A - 4 (CD49d), anti-ICAM-1 (CD54), anti-Lselectin ( C D 6 2 L ) or combination o f 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 o f 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 ( M S ) 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 ± S D percentage o f 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  30 -  HH  20 10 0 -  m A b s used Figure 3.9. M N C from three different R R - M S patients were coincubated with anti-LFA-1 (CD11/CD18), anti-VLA-4 (CD49d), anti-ICAM-1 (CD54), anti-L-selectin ( C D 6 2 L ) 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 I F N - p on P W M - i n d u c e d I g G Secretion i n Healthy Controls (a) and in s R R - M S (b)  • Avonex • Betaseron Rebif  Concentration o f IFN-P ( ng/ml) used  F i g u r e 3.10a a n d b. Suppression o f PWM-induced IgG secretion by I F N - p . B l o o d M N C were stimulated with P W M and co-incubated without and with indicated concentrations (ng/ml) o f IFN-p from A v o n e x ™ , Betaseron® and Rebif®. After 7 days at 37°C, the IgG content o f the supernatants were measured by an E L I S A . These figures show a dosedependent inhibition (mean ± S E M ) o f PWM-induced IgG secretion by different preparations o f 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  30  1  1  6  1  1  1.2  1  1  0.24  1  :  0.048  i  1  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 A v o n e x ™ (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 C o m p a r i s o n of A v o n e x ™ , B e t a s e r o n ® and R e b i f ® on Inhibition of P W M - i n d u c e d I g G Secretion  312  31.2  3.12  Concentration of IFN-P ( ng/ml) used  F i g u r e 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 A v o n e x ™ , 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. A t 312 ng/ml, no significant differences were seen between different IFN-p in inhibition of P W M - i n d u c e d IgG secretion. A t 31.2 and 3.12 ng/ml, A v o n e x ™ or Rebif® inhibited significantly more IgG secretion than that of Betaseron® (p<0.05). Significance p<0.05 * A v o n e x ™ versus Betaseron® + Rebif® versus Betaseron®  92  P e r Dose C o m p a r i s o n of A v o n e x ™ , B e t a s e r o n ® and R e b i f ® on I n h i b i t i o n of P W M - i n d u c e d I g G Secretion  Concentration o f IFN-B ( ng/ml) used  F i g u r e 3.12. Effects o f A v o n e x ™ , Betaseron® and Rebif® on inhibition of PWM-induced IgG secretion were compared according to their proportion o f daily recommended doses. Each column indicates mean ± S E M inhibition o f PWM-induced IgG secretion by different preparation o f IFN-B. N o significant differences was found between the three treatment groups.  93  a  ro  o  "2 2  fl >  • ta •PM  "fl "+3  JS  c c  •S s •v •<fl3 60 -fl fe ~ n-  TOO  o  <fe  _Q V  60  O  .s !O  iooo  I*  -B C «  OO  rrO  'oo 3 O T3  60 C  C  ta  T0000  s- o  I £ W  C/3  s  O  H  r—I  60  _o £0-31  V  -a  o  3  C uw o e  o S  90-HI  a  O  •S fl «  CD c/3  O Q (D (D  C+H  S  O  fl o  5  I0-HI  °  -2  60  CD  -fl  fl fl fl  • rH fe  CD cd  -fl CD  @> ^ C 0  rf  CD CO  +  80-31  Q  fe  1  s" o i= 8  0A  m  O  60-31  CD C+H  i  (U  8  8  8  4?  o  CN  uopaioas O^I JO uoniqnruT  o  q  CN  £  «  - f l  >  C  c  CO  fe  S  £ 8  01-31  §  CD  S-i  ro 2H  a  '  CD  ro o  ^  o  0  fl > Us < 11  ^  o CU  U  co 3  3 60 2; ^ 5 ^ fl T3  O  ~o  O CO.  CD  VI  fl  r * 3 ;  * &  U  ca fe  CD  fl cij  ca3 * o  fl  CN  e  CO  OXJO  fe  fe ^ £  ON  CHAPTER FOUR DISCUSSION 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 SUBSETS IN H A M , HTLV-I C A R R I E R A N D H E A L T H Y CONTROLS  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 o f adhesion o f H A M blood M N C to activated H U V E C  101  4.2.2  Expression o f 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 4.3.2  Mechanism o f adhesion o f R R - M S and healthy blood M N C to H U V E C Mechanism o f adhesion of R R - M S blood M N C to unstimulated and IFN-y-stimulated H U V E C C O M P A R I N G A D H E S I O N PROPERTIES OF H U V E C A N D ECV-304 F O R B L O O D M N C  106  4.4  4.5  4.5.1 4.5.2 4.5.3  T H E EFFECTS 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 SUBSETS A N D FUNCTION IN M S Lymphocyte subsets in a R R - M S patient participating in Schering-Plough trial Lymphocyte subsets in R R - M S patients participating in I C O S trial P W M - i n d u c e d IgG secretion in s R R - M S patients and healthy controls  108 109  Ill  Ill Ill  and the effects o f IFN-P on this function  113  Comparing the effects of different preparations of IFN-P on P W M - i n d u c e d IgG secretion  114  4.5.5  Effects o f 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 CONCLUSIONS  117  4.7  FUTURE E X P E R I M E N T A L CONSIDERATIONS  120  4.5.4  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 C D 8 + 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. W e 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 C D 2 9 expression, which is paralleled by a downregulation o f C D 4 5 R A expression (Akbar et al., 1988). C D 4 + C D 2 9 + cells have also been called helper-inducer since they produce a variety o f 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 o f H A M and H T L V - 1 carriers (Itoyama et al., 1988; Yasuda et al., 1986; Geroni et al., 1988; L i n k et al., 1989; Osame et al., 1987; M o r i et al., 1988). We observed significantly higher levels o f CD8+CD57+ cells in H A M compared with both carriers and controls. Increased number o f 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 o f 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 o f functions has been proposed for C D 8 + C D 5 7 + cells. Some o f 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 o f the generation of cytotoxic T lymphocytes (Wang et al., 1994). If we assume a cytotoxic role for CD8+CD57+ cells, increased C D 8 + C D 5 7 + could explain the high levels o f 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 o f C D 3 + C D 2 7 - cells only in H A M patients and not in carriers. The C D 2 7 molecule is a member o f the tumor necrosis factor superfamily (Goodwin et al., 1993). T cell activation studies in vitro have shown that C D 2 7 - cells arise from C D 4 5 R A - , C D 4 5 R O + , CD27+ T cells after prolonged restimulation (De Jong et al., 1992; Hintzen et al., 1993). A n increased percentage o f C D 4 + C D 2 7 - cells have also been reported in peripheral blood, synovial fluid, and synovial tissue o f 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 o f lymphocytes to endothelial cells seen in H A M patients (Ichinose et al., 1992). This is also consistent with recent findings o f high levels o f the soluble form o f C D 2 7 in the cerebrospinal fluid o f patients with H A M and multiple sclerosis (Hintzen et al., 1999).  97  Our study revealed that the percentage o f both C D 4 + and C D 8 + 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 C D 2 5 , is an a-chain o f the receptor for interleukin-2 (Uchiyama et al., 1981) and here we showed that it is upregulated on C D 4 + cells o f 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 o f C D 2 5 + and H L A D R + 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 o f the H T L V - I proviral genome has been shown to induce the expression o f 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 o f IL-2 and its receptor gene by this transregulatory protein. However, the enhanced expression o f H L A - D R on C D 4 + and C D 8 + cells and upregulation o f C D 2 5 molecules on C D 4 + cells have not been observed in American asymptomatic H T L V - I carriers (Prince, 1990). We also found a high percentage o f activation markers C D 3 8 , H L A - D R , and C D 2 5 on CD8+ cells o f H A M patients after 2 days in culture. This is o f particular interest and potentially relevant to the pathological findings o f H A M patients in whom lymphocytes infiltrating the central nervous system were predominantly C D 8 + (Jacobson et al., 1992). The levels o f CD8+CD38+ cells have been reported to have prognostic value for H I V disease progression (Ho et al., 1993). H i g h levels o f  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 o f progression o f the infectious process or disease development. The levels o f early activation antigen C D 6 9 expressed on C D 3 + (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 o f H T L V - I infected individuals have not been activated recently. It is possible, however, that recently activated lymphocytes expressing C D 6 9 may be sequestered in lymph node or other organs.  4.2  BLOOD MNC-HUVEC ADHESION IN H A M  In this study, we used primary and secondary culture o f human umbilical vein endothelial cells ( H U V E C ) monolayers as a source o f 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 o f 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 o f 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. W e 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 o f lymphocytes to the brain microvascular endothelium, which form the blood-brain barrier, is a critical step in the initiation o f the inflammatory response in the C N S and therefore probably plays an essential role in the pathogenesis o f 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 o f adhesion molecules on endothelial cells is regulated by several cytokines ( Y u 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 o f 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 o f 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 o f 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 o f H T L V - I carriers or healthy controls, and this is highly relevant to the pathogenesis o f H A M . The  100  greater binding o f 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; V o r a et al., 1995). It has been shown that the activation o f T cells with P M A increases the affinity o f 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 o f 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 o f 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 o f H A M cells to H U V E C . This is in agreement with previous findings that effective blocking o f 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 o f 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 K o o y k et al., 1993). We also have shown that antibodies to L-selectin block the binding o f 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 o f both lymphocytes and H U V E C . This inhibition could be attributed to L-selectin recognition o f 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 o f L-selectin other than rolling, has shown that E-selectin, a ligand for L-selectin inhibits transmigration o f 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). W e 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 o f M N C o f healthy controls and  102  multiple sclerosis patients to E C 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 I C A M - 1 is upregulated i n T cell lines carrying H T L V - I and i n lymphocytes o f patients with adult T cell leukemia ( A T L ) (Yamamoto et al., 1982; Yamamoto and Hinuma, 1985). It is known that cell-free H T L V - I exhibits very l o w infectivity and cell-to-cell infection is regarded as the major route o f H T L V - I transmission both in vitro and in vivo (Weiss et al., 1985; Yamamoto and Hinuma, 1985). Therefore, constitutive expression o f I C A M - 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 o f soluble I C A M - 1 have also been reported i n the sera o f multiple sclerosis and H A M patients (Sharief et al., 1993; Tsukada et al., 1993b). Analysis o f adhesion molecules on lymphocytes at isolation showed that the percentage o f C D 4 + and CD8+ cells expressing V L A - 4 was generally higher i n both patients with H A M and carriers than i n controls. Therefore, it is likely that V L A - 4 does not contribute much to the increased binding o f lymphocytes to H U V E C seen i n 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 o f peripheral blood M N C o f s R R - M S and S P - M S to H U V E C . Blood M N C from S P - M S patients exhibited significantly higher adhesion capacity than M N C from normal donors. This is consistent with the previous published report indicating increased adhesion o f 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 o f L F A - 1 on circulating blood M N C than on those o f normal controls (Lou et a l , 1997). Increased adhesion o f S P - M S blood M N C to H U V E C is also probably due to increased levels o f activated lymphocytes seen in circulating peripheral blood o f 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 o f adhesion molecules ( Y u 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 o f clinically stable R R - M S blood M N C was generally higher than that o f healthy subjects (although not statistically significant). This is probably because a certain proportion o f 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 o f blood brain barrier from R R - M S to secondary progressive M S (McLean et a l , 1993). Increased binding o f R R - M S blood M N C to endothelial monolayers have been previously reported, but only during the clinically active phase o f the disease (Tsukada et al., 1993a). This might also imply that the expression o f 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 o f heterogeneous populations o f 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 o f H U V E C (Vora et al., 1996). Such differences were not seen with resting H U V E C . However, in studies o f Tsukada et al. (1993a), no significant differences were found between adhesion o f M N C from clinically active R R - M S and healthy control after T N F - d treatment o f cerebral endothelium.  