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Regulation of immunoproteasome expression by human brain endothelium : functional significance in MHC… Lau, Henry H. W. 2005

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REGULATION OF IMMUNOPROTEASOME EXPRESSION BY HUMAN B R A I N E N D O T H E L I U M : F U N C T I O N A L S I G N I F I C A N C E IN M H C C L A S S I E X P R E S S I O N A N D C D 8 + T C E L L A D H E S I O N TO THE C E R E B R A L ENDOTHELIUM by H E N R Y H.W. L A U  B.Sc. (Medical Laboratory  Science), The University  of British Columbia,  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 L M E N T THE REQUIREMENTS FOR THE D E G R E E OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Pathology and Laboratory Medicine)  T H E UNIVERSITY O F BRITISH C O L U M B I A June 2005  © Henry Hin Wai Lau, 2005  O F  2001  ABSTRACT Brain inflammatory d i s e a s e s are characterized by the entry of blood-borne leukocytes into the brain a c r o s s the blood-brain barrier ( B B B ) that normally restricts their entry into the  central  nervous  system.  Recent  studies  indicate  that  infiltrating  brain  antigen-specific C D 8 + T cells clonally e x p a n d in multiple s c l e r o s i s (MS) lesions. Previous studies from this laboratory s h o w e d that cytokines induce human  brain  m i c r o v e s s e l endothelial cells ( H B M E C ) to e x p r e s s or upregulate s e v e r a l endothelial cell a d h e s i o n m o l e c u l e s ( I C A M - 1 , V C A M - 1 , P E C A M - 1 a n d E-selectin), which mediate leukocyte a d h e s i o n and transendothelial migration.  In addition, cytokine stimulated  H B M E C are induced to e x p r e s s c l a s s II M H C and co-stimulatory m o l e c u l e s and provide s e c o n d a r y s i g n a l s for T cell proliferation.  In the present study, w e investigated the role  of the p r o t e a s o m e in the regulation of M H C c l a s s I e x p r e s s i o n by cerebral m i c r o v e s s e l endothelial cells and C D 8 + T cell a d h e s i o n to the B B B endothelium.  Primary cultures  of H B M E C w e r e treated with: 1) IFN-y to i n c r e a s e the cellular population of the 2 0 S i m m u n o p r o t e a s o m e , a protease that generates M H C c l a s s I specific peptides and 2) T N F - a to upregulate a d h e s i o n m o l e c u l e s . Interferon-Y induced upregulation of the i m m u n o p r o t e a s o m e subunits (31 i and (35i at the m R N A a n d the protein levels.  The  constitutive e x p r e s s i o n of c l a s s I M H C by H B M E C w a s upregulated after treatment with IFN-Y for 4 8 h .  Treatment with the p r o t e a s o m e inhibitor lactacystin downregulated the  IFN-y-induced c l a s s I upregulation.  T h e minimal a d h e s i o n of C D 8 + T cells to H B M E C  w a s significantly upregulated by IFN-y treatment of H B M E C and downregulated by both lactacystin and M H C c l a s s I blocking antibodies.  In addition, both the e x p r e s s i o n of  I C A M - 1 , V C A M - 1 and E-selectin and C D 8 + T cell a d h e s i o n to H B M E C w e r e upregulated by T N F - a and downregulated by co-incubation with lactacystin.  T h e s e studies indicate  that upregulation of the inducible p r o t e a s o m e subunits in H B M E C by proinflammatory cytokines l e a d s to upregulation of M H C c l a s s I e x p r e s s i o n a n d antigen non-specific a d h e s i o n of C D 8 + T cells to cerebral endothelium.  A d h e s i o n of C D 8 + T cells is  mediated by a d h e s i o n m o l e c u l e s e x p r e s s e d by H B M E C , which are a l s o downregulated following p r o t e a s o m e inhibition.  W e therefore s u g g e s t that the p r o t e a s o m e plays an  important role in the regulation of C D 8 + T cell a d h e s i o n to the blood-brain barrier endothelium during brain inflammatory d i s e a s e s .  ii  TABLE OF CONTENTS  Abstract  ii  Table of C o n t e n t s  iii  List of Tables  vi  List of Figures  vi  List of Abbreviations  vii  Acknowledgements  ix  C H A P T E R 1: INTRODUCTION 1.1  The immune system and Inflammation  1  1.2  The Central Nervous System (CNS)  2  1.2.1  T h e B l o o d Brain Barrier ( B B B )  2  1.2.2  Inflammation in the C N S  4  1.2.3  C y t o k i n e s in C N S Inflammation  5  1.2.3.1  T u m o u r N e c r o s i s Factor (TNF)-a  5  1.2.3.2  Interferon (IFN)-y  6  1.2.4  1.2.5  1.2.6 1.3  C N S C e l l s Participating in C N S Inflammation  7  1.2.4.1  Astrocytes  7  1.2.4.2  Microglia  7  1.2.4.3  Perivascular Macrophages/Dendritic Cells  8  L y m p h o c y t e Trafficking in the C N S  9  1.2.5.1  T Lymphocytes  9  1.2.5.2  C D 8 + T Lymphocytes  11  1.2.5.3  Cell Adhesion Molecules  11  1.2.5.4  Immunological S y n a p s e  12  1.2.5.5  A n t i g e n Recognition and T C e l l Transmigration  13  I m m u n o p a t h o g e n e s i s of C N S A u t o i m m u n e Disorders  14  MHC class I antigen processing and presentation  16  1.3.1  Structure of M H C c l a s s I m o l e c u l e s  16  1.3.2  M H C c l a s s I antigen presentation  17  1.3.2.1  The Classical Pathway  17  1.3.2.2  T h e Cross-presentation P a t h w a y  19  iii  1.4  1.5  1.6  The Proteasome and Immunoproteasome  20  1.4.1  T h e Ubiquitin/Proteasome S y s t e m  20  1.4.2  Structure  21  1.4.3  T h e Immunoproteasome  22  1.4.4  P r o t e a s o m e Inhibitors  23  Human Brain Microvessel Endothelial Cells (HBMEC)  24  1.5.1  Endothelial C e l l Heterogeneity  24  1.5.2  R o l e of Endothelial C e l l s in Inflammation  25  1.5.3  In vitro M o d e l of the H u m a n B l o o d - B r a i n Barrier  Objective and Specific Aims  26  1.6.1  Hypothesis  26  1.6.2  Specific A i m s  28  C H A P T E R 2: MATERIALS A N D 2.1  25  METHODS  Endothelial Cell Cultures  29  2.1.1  Isolation of H u m a n Brain M i c r o v e s s e l Endothelial C e l l s ( H B M E C )  29  2.1.2  Isolation of H u m a n Umbilical V e i n Endothelial C e l l s ( H U V E C )  29  2.1.3  Culture C o n d i t i o n s  30  2.2  Cytokine and Proteasome Inhibitor Treatments  30  2.3  Antibodies  31  2.4 Semi-quantitative Reverse Transcription P C R (RT-PCR)  32  2.5  Cell Viability A s s a y s  33  2.5.1  M T T (Thiazolyl B l u e Tetrazolium Bromide) Cytotoxicity A s s a y  33  2.5.2  L I V E / D E A D ® C e l l - M e d i a t e d Cytotoxicity A s s a y  34  2.6  Immunocytochemistry 2.6.1  34  Immunogold Silver Staining ( I G S S ) for S u r f a c e Localization of M H C c l a s s I and E C a d h e s i o n m o l e c u l e s  2.6.2  Immunogold Silver Staining ( I G S S ) for Intracellular Localization of Proteasome Subunits  2.7  34 35  Enzyme-Linked Immunosorbent A s s a y (ELISA)  35  2.7.1  Surface Expression  35  2.7.2  Intracellular E x p r e s s i o n  36  2.8  Isolation and Characterization of CD8+ T Lymphocytes  36  2.9  CD8+ T Lymphocyte Adhesion to H B M E C Monolayers  37  2.10Data Collection and Statistics  38  Chapter 3: R E S U L T S 3.1  Human Brain Microvessel Endothelial Cells (HBMEC) iv  38  3.2  H u m a n Umbilical Vein Endothelial Cells ( H U V E C )  39  3.3  P r o t e a s o m e Subunit Expression by H B M E C  39  3.3.1  R N A E x p r e s s i o n Detected by Semi-quantitative R T - P C R  39  3.3.2  Intracellular Protein E x p r e s s i o n by H B M E C  40  3.4  Effect of Lactacystin on Cell Viability  41  3.4.1  M T T Cytotoxicity A s s a y  41  3.4.2  L I V E / D E A D ® C e l l - M e d i a t e d Cytotoxicity A s s a y  42  3.5  Surface Expression of M H C C l a s s IMolecules by H B M E C  42  3.6  Surface E x p r e s s i o n of A d h e s i o n Molecules by H B M E C  43  3.7  C D 8 + T L y m p h o c y t e A d h e s i o n to H B M E C  44  C H A P T E R 4: D I S S C U S S I O N  in vitro M o d e l  4.1  H B M E C as an  of the B B B  4.2  Expression of P r o t e a s o m e Subunits by H B M E C  45  4.3  M H C C l a s s IMolecule Expression  49  4.4  A d h e s i o n M o l e c u l e E x p r e s s i o n at the B B B  51  4.5  C D 8 + T L y m p h o c y t e A d h e s i o n  53  44  C H A P T E R 5: C O N C L U S I O N S 5.1  S u m m a r y and Significance  55  5.2  Future Directions  58  R E F E R E N C E S  60  V  LIST O F T A B L E S Table 1  P C R Primer Sequences  33  LIST O F F I G U R E S Figure 1  Primary Cultures of H u m a n Brain M i c r o v e s s e l Endothelial C e l l s  73  Figure 2 a  R N A E x p r e s s i o n of P r o t e a s o m e Subunits by H B M