4.3.1 Mechanism of adhesion of RR-MS and healthy blood MNC to HUVEC We also compared the relative contribution o f major adhesion molecules in binding blood M N C o f R R - M S and o f 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 o f 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 K o o y k et al., 1993), although there is some dispute (Hughes, 1996). Previous studies has shown that adhesion o f  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 o f 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 C o n A , 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 o f the antibody to V L A - 4 to induce aggregation o f C o n A activated lymphocytes in vitro. Lymphocyte aggregates are then easier to remove during the washing stage o f the assay, thus producing an apparent reduction in binding. Combinations o f L F A - 1 , I C A M - 1 , V L A - 4 and L-selectin m A b s failed to show greater inhibition than seen with the L F A - 1 m A b alone, indicating that the L F A - 1 predominates  pathway  i n mediating adhesion o f R R - M S and healthy subjects to untreated  H U V E C . W e further have shown that the major adhesion molecules investigated in this study are equivalently involved in adhesion o f 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 o f L F A - 1  adhesion  pathway in binding o f 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 IFNy-stimulated HUVEC To  characterize  further  the  adhesion  molecule-ligand pairs  involved i n  inflammation, we extended this study to test antibodies directed against various adhesion molecules to block the adhesion o f 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 o f 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 o f L F A - 1 and I C A M - 1 on binding o f 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 o f 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 o f 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 o f V C A M 1 on H U V E C (Lindington et a l , 1999). A n increase in levels of expression o f 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 o f a n t i - V L A -  108  4 antibody alleviated the clinical and pathological symptoms o f E A E (Yednock et al., 1992). In the present study antibody to V L A - 4 inhibited M N C adhesion to activated HUVEC  but to a lesser extend than did anti-LFA-1. This may be explained by  differences in the distribution o f 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 o f the cells (Shimizu et al., 1990). We were not able to completely inhibit M N C adhesion to HUVEC  using a combination o f the studied mAbs. This indicates the potential  involvement o f other receptor/ligand interactions. Some other candidate that might be involved in adhesion include C D 4 4 , which has been implicated in the adhesion o f 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 o f this project, we also assessed the suitability o f 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 E C V - 3 0 4 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 o f E C V - 3 0 4 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 o f adhesion molecules expression and subsequent functional enhancement for M N C adhesion to E C V - 3 0 4 . Monoclonal antibody blocking studies demonstrated that adhesion pathways o f RR-MS  and healthy controls to untreated H U V E C  and E C V - 3 0 4 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 E C V - 3 0 4 . Our results agree with recent observations that indicate E C V - 3 0 4 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 E C V - 3 0 4 with T N F - a does not result in the induction o f V C A M - 1 expression (Lindington et al., 1999). Our data however, conflicts with a report that demonstrated constitutive expression o f 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 differences  in adhesion  molecule expression  in response  to  different  in antibodies utilized and in the sensitivity o f the detection  employed.  110  activators, methods  4.5  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 DRUGS O N L Y M P H O C Y T E SUBSET A N D F U N C T I O N IN M S  4.5.1  Lymphocyte subsets in a RR-MS patient participating in Schering-Plough trial Analyzing lymphocytes subset o f a R R - M S patient who participated i n 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 W e 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 Clinic. Due to the limited number o f participants, statistical analysis was not possible and all changes in lymphocyte subsets cannot be discussed rationally. H u 2 3 F 2 G treatment had no major effect on the number o f circulating CD3+, C D 4 + or CD8+ T cells. Focusing only on few lymphocyte subsets, we observed a major reduction of C D 3 + C D 2 6 + (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. A t 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 o f C D 4 + and C D 8 + cells expressing V L A - 4 adhesion molecules. O n the contrary, in a placebo treated patient the percentage o f C D 4 + and CD8+ cells expressing V L A - 4 and L-selectin increased while that o f CD3+CD26+ remained unchanged.  Furthermore, in a RPv-MS patient  treated with intravenous methylprednisolone no major reduction o f the discussed adhesion molecules was observed. In contrast to high dose H u 2 3 F 2 G treated patient, the percentage o f C D 4 + and C D 8 + cells expressing L-selectin increased. These results, with some degree o f confidence, indicated that the observed shifts with respect to the discussed lymphocyte subsets are likely due to immunotherapy with H u 2 3 F 2 G rather than spontaneous. Moreover, our data particularly in the R R - M S patient treated with high dose H u 2 3 F 2 G indicates that the immunotherpay with H u 2 3 F 2 G achieved its desired objectives o f reducing activation and adhesion related antigens on peripheral blood lymphocytes. The reduction o f activation and adhesion related antigen on peripheral blood lymphocytes is relevant i n C N S inflammatory cell infiltration in M S since it is these cells that are likely to be involved in the process o f endothelial adhesion and extravasation to sites of inflammation (Wekerle et al., 1986; Estess et al., 1999). The significance o f adhesion molecules in M S pathology has already been addressed in the Introduction. Furthermore, in peripheral blood o f active M S patients' lymphocytes were found to have higher expression o f the activation marker C D 2 6 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  H u 2 3 F 2 G treated M S patients. The reason for clinical inefficacy of H u 2 3 F 2 G in R R - M S patients is not clear (Lublin 1999). However, we have to bear in mind that the treatment  112  schedule with H u 2 3 F 2 G is for relapses of M S and this might be too late once the inflammation is irretrievably established in the C N S . Therefore, H u 2 3 F 2 G might achieve its desired effects i f given at very early stage o f the M S relapse. A note o f caution should also be raised and stressed here in the interpretation o f fluctuations o f lymphocyte subsets in the studied patients, as the number o f 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 W e 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 o f 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 i n M S is believed to be defective function o f 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 o f IgG secretion induced by P W M in peripheral blood M N C of high responders i n 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 ' G o r m a n 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 P W M - i n d u c e d 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 P W M - i n d u c e d 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 W e also directly compared  the in vitro biological activity of I F N - p from  A v o n e x ™ , 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 A v o n e x ™ and Rebif® had higher biologic activity compared with B e t a s e r o n ® when used at a similar concentration.  The difference  was particularly evident when we  compared A v o n e x ™ (IFNp-la) with Betaseron® ( I F N p - l b ) . A v o n e x ™ and Rebif® amino-acid sequence and glycosylation pattern are identical to those of endogenous human I F N - 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 A v o n e x ™ and Rebif® (Runkel et al., 1998). We did not expect generally a higher inhibitory effects for A v o n e x ™ 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 A v o n e x ™ and Rebif®. We do not have a clear explanation to our observation. The differences in formulation might explain this phenomenon. A v o n e x ™ is formulated in a higher concentration o f 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 A v o n e x ™ 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 o f A v o n e x ™ 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 ) o f A v o n e x ™ and Rebif® was administrated intramuscularly to healthy volunteers, the serum neoptrin concentration was higher in A v o n e x ™ treated individuals ((Alam et al., 1997). When we corrected for specific activity ( M I U ) and weekly dose and expressed concentration as a fraction of the M I U o f IFN-P activity injected per week, Betaseron®, A v o n e x ™ and Rebif® had similar activity with respect to their influence on inhibition o f 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 (DhibJalbut 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 o f 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 o f  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 o f M N C with I F N p - l b results in significant reduction o f M N C - H U V E C adhesion. This effect was not a result o f 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 o f I F N P - l b on adhesion may be cell-specific. This explanation is supported by the finding that IFN-P is capable o f downregulating IFN-y-induced expression o f 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 o f 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 o f epidermal carcinoma cell line with closely related I F N - a results in a significant reduction o f their binding to H U V E C (Dao et al., 1995). Our results showing the effects o f 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 i n migration o f 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 o f activated T cells through fibronectin, by acting on a matrix metalloproteinase ( M M P - 9 ) produced by the lymphocytes (Stuve et al., 1996). Recently it has also been shown that the migration across  fibronectin-coated  membranes o f 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 o f the possible mechanisms for the inhibitory effects o f 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 o f adhesion molecule expression on M N C . W e did not  116  address this question in our study; however, it has been previously shown that pretreatment o f M N C with IFN-P results in lower basal and IFN-y-induced expression o f VLA-4  (Soilu-Hanninen et  al,  1995). Downregulated expression  lymphocytes has also been shown in R R - M S  of V L A - 4  on  patients after treatment with I F N p  (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 o f expression or function of other adhesion molecules involved in M N C - H U V E C binding. In fact, a decrease in expression o f C D 18 (P chain o f L F A - 1 ) on M N C o f I F N p l b treated M S patients has been previously reported (Corsini et al., 1997, Gelati et al., 1999). The effects o f I F N p - l b on the functional activity o f adhesion molecules might also be responsible for the decreased adhesiveness o f M N C to H U V E C since quantitative changes in the expression o f cell surface adhesion molecules are not always consistent with the level o f cell adhesion (Piela and K o r n , 1990; Gamble and Vdas, 1991). In vitro treatment with I F N p - l b has also been shown to downregulate the expression o f 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 o f H A M and therefore the  117  assessment o f these lymphocyte subsets may be of value in detecting or evaluating inflammatory diseases and monitoring treatment. We have also demonstrated increased adhesion o f H A M and clinically active M S blood M N C to H U V E C . Our data and that o f others lend support to the view that infiltration of M N C across the B B B into the C N S i n H A M and M S is due to increased interaction between blood M N C and endothelium. W e also speculate that in both H A M and M S chronic and systemic activation o f 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 o f 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 / I C A M - 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 o f H U V E C . These findings are relevant to better understanding  the  mechanisms  o f adhesion  inflammatory conditions.  118  under  both  inflammatory and  non-  We have also compared adhesion properties o f 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, E C V - 3 0 4 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 , E C V - 3 0 4 does not respond to INF-y and L-929 supernatant for enhancing their adhesion to M N C adhesion. Therefore, E C V - 3 0 4 may not be useful for some adhesion assays studies. W e demonstrated increased IgG secretion i n stable R R - M S i n response to P W M . This increase i n T cell dependent B cell activity further adds to a spectrum o f immune abnormality that has already been reported i n this disease. This also supports the view that clinical stability i n M S does not necessarily translate into normal functional immune responses. W e further demonstrated that IFN-P is capable o f significantly inhibiting mitogen-induced IgG secretion. This indicates that another beneficial mechanism o f actions o f IFN-P i n M S might be due to the capacity o f IFN-P to downregulate B cell activity and IgG secretion. W e also compared the biological activity o f different preparations o f IFN-P according to their capacity in inhibiting PWM-induced IgG secretion. The results indicated that A v o n e x ™ had the highest in vitro activity followed by Rebif® and Betaseron®, respectively, when used at the same mass dose or proportion o f their daily recommended dose. However, when we calculated the amount o f different IFN-P used in vitro as a fraction o f their weekly injection using their antiviral activity i n units, these differences disappeared. These in vitro effects o f A v o n e x ™ Rebif® and Betaseron® were more consistent with their antiviral activity and therefore may be used as an  119  alternative assay i n evaluating the biological activities o f IFN-B. Direct comparative clinical trials have yet to be conducted, but i f in vitro biologic activity o f each o f these preparations o f IFN-B is representative o f 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 o f administration.  4.7  F U T U R E E X P E R I M E N T A L CONSIDERATIONS  In the current project, we found that a number o f lymphocyte subsets were significantly altered in H A M patients. In order to further elucidate the role o f the altered lymphocyte subsets i n immunopathology o f H A M and examine a potential association between the lymphocyte subsets and disease progression, it would be o f significant interest to perform a long follow-up study o f early H A M patients. Our understanding o f the pathomechanisms o f H A M could be further enhanced by defining the functional properties o f the altered lymphocyte subsets in terms o f 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 o f HAM  than cells o f the peripheral blood. Therefore, a comprehensive analysis o f  lymphocyte subsets i n the C S F o f H A M patients is another avenue that needs to be explored. Longitudinal analysis o f 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 o f the disease.  120  In this project endothelial cells from umbilical veins were used to support the binding o f 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 o f 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 o f the adhesion and activation related antigens o f 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 ( S P - M S ) was generally higher compared to that o f clinically stable M S ( s R R - M S ) . This might indicate that the increased adhesion o f 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 o f the disease activity in R R - M S patients during both relapse and remission be better in delineating the precise sequence o f events. We have also shown that in vitro treatment o f 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 o f 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 o f M N C with IFN-B and subsequent analysis o f the expression o f markers o f activation. Another area of study would be to examine the effects o f 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 o f IFN-P in downregulation of activated M N C might be more easily observed i n H A M . We have evaluated the in vitro biological activity o f the three preparations o f IFN-p, namely A v o n e x ™ , Betaseron®, and Rebif® and found that A v o n e x ™ 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 o f the different preparations o f IFN-P. For example, longitudinal measurement o f 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 A v o n e x ™ , Betaseron® or Rebif® at the current recommended frequencies, doses, and routes o f administration. Using the same assay, in vivo biological activity o f the different IFN-P preparations could be further compared i n M S patients or healthy volunteers when used at the similar specific activity, frequency and route o f administration.  122  REFERENCES:  Akbar A , Terry L , Timms A , Beverley P, Janossy G . Loss o f C D 4 5 R A and gain o f U C H L 1 reactivity is a feature o f primed T cells. J. Immunol. 140: 2171-2178; 1988. A l a m J, Goelz S, Rioux P, Scaramucci J, Jones W , McAllister A , Campion M , Rogge M . Comparative pharmacokinetics and pharmacodynamics o f two recombinant human interferon beta-la (IFNB-la) products administered intramuscularly in healthy male and female volunteers. Pharm. Res. 14: 546-549; 1997. Antel J, Bania M , Noronha A , Neely S. Defective suppressor cell function mediated by T8+ cell lines from patients with progressive multiple sclerosis. J. Immunol. 137: 34363439; 1986. Antel J, Rosenkoetter M , Reder A , Oger J, Arnason G . Multiple sclerosis: relation o f in vitro IgG secretion to T suppressor cell number and function. Neurology. 34: 1155-1160; 1984. A p l i n A , Howe A , Alhari S, Juliano R. Signal transduction and signal modulation by cell adhesion receptors: the role o f integrins, cadherins, immunoglobulin molecules, and selectins. Pharm. Rev. 50: 197-263; 1998. Autran B , Leblond V , Sadat-Sowti B , Lefrance E , Got P, Sutton L , Binet J, Debre P. A soluble factor released by CD8+, CD57+ lymphocytes from bone morrow transplant patients inhibit cell mediated cytolysis. Blood. 77: 2237-2241; 1991. Bacon K . Chemokine, leukocyte trafficking, and inflammation. Curr. Opin. Immunol. 6: 865-873; 1994. Baggiolini M . Chemokines and leukocyte traffic. Nature 392: 565-568; 1998. Baig S, Olsson T, Yu-ping J, Hojeberg B , Cruz M , L i n k H . Multiple sclerosis: cells secreting antibodies against myelin-associated glycoprotien are present i n cerebrospinal fluid. Scan.. J. Immunol. 33: 73-79; 1991 Baker C , Billingham R. Immunologically priviledged sites. A d v . Immunol. 25: 1-54; 1977. Baron J, Reich E , Visintin I Janeway C . The pathogenesis o f adoptive murine autoimmune diabetes requires an interaction between oc4 integrins and vascular cell adhesion molecule-1. J. C l i n . Invest. 93: 1700-1708; 1994.  123  Beck J, Rondot P, Catinot L , Falcoff E , Kirchner H , Wietzerbin J. Increased production of interferon gamma and tumor necrosis factor precedes clinical manifestation i n multiple sclerosis: do cytokines trigger off exacerbations? A c t a N e r u o l . Scand. 78: 318-323; 1988. Becker C , Gidal B , Fleming J. Immunotherapy in multiple sclerosis, part 2. A m . J. Health-Syst. Parm. 52: 2105-2120; 1995. Bellamy A , Cadler V , Feldmann M , Davison A . The distribution o f interleukin-2 receptor bearing lymphocytes i n multiple sclerosis: evidence for a key role o f activated lymphocytes. C l i n . E x p . Immunol. 61: 248-256; 1985. Berlin C , Bargatz R, Campbell J, von Andrian U , Szabo M , Hasslen S, Nelson R, Berg E , Erlandsen S, Butcher E . cc4 integrins mediate lymphocyte attachment and rolling under physiologic flow. C e l l . 80: 413-422; 1995. Bernard C , M n d e l T, Mackay I. Experimental models o f human autoimmune disease: Overview and prototypes. In: Rose N , Mackay I. The autoimmune diseases II. San Diego: Academic press, 47-106; 1992. Bertram J, Kuwert E . H L A antigen frequencies in multiple sclerosis. Eur. J. Neurol. 7: 74-791; 1982. Bevilacqua M , Nelson R. Selectins. J. C l i n . Invest. 91: 379-387; 1993. Bianchi E , Bender J, Biasi F, Pardi R. Through and beyond the wall: late steps in leukocyte transendothelial migration. Immunol. Today. 18: 586-591; 1997. B o L , Peterson J, M o r k S, Hoffman P, Gallatin W , Ransohoff R, Trapp B . Distribution o f immunoglobulin superfamily members I C A M - 1 , -2, -3, and the beta 2 integrin L F A - 1 in multiple sclerosis lesions. J. of Neuropath. E x p . Neurol. 55: 1060-72; 1996 Borthwick N , B o f i l l M , Gombert W , Akbar A , Medina E , Sagawa K , Lipman M , Johnson M , Janossy G . Lymphocyte activation in H I V - I infection. II. Functional defects o f C D 2 8 T cells. A I D S . 8: 431-441; 1994. Bowie A , Moynagh P, O ' N e i l l L . The human endothelial cell line E C V - 3 0 4 as a model o f endothelial cell activation by interleukin-1. Biochem. Soc. Trans. 23: S I 0 9 ; 1995. Bratt K , Guthikonda P, Goodman A , Mattson D . The effects o f in vitro and in vivo interferon-b-lb on immunoglobulin G synthesis by peripheral blood lymphocytes. Neruology. 46: A 1 3 6 - A 1 3 7 ; 1996. Brochet B , Dousset V . Pathological correlates o f magnetization transfer imaging abnormalities in animal models and human with multiple sclerosis. Neurology. 53: SI 2S17; 1999.  124  Brosnan C , Cannella B , Battistini L , Raine C . Cytokine localization in multiple sclerosis lesions: correlation with adhesion molecules expression and reactive nitrogen species. Neurology. 45: S16-S21; 1995. B r o w n K , V o r a A , Biggerstaff J, Edgell J, Oikle S, Mazure G , Taub N , Meager A , H i l l T, Watson C . Application o f an immortalized human endothelial cell line to the leukocyte: endothelial adherence assay. J. Immunol. Meth. 163: 13-22; 1993. Burns C , Tsai V , Zvaifler N . H i g h percentage of CD8+, Leu-7+ cells in rheumatoid arthritis synovial fluid. Arthritis Rheum. 35: 865-873, 1992. Butcher E . Leukocyte-endothelial cell recognition: three (or more) steps in specificity and diversity. C e l l : 67: 1033-1036; 1991 Calabresi P, Pelfrey C , Tanquill L , Maloni H , McFarland H . V L A - 4 expression on peripheral blood lymphocytes is downregulated after treatment o f multiple sclerosis with interferon beta. Neurology. 49: 1111-1116; 1997. Campbell J, Hedrick J, Zlotnik A , Siani M , Thmpson D , Butcher E . Chemokines and the arrest o f lymphocytes rolling under flow conditions. Science. 279: 381-384; 1998. Cannella B , Cross A , Raine C . Anti-adhesion molecule therapy in autoimmune encephalomyelitis. J. Neuroimm. 46: 43-55; 1993.  experimental  Canella B , Raine C . The adhesion molecule and cytokine profile o f multiple sclerosis. Ann. Neurol. 37: 424-435; 1995. Capron M , Kazatchkine M , Fischer E , Butterworth A , Joseph M , Kusnierz J, Prin L , Papin J, Capron A . Functional role of the a-chain o f complement receptor type-3 in human eosinophil-dependent antibody mediated cytotoxicity against schistosomes. J. Immunol. 139: 2059-2065; 1987. Carley W , M i l i c i A , Madri A . Extracellular matrix specificity for the differentiation o f capillary endothelial cells. E x p . Cell Res. 178: 422-434; 1988. Carlos T, Harlan J. Leukocyte-endothelial adhesion molecules. Blood. 84: 2068-2101; 1994. Ceroni M , Piccardo P, Rogers-Johnson P, M o r a C , Asher D , Gajdusek C , Gibbs C . Interathecal synthesis o f IgG antibodies to H T L V - I supports an etiological role for H T L V - I in tropical spastic paraparesis. A n n . Neurol. 23: S188-S191; 1988. Challoner P, Smith K , Parker J, et al. Plaque-associated expression o f human herpesvirus 6 in multiple sclerosis. Pore. Natl. Acad. Sci. U S A . 92: 7440-7444; 1995.  