E C  74  Figure 2b  Semi-quantitative A s s e s s m e n t of P r o t e s o m e Subunit E x p r e s s i o n by H B M E C by P C R  75  Figure 2 c  R N A E x p r e s s i o n of P r o t e a s o m e Subunits by H U V E C  76  Figure 2d  Semi-quantitative A s s e s s m e n t of P r o t e s o m e Subunit E x p r e s s i o n by H U V E C by P C R  77  Figure 3 a  Intracellular Localization of P r o t e a s o m e Subunits in H B M E C  78  Figure 3b  Intracellular Localization of P r o t e a s o m e Subunits in H B M E C  79  Figure 4 a  M T T Viability A s s a y Performed on H B M E C by M T T  80  Figure 4b  L I V E / D E A D ® C e l l - M e d i a t e d Cytotoxicity Kit P e r f o r m e d on H B M E C  81  Figure 5  E x p r e s s i o n of M H C c l a s s I by H B M E C following IFN-y Stimulation  82  Figure 6 a  A d h e s i o n M o l e c u l e s E x p r e s s i o n by H B M E C following T N F - a Stimulation  83  Figure 6b  S u r f a c e Localization of A d h e s i o n M o l e c u l e s on H B M E C  84  Figure 7 a  Quantitation of C D 8 + T Lymphocyte A d h e s i o n to H B M E C  85  Figure 7b  Quantitation of C D 8 + T Lymphocyte A d h e s i o n to H B M E C  86  vi  ABBREVIATIONS a.a.  a m i n o acid(s)  Ab  antibody  ANOVA  a n a l y s i s of v a r i a n c e  APC  antigen presenting cell  B m  beta 2 microglobulin  BBB  blood-brain barrier  CNS  central nervous s y s t e m  CSF  c e r e b r o s p i n a l fluid  CTL  cytotoxic T lymphocyte  DC  dendritic cell(s)  EAE  experimental allergic encephalomyelitis  EC  endothelial cell(s)  ER  e n d o p l a s m i c reticulum  ERAP1  e n d o p l a s m i c reticulum a m i n o p e p t i d a s e 1  FITC  fluorescein Isothiocyanate  HBMEC  h u m a n brain m i c r o v e s s e l endothelial cell(s)  HIV-1  h u m a n immunodeficiency virus type 1  HLA  h u m a n leukocyte antigen  HRP  h o r s e r a d i s h peroxidase  HUVEC  h u m a n umbilical vein endothelial cell(s)  ICAM-1  intercellular a d h e s i o n m o l e c u l e - 1  IFN-B  Interferon beta  IFN-y  Interferon g a m m a  IGSS  i m m u n o g o l d silver staining  2  vii  IS  immunological s y n a p s e  LC  lactacystin  LCA  leukocyte c o m m o n antigen  LFA-1  leukocyte function-associated antigen - 1  LPS  lipopolysaccharide  MAF  m a c r o p h a g e activating factor  MBP  myelin b a s i c protein  MHC  major histocompatibility c o m p l e x  MS  multiple s c l e r o s i s  MTT  T h i a z o l y l Blue Tetrazolium B r o m i d e  NK  natural killer (cells)  NOD  n o n - o b e s e diabetic  PBMC  peripheral blood m o n o n u c l e a r cells  PE  R-Phycoerythrin  PerCP  Peridinin-chlorophyll-protein  RT-PCR  reverse-transcriptase p o l y m e r a s e chain reaction  SP  single positive  TAP  transporters a s s o c i a t e d with antigen presentation  TCR  T cell receptor(s)  TNF-a  tumor n e c r o s i s factor alpha  VCAM-1  v a s c u l a r cell a d h e s i o n molecule - 1  VLA  very late activation antigen  viii  ACKNOWLEDGEMENTS  T h e r e are a n u m b e r of p e o p l e I would like to thank during the journey of my graduate study.  F o r t h o s e w h o h a v e supported m e during all t h e s e y e a r s and t h o s e w h o provide  invaluable l e s s o n s a n d w o r d s will all a l w a y s be r e m e m b e r e d for the rest of my life.  To  my committee m e m b e r s : Drs. Walker, Mui and M o o r e - d e e p appreciation for all your help. friend!  To K e n - your k n o w l e d g e w a s always the best help a n d thanks for being a great R u k m i n i - y o u s h a p e d most of my practical training e x p e r i e n c e ; To R e z a : for all  your technical s u g g e s t i o n s a n d i d e a s ; A n d last, but not least, Dr.Zis - your g u i d a n c e o p e n e d my e y e s on not only how to a c h i e v e to be s u c c e s s f u l a s a scientist but also how to be a more careful a n d thorough student.  May 2005  ix  C H A P T E R 1: I N T R O D U C T I O N  1.1  THE IMMUNE SYSTEM AND INFLAMMATION T h e i m m u n e s y s t e m provides d e f e n c e against infections a n d thus contributes to the  survival of the s p e c i e s .  P a t h o g e n s s u c h a s v i r u s e s , bacteria a s well a s toxins are  handled by the i m m u n e s y s t e m .  T h e r e are two a r m s of the i m m u n e s y s t e m : 1) the  innate immunity a n d 2) the adaptive immunity.  Innate immunity constitutes the first  d e f e n c e against p a t h o g e n s a n d consists of c o m p l e x reactions initiated by local cells and proteins attempting to prevent any further d a m a g e , known a s inflammation. C o m m o n constituents on bacterial s u r f a c e s activate local cells s u c h a s m a c r o p h a g e s , which b e c o m e activated a n d secrete cytokines, a diverse group of proteins that play an important role in the initiation a n d regulation of the i m m u n e r e s p o n s e .  A l o n g with other  secreted mediators, t h e s e cytokines i n c r e a s e the permeability of blood v e s s e l s and the e x p r e s s i o n of a d h e s i o n m o l e c u l e s on endothelial cells ( E C ) thus  leading to  recruitment of circulating leukocytes to the site of the i m m u n e r e s p o n s e . has b e e n traditionally classified a s acute or chronic.  the  Inflammation  A c u t e inflammation is a n early  r e s p o n s e to a stimulus, s u c h a s bacterial or viral infections, a n d is characterized by: 1) Vasodilation a n d i n c r e a s e d v a s c u l a r wall permeability. 2) Extravasation of p l a s m a protein a n d p o l y m o r p h o n u c l e a r leukocytes into the surrounding t i s s u e s .  Chronic  inflammation is a m u c h more specific r e s p o n s e that follows the acute r e s p o n s e . It is d e p e n d e n t upon antigen presentation and recognition, cell to cell interactions, and is a s s o c i a t e d with prolonged i n c r e a s e d v a s c u l a r permeability a n d infiltration of chronic inflammatory cells (lymphocytes, m a c r o p h a g e s , a n d p l a s m a cells).  Inflammation is part  of a physiological p r o c e s s initiated by innate immunity intended to control pathogen d a m a g e a n d repair, however, it c a n c a u s e s e v e r e t i s s u e d a m a g e w h e n this p r o c e s s l  persists.  T h u s , the body c a n eliminate persistent p a t h o g e n s or p a t h o g e n s frequently  encountered by the host through the adaptive i m m u n e s y s t e m that activates humoral (antibody mediated) a n d /or cell-mediated (B a n d T lymphocyte) immunity.  T h e body  initiates this reaction during the first e x p o s u r e to a p a t h o g e n a n d eventually reacts more efficiently in s u b s e q u e n t encounters.  T h u s , the body is protected by both innate a n d  acquired immunity [1].  T H E C E N T R A L N E R V O U S S Y S T E M  1.2 1.2.1  The Blood-Brain Barrier The  (CNS)  (BBB)  B B B is a n important biological barrier, the a n a t o m i c a l b a s i s of which is the  endothelium that lines the brain m i c r o v e s s e l s .  T h i s barrier s e r v e s to protect the brain  from toxic s u b s t a n c e s whilst at the s a m e time allowing a c c e s s to e s s e n t i a l nutrients a n d chemical signals. 1885  T h e G e r m a n bacteriologist P a u l Ehrlich w a s the first to o b s e r v e in  that following injection of d y e s into the s y s t e m i c circulation, all o r g a n s w e r e stained  except the brain.  H e postulated that the brain had a lower affinity for d y e s .  His later  studies in 1909 a n d 1913 with his student, E d w i n E. G o l d m a n n , s h o w e d that direct injection of trypan blue into the brain stained only brain t i s s u e but not other peripheral organs.  This indicated the p r e s e n c e of a barrier b e t w e e n the brain a n d the blood.  In  the 1960's, R e e s e a n d K a r n o v s k y performed ultrastructural studies using e x o g e n o u s horseradish p e r o x i d a s e a n d s h o w e d conclusively that the cerebral E C in adult mice w e r e the anatomical b a s i s of the B B B [2]. A better understanding of the B B B has b e e n a c h i e v e d over the y e a r s . T h e primary role of the B B B is to maintain the C N S in a chemically b a l a n c e d environment in order to allow optimal neuronal activity.  A n o t h e r important function of the B B B is to regulate the  p a s s a g e of h a e m a t o g e n o u s cells from blood into the brain. 2  T h e B B B is present  throughout the C N S with the exception of the choroid plexus, pituitary, a r e a postrema, pineal g l a n d , a n d hypothalamus. through  a  fenestrated  h o m e o s t a s i s [3].  S o l u t e s a n d h o r m o n e s c a n freely m o v e in and out  endothelium  in  these  areas,  thus  maintaining  peripheral  E C lining the cerebral blood v e s s e l s h a v e two unique morphological  features that e n a b l e t h e m to regulate the m o v e m e n t of s u b s t a n c e s into and out of the brain: 1) p r e s e n c e of tight junctions between adjacent cells that form a barrier to the paracellular m o v e m e n t of ions, proteins and leukocytes from blood into the brain.  2)  Paucity of c y t o p l a s m i c v e s i c l e s that restrict s u b s t a n c e s from entering the brain via vesicular transport a c r o s s the cell [3]. transmembrane  (junctional  T h e tight junctions are an intricate c o m p l e x of  a d h e s i o n m o l e c u l e - 1 , platelet endothelial cell a d h e s i o n  molecule-1, o c c l u d i n , a n d claudins) a n d c y t o p l a s m i c (zonula occludens-1 and - 2 , cingulin, A F - 6 , a n d 7 H 6 ) proteins linked to the actin cytoskeleton.  T h e e x p r e s s i o n and  subcellular localization of tight junction proteins are m o d u l a t e d by s e v e r a l intrinsic signaling pathways, including those involving c a l c i u m , phosphorylation, a n d G-proteins. Tight junctions between cerebral E C limit the interendothelial s p a c e to 12 angstroms, a size that is far s m a l l e r than most proteins.  L o c a t e d along the lateral endothelial p l a s m a  m e m b r a n e , t h e s e junctions prevent m a c r o m o l e c u l e s from diffusing paracellularlly. property is referred to a s the gate function.  This  A n o t h e r function of the tight junctions is the  f e n c e function which is the separation of proteins a n d lipids at the a p i c a l d o m a i n from the basolateral d o m a i n of the p l a s m a m e m b r a n e .  Both properties of tight junctions  constitute the m o l e c u l a r b a s i s of "tightness" of the junctions that isolates the apical from the basolateral d o m a i n s [4].  P r e v i o u s studies on frog a n d rat brains demonstrated that  cerebral m i c r o v e s s e l s h a v e a transendothelial electrical r e s i s t a n c e in the order of 1000 2 0 0 0 Q . c m , a tightness similar to that of tight epithelial barriers. T h e resistance is m u c h 2  higher than that of peripheral endothelium, which is approximately 10 Q . c m 3  2  [5].  In  addition to its barrier function, the B B B through specific e n z y m e s , transporters a n d receptors o n the E C m e m b r a n e s , allows e s s e n t i a l nutrients, electrolytes s u c h a s g l u c o s e , amino a c i d s (a.a.) a n d ions to enter the brain [6].  In addition,  P-glycoprotein a n d  multidrug resistance proteins prevent drugs from c r o s s i n g the endothelial barrier [7]. P o t a s s i u m ions a n d glycine a r e also active-transported b a c k to the brain by the transporter proteins [6].  1.2.2 Inflammation in the C N S Brain  inflammation  is the culmination  of  numerous  processes.  Similar to  inflammatory  r e s p o n s e s in peripheral t i s s u e s , cell types that participate  inflammation  include neutrophils, m a c r o p h a g e s , mast cells, lymphocytes, platelets,  dendritic cells a n d E C .  in C N S  C h e m o t a x i s a p p e a r s to be a n important event in the recruitment  of leukocytes to sites of inflammation.  T h e p r e s e n c e of c h e m o t a c t i c factors a n d  e x p r e s s i o n of c h e m o t a c t i c receptors on leukocytes a s well a s the e x p r e s s i o n of a d h e s i o n m o l e c u l e s on v a s c u l a r endothelium a n d their ligands o n leukocytes lead to leukocyte a d h e s i o n a n d migration a c r o s s the endothelium a n d a c c u m u l a t i o n in the inflammatory site.  In addition  to the chemotactic factors  and adhesion molecules,  cytokine  stimulation a n d antigenic interaction between leukocytes a n d endothelium contribute to inflammatory cell activation a n d specific elimination of p a t h o g e n s respectively. For a long time, it w a s believed that the C N S w a s a n immunologically privileged site, d u e to the p r e s e n c e of the B B B .  Transplanted tissue in the C N S is not rejected, unlike  transplants in peripheral o r g a n s .  Later studies s h o w e d that rejection d o e s indeed o c c u r  in the C N S , albeit at a m u c h slower rate [8]. Anatomically, a c l a s s i c a l lymphatic s y s t e m d o e s not exist in the C N S .  Despite this,  it a p p e a r s that the m o v e m e n t of cerebrospinal fluid ( C S F ) a n d brain extracellular fluid  allows for d r a i n a g e of p o s s i b l e antigenic material into identifiable lymphatics, which are found under the cribriform plate and along cranial a n d spinal nerve roots [9-11] and into the cervical lymph n o d e s . i m m u n e surveillance.  Lymphocyte traffic a l s o a p p e a r s to h a v e s o m e role in  T h e data suggest that activated T a n d B cells have the ability to  enter the brain in a more or less random m a n n e r [12].  Furthermore, there is e v i d e n c e  suggesting that perivascular cells a r e fully c a p a b l e of presenting C N S antigens to T lymphocytes [13, 14]. T h e a b o v e indicate that immune surveillance d o e s indeed occur, albeit at a slower a n d more limited rate than at the periphery.  1.2.3 C y t o k i n e s in C N S I n f l a m m a t i o n S e v e r a l cytokines h a v e b e e n implicated in C N S inflammation d u e to their ability to mediate i m m u n e r e s p o n s e s .  Activated infiltrating l y m p h o c y t e s a s well a s glial cells,  both e x p r e s s a n d r e s p o n d to cytokines for intercellular c o m m u n i c a t i o n p u r p o s e s under inflammatory conditions.  T h e r e is e v i d e n c e s u g g e s t i n g that peripheral cytokines m a y  activate C N S cells e v e n in the p r e s e n c e of the blood-brain barrier [15-17]. an and  Brain E C are  attractive target in studies investigating interactions b e t w e e n infiltrating leukocytes the C N S [18].  1.2.3.1 Tumor Necrosis Factor (TNF)-a T N F - a is a type II t r a n s m e m b r a n e protein with 1 5 7 a . a . r e s i d u e s that exists in soluble form a s homotrimer.  Its e x i s t e n c e w a s first s u s p e c t e d more than a century a g o  by the G e r m a n p h y s i c i a n Dr. P. B r u n e s w h o o b s e r v e d r e g r e s s i o n of tumors in his patients following a c u t e bacterial infections [19].  Later s t u d i e s confirmed its existence  by showing that endotoxin isolated from gram-negative bacteria led to tumor regression after injection into tumor-bearing mice.  T N F - a is mainly p r o d u c e d by m o n o c y t e s a n d 5  m a c r o p h a g e s , but T cells, natural killer (NK) cells, Kupffer cells, smooth m u s c l e cells and epidermal cells c a n also produce this cytokine.  Sensitive inducers of T N F - a include  calcium ionophores, immunopotentiating cytokines s u c h a s IL-1, IL-2, interferons a n d lipopolysaccharide ( L P S ) .  T N F - a h a s the ability to upregulate its o w n production  through the activation of the NFKB transcription factor. cytokine that i n d u c e s phagocytosis by neutrophils. in structural  reorganization,  upregulation  T N F - a is a n inflammatory  Stimulation of E C by T N F - a results  of a d h e s i o n  and M H C molecules, and  e x p r e s s i o n of c h e m o k i n e s and costimulatory m o l e c u l e s [19]. microglia a n d neurons c a n also produce this cytokine.  In the C N S , astrocytes,  T h e p r e s e n c e of T N F - a h a s  been d o c u m e n t e d in acute multiple sclerosis ( M S ) lesions a n d s h o w n to b e produced by hypertrophic astrocytes [20].  1 2 . 3 . 2 Interferon (IFN)-y IFN-y w a s originally d i s c o v e r e d by two groups of investigators and given two n a m e s : IFN-y a n d m a c r o p h a g e activating factor ( M A F ) .  It is a type II interferon or immune  interferon w h i c h , similar to the type I interferons (IFN-a and IFN-(3), c a n "interfere" with viral infections.  