125  Chang J, Weiner H , Hafler D . Autoreactive T cells in multiple sclerosis. Int. Rev. Immunol. 9: 183-201; 1992. Chen Y , Kuchroo V , Inobe J, Hafler D , Weiner H . Regulatory T cell clones induced by oral tolerance: suppression o f autoimmune encephalomyelitis. Science. 265: 1237-1240; 1994. Chofflon M , Juillard C , Juillard P, Gauthier G , Grau G . Tumor necrosis factor alpha production as a possible predictor o f relapse in patients with multiple sclerosis.Euro. Cytokine Netw. 3: 523-531; 1992. Clark E , Brugge J. Integrins and signal transduction pathways: the road taken. Science. 268: 233-239; 1995. Coons A , Kaplan M . Localization o f antigen in tissue cells: Improvement in a method for the detection o f antigen by means of fluorescent antibody. J. Exp. M e d . 91: 1-10; 1950. Corley R, LoCascio N , Ovnic M , Haughton G . Two separate functions o f class II l a molecules: T cell stimulation and B cell excitation. Proc. Natl. Acad. Sci. U S A . 82: 516520, 1985. Correale J, Gilmore W , M c M i l l a n M , L i S, McCarthy K , Le T, Weiner L P . Patterns o f cytokine secretion by autoreactive proteiolipid proteins-specific T cell clones during the course o f multiple sclerosis. J. Immunol. 154: 2959-2968; 1995. Corsini E , Gelati M , Dufour A , Massa G , Nespolo A , Ciusani E , Milanese C , L a Manita L , Salmaggi A . Effects o f B - I F N - l b treatment in M S patients on adhesion between P B M N C , H U V E C and M S - H B E C : and in vivo and in vitro study. J. Neuroimmunol. 79: 76-83; 1997. Cserr H , K n o p f P. Cervical lymphatics, the blood-brain barrier and the immunoreactivity of the brain: a new view. Immunol Today. 13: 507-12; 1992. Dal Canto M , M e l v o l v R, K i m B , M i l l e r S. Two models o f multiple sclerosis: Experimental allergic encephalomyelitis ( E A E ) and Theiler's murine encephalomyelitis virus ( T E M V ) infection. A pathological and immunological comparison. M i c r o . Res. Tech. 32: 215-229; 1995. Dao T, Iwaki K , Taeuchi M , Oahshi K , Fukuda S, Kurimoto M . Natural human interferon-a inhibits the adhesion o f human carcinoma cell line to human vascular endothelium. J. Interferon. Cytokine Res. 15: 869-876; 1995. Davignon D , Martz E , Reynolds T, Kurzinger K , Springer T. Monoclonal antibody to a novel lymphocyte function-associated antigen ( L F A - 1 ) : mechanism o f blocking o f T lymphocyte-mediated killing and effects on other T and B lymphocyte function. J. Immunol. 127: 590-595; 1981.  126  De Jong R, Brouwer M , Hooibrik B , Pouw-Kraan T, Miedema F, van Lier R. The C D 2 7 subset o f peripheral blood memory C D 4 + lymphocytes contains functionally differentiated T lymphocytes that develop by persistent antigenic stimulation in vivo Eur. J. Immunol. 22: 993-999; 1992. Dekaban G , Oger J, Foti D , K i n g E , Waters D , Picard F, A r p J, Werker D , Rice G . H T L V - 1 associated with disease in Aboriginal Indians from British Columbia: a serological and P C R analysis. C l i n . Diagn. V i r o l . 2:67-78; 1993. Dhawan S, Weeks S, Abbasi S, Gralnick H , Notkins A , Klotman M , Yamada K , Klotman P. Increased expression o f a 4 B l and a 5 B l integrins on HTLV-I-infected lymphocytes. Virology. 197: 778-781; 1993. Dhib-Jalbut S, Jiang H , Williams G . The effect o f interferon-B-lb on lymphocyteendothelial cells adhesion. J. Neuroimmunol. 71: 215-222; 1996. Dirks W , M a c L e o d R, Drexler H . E C V - 3 0 4 (endothelial) is really T24 (bladder carcinoma): cell line crosscontamination at source. In vitro cell. Dev. B i o l . - A n i m a l . 35: 558-559; 1999. Dobbie M , Hurst R, K l e i n N , Surtees R. Upregulation o f intercellular adhesion molecule1 expression on human endothelial cells by tumor necrosis factor-a in an in vitro model of the blood-brain barrier. Brain research. 830: 330-336; 1999. Dolman D , Anderson P, Rollinson C , Abbott N . Characterization o f a new i n vitro model of the blood-brain barrier ( B B B ) . J. Physiol. 505: 56-57; 1997. Dore-Duffy P, Newman W , Balabanov R. Lisak R, Mainolfi E , Rothlein R, Peterson M . Circulating, soluble adhesion proteins in cerebrospinal fluid and serum o f patients with multiple sclerosis: correlation with clinical activity. A n n . Neurol. 37: 55-62; 1995. Dorovini-Zis K , Prameya R, Bowman P. Culture and characterization o f microvascular endothelial cells derived from human brain. Lab. Invest. 64: 425-436; 1991. Doukas J, Pober J. IFN-y enhances endothelial activation induced by T N F - a but not IL-1. J. Immunol. 145: 1727-1733; 1990. Droogan A , M c M i l l a n S, Douglas J, Hawkins S. Serum and cerebrospinal fluid levels o f soluble adhesion molecules in multiple sclerosis: predominant intrathecal release o f vascular adhesion molecules-1. J. Neuroimmunol. 64: 185-191; 1996. Drulovic J, Mostarica-Stojkovic M , Levic Z , Stojsavljevic N , Pravica V , Mesaros S. Interleukin-12 and tumor necrosis factor-a levels in cerebrospinal fluid o f multiple sclerosis patients. J . N e u r o l . Sci. 147: 145-150; 1997.  127  Duquette P, Pleines J, Girard M , Charest L , Senecal-Quevillon M , Masse C . The increased susceptibility in women to multiple sclerosis. . Can. J. Neurol. Sci. 19: 466471;1992. Dustin M , Springer T. T cell receptor crosslinking transiently stimulates through L F A - 1 . Nature. 341: 619-624; 1989.  adhesiveness  Dustin M L , Springer T A . Role of lymphocyte adhesion receptors in transient interactions and cell locomotion. A n n u Rev Immunol. 9:27-66; 1991. Elovaara, I., Koenig, A . , Brewah, R., Woods, R., Lehky, T. Jacobson, S. H i g h human Tcell lymphotropic virus type I (HTLV-I)-specific precursor cytotoxic T lymphocyte frequencies in patients with HTLV-I-associated neurological disease. J. E x p . M e d . 177: 1567-1573, 1993. Estess P, Nandi A , Mohmadzadeh M , Siegelman M . Interleukin 15 induces endothelial hyaluronan expression in vitro and promotes activated T cell extravasation through a CD44-dependent pathway in vivo. J. Exp. M e d . 190: 9-19; 1999. Etzioni A , Frydman M , Pollack S, A v i d o r I, Phillips M , Paulson J, Gershoni-Baruch R. Recurrent severe infections caused by a novel leukocyte adhesion deficiency. N . Engl. J. M e d . 327: 1789-1792; 1992. European Study Group on interferon b - l b in secondary progressive M S . Placebocontrolled multicentre randomized trial o f interferon b - l b in treatment o f secondary progressive multiple sclerosis. Lancet. 352: 1491-1497; 1998. Fauci A , Whalen G , Burch C . Activation o f human B lymphocytes. Cellular requirements, interactions and immunoregulation o f pokeweed-induced totalimmunoglobulin producing plaque-forming cells in peripheral blood. C e l l . Immunol. 54: 230-240; 1980. Frelinger A , L a m S, Plow E , Smith M , Loftus J, Ginsberg M . Occupancy o f an adhesive glycoprotein receptor modulates expression o f an antigenic site involved in cell adhesion. J B i o l Chem. 263:12397-12402; 1988. Fukodome K , Furuse M , Fukuhara N , Orita S, Imai T, Takagi S, Nagira N , Hinuma Y , Yoshie O . Strong induction o f I C A M - 1 i n human T cells transformed by human T-cellleukemia virus type-1 and exression o f I C A M - 1 or L F A - 1 in adult T cell leukemiaderived cell lines. Int. J. Cancer. 52: 418-427; 1992. Gamble J, Vdas M . Endothelial cell adhesiveness for human T lymphocytes in inhibited by transforming growth factor-p. J. Immunol. 146: 1149-1154; 1991. Gessain A , Barin F , Vernant J, Gout O, Calender A . Antibodies to human T lymphotropic virus type-1 in patients with tropical spastic paraparesis. Lancet. 2: 407-409; 1985.  128  Gessain A . Epidemiology o f H T L V - I and associated disease. I N : Hollsberg P, Hafler D . ed. Human T cell lymphotropic virus type I. Chichester: Wiley & Sons, 33-64; 1996. Gelati M , Corsini E , Dufour A , Massa G , L a Manita L , Milanese C , Nespolo A , Salmaggi A . Immunological effects of in vivo interferon-b-lb treatment in ten patients with multiple sclerosis: a 1-year follow up. J. Neurol. 246: 569-573; 1999. Genain C , Nguyen M , Letvin N , Pearl R, Davis R, Adelman M , Lees M , Linington C , Hauser S. Antibody facilitation o f multiple sclerosis-like lesions in a nonhuman primate. J. C l i n . Invest. 96: 2966-2974; 1995. Geroni M , Piccardo P, Rodgers-Johnson P, M o r a C , Asher D , Gajdusek D , Gibbs C. A n n . Neurol. Intrathecal synthesis o f IgG antibodies to H T L V - I supports an etiological role for H T L V - I i n tropical spastic paraparesis. 23: S188-S191; 1988. Giraudon P, Buart S, Bernard A , Thomasset N , Belin M . Extracellular matrix-remodeling metalloproteinases and infection o f the central nervous system with retrovirus human Tlymphotropic virus type I ( H T L V - I ) . Prog. Neurobiol. 49: 169-84; 1996. Glynn P, Linington C. Cellular and molecular mechanisms o f autoimmune demyelination in the central nervous system. Crit. Rev. Neurobiol. 4: 367-387; 1989. Godoy A , K i r a J, Hasuo K , Goto I. Characterization o f cerebral white matter lesions o f HTLV-I-associated Myelopathy/tropical spastic paraparesis in comparison with multiple sclerosis and collagen-vasculitis: a smiquantative M R I study. J. Neurol. Sci. 133: 102111;1995. Goldenberg P, K w o n E , Benjamins J, Whitaker J, Quarles R, Prineas J. Opsonization o f normal myelin by anti-myelin antibodies and normal serum. J. Neuroimmunol. 23: 157-:; 1989. Goldstein G , Betz A . Recent advances in understanding brain capillary function. A n n . Neurol. 14: 389-395; 1983. Goodwin R, Alderson M , Smith C , Armitage R, VandenBos T, Jerzy R, Tough T, Shoenborn M , Davis-Smith T, Hennen K . Molecular and biological characterization o f a ligand for C D 2 7 defines a new family o f cytokines with homology to tumor necrosis factor. C e l l . 73: 447-456; 1993. Goust J, Hogan E , Arnaud P. Abnormal regulation o f IgG production in multiple sclerosis. Neurology. 32: 228-234; 1982. Gran B , Hemmer B , Vergelli M , McFarland H F , Martin R Molecular mimicry and multiple sclerosis: degenerate T-cell recognition and the induction o f autoimmunity. A n n Neurol. 45: 559-67; 1999.  129  Greenwood J, Wang Y , Calder V . Lymphocyte adhesion and transendothelial migration in the central nervous system: the role of L F A - 1 , I C A M - 1 , V L A - 4 and V C A M - 1 . Immunology. 86: 408-415; 1995. Hafler D , Fox D , Manning M , Schlossman S, Reinherz E , Weiner H . In vivo activated T lymphocytes i n the peripheral blood and cerebrospinal fluid o f patients with multiple sclerosis. N . Eng. J. M e d . 312: 1405-1411; 1985. Hafler D , Weiner H . M S : a C N S and systemic autoimmune disease. Immunol. Today. 10: 104-107; 1989. Hara T, Jung L , Bjorndahl J, F u S. Rapid induction of a phosphorylated 28 kD/32 k D disulfide-linked early activation antigen ( E A 1) by 12-o-tetradeanoyl phorbol-13-acetate, mitogens, and antigens. J. Exp. M e d . 164: 1988-2005; 1986. Hartung H , Hughes R, Taylor W , Heininger K , Reiners K , Toyka K . T cell activation in Guillain-Barre syndrome and in M S : elevated serum levels of soluble IL-2 receptors. Neurology, 40: 215-218; 1990. Hartung H , Reiners K , Archelos J, Michels M , Seeldrayers P, Heidenreich F , Pflungupt K , Toyka K . Circulating adhesion molecules and tumor necrosis factor receptor in multiple sclerosis: correlations with magnetic resonance imaging. A n n . Neurol. 38: 186193; 1995. Hauser S, A u l t K , Johnson D , Hoban C , Weiner H . Increased IgG secretion by unstimulated mononuclear cells in active multiple sclerosis and functional assessment o f the T8 subset. C l i n . Immunol. Immunopathol. 37: 312-323; 1985. Hess D , Bhutwala T, Sheppard J, Zhao W , Smith J. I C A M - 1 expression on human brain microvascular endothelial cells. Neurosci. Lett. 168: 201-204; 1994. Hickey W , H s u B , K i m u r a H . T lymphocyte entry into the central nervous system. J. Neurosci. Res. 28: 254-260; 1991. Hintzen R, de Jong R, Lens S, Brouwer M , Baars P, van Lier R. Regulation o f C D 2 7 expression on subsets o f mature T-lymphocytes. J. Immunol. 151: 2426-2435, 1993. Hintzen R, Paty D , Oger J. Cerebrospinal fluid concentrations o f soluble C D 2 7 in H T L V I associated myelopathy and multiple sclerosis. J Neurol Neurosurg Psychiatry. 66:791793;1999. Ho H , Hultin L , Mitsuyasu R, Matud J, Hausner M , Bockstoce D , Chou C , O ' R o u k e S, Taylor J, Giorgi J. Circulating HIV-specific C D 8 cytotoxic T cells express C D 3 8 and H L A - D R antigens. J. Immunol. 150: 3070-3079; 1993.  130  Hofman F, von Hanwehr R, Dinrello C , M i z e l S, Hinton D , Merrill J. Immunoregulatory molecules and IL-2 receptors identified in multiple sclerosis brain. J. Immunol. 136: 3239-3245; 1986. Hogg N , Landis R. Adhesion molecules in cell interactions. Curr. Opin. Immunol. 