IFN-y c o n s i s t s of 166 a n d 1 5 5 a . a . . T h e primary producers of this  cytokine a r e N K cells a n d activated T lymphocytes, but it c a n b e e x p r e s s e d by almost every cell type a s d o e s its receptor.  Both e x o g e n o u s and e n d o g e n o u s stimuli including  L P S , bacterial superantigens, " n o n - s e l f s u b s t a n c e s , IL-12 a n d IL-18 a r e c a p a b l e of inducing IFN-y e x p r e s s i o n .  It is considered a potent activator of m a c r o p h a g e s and E C .  It is responsible for the upregulation of c h e m o k i n e s , co-stimulatory m o l e c u l e s and M H C c l a s s II m o l e c u l e s in E C .  C e l l types responsible for producing IFN-y in the C N S include  microglia a n d astrocytes [21, 22],  6  1.2.4 C N S C e l l s Participating in C N S  Inflammation  1.2.4.1 Astrocytes A s t r o c y t e s are glial cells that have a major role in the m a i n t e n a n c e of C N S homeostasis.  A s t r o c y t e s help regulate neuronal function a n d development, play a n  important role in neurotransmitter metabolism, and regulate extracellular p H a n d ion levels. of  Furthermore, there is e v i d e n c e suggesting that astrocytes i n c r e a s e the number  mature,  functional  s y n a p s e s on  m a i n t e n a n c e in vitro [23].  C N S neurons  and  are  required  for  synaptic  In addition to homeostatic functions, t h e s e cells contribute to  the m a i n t e n a n c e of the B B B through the c l o s e interaction of perivascular astrocytic endfeet a n d cerebral endothelium [24].  In the context of C N S d a m a g e , astrocytes play  a similar role to fibroblasts in the periphery by undergoing proliferation in a p r o c e s s termed astrogliosis. T h e role of astrocytes in C N S immune function is relatively unclear.  T h e r e is  e v i d e n c e that t h e s e cells are c a p a b l e of producing specific cytokines a n d c h e m o k i n e s under inflammatory conditions. is less well e s t a b l i s h e d .  Their role a s antigen presenting cells ( A P C ) in the C N S  T h e e x p r e s s i o n of c l a s s II M H C m o l e c u l e s is debatable and the  e x p r e s s i o n of various costimulatory controversial  [25].  m o l e c u l e s ( C D 4 0 , C D 8 0 , a n d C D 8 6 ) remains  R e c e n t post mortem  immunohistochemical  studies s h o w that  astrocytes e x p r e s s M H C c l a s s I m o l e c u l e s in active l e s i o n s of chronic M S [26].  1.2.4.2 Microglia Microglial cells k e e p a rather quiescent profile in the normal C N S e v e n though they s h a r e the s a m e myeloid progenitor with m a c r o p h a g e s at the periphery. In r e s p o n s e to physiological or s t r e s s stimuli, t h e s e cells undergo a maturation p r o c e s s or activation which results in the acquisition of various m a c r o p h a g e properties. 7  Activated microglia  e x p r e s s a n u m b e r of pattern recognition receptors: F c a n d c o m p l e m e n t receptors; cytokines a n d their receptors; c h e m o k i n e s a n d their receptors; a n d prostanoids a n d prostanoid receptors.  Furthermore, during C N S inflammation,  activated  microglia  e x p r e s s c l a s s II M H C m o l e c u l e s and adhesion/costimulatory m o l e c u l e s (such a s C D 1 1 a , C D 4 0 , C D 5 4 , C D 5 8 , C D 8 0 , a n d C D 8 6 ) [27, 28].  Studies in experimental allergic  encephalomyelitis ( E A E ) , the experimental m o d e l of multiple s c l e r o s i s (MS) s h o w upregulation of n o n - c l a s s i c a l c l a s s I M H C ( C D 1 ) e x p r e s s i o n , s u g g e s t i n g that microglia may  participate  inflammation [29].  in broadening the  presented  repertoire  of antigens during C N S  T h e r e is e v i d e n c e that microglial activation plays a major role in  C N S pathophysiology.  1.2.4.3 Perivascular Macrophages/Dendritic Cells P e r i v a s c u l a r m a c r o p h a g e s , a l s o known a s p e r i v a s c u l a r microglial cells or 'Mato' cells, are b o n e marrow-derived cells that continuously enter the C N S a s part of normal physiology a n d m a y s e r v e a s the principal antigen-presenting cell in the C N S [13, 14]. T h e r e is s o m e e v i d e n c e that this cell type returns to the lymph n o d e s a n d s p l e e n after acquiring C N S material  [30].  However, other studies contradict this view, s i n c e  heat-killed bacilli within m a c r o p h a g e s in the C N S fail to elicit a n i m m u n e r e s p o n s e [31]. T h e concept of the critical nature of the perivascular m a c r o p h a g e is a p p e a l i n g , however definitive e v i d e n c e for this role is not yet available. Dendritic cells ( D C ) are derived from haematopoietic s t e m cells in the bone marrow a n d are found a s immature cells in virtually all t i s s u e s .  D C readily uptake, p r o c e s s a n d  present antigen (via both M H C c l a s s I and M H C c l a s s II m o l e c u l e s ) , following which they mature a n d migrate to lymphoid organs, where they interact with n a i v e T cells. Their maturation results in alteration in their c o m p l e m e n t of surface receptors a n d secreted 8  m o l e c u l e s [32]. T h e question r e m a i n s a s to their p r e s e n c e in the normal C N S .  M a t y s z a k a n d Perry  (1996) w e r e able to o b s e r v e a small number of cells in the choroid plexus a n d m e n i n g e s [33].  M c M e n a m i n et al (1999) demonstrated that D C consititutively e x p r e s s M H C c l a s s  II, a n d are located predominantly around blood v e s s e l s , dura matter, leptomeninges and choroid plexus [34].  Mature D C injected into the C N S p a r e n c h y m a migrate via olfactory  nerves to conventional afferent lymphatics and c a n potentially induce a primary T-cell r e s p o n s e [35]. In contrast, other studies failed to s h o w t h e p r e s e n c e of D C s in the normal brain [33, 36, 37]. DC [38-41]. MS  have recently b e e n identified in experimental C N S inflammation a n d in M S It h a s b e e n s u g g e s t e d that myelin antigens are r e l e a s e d into the C S F during  e x a c e r b a t i o n s a n d captured by D C located in C S F , m e n i n g e s a n d choroids plexus.  Following antigen capture, D C s migrate to lymph n o d e s a n d initiate T cell r e s p o n s e s against  myelin  [42].  However,  the idea  is controversial  a n d requires  further  investigation.  1.2.5 L y m p h o c y t e Trafficking in the C N S 1.2.5.1 T Lymphocytes T h e r e h a s b e e n c o n s i d e r a b l e interest in the role that lymphocytes, particularly T cells, play in C N S inflammation,  under a variety of conditions.  M u c h of this is  rationalized by the postulated role of T cells in the pathophysiology of multiple sclerosis (MS)  a n d its experimental m o d e l , E A E and of viral encephalitis. T lymphocytes are derived from hematopoietic s t e m cells p r o d u c e d in the fetal liver  and the adult b o n e marrow.  Progenitor cells committed to T lymphocytes migrate to the  thymus a n d rearrange their T cell receptor g e n e profile w h i c h is highly polymorphic. 9  O n c e committed to T cell lineage, thymocytes initially e x p r e s s both C D 4 and C D 8 molecules.  9 5 % of t h e s e double positive lymphocytes die during the positive a n d  negative selection to g e n e r a t e a complete repertoire of T cells found in the periphery. Double positive T cells that have affinity towards self M H C c l a s s II m o l e c u l e s are positively s e l e c t e d to b e c o m e C D 4 + C D 8 - single positive ( S P ) T cells a n d T cells with affinity towards self M H C c l a s s I m o l e c u l e s b e c o m e C D 4 - C D 8 + S P T cells. undergo clonal deletion if they bind too tightly to self M H C m o l e c u l e s . lymphocytes leave the thymus a n d are c o n s i d e r e d n a i v e T cells.  S P T cells Surviving T  T h e y are first primed  to antigens presented by dendritic cells at lymphatic tissue, upon which they are c o n s i d e r e d m e m o r y T cells that circulate in the body a n d scout for antigens [1]. M a n y groups h a v e s h o w n that the C N S is under constant i m m u n e surveillance by T cells, but the question remains a s to the d e g r e e of surveillance.  In vivo C N S trafficking  studies using radioactively labelled activated T cells s h o w that f e w e r T cells gain entry to C N S c o m p a r e d to other t i s s u e s [43].  This m a y be e x p l a i n e d by the w e a k e x p r e s s i o n of  a d h e s i o n m o l e c u l e s in the normal brain a s c o m p a r e d to other o r g a n s .  Interestingly,  recent studies h a v e s h o w n that T lymphocyte infiltration is greatest in the spinal cord and least in the c e r e b r u m [43, 44].  T h u s , the minimal leukocyte traffic o b s e r v e d in the  normal C N S supports the concept that the C N S is a relatively immunologically privileged site. It is n o w well d o c u m e n t e d that activated, but not resting, T cells, irrespective of their antigen specificity, c a n c r o s s the B B B [45].  H i c k e y et al [46] demonstrated that T  lymphoblasts enter the C N S a n d other tissues in an apparently r a n d o m m a n n e r while T cells not in the blast p h a s e w e r e e x c l u d e d .  T h e y found that the activation state a p p e a r s  to be the major determining factor in the ability to enter the C N S .  Furthermore, the  retention of activated lymphocytes in the C N S is d e p e n d e n t on M H C - r e s t r i c t e d antigen 10  recognition [46]. A l t h o u g h a d h e s i o n and migration of T cells a c r o s s the endothelium require a d h e s i o n m o l e c u l e s , the m e c h a n i s m of retention of antigen specific T cells in the C N S is largely u n k n o w n [12, 4 7 , 48]. In addition, at the present time there is no e v i d e n c e that specific "homing receptors" or "molecular a d d r e s s i n s " promote interactions between T l y m p h o c y t e s a n d brain endothelium.  1.2.5.2 CD8+ Tcells N a i v e C D 8 + T cells a r e stimulated by antigen-presentating  cells ( A P C ) (e.g.  dendritic cells in lymph nodes) via 2 signals: 1) A signal initiated from s u c c e s s f u l binding of M H C c l a s s l-peptide c o m p l e x to their T C R , 2) A signal from C D 8 0 a n d C D 8 6 binding to their C D 2 8 receptor.  O n c e they a r e activated, C D 8 + T cells secrete IL-2 a n d  upregulate the IL-2 receptor.  T h i s leads to proliferation a n d differentiation of the T cells  towards a r m e d effector cell status.  Effector C D 8 + T cells require only s u c c e s s f u l  binding of M H C c l a s s l-peptide c o m p l e x in order to initiate their cytotoxic function.  They  first bind I C A M - 1 o n target cells, allowing the T cell to remain in contact with the target cell and s c a n its surface for the p r e s e n c e of specific M H C c l a s s I p e p t i d e - c o m p l e x e s . T h e T cell d i s e n g a g e s the target cell if the later doesn't carry the specific antigen. Otherwise, C D 8 + T cells r e l e a s e their granules containing s p e c i a l i z e d l y s o s o m e s a n d cytotoxic proteins toward their target cells to induce a p o p t o s i s . c l a s s I e x p r e s s i o n of different  Therefore, the M H C  cell types is critical in C D 8 + T cell action  during  inflammation [1].  1.2.5.3 Cell adhesion molecules Adhesion  molecules  play  leukocyte-endothelial interaction.  an  important  role  during  the initial  stage  of  Trafficking of T l y m p h o c y t e s a c r o s s the B B B involves  n  rolling along the cerebral endothelium, followed by firm a d h e s i o n mediated by E C a d h e s i o n m o l e c u l e s and their ligands on T lymphocytes.  A d h e s i o n is followed by  transmigration which takes place either between adjacent E C or through the cell body. During inflammation, pro-inflammatory cytokines s u c h a s IFN-y a n d T N F - a are r e l e a s e d . Previous work from this laboratory has s h o w n that the cerebral endothelium is induced by inflammatory  cytokines to e x p r e s s de  novo or upregulate the e x p r e s s i o n of  intercellular a d h e s i o n molecule-1 (ICAM-1), v a s c u l a r cell a d h e s i o n molecule-1 ( V C A M - 1 ) and E-selectin that bind to their corresponding ligands on T lymphocytes ( L F A - 1 , very late activation antigen ( V L A - 4 ) and sialyl L e w i s X , respectively) [49, 50].  T N F - a is  believed to be o n e of the cytokines responsible for the upregulation of these a d h e s i o n molecules.  H u m a n g e n e s of E-selectin, V C A M - 1 a n d I C A M - 1 are known to have  promoter regions for the transcription factor NFKB binding region.  NFKB (p50/p65) is a  ubiquitous transcription factor and pleiotropic regulator of m a n y g e n e s involved in immune  and  inflammatory  responses.  NFKB  is activated  by degradation  of  a  cytoplasmic inhibitor, k B a , which in resting cells prevents nuclear localization of NFKB. T h e p r o t e a s o m e is responsible for NFKB activation by d e g r a d i n g k B a .  Inhibition of the  p r o t e a s o m e prevents NKKB activation and thus results in blocking the T N F - a - i n d u c e d e x p r e s s i o n of E-selectin, V C A M - 1 , and I C A M - 1 .  L y m p h o c y t e a d h e s i o n to stimulated  endothelial m o n o l a y e r s is largely reduced d u e to the inhibition [51].  1.2.5.4 Immunological Synapse Initiation of a s u c c e s s f u l immune r e s p o n s e requires not only a strong binding between M H C - p e p t i d e c o m p l e x e s and the T cell receptor, but a l s o a stable environment which is provided by the immunological s y n a p s e .  Interaction of C D 4 + T cells with their  target A P C activates signaling events in the C D 4 + T cell which result in new protein 12  synthesis, cell differentiation a n d cell proliferation.  U p o n recognition of the target, the  C D 4 + T cells s u r f a c e m e m b r a n e u n d e r g o e s rapid reorganization, e s p e c i a l l y of proteins located at the contact site.  T cells forms a specific topological rearrangement which is  known a s the "immunological s y n a p s e " (IS) [52]. A d h e s i o n m o l e c u l e s in the C D 8 + T cell m e m b r a n e s u c h a s leukocyte function-associated antigen - 1 (LFA-1) concentrate at the site of cell.cell contact a n d bind to ligands on the A P C s u c h a s I C A M - 1 , forming a tight bond b e t w e e n the two cells.  T-cell receptor ( T C R ) - p e p t i d e - M H C interactions  were first s e e n to localize in a ring surrounding a central cluster of L F A - 1 - I C A M - 1 interactions, creating a n immature T-cell s y n a p s e , w h i c h later matures s o that a ring of integrins s u r r o u n d s the M H C - T C R cluster located at the centre. forms  only  upon  recognition  of agonist  A mature T-cell IS  p e p t i d e - M H C , implying  that  TCR-MHC  interactions m a y i n c r e a s e T cell a d h e s i o n d u e to IS formation.  1.2.5.5 Antigen Recognition and T Cell Transmigration R e c e n t e v i d e n c e s u g g e s t s that i n c r e a s e d c o g n a t e recognition between M H C c l a s s I a n d T C R of C D 8 + T cells e n h a n c e s migration of antigen-specific C D 8 + T cells a c r o s s E C m o n o l a y e r s [53]. TCR-mediated  T h e m e c h a n i s m is not yet fully understood but it m a y involve  activation  of signalling  pathways  e x p r e s s i o n a n d cytoskeleton rearrangement. motility m a y s e r v e two p u r p o s e s .  that  control  adhesion  molecule  T C R - m e d i a t e d control of lymphocyte  First, it renders the p r o c e s s of recruitment to the  antigenic site of inflammation more efficient by facilitating transmigration of T cells with antigen specificity. APC.  S e c o n d , it helps mobile T cell to stop thus facilitating binding to the  S e v e r a l studies using self-reactive T cells support this idea [54, 55].  Although there is e v i d e n c e indicating that C D 8 + T cells enter the C N S for either 13  survillence p u r p o s e s [56] or clonal e x p a n s i o n resulting in direct d a m a g e to brain tissue [35, 57, 58], the m e c h a n i s m of C D 8 + interaction with a n d a d h e s i o n to brain endothelium is poorly u n d e r s t o o d .  In a m o u s e model of inflammation, P-selectin w a s s h o w n to have  a critical role for C D 8 + T cell a d h e s i v e n e s s to inflammed brain v e n u l e s [59].  Therefore  the a d h e s i o n of C D 8 + T C to brain v e s s e l endothelium m a y be the first p r o c e s s during inflammatory  r e s p o n s e s in the  CNS.  