5: 383390;1993. Hughes S. Functional characterization o f the spontaneously transformed human umbilical vein endothelial cell line E C V - 3 0 4 : use in an in vitro model o f angiogenesis. E x p . C e l l Res. 225: 171-185; 1996. Hughes C , Male D , Lantos L . Adhesion o f lymphocytes to cerebral microvascular cells: effects o f interferon-gamma, tumour necrosis factor and interleukin-1. Immunology. 64: 677-81; 1988. Huitanga I, Damoiseaux J, Dopp E , and Dijkstra C . Treatment with anti-CR3 antibodies E D 7 and E D 8 suppresses experimental allergic encephalomyelitis in Lewis rats. Eur. J. Immunol. 23: 709-715; 1993. Huynh H , Oger J, Dorovini-Zis K . Interferon-B downregulates interferon-y-induced class II M H C molecules expression and morphological changes i n primary culture o f human brain microvessel endothelial cells. J. Neuroimmunol. 60: 63-73; 1995. Ichinose K , Nakamura T, Kawakami A , Egyuchi K , Nagasato K , Shibayama K , Tsujihata M , Nagataki M . Increased adherence of T cells to human endothelial cells in patients with human T-cell lymphotropic virus type I-associated myelopathy. Arch. Neurol. 49: 74-76; 1992. Ijichi S, Eiraku N , Osame M , Izumo S, Kubota R, Maruyama I, Matsumoto M , N i i m u r a T, Sonoda S. Activated T lymphocytes in cerebrospinal fluid of patients with H T L V - I associated myelopathy ( H A M / T S P ) . J. Neuroimmunol. 25: 251-254; 1989. Imai T, Hieshima K , Haskell C , Baba M , Nagira M , Nishimura M , K a k i z a k i M , Takagi S, Nomiyama H , Schall T, Yoshie O. Identification and molecular characterization o f fractalkine receptor C X C R 1 , which mediates both leukocyte migration and adhesion. C e l l . 91: 521-530; 1997. Imai T, Tanaka Y , Fukudome K , Takagi S, A r a k i K , Yoshie O. Enhanced expression o f L F A - 3 on human T cell lines and leukemic cells carrying human T-cell leukemia virus type-1. Int. J. Cancer. 55: 811-816; 1993. Imhof B , Dunon D . Leukocyte migration and adhesion. A d v Immunol. 58: 345-415; 1995.  131  Ishikawa T, Imura A , Tanaka K , Shirane H , Okuma M , Uchiyama T. E-selectin and vascular cell adhesion molecule-1 mediate adult T-cell leukemia cell adhesion to endothelial cells. Blood. 82: 1590-1598; 1993. Itoyama Y , Minato S, K i r a J, Goto I, Sato H , Okochi K , Yamamoto N . Altered subset o f peripheral blood lymphocytes in patients with H T L V - 1 -associated myelopathy ( H A M ) . Neurology. 38: 816-818; 1988a. Itoyama Y , Minato S, K i r a J, Goto I, Sato H , Okochi K , Yamamoto N . Spontaneous proliferation o f peripheral blood lymphocytes increased in patients with H T L V - 1 associated myelopathy. Neurology. 38: 1302-1307; 1988b. Iwasaki Y , Ohara Y , Kobayashi I, and A k i z u k i I. Infiltration o f helper/inducer T lymphocytes heralds central nervous system damage in human T - c e l l leukemia virus infections. A m . J. Pahol. 140: 1003-1008; 1992. Jacobs L , Cookfair D , Rudick R, Herndon R M , Richert JR, Salazar A M , Fischer JS, Goodkin D E , Granger C V , Simon J H , A l a m JJ, Bartoszak D M , Bourdette D N , Braiman J, Brownscheidle C M , Coats M E , Cohan S L , Dougherty D S , K i n k e l R P , Mass M K , Munschauer F E 3rd, Priore R L , Pullicino P M , Scherokman B J , Whitham R H , Intramuscular interferon B - l a for disease progression in relapsing multiple sclerosis. A n n . Neurol. 39: 285-294; 1996. Jacobson S, Zainovic V , M o r a C , Rodger-Johnson P, Sheremato W , Gibbs C , Gadjusek C , M c F a r l i n D . Immunological findings in neurological diseases associated with antibodies to H T L V - 1 : activated lymphocytes in tropical spastic paraparesis. Ann. Neurology. 23: S196-200; 1988. Jacobson S, Shida H , M c F a r l i n D , Fauci A , Koeing S. Circulating C D 8 + cytotoxic T lymphocytes specific for H T L V - I p X in patients with H T L V - I associated neurological disease. Nature. 348: 245-248, 1990a. Jacobson S, Gupta A , Mattson D , M i n g i o l i E , M c F a r l i n D . Immunological studies in tropical spastic paraparesis. A n n . Neurol. 27: 149-156; 1990b. Jacobson S, M c F a r l i n D , Robinson S, Voskuhl R, Martin R, Brewah A , N e w e l l A , Koenig S. Demonstration o f H T L V - I specific cytotoxic T-lymphocytes in the cerebrospinal fluid o f patients with HTLV-I-associated neurological disease. A n n . Neurol. 32:651-657; 1992. Jaffe E , Nachman R, Becker C . M i n i c k R. Culture o f human endothelial cells derived from umbilical veins: identification by morphologic and immunological criteria. J. C l i n . Invest. 52: 2735-2756; 1973.  132  James S, Neckers L , Graeff A , Cossman J, Balch C , Strober W . Suppression o f immunoglobulin synthesis by lymphocyte subpopulations in patients with Crohn's disease. Gastroenterology. 86: 1510-1518; 1984. Johnson B , Issekutz T, Kubes P. The alpha 4-integrin supports leukocyte rolling and adhesion in chronically inflamed postcapillary venules in vivo. J. E x p . M e d . 183: 19952006; 1996. Johnson K , Nelson B . Multiple sclerosis: diagnostic usefulness o f cerebrospinal fluid. A n n Neurol. 2: 425-431; 1977. Joly P, Guillon J, Mayaud C , Plata F, Theodorou I, Denis M , Debre P, Autrant B . C e l l mediated suppression o f HIV-specific cytotoxic T lymphocytes. J. Immunol. 143: 21932001;1989. Joo F. The blood-brain barrier in vitro: the second decade. Neurochem. Int. 23: 499-521; 1993. Jung T, Dailey M . Rapid modulation o f homing receptors (gp90Mel-14) induced by activation o f protein kinase C . Receptors shedding due to accelerated proteolytic cleavage at the cell surface. J. Immunol. 144: 3130-3136; 1990. Kansas G . Selectins and their ligands: current concept and controversies. Blood. 88: 3259-3287; 1996. Kaplan J, Osame M , Kubota H . The risk o f development o f H T L V - I associated myelopathy/tropical spastic paraparesis ( H A M / T S P ) among persons infected with H T L V - I . J. Acquir. Immune. Defic. Syndr. 3: 1096-1001; 1990. Karpas A , K a m p f U , Siden A , K o c h M , Poser S. Lack of evidence for involvements o f know human retroviruses in multiple sclerosis. Nature. 322: 177-178; 1986. K e l l y R, Ellison G , Myers L , Goymerac V , Tarrick S, K e l l y C . Abnormal regulation o f in vitro IgG production in multiple sclerosis. A n n . Neurol. 9: 267-272; 1981. Kermode A , Thompson A , Tofts P, et al., Breakdown o f the blood-brain barrier precedes symptoms and other M R I signs of new lesions in multiple sclerosis. Pathogenetic and clinical implications. Brain. 113: 1477-1489; 1990. K i r a J, Tobimatsu S, Goto I, Hasuo. Primary progressive versus relapsing remitting multiple sclerosis patients in Japanese patients: a combined clinical, magnetic resonance imaging and multimodality evoked potential study. J. Neurol. Sci. 117: 179-185; 1993. Kishimoto T, Jutila M , Berg E , et al. The neutrophil Mac-1 and M E L - 1 4 glycoproteins are inversely regulated by chemotactic factors. Science 245: 1238-1241; 1989.  133  Kohem C , Brezinschek R, Wisbey H , Tortorella C , Lipsky P. Enrichment o f differentiated C D 4 5 R B , C D 2 7 - memory T cells in the peripheral blood, synovial fluid, and synovial tissue o f patients with rheumatoid arthritis. Arthr. Rheum. 139: 844-854, 1996. Kotovuori P, Tontti E , Pigott R, Shepherd M , K i s o M , Hasegawa A , Renkonen R, Nortamo P, Altieri D C , Gahmberg C G . . The vascular E-selectin binds to the leukocyte integrins C D 1 1 / C D 1 8 . Glycobiology. 3: 131-136; 1991. Krensky A , Sanchez-Madrid F , Robbins E , Nagy J, Springer T, Burakoff S. The functional significance, distribution, and structure o f L F A - 1 , L F A - 2 , and L F A - 3 : cell surface antigens associated with CTL-target interactions. J. Immunol. 131: 611-616; 1983. Kuchler-Bopp S, Delaunoy J, Artault J, Zaepfel, Dietrich J. Astrocytes induce several blood-brain barrier properties in non-neural endothelial cells. NeruroReport. 10: 13471353; 1999. Kuijpers T, Hoogerwerf M , V a n der Laan L , Nagel G , van der Schoot C , Grunert F , Roos , D . C D 6 6 nonspecific cross-reacting antigens are involved in neutrophil adherence to cytokine-activated endothelial cells. J. Cell B i o l . 118: 457-466; 1992. Kurtzke J. Disability rating scales in multiple sclerosis. A n n . N Y . Acad. S c i . 346: 347360; 1984. Landay A , Bauer K . F l o w cytometric analysis o f cells o f the immune system. J. Immunol. Immunopharmacol. 8: 166-171; 1988. Larson R, Springer T. Structure and function of leukocyte integrins. Immunol. Rev. 114: 181-217; 1990. Lassmann H , Raine C , Antel J, Prineas J. Immunopathology o f multiple sclerosis: Report on an international meeting held at the institute of neurology o f the university o f Vienna. J. Neuroimmunol. 86: 213-217; 1998. Lawrence M , Springer T. Leukocyte roll on a selectin at physiologic flow rates: distinction from and prerequisite for adhesion through integrins. C e l l . 65: 859-873; 1991. Lehmann P, Forsthuber T, M i l l e r A , Sercarz E . Spreading of T-cell autoimmunity to cryptic determinants o f autoantigen. Nature. 558: 155-157; 1992. Leppert D , Waubant E , Burk M , Oksenberg J, Hauser S. Interferon Beta-lb inhibits gelatinase secretion and in vitro migration o f human T cells: a possible mechanism for treatment efficacy in multiple sclerosis. A n n . Neurol. 40: 846-852; 1996.  134  Leroy, E . , Calvo, C , Divine, M . , Gourdin, M . , Baujean, F., Ben Ariba, M . , Mishal, Z . , Vernant, J., Farcet, J., and Senik, S. Persistence o f surviving patients after allogeneic bone morrow transplantation. J. Immunol. 137: 2180-2189; 1986. Levin M , Jacobson S. H T L V - I associated myelopathy/tropical spastic paraparesis ( H A M / T S P ) : a chronic progressive neurological disease associated with immunologically mediated damage to the central nervous system. J. Neurovirol. 3: 126-140; 1997. Levinson A , Sandberg-Wollheim M , Lisak R, Zweiman B , Sjogren K , Laramore C , Moskovitz A , Hedstrom S. Analysis o f B-cell activation o f cerebrospinal fluid lymphocytes in multiple sclerosis. Neurology. 33:1305-10; 1983. Levitt D , Griffin N , Egan M . Mitogen-induced plasma cell differentiation in patients with multiple sclerosis. J. Immunol. 124: 2117-2121; 1980. Lewis D , Puck J, Babock G , R i c h R. Disproportionate expansion o f a minor T cell subset in patients with lymphoadenopathy syndrome and acquired immunodeficiency syndrome. J. Infect. D i s . 151: 555-559; 1985. Lindington E , Moyes L , M c C o r m a c k A , Rose M . A comparison o f primary endothelial cells and endothelial cell lines for studies of immune interactions. Transpl Immunol. 7: 239-246; 1999. L i n k H , Cruz M , Gessain A , Gout O, Kam-Hansen S. Chronic progressive Myelopathy associated with H T L V - I : Oligoclonal IgG and a n t i - H T L V - I IgG antibodies i n the cerebrospinal fluid and serum. Neurology. 39: 1566-1572; 1989. Link J, Soderstorm M , Olsson T, Hojeberg B , Ljungdahl A , L i n k H . Increased transforming growth factor b, increased IL-4, and interferon-g in multiple sclerosis. A n n . Neurol. 36: 379-386; 1994. L o u J, Dayer M , Grau E , Burger D . Direct cell/cell contact with stimulated T lymphocytes induces the expression o f cells adhesion molecules and cytokines by human brain microvascular endothelial cells. Eur. J. Immunol. 26: 3107-3113; 1996. L o u J, Chofflon M , Juillard C , Donati Y , M i l i N , Siergrist C , Grau G . Brain microvascular endothelial cells and leukocytes derived from patients with multiple sclerosis exhibit increased adhesion capacity. NeuroReport. 8: 629-633; 1997. L o u J, Y v a n G , Zheng L , Giroud C , Morel P, Clements J, Ythier A , Grau G . InterferonP inhibits activated leukocyte migration through human brain microvascular endothelial cell monolayer. Lab. Invest. 79: 1015-1025; 1999. Lublin F, Reingold S. Defining the clinical course o f multiple sclerosis: Results o f an international survey. Neurology, 46: 907-911; 1996.  135  Lublin F. A phase II trial o f anti-CD 11/CD 18 monoclonal antibody in acute exacerbation of multiple sclerosis. Neurology. 52(suppl 2): A290; 1999. Lucchinetti C , Bruck W , Rodriguez M , Lassmann H . Distinct patterns o f multiple sclerosis pathology indicates heterogeneity in pathogenesis. Brain Pathol. 6: 259-274; 1996. Luster A . Chemokines-chemotactic cytokines that mediate inflammation. N e w Engl. J. M e d . 338: 436-445; 1998. Maher P, O'Toole C , Wreghitt T, Spiegelhalter D , English T. Cytomegalovirus infection in cardiac transplant recipients associated with chronic T cell subset ratio inversion with expansion of a Leu-7+ TS-C+ subset. C l i n . Exp. Immunol. 62: 515-524; 1985. Mainolfi L , Rothlein R. Elevated levels o f s I C A M - 1 in patients with human T-cell leukemia virus type I associated myelopathy and adult T-cell leukemia. Blood. 80: 24342435;1992. Makgoba M , Sanders M , Shaw S. The C D 2 - L F A - 3 and L F A - 1 - I C A M - 1 relevance to T-cell recognition. Immunol. Today. 