P r e v i o u s studies demonstrated  a  general  a d h e s i v e n e s s of C D 8 + T C to M H C c l a s s I m o l e c u l e s possibly v i a C D 8 binding to the non-peptide g r o o v e compartment  of M H C [60-62].  It is important,  therefore,  to  investigate the role of M H C c l a s s I and E C a d h e s i o n m o l e c u l e s in the a d h e s i o n of C D 8 + T C to the cerebral endothelium.  1.2.6 Immunopathogenesis of CNS Autoimmune Disorders S e v e r a l d i s o r d e r s of the C N S including inflammatory, infectious a n d autoimmune d i s e a s e s are c h a r a c t e r i z e d by infiltration of inflammatory cells into the brain and an a s s o c i a t e d i n c r e a s e d permeability of the B B B to ions, proteins a n d water.  Brain a n d  spinal cord lesions in a u t o i m m u n e demyelinating d i s e a s e s s u c h a s multiple s c l e r o s i s (MS) are c h a r a c t e r i z e d by infiltration of both C D 4 + a n d C D 8 + T lymphocytes a n d s u b s e q u e n t demyelination [56, 57, 63].  T h e m e c h a n i s m s involved in the a d h e s i o n a n d  migration of inflammatory cells a c r o s s the B B B are presently not fully defined.  Both  environmental a n d genetic factors have b e e n implicated in the p a t h o g e n e s i s of this disease.  Environmental factors including g e o g r a p h i c a l a n d temporal correlations are  possibly contributing but exact e v i d e n c e is difficult to obtain [64].  Individuals with the  H L A - D R 2 allele a n d s e v e r a l other polymorphisms with H L A - D R a n d D Q have increased susceptibility to M S [64, 65]. To date, data from both M S and E A E point towards a g e n e r a l m o d e l explaining the 14  molecular events that contribute to the d e v e l o p m e n t of demyelination [64].  Thus,  studies in E A E h a v e s h o w n that the C D 4 + Th1 phenotype h a s a critical role in driving the disease  phenotype.  myelin-associated proteolipid  protein  Autoantigens  protein (PLP)  (MAG), [66,  such  myelin 67],  as  myelin  basic  oligodendrocyte  aB-crystallin  [68];  protein  (MBP),  glycoprotein  (MOG),  2',3'-cyclic  nucleotide  3 ' - p h o s p h o d i e s t e r a s e ( C N P ) [69] have a l s o b e e n implicated in the p a t h o g e n e s i s of E A E . Two theories h a v e b e e n put forth in order to explain the generation of autoimmune T cells in the C N S : the m o l e c u l a r mimicry a n d the "mistaken s e l f m o d e l s .  T h e molecular  mimicry theory p r o p o s e s that myelin-reactive T cells are activated by bacterial or viral agents that s h a r e epitopes with self antigens; or that foreign antigen-reactive T cells recognize self antigens [70].  T h e "mistaken s e l f c o n c e p t p r o p o s e s that a stress protein,  aB-crystallin, is e x p r e s s e d de novo in infected cells at the periphery a n d that activated T cells against the aB-crystallin will target the aB-crystallin in myelin a n d oligodendrocytes and further lead to myelin d a m a g e [68]. W h i l e the main f o c u s of research thus far h a s b e e n on characterizing the role of C D 4 + T cells in a u t o i m m u n e d i s e a s e s , H u s e b y a n d c o l l e a g u e s h a v e s h o w n that C D 8 + T cell c l o n e s , specific for a c l a s s I restricted-fragment of the M B P 7 9 - 8 7 , produce s e v e r e E A E with paralysis a n d other neurological deficits [71].  In addition, C D 8 + T cells  reactive  35-55  to  myelin  oligodendroglial  glycoprotein  MOG  induced  massive  inflammation a n d demyelination in the central nervous s y s t e m [57, 72]. S i n c e cerebral E C are the first native cells in the C N S to encounter circulating lymphocytes, it is tempting to h y p o t h e s i z e that antigen recognition by m e m o r y T-cells a n d s u b s e q u e n t activation a n d migration o c c u r s at the B B B .  15  1.3  M H C C L A S S IA N T I G E N P R O C E S S I N G A N D  P R E S E N T A T I O N  T h e study of M H C m o l e c u l e s initially f o c u s e d on tissue transplant biology.  It w a s  first noticed in 1916 by Little and T y z z e r that s e v e r a l dominant g e n e s influenced the outcome  of  allogeneic  tumour  grafts.  It  was  later  confirmed  by  successful  transplantation of skin grafts between identical twins that the, transplantation w a s influenced by genetic factors.  outcome  Later o n , with the a d v a n c e s of serological typing  t e c h n i q u e s , the h u m a n leukocyte antigen (HI_A) s y s t e m w a s d i s c o v e r e d with 3 allelic series: A , B and C , w h i c h c o d e the c l a s s I of the M H C c o m p l e x .  With recent D N A b a s e d  typing t e c h n i q u e s , m a n y new polymorphic loci within the M H C h a v e b e e n identified, including the c l a s s II M H C or the H L A - D loci. is to present peptides to lymphocytes.  T h e main function of the M H C m o l e c u l e s  M H C c l a s s I is e x p r e s s e d by the vast majority of  nucleated cells, allowing circulating C D 8 + cytotoxic T cells to s u r v e y them for possible infection or tumor specific protein e x p r e s s i o n during tumorigenesis.  T h e T C R on  lymphocytes requires s u c c e s s f u l binding to the M H C - a n t i g e n i c peptide c o m p l e x in order to b e c o m e activated.  Studies s u g g e s t that M H C p o l y m o r p h i s m  or the  presented  antigenic peptide contribute to the T cell r e s p o n s e , s u g g e s t i n g a m e c h a n i s m for the pathogenesis of a u t o i m m u n e d i s e a s e [1].  1.3.1  Structure of M H C class I  molecules  T h e overall structure of M H C c l a s s I m o l e c u l e s is similar to that of M H C c l a s s II. T h e y have similar immunoglobulin d o m a i n s near the cell s u r f a c e and distally they both have two d o m a i n s folded together, creating a long cleft for peptide binding. I consists of two polypeptide chains. composed polymorphic  of the  T h e a c h a i n , e n c o d e d in the M H C locus, is  a 1 , a.2 and a 3 d o m a i n s .  peptide  binding  cleft.  M H C class  Alphal  and  T h e B chain or the 16  a.2 d o m a i n s form  ^-microglobulin  (p^m)  the is  covalently bound to the a chain near the cell surface but it is not encoded in the M H C locus and is not polymorphic.  Peptides bound to M H C class I are resticted to a length  of 8-10 a.a. because the pocket-shaped structure at both ends of the cleft restricts binding of oversized peptides.  Bound peptides are required to have certain a.a. that  serve as anchor residues for both ends of the cleft and they cannot move freely once bound to the cleft [73]. MHC  O n c e successfully bound, peptides improve the stability of the  class I molecules.  In addition, polymorphism at the a1 and a2 chains affects  which a.a. line at the end of the peptide binding cleft and thus affect the specificity of bound peptides.  Together, the M H C class l-peptide complexes are highly specific for T  cell clones bound to them and stably expressed on the cell surface for successful T C R binding.  1.3.2  M H C class Iantigen  presentation  1.3.2.1 The Classical Pathway  Nature Immunology, vol. 5, p.661 (2004)  17  M H C c l a s s I antigen presentation  requires: i) selective b r e a k d o w n  of  intracellular  proteins into peptides with suitable length, ii) s u c c e s s f u l binding of the peptides to suitable a n c h o r sites in the binding groove of M H C c l a s s I.  C e l l s under stress s u c h a s  virus infected cells normally have i n c r e a s e d M H C c l a s s I e x p r e s s i o n a s s o c i a t e d with viral protein derived antigens.  A n important candidate for producing the right s i z e of  antigens for binding to M H C c l a s s I is a giant carboxyl p e p t i d a s e c o m p l e x called the proteasome.  S i n c e M H C c l a s s I g e n e s are highly polymorphic, the proteins e n c o d e d by  e a c h allele c a n bind only o n e specific set of peptides. T h e p r o t e a s o m e is responsible for antigenic peptide production through its preference for cleaving at the carboxyl (C) terminus of peptides suitable for M H C c l a s s I a n c h o r r e s i d u e s (hydrophobic a n d b a s i c residues) [74].  U n d e r normal conditions, the p r o t e a s o m e is mainly responsible for the  degradation of transcriptional factors, misfolded & expired proteins in the cytoplasm a n d nucleus [75].  T h e products of the p r o t e a s o m e range from 4 - 2 0 a . a . in length.  