10: 417-422; 1989.  pathways:  Male K , Pryce G . Kinetics o f M H C gene expression and m R N A synthesis in brain endothelium. Immunology. 63: 37-42; 1988. Maloney E , Cleghorn F , Morgan O. Incidence o f H T L V - I associated myelopathy/tropical spastic paraparesis ( H A M / T S P ) in Jamaica and Trinidad. J Acquir Immune Defi Syndr Human Retrovirol. 17: 167-170; 1998 Mantovani A , Bussolino F, Dejana E . Cytokine regulation o f endothelial cell function. F A S E B J . 6:2591-2599; 1992. Marrosu M , Muntoni F, Murru M Spinicci G , Pischedda M P , Goddi F, Cossu P, Pirastu M . Sardinian multiple sclerosis is associated with H L A - D R 4 . Neurology, 38: 1749-1753; 1988. Martin R., Hohlfeld R., McFarland H . Infections and inflammatory diseases: multiple sclerosis. In: Brandt T, Caplan R, Dichgans J, eds. Neurologic disorder course and treatment. N e w York: Academic Press, 483-505; 1996. Martin R, McFarland H , M c F a r l i n D . Immuological aspects o f demyelinating A n n . Rev. Immunol. 10: 153-187; 1992.  diseases.  Martz E . L F A - 1 and other accessory molecules functioning in adhesions o f T and B lymphocytes. H u m . Immunol. 18: 3-10; 1987.  136  Massacesi L , Genain C , Lee-Parritz D , Canfield D , Hauser S. Active and passively induced experimental autoimmune encephalomyelitis in common marmosets: a new model for multiple sclerosis. A n n . Neurol. 37: 519-530; 1995. Matsuda M , Tsukada N , Miyagi K , Yanagisawa N . Increased levels o f soluble vascular cell adhesion molecule-1 ( V C A M - 1 ) in the cerebrospinal fluid and sera o f patients with multiple sclerosis and human T lymphotropic virus type-1-associated myelopathy. J. Neuroimmunol. 59: 35-40; 1995a. Matsuda M , Tsukada N , Miyagi K , Yanagisawa N . Adhesion o f lymphocytes to endothelial cells in experimental allergic encephalomyelitis before and after treatment with endotoxin lipopolysaccharide. Int. Arch. Allergy Immunol. 106: 335-344; 1995b. McFarland H . The multiple sclerosis lesion. A n n . Neurol. 77: 419-420; 1995. M c L e a n B , Zeman A , Barnes D , Thompson E . Pattern o f blood-brain barrier impairment and clinical features in multiple sclerosis. J. Neurol. Nerurosurg. Psychiatry. 56: 356-360; 1993. Merrill J, Benveniste E . Cytokines in inflammatory brain lesions: helpful and harmful. Trends Neurosci. 19: 331-338; 1996. Miedema F, Tetteroo P, Terpstra F, Kiezer G , Roos M , Weemomg R, Weemacs C , Roos D , M e l i e f C . Immunological studies o f L F A - 1 and Mol-deficient lymphocytes from patients with recurrent bacterial infections. J. Immunol. 34: 3075-3081; 1985. M i n g Wang J, M c V i c a r D , Oppenheim J, K e l v i n D . Identification o f R A N T E S receptors on human monocytic cells: competition for binding and characterization by homologous chemotactic cytokines. J. E x p . M e d . 177: 669-705; 1993. Mobner R, Fassbender K , Kuhnen J, Schwartz A , Hennerici M . Circulating L-selectin in multiple sclerosis patients with active, gadolinium-enhancing brain plaques. J. Neuroimmunol. 65: 61-65; 1996. Mokhtarian F, M c F a r l i n D , Raine C . Adoptive transfer o f myelin basic protein-sensitized T cells produces chronic relapsing demyelinating disease in mice. Nature. 309: 356-358; 1984. Monteyne P, Bureau J, Brabic T. Viruses and multiple sclerosis. Curr. Opin. Neurol. 11: 287-291; 1998. Moore G , Traugott U , Scheinberg L , Raine C . Tropical spastic paraparesis: a model o f virus induced, cytotoxic mediated demyelination ? A n n Neurol. 26: 523-530; 1989.  137  M o r i M , Kinoshita K , Ban N , Yamada Y , Shiku H . Activated T lymphocytes with polyclonal gammopathy in patients with human T-lymphotropic virus type I-associated myelopathy. A n n . Neurol. 24: 280-282; 1988. Morimoto C , Letvin N , Boyd A , Hagan M , Brown H , Kornacki M , Schlossman S. The isolation and characterization o f the human suppressor-inducer T cell subset. J. Immunol. 134: 3762-3769, 1989. Mosley K , Cuzner M . Receptor-mediated phagocytosis o f myelin by macrophages and microglia: effects o f opsonization and receptor blocking agents. Neurochem. Res. 21: 481-487; 1996. Mosmann T, Moore K . The role o f IL-10 in cross-regulation o f T h l and Th2 responses. Immunol. Today. 12: A 4 9 - A 5 3 ; 1991. Mukae H , Kohno S, Nobuo M , Morikawa N , Kadota J, Matsukura S, Hara K . Increase in T-cells bearing C D 2 5 in bronchoalveolar lavage fluid from H A M / T S P patients and H T L V - I carriers. Microb. Immunol. 38: 55-62; 1994. Mulligan M , Varani J, Warren J, T i l l G , Smith C , Anderson D , Todd R, Ward P. Roles o f B2 integrins o f rat neutrophils in complement-and oxygen radical-mediated acute inflammatory injury. J. Immunol. 148: 1847-1857; 1992. Nagai M , Yashiki S, Fujiyoshi T, Fujiyama C , Kitze B , Izumo S, Osame M , Sonaoda S. Characterization o f a unique T-cell clone established from a patient with H A M / T S P which recognized HTLV-I-infected T-cell antigens as well as spinal cord tissue antigens. J. Neuroimmunol. 65: 97-106; 1996. Nakagawa M , Izumo S, Ijichi S, Kubota J, Arimura K , Kawabata M , Osame M . H T L V - I associated Myelopathy: Analysis o f 213 patients on clinical features and laboratory findings. J. Neurovirol. 1: 50-61; 1995. Nilsen E , Johansen F, Jahnsen F, Lundin K , Scholz T, Brandtzaeg P, Haraldsen G . Cytokine profiles o f cultured mircovascular endothelial cells from the human intestine. Gut. 42: 635-642; 1998. Nishiura Y , Nakamura T, Ichinose K , Nagasato K , Ohishi R, Watanabe H , Tsujihata M , Nagataki M . Antiproliferative factor against the human glioblastoma cell line T 9 8 G identified in culture supernatant o f C D 4 + cells from patients with HTLV-1-associated myelopathy. J. Neurol. Sci. 122: 214-219; 1994. Noronha A , Richman D , Arnason B . Detection o f in vivo stimulated cerebrospinal fluid lymphocytes by flow cytometry in patients with multiple sclerosis. N . Eng. J. M e d . 303: 713-717; 1980.  138  O'Gorman M , Oger J, Kastrukoff L . Reduction o f immunoglobulin G secretion in vitro following long term lymphoblastoid interferon (Wellferon) treatment i n multiple sclerosis patients. C l i n . E x p . Immunol. 67: 66-75; 1987. Oen K , Danell G , Stewart S, Wilkins J, Tazumi K , Jacobson K . Adhesion o f peripheral blood lymphocytes of children with arthritis to human umbilical vein endothelial cells. C l i n . E x p . Immunol. 95: 415-423; 1994. Oger J, Dekaban G . H T L V - I associated myelopathy: autoimmunity. Autoimmunity. 21: 151-159; 1995.  A  case  o f viral-induced  Oger J, Arnason B , Pantazis N , Lehrich J, Young M . Synthesis o f nerve growth factor by L a n d 3 T 3 cells in culture. Proc. Natl. Acad. Sci. U S A . 71: 1554-1558; 1974. Oger J, Mattson D , Roos R, Antel J, Arnason B . Isoelectric focusing o f IgG secreted by human peripheral blood lymphocytes in Multiple Sclerosis and controls. Neurol. 31: 144; 1981. Oger J, O'Gorman M , Willoughby E , L i D , Paty D . Changes i n immune function i n relapsing multiple sclerosis correlate with disease activity as assessed by magnetic resonance imaging. A n n . N . Y . Acad Sci. 540:597-601; 1988 Oger J, Werker D , Foti D , Dekaban G . HTLV-1-associated myelopathy: an endemic disease o f Canadian Aboriginal o f the Northwest Pacific. Can. J. Neuro. S c i . 20: 302-306; 1993. O i V , Glazer A , Stryer L . Fluorescent phycobiliprotein conjugates for analyses o f cells and molecules. J C e l l B i o l . 93: 981-986; 1982. Oldstone M . Molecular mimicry and autoimmune disease. Cell., 50: 819-820; 1987 Olsson T, Baig S, Hojeberg B , L i n k H . Animyelin basic protein and antimyelin antibodyproducing cells in multiple sclerosis. A n n . Neurol. 27: 132-136; 1990. Olsson T. Critical influences o f the cytokine orchestration on the outcome o f myelin antigen-specific T-cell autoimmunity in experimental autoimmune encephalomyelitis and multiple sclerosis. Immunol. Rev. 144: 245-268; 1995. Oppenheimer-Marks N , Davis L , Boque D , Ramberg J, Lipsky P. Differential utilization of I C A M - 1 and V C A M - 1 during the adhesion and transendothelial migration o f human T lymphocytes. J. Immunol. 147: 2913-2921; 1991. Oppenheimer-Marks N , Davis L , Lipsky P. Human T lymphocyte adhesion to endothelial cells and transendothelial migration: alteration o f receptor use relates to the activation status o f both the T cell and the endothelial cell. J. Immunol. 145: 140-148; 1990.  139  Osame M , Matsumoto M , Usuku K , Izumo S, Ijichi N , Amitani H , Tara M , Igata A . Chronic progressive myelopathy associated with elevated antibodies to human Tlymphotropic virus type I and adult T-cell leukemialike cells. A n n . Neurol. 21: 117-122; 1987. Osame M , Usuku K , Izumo S, Ijichi N , Amitani H , Tara M , Igata A . H T L V - I associated myelopathy, a new clinical entity. Lancet. 1: 1031-1032, 1986. Osame M , Izumo S, Igata A . Blood transfusion and HTLV-I-associated myelopathy. Lancet. 2: 104-105; 1990. Osborn L . Leukocyte adhesion to endothelium in inflammation. Cell 62: 3-6; 1990. Panitch H , Hirch R, Schindler J, et al. Treatment o f multiple sclerosis with g interferon: exacerbation associated with activation o f the immune system. Neurology. 37: 10971120;1987. Panitch H . Influence of infection on exacerbations o f multiple sclerosis. A n n . Neurol. 36: S25-S28; 1994. Pardia R, Bender J, Dettori C , et al. Herterogeneous distribution and trnasmembrane signaling properties o f lymphocyte function associated antigen ( L F A - 1 ) in human lymphocyte subsets. J. Immunol. 143: 3157-3166; 1989. Pardridge W . Recent advances in blood-brain barrier transport. A n n . Rev. Pharmacol. Toxicol. 28: 25-39; 1988. Paty D , L i K , The U B C M S / M R I Study group, and the IFNB Multiple Sclerosis Study Group. Interferon P-lb is effective in relapsing-remitting multiple sclerosis. II. M R I analysis. Neurology. 43: 662-667; 1993. Phillips J, Lanier L . Lectin-dependent and anti-CD3 induced cytotoxicity are preferentially mediated by peripheral blood cytotoxic T lymphocytes expressing Leu-7 antigen. J.Immunol. 136: 1579-1585; 1986. Piali L , Hammel P, Uherek C , Bachmann F, Gisler R, Dunon D , Imhof B . C D 3 1 / P E C A M - 1 is a ligand for avb3 integrin involved in adhesion o f leukocytes to endothelium. J. C e l l . B i o l . 130: 451-460; 1995. Picker L , Warnock R, Burns A , Doerschuk C , Berg E , Butcher E . The neutrophil selectin L E C A M - 1 presents carbohydrate ligands to the vascular selectins E L A M - 1 and G M P 140. C e l l , 66: 921-933; 1991. Piela T, K o r n J. ICAM-1-dependent fibroblast-lymphocyte adhesion: discordance between surface expression and function of I C A M - 1 . C e l l . Immunol. 129: 125-137; 1990.  140  Pober J, Cotran R. Cytokine-mediated activation o f vascular endothelium Physiol. Rev. 70:427-451; 1990. Pober J. Cytokine-mediated activation o f vascular endothelium. A m . J. Pathol. 133: 426433;1988. Poser's C M , Paty D , Scheinberg L , M c D o n a l d W , Davis F, Ebers G , Johnson K , Sibley W , Silberberg D , Tourtellotte W . N e w diagnostic criteria for multiple sclerosis: guidelines for research protocols. A n n N e r u r o l . 13: 227-231; 1983. Prat A , A l - A s m i A , Duquette P, Antel J. Lymphocyte migration and multiple sclerosis: relation with disease course and therapy. A n n . Neurol. 46: 253-256; 1999. Prince H . American blood donor seropositive for human T-lymphotropic virus type I/II exhibit normal lymphocyte subsets. Transfusion. 30: 787-790; 1990. P R I S M S Study Group. Randomised double-blind placebo-controlled study o f interferon b - l a on neurological disability in relapsing/remitting multiple sclerosis. Lancet. 352: 1498-1504; 1998. Raine C , Canella B , Duyvesteyn A , Cross A . Homing to central nervous system vasculature by antigen specific lymphocytes. II Lymphocyte endothelial cell adhesion during the initial stages of autoimmune demyelination. Lab. Invest. 63: 476-489; 1990. Raine C , Canella B , Hauser S, Genain C. Demyelination in primate autoimmune encephalomyelitis and acute multiple sclerosis lesions: a case for antigen-specific antibody mediation. A n n . Neurol. 46: 144-160; 1999. Raine C . The Dale E . M c F a r l i n Memorial Lecture: the immunology o f the multiple sclerosis lesion. A n n . Neurol. 36(suppl): S61-S72; 1994. Raine C , Canella B , Duyvesteyn A , Cross A . Homing to central nervous system vasculature by antigen specific lymphocytes. II Lymphocyte endothelial cell adhesion during initial stages o f autoimmue demyelination. Lab. Invest. 63: 476-489; 1990. Richardson J, Edwards A , Cruickshank J, Rudge P, Dalgleish A . In vivo cellular tropism of human T-cell leukemia virus type I. J. V i r o l . 