Several  other a m i n o p e p t i d a s e s m a y further trim these peptides to the right s i z e for transportation to the e n d o p l a s m i c reticulum ( E R ) .  S i n c e c y t o p l a s m i c peptides cannot diffuse a c r o s s  m e m b r a n e s , trimmed peptides are transported to the E R through the A T P c o n s u m i n g transporters a s s o c i a t e d with antigen presentation (TAP) - a n E R - r e s i d e n t , heterodimeric protein [76].  O n c e inside the E R , t h e s e peptides bind to a proper M H C c l a s s I a heavy  chain and c o m p l e t e the folding of the molecule followed by transportation to the cell surface.  Lengthy peptides may be selectively cut d o w n to b e t w e e n 8 to 10 a.a by an  E R resident protease called the e n d o p l a s m i c reticulum a m i n o p e p t i d a s e 1 ( E R A P 1 ) in order to fit in the binding groove [77].  E v i d e n c e that E R A P 1 w o r k s closely with the  p r o t e a s o m e to p r o d u c e correct-size M H C c l a s s I antigens is b a s e d on the: 1) strong preference on substrates with 9-16 a . a , 2) product s i z e with 8-9 a . a , 3) preference on peptides with hydrophobic  C-terminus, a n d 4) IFN-y inducibility. 18  Efficient  peptide  matching to pre-mature M H C c l a s s I a l s o is d o n e by the T A P transporters.  This  c o m p l e x clusters the relevant m o l e c u l e s that are involved in M H C c l a s s I loading in one location.  E a c h c o m p l e x includes 3 to 4 M H C c o p i e s of the peptide-receptive M H C c l a s s  I m o l e c u l e s a n d stabilizes by a dedicated molecular c h a p e r o n e , t a p a s i n , a n d one c o p y of general p u r p o s e m o l e c u l a r c h a p e r o n e s , calreticulin a n d E R p 6 0 [78].  Tapasin is critical  for optimal peptide binding to M H C c l a s s I m o l e c u l e s s i n c e it not only interacts directly with M H C c l a s s I m o l e c u l e s but a l s o simultaneously interacts with E R p 6 0 [79].  ERp60  is a thioreductase that supports the formation of a disulphide bridge that connects the 'walls' of the M H C c l a s s I peptide-binding groove with its b a s e [80]. with calreticulin that further supports the w h o l e binding p r o c e s s .  E R p 6 0 interacts  S u c c e s s f u l peptide  binding r e l e a s e s the M H C c l a s s I molecule from the M H C c l a s s - l - l o a d i n g c o m p l e x for delivery to the cell surface through the standard golgi apparatus secretory pathway. Therefore, the generation of M H C c l a s s I peptides by the p r o t e a s o m e is very important for M H C c l a s s I antigen presentation [81, 82].  1.3.2.2  The Cross-presentation Pathway  M H C c l a s s I m o l e c u l e s are primarily important for i m m u n e surveillance by both N K cells a n d C D 8 + T cells.  O n the other hand, A P C s s u c h a s D C prime a n d activate C D 8 +  T cells through M H C c l a s s I e x p r e s s i o n after acquiring p a t h o g e n s through direct infection. In the c l a s s i c presentation pathway, the antigens p r e s e n t e d to C D 8 + T cells are restricted to e n d o g e n o u s proteins and peptides d e g r a d e d in the cytosol.  However,  there are p a t h o g e n s which D C c a n p r o c e s s a n d present to C D 8 + T cells without being infected  [83].  Recent  studies  show  that  a  specialized  pathway  called  cross-presentation pathway, allows D C a n d possibly m a c r o p h a g e s to acquire antigens from infected or a b n o r m a l cells [84].  E x o g e n o u s antigens are p h a g o c y t o s e d into 19  p h a g o s o m e s w h i c h are formed by fusion of p l a s m a a n d E R m e m b r a n e s .  Inside the  p h a g o s o m e , a E R surface transporter, S e c 6 1 pore c o m p l e x , transports large e x o g e n o u s fragments to the cytosolic p r o t e a s o m e for degradation.  Similar to the c l a s s i c a l pathway,  c l e a v e d peptides with the right s i z e are then transported b a c k to the p h a g o s o m e by the TAP  transporter.  Transported peptides are s c r e e n e d by the M H C c l a s s I m o l e c u l e s  a s s o c i a t e d with the T A P protein.  C h a p e r o n e proteins s u c h a s t a p a s i n , calreticulin and  E R p 5 7 and E R a m i n o p e p t i d a s e are all present to optimize M H C - p e p t i d e binding [85, 86]. S i n c e the p r o t e a s o m e is a s s o c i a t e d with this s p e c i a l p h a g o s o m e , it is believed that the entire unit alone is sufficient to mediate cross-presentation [87].  T h e exact m e c h a n i s m  for resurfacing s u c c e s s f u l l y b o u n d M H C c l a s s I - peptide c o m p l e x e s with this pathway is not clear.  It is p o s s i b l e that the p h a g o s o m e returns to the E R and enters the normal  secretory  pathway  since  cross-presentation [88].  inhibitors  of  the  golgi  -  ER  transport  can  inhibit  Although the m e c h a n i s m is different, the p r o t e a s o m e s e e m s to  play a similarly important role a s in the c l a s s i c a l pathway.  Presentation of e x o g e n o u s  antigens is highly sensitive to specific inhibitors of the p r o t e a s o m e , indicating that cytosolic  proteolysis  is  still  the  primary  method  for  epitope  generation  in  the  cross-presentation pathway [89].  1.4  T H E P R O T E A S O M E A N D  1.4.1 T h e U b i q u i t i n / P r o t e a s o m e  I M M U N O P R O T E A S O M E S y s t e m  T h e 2 6 S p r o t e a s o m e is involved in key biological p r o c e s s e s s u c h a s cell-cycle regulation, a p o p t o s i s , m o r p h o g e n e s i s , a n d metabolic control.  T h e r e are 8 0 0 0 0 0 c o p i e s  of the 2 6 S p r o t e a s o m e per cell a n d they are located mostly in the cytosol a n d nucleus. E a c h c o p y h a s a n efficiency of 11 substrates per s e c o n d in a v e r a g e . is  a carboxyl p e p t i d a s e that belongs to the ubiquitin/proteasome 20  The proteasome s y s t e m which is  responsible for the selective degradation of polyubiquitinated proteins [90].  Substrates  for proteasome require conjugation with multiple units of ubiquitin to begin degradation. Ubiquitin is a highly conserved 76 a.a. protein, which is activated in an ATP-dependent process by ubiquitin-activating enzyme (E1), followed by conjugation with other units by ubiquitin-conjugating enzyme (E2). the ubiquitin-protein ligase (E3).  Finally, conjugation to the target protein is done by  Polyubiquitinated chains act as a signal recognized by  the proteasome [91].  1.4.2 Structure Constitutive proteasome  Immunoproteasome  Nature Immunology Vol.5, p.663 (2004)  The 2 6 S proteasome complex is formed by the catalytic 2 0 S core proteasome and two  19S regulatory  complexes primarily  ubiquitinated substrates [92].  responsible for binding  and unfolding  The 2 0 S core proteasome consists of 14 structurally  different subunits forming 4 seven-member rings stacked on each other.  The outer two  rings are composed of a subunits (a1-a7) that control substrate access, whereas the inner two rings are formed by seven (3 subunits (B1-B7). only three, 61, 62 and 65, are proteolytically active.  Of the seven B-type subunits,  Potentially all three subunits can  cleave peptides between all a.a. residues, but each subunit has its own preferred activity towards its substrates: Subunit B1 has chymotrypsin-like activity, which cleaves after hydrophobic residues; (32 has trypsin-like activity, which cleaves after basic residues;  21  a n d 65 h a s c a s p a s e - l i k e or peptidylglutamyl peptide hydrolyzing ( P D P H ) - l i k e activity, which c l e a v e s after acidic residues.  T h e s e B subunits h a v e activities similar to e a c h  other a n d are mainly r e s p o n s i b l e for degrading proteins [93-95].  T h e 1 9 S regulatory  c o m p l e x e s c o m p o s e d of six A T P a s e subunits a n d nine to ten n o n - A T P a s e subunits, form a n attached structure to the 2 0 S p r o t e a s o m e .  T h e y act a s 'gates' that control the  a c c e s s of substrates to the inner catalytic c h a m b e r [96].  1.4.3 T h e  I m m u n o p r o t e a s o m e  T h e constitutive p r o t e a s o m e c l e a v e s after acidic, b a s i c a n d hydrophobic residues without specific preference a n d its proteolytic inhibitors [97].  