64: 5682-5687, 1990. Rieckmann P, Albrecht M , Kitze B . Cytokine m R N A levels in mononuclear blood cells from patients with multiple sclerosis. Neurology. 44: 1523-1526; 1994a. Rieckmann P, Martin S, Wechselbraun I, Albrecht M , Kitze B , Weber T, Tumani H , Broocks A , Luer W , Helwig A , Poser S. Serial analysis o f circulating adhesion molecules and T N S receptor in serum from patients with multiple sclerosis: Cicam-1 is an indicator for relapse. Neurology, 44: 2367-2372; 1994b.  141  Romagnani S. The T h l / T h 2 paradigm. Immunol. Today. 18: 263-265; 1997. Roman G , Osame M . Identity o f HTLV-I-associated tropical spastic paraparesis and HTLV-I-associated Myelopathy. Lancet 1: 651; 1988. Romanic A , Madri J. The induction o f 72-kD gelatinase in T cells upon adhesion to endothelial cells is V C A M - 1 dependent. J. Cell B i o l . 125:1165-78; 1994. Romanic A , Graesser D , Baron J L , Visintin I, Janeway C A Jr, M a d r i J A . T cell adhesion to endothelial cells and extracellular matrix is modulated upon transendothelial cell migration. Lab Invest.76:11-23; 1997. Roos R. Viruses and demyelinating disease o f the central nervous system. Neurol. C l i n 1: 681-700; 1983. Rosenkoetter M , Reder A , Oger J, Antel J. T cell regulation o f polyclonal induced immunoglobulin secretion in humans. J. Immunol. 132: 1779-1783; 1984. Rott O, Fleischer B , Cash E . Interleukin-10 prevent the development o f E A E in mice. Eur. J. Immunol. 24: 1434-1440; 1994. Runkel L , Meier W , Pepinsky R, Karpusas M , Whitty A , K i m b a l l K , Brickelmaier M , Muldowney C , Jones W , Goelz S. Structural and functional differences between glycosylated and non-glycosylated forms o f human interferon b (IFN-P). Pharm. Res. 15(4): 641-649; 1998. Sadovnick A , Ebers G . Epidemiology o f multiple sclerosis: A critical overview. Can. J. Neurol. Sci. 20: 17-29; 1993. Sanders V , Waddell A , Felisan S, L i X , Conard A . Herpes simplex virus in postmortem multiple sclerosis brain tissue. Arch. Neurol. 53: 123-124; 1996. Sanders M , Makgoba M , Shaw S. Human naive and memory T cells. Reinterpretation o f helper-inducer and suppressor-inducer subsets Immunol. Today. 9: 195-199; 1988. Schall T, Bacon K , Toy K , Goeddel D . Selective attraction o f monocytes and T lymphocyte o f the memory phenotype by cytokine R A N T E S . Nature. 347: 669-671; 1990. Schall T, Bacon K . Chemokine, leukocyte trafficking, and inflammation. Curr. Opin. Immunol. 6: 865-873; 1994. Schluesener H , Meyermann R. Intercrines in brain pathology: expression o f intercrines in multiple sclerosis and Morbus Creutzfeldt-Jacob lesion. Acta Neruopathol. 86: 393-396; 1993.  142  Scholding N , Zajicek J, W o o d N , Compston D . The pathogenesis o f demyelinating disease. Prog. Neurobiol. 43: 143-173; 1994. Sharief M , N o o r i M , Ciardi M , Cirelli A , Thompson E . Increased levels o f circulating I C A M - 1 in serum and cerebrospinal fluid o f patients with active multiple sclerosis. Correlation with T N F - a and blood-brain barrier damage. J. Neuroimmunol. 43: 15-21; 1993. Schering-Plough. Further studies on the biological properties o f interleukin-10. Data on file as P-5806. Schering-Plough. Single dose therapy with IL-10 in mice with experimental allergic encephalomyelitis ( E A E ) . Data on file as D-27219. Shimizu Y , van Seventer G , Horgan K , Shaw S. Roles o f adhesion molecules i n T-cell recognition: fundamental similarities between four integrins on resting human T cells ( L F A - 1 , V L A - 4 , V L A - 5 , V L A - 6 ) in expression, binding, and costimulation. Immunol. Rev. 114:109-43; 1990. Shimizu Y , Newman W , Gopal V , Horgan K , Graber N , Beall D , van Seventer G , Shaw S. Four molecular pathways of T cell adhesion to endothelial cells: role o f L F A - 1 , V C A M - 1 , and E L A M - 1 , and changes in pathway hierarchy under different activation conditions. J. C e l l . B i o l . 113: 1203-1212; 1991. Shimizu Y , Newman W , Tanaka Y , Shaw S. Lymphocyte interactions with endothelial cells. Immunol. Today. 13: 106-112; 1992. Siegel D , Le J, Vilcek J. Modulation of lymphocyte proliferation an immunoglobulin synthesis by interferon-y and type-I interferons. Cell. Immunol. 101: 380-390; 1986. Simon J, Jacobs L , Campion M , Wende K , Simonian N , Cookfair D , Rudick R, Herndon R, Richert J, Salazar A , A l a m J, Fischer J, Goodkin D , Granger C , Lajaunie M , MartensDavidson A , Meyer M , Sheeder J, Choi K , Scherzinger A , Bartoszak D , Bourdette D , Braiman J, Brownscheidle C , Whitham R. Mangetic resonance studies o f intramuscular interferon B - l a for relapsing multiple sclerosis. A n n . Neurol. 43: 79-87; 1998. Smeltz R, Swanborg R. Concordance and contradiction concerning cytokines and chemokines i n experimental demyelinating disease. J. Neursci. Res. 51: 147-153; 1998. Smith S. IL-10 does not prevent the development o f E A E in mice. F A S E B . 8: A994; 1994. Sobel R, Mitchell M , Fondren G . Intercellular adhesion molecule-1 ( I C A M - 1 ) in cellular immune reactions in the human central nervous system. A m . J. Pathol. 136: 1309-1316; 1990.  143  Soilu-Hanninen M , Salmi A , Salonen R. Interferon-P downregulates expression of V L A 4 antigen and antagonizes interferon-g-induced expression of H L A - D Q i n human peripheral blood monocytes. J. Neuroimmunol. 60: 99-106; 1995. Spertini O, Luscinskas F, Kansas G , Munro J, Griffin J, Gimbrone M , Tedder T. Leukocyte adhesion molecule-1 ( L A M - 1 , L-selectin) interacts with an inducible endothelial cell ligand to support leukocyte adhesion. J. Immunol. 147: 2565-2573; 1991. Springer T. Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration. A . Rev. Physiol. 57: 827-872; 1995. Springer T. Adhesion receptors of the immune system. Nature. 346: 425-434; 1990. Springer T. Traffic signals for lymphocyte recirculation and leukocyte emigration: The multistep paradigm Cell. 76: 301-314; 1994. Staunton D , Dustin M , Springer T. Functional cloning of I C A M - 2 , a cell adhesion ligand for L F A - 1 homologous to I C A M - 1 . Nature. 339: 61-64; 1989. Stins M , Gilles F, K i m K . Selective expression of adhesion molecules on human brain microvascular endothelial cells. J. Neuroimmunol. 76: 81-90; 1997. Stuve O, Dooley N , H u m J, Antel J, Francis G , Williams G , Y o n g V . Interferon-P-lb decreases the migration of T-lymphocytes in vitro: effects on matrix metalloproteinase-9. Ann. Neurol. 40: 853-863; 1996. Sun J, Olsson T, Wang W , et al., Autoreactive T and B cells responding to myelin proteolipid protein in multiple sclerosis and controls. Eur. J. Immunol. 21: 1461-1468; 1991. Svenningsson A , Hansson G K , Andersen O, Andersson R, Patarroyo M , Stemme S. Adhesion molecule expression on cerebrospinal fluid T lymphocytes: evidence for common recruitment mechanisms in multiple sclerosis, asceptic meningitis, and normal controls. A n n . Neurol. 34: 155-161; 1993. Swanborg R. Experimental autoimmune encephalomyelitis i n rodents as a model for human demyelinating disease. C l i n . Immunol. Immunopathol. 77: 4-13; 1995. Takahashi K , Sawasaki Y , Hata J, M u k a i K , Goto T. Spontaneous transformation and immortalization of human endothelial cells. In Vitro Cell. Dev. B i o l . 25: 265-274; 1990. Takeichi M . Morophogenic roles of classic cadherins. Curr. Opin. Cell B i o l . 7: 619-627; 1995.  144  Tanaka Y , Adams D , Huscher S, Hirano H , Siebenlist U , Shaw S. T-cell adhesion induced by proteoglycan-immobolized cytokine MIP-1 beta. Nature. 361: 79-82; 1993. Tatewaki, M . , Yamaguchi, K . , Matsuoka, M . Ishii, T., Miyasaka, M . , M o r i , S., Takatsuki, K . and Watanbe, T. Constitutive overexpression o f the L-selectin gene in fresh leukemic cells o f adult T-cell leukemia that can be transactivated by human T-cell lymphotropic virus type 1 Tax. Blood. 86: 3109-3117; 1995. Taub D , C o n l o n K , L i o y d A , Oppenheim J, K e l v i n D . Preferential migration o f activated C D 4 + and C D 8 + T cells in response to MIP-1 alpha and MIP-1 beta. Science. 260: 355358;1993. Tedder T, Penta A , Levine B , Freedman A . Expression o f the human leukocytes adhesion molecule L A M - 1 : Identity with TQ1 and L e u 8 differentiation antigens. J. Immunol. 144: 532-540, 1990. Tedder T, Steeber D , Chen A , Engel P. The selectins: vascular adhesion molecules. F A S E B . 9: 866-873; 1995. Tender C , Greenberg S, Blattner W , Manns A , Murphy E , Fleisher T, Hanchard B , Morgan O, Burton J, Nelson D , Waldmann T. Transactivation o f interleukin 2 and its receptor induces immune activation in human T-cell lymphotropic virus type I-associated myelopathy: pathogenic implications and a rationale for immunotherapy. Proc. Natl. Acad. Sci. U . S . A . 87: 5218-5222; 1990. The IFNp Multiple Sclerosis Study Group and the U B C M S / M R I Analysis Group. Interferon B - l b in the treatment o f multiple sclerosis: final outcome o f the randomized controlled trial. Neurology. 45: 1277-1285; 1995. The IFNP Multiple Sclerosis Study Group: Interferon P-lb is effective in relapsingremitting multiple sclerosis. I. Clinical results. Neurology. 43: 655-661; 1993. Thornhill M . Wellicome S. Mahiouz D . Lanchbury J. Kyan-Aung U . Haskard D . Tumor necrosis factor combines with IL-4 or IFN-gamma to selectively enhance endothelial cell adhesiveness for T cells. The contribution o f vascular cell adhesion molecule-1dependent and -independent binding mechanisms. J. Immunol. 146: 592-598; 1991. Tonetti M , M i l l o E , Sturla L , Bisso A , De Flora A . Effects o f the murine L929 and L1210 cell lines on nitric oxide and T N F - a production by R A W 264.7 murine macrophages. Biochem. Biophy. Res. Commun. 230: 636-640; 1997. Traganos F. F l o w cytometry: Principle and applications. Cancer Invest. 2: 149-163; 1984. Traugott U , Reinherz E , Raine C . Multiple sclerosis: Distribution o f T cells, T cell subsets, and la-positive macrophages in lesions o f different ages. J. Neuroimmunol. 4: 201-221; 1983.  145  Tsujino A , Nakamura T, Furuya T, Goto H , Nishiura Y , Shirabe S, Nakane S, Motomura M , Nagataki S. Elevated serum levels o f soluble E - and L-selectin in patients with human T-cell lymphotropic virus type I-associated myelopathy. J. Neurol. Sci. 155: 76-79; 1998. Tsukada N , Matsuda M , M i y a g i K , Yanagisawa N . In vitro intercellular adhesion molecule-1 expression on brain endothelial cells in multiple sclerosis. J. Neuroimmnol. 49: 181-187; 1994 Tsukada N , Matsuda M , Miyagi K , Yanagisawa N . Adhesion o f cerebral endothelial cells to lymphocytes from patents with multiple sclerosis. Autoimmunity. 14: 329-333; 1993a. Tsukada N , M i y a g i K , Matsuda M , Yanagisawa N . Increased levels o f circulating intercellular adhesion molecule-1 in multiple sclerosis and human T-lymphotropic virus type I-associated myelopathy. A n n . Neurol. 33(6): 646-9; 1993b. Tuomanen E , Saukkonen K , Sande S, Cioffe C , Wright S. Reduction o f inflammation, tissue damage, and mortality in bacterial meningitis in rabbits treated with monoclonal antibodies against adhesion-promoting receptors o f leukocyte. J. E x p . M e d . 170: 959969; 1989. Uchiyama T, Nelson D , Fleisher T, Waldmann T. A monoclonal antibody (anti-Tac) reactive with activated and functionally mature human T cells. II. Expression o f Tac antigen on activated cytotoxic killer T cells, suppressor cells, and on one o f two types o f helper T cells. J. Immunol. 126: 1398-1403; 1981. Ulvestad E , Williams K , Wedeler C , Antel J, Nyland H , M o r k S, Matre R. Reactive microglia in multiple sclerosis lesions have an increased expression o f receptors for the Fc part o f IgG. J. Neurol. Sci. 121: 125-131; 1994. Umehara F , Izumo S, Takeya M , Takahashi K , Sato E , Osame M . Expression o f adhesion molecules and monocyte chemoattractant protein -1 ( M C P - 1 ) in the spinal cord lesions in HTLV-I-associated myelopathy. Acta Neuropathol. 91:343-350; 1996. Umehara F , Izumu S, Nakagawa M , Ronquillo A , Takahashi K , Matsumoro K , Sato E , Osame M . Immunocytochemical analysis of the cellular infiltrates in the spinal cord lesions in H T L V - I associated myelopathy. J. Neuropathol. E x p . Neurol. 52: 424-430; 1993. Utz U , McFarland H F . The role o f T cells in multiple sclerosis: implications for therapies targeting the T cell receptor. J. Neruopathol. E x p . Neurol. 53: 351-358; 1994. V a n K o o y k Y , Kemenade E , Weder P, V a n de Wiel-van Kemenad E , Huijbens R, Figdor C. Lymphocyte function-associated antigen dominates very late antigen 4 in binding o f activated T cells to endothelium. J. E x p . M e d . 177: 185-190; 1993.  146  V a r k i A . Selectin ligands: w i l l the real ones pleas stand up ? J. C l i n . Invest. 100: S31 S35; 1997. V o r a A , Perkin G , M c C o y T, Dumonde D , Brown K . Enhanced binding o f lymphocytes from patients with multiple sclerosis to tumor necrosis factor-alpha-treated endothelial monolayers: associations with clinical relapse and adhesion molecule. C l i n . E x p . Immunol. 105: 155-162; 1996. V o r a A , Perry M , Hobbs C , Dumonde D , Brown K . Selective binding o f peripheral blood lymphocytes to the walls o f cerebral vessels in frozen sections o f human brain. J. Immunol. Meth. 180: 165-180; 1995. Waksman B . Multiple Sclerosis. Curr. Opin. Immunol. 1: 733-739; 1989. Wang E , Lehner P, Graham S, Borysiewicz L . C D 8 (C57+) T cells in normal, healthy individuals specifically suppress the generation o f cytotoxic T cells to Epstein Barr virustransformed B cell lines. Eur. J. Immunol. 24: 2903-2909; 1994. Washington R, Burton J, Todd R, Newman W , Dragovic L , Dore-Duffy P. Expression o f immunologically relevant endothelial cell activation antigens on isolated central nervous system microvessels from patients with multiple sclerosis. A n n . Neurol. 35: 89-97; 1994. Watson C , Whittaker S, Smith N , Vora A , Dumonde D , Brown K . IL-6 acts on endothelial cells to preferentially increase their adherence for lymphocytes. C l i n . E x p . Immunol. 105: 112-119; 1996. Weiss R, Clapham P, Nagy K , Hoshino H . Envelope properties o f human T-cell-leukemia viruses. Curr. Topics Microbiol. Immunol. 115: 235-246; 1985. Wekerle H , Linington C , Lassmann H , Meyermann R. Cellular reactivity within the C N S . Trends Neurol. Sci. 4: 271-277; 1986. Weller R, Englehardt B , Phillips M . Lymphocyte targeting o f the central nervous system: a review o f afferent and efferent CNS-immune pathways. Brain Pathol. 6: 275-288; 1996. Welsh C , Rose J, H i l l K , Townsend J. Augmentation o f adoptively transferred experimental allergic encephalomyelitis by administration o f a monoclonal antibody specific for L F A - 1 . J. Neuroimmunol. 43: 161-167; 1993. Whitcup S, DeBarge R, Caspi R, Harning R, Nussenblatt R, Chan C . Monoclonal antibodies against I C A M - 1 (CD54) and L F A - 1 ( C D 11 a/CD 18) inhibit experimental autoimmune uveitis. C l i n . Immunol. Immunopathol. 67: 143-150; 1993. Wilkins J , Stupack D , Stewart S, Ciaxia S. (31 integrin-mediated lymphocyte adherence to extracellular matrix is enhanced by phorbol ester treatment. Eur. J. Immunol. 21: 517522; 1991.  147  Willenborg D , Prowse S. Immunoglobulin-deficient rats fail to develop experimental allergic enchephlomyelitis. J. Neuroimmunol. 5: 99-109; 1983. Wong D , Dorovini-Zis K . Expression o f vascular cell adhesion molecule-1 ( V C A M - 1 ) by human brain microvessel endothelial cells in primary culture. Microvasc. Res. 49: 325339; 1995. W o n g D , Dorovini-Zis K . Regulation by cytokines and lipopolysaccharide o f E-selectin expression by human brain microvessel endothelial cells in primary culture. J. Neuropathol. E x p . Neurol. 55: 225-235; 1996. Wong D , Prameya R, Dorovini-Zis K . J. Neuropathol. In vitro adhesion and migration o f T lymphocytes across monolayers o f human brain microvessel endothelial cells: regulation by I C A M - 1 , V C A M - 1 , E-selectin and P E C A M - 1 . E x p . Neurol. 58: 138-152; 1999. Woodroofe M , Cuzner M . Cytokine m R N A expression in inflammatory multiple sclerosis lesions: Detection by nonradioactive in situ hybridization. Cytokine. 5: 583-588; 1993. Wucherpfennig K , Strominger J. Molecular mimicry in T-cell mediated autoimmunity: viral peptides activate human T cell clones specific for myelin basic protein. C e l l . 80: 695-705; 1996. Yamamoto N , Hinuma Y . V i r a l aetiology o f adult T-cell leukemia. J. Gen. V i r o l . 66: 1641-1660; 1985. Yamamoto N , Okada M , Koyanagi Y , Kannagi Y , Hiunuma Y . Transformation o f human leukocytes by co-cultivating with an adult T-cell leukemia-virus produce cell line. Science. 217: 737-739; 1982. Yap A , Brieher W , Gumbiner B . Molecular and functional analysis o f cadherin-based adherens junctions. A n n . Rev. Cell. Dev. B i o l . 13: 119-146; 1997. Yasuda Y , Yoshitatsu S, Mitchel M , Sei Y , Yokoyama M , Tanaka K , Hara A . Healthy H T L V - I carriers in Japan: the haematological and immunological characteristics. B r . J. Haemat. 64: 195-203; 1986. Yednock T, Cannon C , Fritz L , Sanchez-Madrid F, Steinman L , Karin N . Prevention o f experimental autoimmune encephalomyelitis by antibodies against a4bl integrin. Nature. 356: 63-66; 1992. Y o n g W , Chabot S, Suve O, Williams G . Interferon beta in the treatment o f multiple sclerosis: mechanisms o f action. Neurology. 51: 682-689; 1998.  148  Yoshida M , Osame M , K a w i H . Increased replication of H T L V - 1 in HTLV-1-associated myelopathy. Ann. Neurol. 26: 331-335; 1989. Y u C , Haskard D , Cavender D , Johnson, Z i f f M . Human gamma interferon increases the binding o f T lymphocytes to endothelial cells. C l i n . Exp. Immunol. 62: 554; 1985. Y u M , Nishiyama A , Trapp B , Tuohy V . Interferon-B inhibits progression o f relapsingremitting experimental autoimmune enchephaloyelitis. J. Neuroimmunol. 64: 91-100; 1996. Y u C , Haskard D , Cavender D , Johnson A , Z i f f M . Human gamma interferon increases the binding o f T lymphocytes to endothelial cells. C l i n . E x p . Immunol. 62: 554-560; 1985. Zeman A , K i d d D , M c L e a n B , Kelly M , Francis D , M i l l e r D , Kendall B , Rudge P, Thompson E , M c D o n a l d W . A study o f oligoclonal band negative multiple sclerosis. J. Neurol. Neurosurg. Psychiatry. 60: 27-30; 1996. Zhang J, Maarkovic S, Raus J, Weiner H , Hafler D . Increased frequency o f IL-2 responsive T cells specific for myelin basic protein and proteolipid protein in peripheral blood and cerebrospinal fluid o f patients with multiple sclerosis. J. E x p . M e d . 179: 973984; 1994.  149  APPENDIX A  A. 1  T H E EFFECTS 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  Lymphocyte subsets in a R R - M S patient participating in Schering-Plough® trial A . l .2 Lymphocyte subsets in R R - M S patients participating in I C O S trial  151  A . 1.1  150  151 151  A.l  T H E EFFECTS OF I M M U N O M O D U L A T O R Y DRUGS O N L Y M P H O C Y T E PHENOTYPE IN MS  A.l.l  Lymphocyte  subsets in a RR-MS  patient participating  in  Schering-Plough®  trial Table A . l shows lymphocyte subsets o f a R R - M S patient who participated in Schering-Plough® trial at pretreatment, and at 2 and 7 days post therapy. The nature o f 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 o f the highlighted change in lymphocyte subsets that occurred either at 2 and/or 7 days post-treatment. Major reduction is seen in the percentage o f C D 19+ (B cells), C D 4 + C D 2 9 + (helper induced), C D 3 + C D 2 6 (activated T cells), C D 4 + C D 6 2 L + (selectin expression on CD4+), C D 8 + C D 6 2 L + (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 o f 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 o f rhuanti-LFA-1 on the clinical course o f R R - M S is now unblinded, therefore, the nature o f treatment is known.  151  Table A . 2 shows lymphocyte subsets in a R R - M S patient who received a single injection o f 2 mg/kg of recombinant and humanized anti-LFA-1 m A b . Some o f 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+,  C D 4 + C D 6 2 L + , and C D 8 + C D 6 2 L + 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 m A b . After a single injection o f 1 mg/kg o f anti-LFA-1, there was a major reduction in the percentage o f 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 i n her percentage of CD3+CD26+, or C D 4 + C D 6 2 L + and C D 8 + C D 6 2 L + cells after the treatment. Table A . 4 shows the lymphocyte subsets o f a R R - M S patient in I C O S 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 o f CD4+CD49d+ and CD8+CD49+  after  placebo  treatment.  The  percentage  of  CD4+CD62L+  and  C D 8 + C D 6 2 L + cells were also moderately increased. Moreover, the percentage o f CD3+CD26+ cells remained unchanged. Table A . 5 shows the lymphocyte subsets o f a R R - M S patient i n I C O S Corp. trial that received intravenous methylprenisolone. Similar to the patient who received placebo (Table A.4), the percentages o f C D 4 + C D 6 2 L + and C D 8 + C D 6 2 L + cells were moderately increased while the percentage  o f CD3+CD26+ remained almost unchanged  treatment. The percentage of CD4+CD49d+ was also unchanged.  152  after  Table A . l Lymphocyte Subsets of a RR-MS Patient Enrolled in Schering Plough Trial Subsets  pretreatment  CD3+ C D 19+ CD16+CD56+ CD3+CD4+ CD3+CD8+ CD4+CD45RA+ CD4+CD29+ CD8+CD28+ CD8+CD57+ CD3+CD27CD4+HLA-DR+ CD8+HLA-DR+ CD4+CD25+ CD8+CD25+ CD4+CD38+ CD8+CD38+ CD3+CD26+ CD3+CD30+ CD3+CD69+ CD4+CD49d+ CD8+CD49d+ CD4+CD62L+ CD8+CD62L+ CD8+CDllb+ CD3+CD54+  76.4 16.7 4.6 55.3 19.4 48.6 39.6 4.2 24.6 86.4 2.4 3.7 0.6 0 6.0 2.8 40.8 0.1 0.5 20.4 42.4 86.4 57.5 14.3 2.4  2 days Post treatment  7 days Post treatment  83.0  72.3 18.8 6.0 50 19.4 46.2 28.2 0.4 32.8 82.8 1.7 2.2 0.4 0.1 3.7 2.4 55.2 0 1  5.0 i 5.0 63.4 18.1 51.5 46.7  2.8 i 42.9| 82.3 2.8 4.3 0.8 0  10.3|  5.0T 17.0^ 0 0 23.5 54.6 90.1 44.1 14.5 1.1  10.8| 31.4  58.4| 29.4| 10.8 3.1  Note. CD3+, C D 19+, CD16+CD56+, CD3+CD4+, and C D 3 + C D 8 + lymphocyte subsets are given as percentage of the total number o f lymphocyte (CD45 " C D 1 4 - ) . Other lymphocyte subsets were calculated as the proportion o f 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 pretreatment. sht  153  Table A . 2 L y m p h o c y t e Subsets of a R R - M S Patient ( D E S #0801) E n r o l l e d i n 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 ( H u 2 3 F 2 G ) Subsets  Pre-treatment  5 days post-treatment  CD3+ CD19+ CD16+CD56+ CD3+CD4+ CD3+CD8+ CD4+CD45RA+ CD4+CD29+ CD8+CD28+ CD8+CD57+ CD3+CD27CD4+HLA-DR+ CD8+HLA-DR+ CD4+CD25+ CD8+CD25+ CD4+CD38+ CD8+CD38+ CD3+CD26+ CD3+CD30+ CD3+CD69+ CD4+CD49d+ CD8+CD49d+ CD4+CD62L+ CD8+CD62L+ CD8+CDllb+ CD3+CD54+  74.8 8.9 8.3 56.3 15.7 53.2 39 11.9 18.8 7.1 5 7.3 3.2 0.1 17 19.4 71.1 0.4 0.6 15.1 34.1 84.1 54.1 25.8 3.6  73.6 5.81 11.3? 51.5 15.9 34.81 24.51 3.81 14.4 3.51 2.71 4.2 J, 1.71 0 5.4J, 14.4 29.41 0 1.5 5.41 121 42.81 31.51 30.9 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 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 pretreatment. n g h t  154  Table A . 3 L y m p h o c y t e Subsets of a R R - M S Patient ( M C E #0802) E n r o l l e d 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 ( H u 2 3 F 2 G ) Subsets  Pre-treatment  CD3+ CD19+ CD16+CD56+ CD3+CD4+ CD3+CD8+ CD4+CD45RA+ CD4+CD29+ CD8+CD28+ CD8+CD57+ CD3+CD27CD4+HLA-DR+ CD8+HLA-DR+ CD4+CD25+ CD8+CD25+ CD4+CD38+ CD8+CD38+ CD3+CD26+ CD3+CD30+ CD3+CD69+ CD4+CD49d+ CD8+CD49d+ CD4+CD62L+ CD8+CD62L+ CD8+CDllb+ CD3+CD54+  79 13.2 4.5 49.8 27.8 23.6 39.7 6.4 12.8 5.4 5.6 9.2 2.5 0.1 19.2 10.3 49.6 0.2 0.9 25.8 26.1 65.9 50.6 13.9 1.8  5 days post-treatment 78.6 10.9 8.2T 49.7 26.8 57.7f 40.5 12.9| 15.6 4.3 3.4| 61 2.6 0.2 14.3 14.3 45.3 0.2 1.1 ll.lj, 13| 64.2 49.3 20.9T ll.lt  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 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 pretreatment. ght  155  Table A.4 L y m p h o c y t e Subsets of a R R - M S Patient ( M O S #0803) E n r o l l e d i n I C O S T r i a l W h o Received Placebo Subsets  Pre-treatment  5 days post-treatment  CD3+ C D 19+ CD16+CD56+ CD3+CD4+ CD3+CD8+ CD4+CD45RA+ CD4+CD29+ CD8+CD28+ CD8+CD57+ CD3+CD27CD4+HLA-DR+ CD8+HLA-DR+ CD4+CD25+ CD8+CD25+ CD4+CD38+ CD8+CD38+ CD3+CD26+ CD3+CD30+ CD3+CD69+ CD4+CD49d+ CD8+CD49d+ CD4+CD62L+ CD8+CD62L+ CD8+CDllb+ CD3+CD54+  78.4 4.9 7 37.4 33 53.3 44.8 5.4 23.9 27.8 4.1 3.1 1.4 0.2 24.8 12.7 35.9 0.1 1.5 18.3 27.5 75.2 44.1 22.7 1.3  75.4  8.4| 14.9T 32.7 33.9 62.2 57.9 nd 29.4 24.5 3.2 2.9  3.3f 0.1  47.2| 21| 35.9 0.1 0.7 29.1  59.9f 84.6 56.9 29.5 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 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 pretreatment. n g h t  156  Table A.5 L y m p h o c y t e Subsets of a R R - M S Patient ( T E S #0804) E n r o l l e d i n I C O S T r i a l W h o Received Intravenous Methylprednisolone Subsets  Pre-treatment  5 days post-treatment  CD3+ C D 19+ CD16+CD56+ CD3+CD4+ CD3+CD8+ CD4+CD45RA+ CD4+CD29+ CD8+CD28+ CD8+CD57+ CD3+CD27CD4+HLA-DR+ CD8+HLA-DR+ CD4+CD25+ CD8+CD25+ CD4+CD38+ CD8+CD38+ CD3+CD26+ CD3+CD30+ CD3+CD69+ CD4+CD49d+ CD8+CD49d+ CD4+CD62L+ CD8+CD62L+ CD8+CDllb+ CD3+CD54+  79.1 14.6 3.7 56 21.2 24.8 36 6.5 10.8 7.3 1.7 5.8 1.1 0.2 8.9 8.1 57.2 0.1 0.5 14.5 nd 77.8 59.7 16.8 3.1  76.1  20.lt 3.8 59.2 17.4  50t 36.4  3.31 10.1  3.9t 1.3 5.4 0.1 0.3 7.7 10.4 62.3 0 1 12 33 90.7 74.7 12.2 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 ' 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. t and I indicate major (>30%) increase and decrease, respectively, versus pretreatment r  157  8ht  

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