activity  is inhibited  by  proteasome  P r e v i o u s studies have s h o w n that a n e w population of p r o t e a s o m e  subunits, called the i m m u n o p r o t e a s o m e subunits, is upregulated through the IFN-y signalling pathway in m a n y cell types in both h u m a n s a n d m i c e [98-100].  Constitutive  p r o t e a s o m e subunits (31, 6 5 a n d 32 are replaced by the i m m u n o p r o t e a s o m e subunits B1 i, B5i  and  B2i after  IFN-y  stimulation.  a s s e m b l e with e a c h other, immunoproteasome  without  forming  Immunoproteasome a new  increasing  the  population total  of  subunits  cooperatively  proteasome  proteasome  called  population  the  [101].  Therefore, a shift of the intracellular p r o t e a s o m e population from consitutive p r o t e a s o m e to i m m u n o p r o t e a s o m e o c c u r s during IFN-y stimulation.  Unlike the constitutive form,  i m m u n o p r o t e a s o m e subunits h a v e high c l e a v a g e specificity towards hydrophobic a n d b a s i c residues, making i m m u n o p r o t e a s o m e a e x o - c a r b o x y l p e p t i d a s e specializing in producing peptides or N-extended peptides optimal for binding to M H C c l a s s I m o l e c u l e s [102, 103].  P r o t e a s o m e inhibitors completely b l o c k e d presentation of uncut peptides  with e v e n a single extra C-terminal residue but failed to block presentation for the size-corrected peptides [104].  This indicates p r o t e a s o m e s are the only p r o t e a s e s in 22  cells that c a n g e n e r a t e the proper C terminus of t h e s e peptides from longer precursors. P r o f e s s i o n a l A P C s u c h a s D C s h o w strong upregulation of the  immunoproteasome  during their antigen p r o c e s s i n g stage [105-107] a n d their ability to present antigens is inhibited  by p r o t e a s o m e  inhibitors  [108].  T h i s strongly  supports the  notion  that  i m m u n o p r o t e a s o m e s play a n important role in M H C c l a s s I antigen presentation a n d C D 8 + T cell priming.  In addition, s e v e r a l subcellular localization studies indicate that  the i m m u n o p r o t e a s o m e localizes around the E R while the constitutive p r o t e a s o m e is located  uniformly  in  the  cytosol  [109-112].  This  further  indicates  that  the  i m m u n o p r o t e a s o m e is m o r e s p e c i a l i z e d in producing peptides for M H C c l a s s I antigen presentation.  Therefore, the i m m u n o p r o t e a s o m e is believed to e n h a n c e M H C c l a s s I  antigen presentation in both h u m a n a n d murine s y s t e m s [102, 103, 113].  1.4.4 P r o t e a s o m e  inhibitors  T h e r e are 2 m a i n categories of p r o t e a s o m e inhibitors: 1) peptide-derived a n d 2) small m o l e c u l e [94].  Peptide-derived inhibitors are synthetic oligopeptides d e s i g n e d to  target the p r o t e a s o m e catalytic subunits.  Their first generation are peptide a l d e h y d e s  that form covalent h e m i a c e t a l s with threonine at the active site.  S i n c e a l d e h y d e groups  also react with active sites of cysteine p r o t e a s e s , the inhibition specificity of these reagents is quite variable a n d a l s o reversible.  A n e x a m p l e of this  type of inhibitor,  ritonivir, a n inhibitor of the protease e n c o d e d by H u m a n Immunodeficiency Virus type 1 (HIV-1), w a s u s e d experimentally to reduce the destruction of C D 4 + T cell killing mediated by cytotoxic T lymphocytes ( C T L ) [114].  It is believed that the specific  inhibition of chymotrypsin-like activity of B subunits is the m e c h a n i s m of its p r o t e a s o m e inhibition [94].  A n o t h e r potent a n d the most specific p r o t e a s o m e inhibitor is lactacystin.  It is a natural S t r e p t o m y c e s metabolite that binds covalently to all B-type subunits a n d 23  can  irreversibly inhibit the chymotryptic- a n d tryptic-like p e p t i d a s e activity.  Lactacystin  s p o n t a n e o u s l y h y d r o l y z e s into B-lactone, which then reacts with the N-terminal threonine of the B5 subunit.  T h i s inhibitor blocks M H C c l a s s I antigen presentation of ovalbumin  but d o e s not affect  presentation of correct peptide epitopes that do not  p r o t e a s o m e p r o c e s s i n g [115].  require  S i n c e it d o e s not affect c y s t e i n e or serine proteases,  which are vital for normal cell function, it is the most popular tool u s e d to study the effect of p r o t e a s o m e inhibition.  1.5  H U M A N  B R A I N M I C R O V E S S E L E N D O T H E L I A L  1.5.1 E n d o t h e l i a l C e l l EC  C E L L S  ( H B M E C )  Heterogeneity  that line the luminal s u r f a c e s of all blood v e s s e l s are involved in both normal  physiological functions a n d pathological conditions, including h o m e o s t a s i s , coagulation, a n g i o g e n e s i s , a n d regulation of inflammatory cell migration. large v e s s e l a n d m i c r o v a s c u l a r endothelium. maintaining  the  v a s c u l a r tone  and  other  T h e r e are two types of E C :  L a r g e v e s s e l E C are responsible for  physiological functions.  Microvascular  endothelium is divided in three groups, b a s e d on morphology: 1) discontinuous, 2) fenestrated,  and  3)  continuous.  Organs  such  as  lymphoid  organs,  spleen,  gastrointestinal tract, choroid plexus a n d kidney g l o m e r u l u s , w h i c h d e a l with high molecular, m a c r o m o l e c u l a r a n d cellular e x c h a n g e , h a v e both the discontinuous a n d fenestrated endothelium [116, 117].  In continuous m i c r o v e s s e l s , adjacent E C are  bound together by tight junctions located along the lateral s u r f a c e s of E C .  T h e B B B is  c o m p o s e d of continuous endothelium responsible for maintaining C N S h o m e o s t a s i s . is  It  now well e s t a b l i s h e d that endothelial cells from different v a s c u l a r b e d s and s p e c i e s  differ in their morphology, function, antigenic, permeability a n d immunological properties [118]. 24  1.5.2 R o l e of Endothelial C e l l s in  Inflammation  It is now well e s t a b l i s h e d that the endothelium plays a n active role in inflammation. P r e v i o u s studies s u g g e s t s that h u m a n peripheral E C h a v e the ability to present antigen to T cells a n d trigger their proliferation a n d diffientiation [119, 120].  In addition, M H C  e x p r e s s i o n by v a s c u l a r E C is significant, suggesting t h e ability for c o g n a t e antigen presention to circulating m e m o r y T cells [121-123].  S e v e r a l studies have s h o w n that  peripheral E C e x p r e s s a d h e s i o n m o l e c u l e s and c h e m o k i n e s for recruitment of circulating leukocytes.  T h e role of brain m i c r o v e s s e l endothelial cells in C N S inflammation is not  presently well d e f i n e d . LPS  P r e v i o u s studies from this laboratory s h o w e d that cytokines a n d  induce d e novo e x p r e s s i o n or upregulation of E C a d h e s i o n m o l e c u l e s by H B M E C  which are important in mediating the a d h e s i o n a n d migration of T lymphocytes a c r o s s brain E C [49, 50, 124-126]. EC  Additional studies have a d d r e s s e d the potential of cerebral  to function a s antigen-presenting cells ( A P C ) by investigating the induced e x p r e s s i o n  of M H C c l a s s II, M H C c l a s s I [26], a n d costimulatory m o l e c u l e s [127-129]. and  Thus, Huynh  Dorovini-Zis d e m o n s t r a t e d de novo e x p r e s s i o n of M H C c l a s s II by H B M E C following  treatment with IFN-y [130] a n d O m a r i et al. w e r e a b l e to s h o w that brain E C c a n b e induced by IFN-y to e x p r e s s or upregulate the costimulatory m o l e c u l e s ( C D 8 0 , C D 8 6 , C D 4 0 a n d L F A - 3 ) a n d i n d u c e co-stimulation a n d proliferation of C D 4 + T cells [129, 131, 132].  U s i n g the s a m e in vitro s y s t e m , S h u k a l i a k et al. confirmed the ability of H B M E C  to s y n t h e s i z e , s e c r e t e a n d bind B-chemokines which are important for the a d h e s i o n and migration of C D 4 + T cell s u b s e t s a c r o s s the endothelium [133, 134].  1.5.3 In v i t r o M o d e l o f t h e H u m a n B l o o d - B r a i n  Barrier  In o u r laboratory, a reproducible m o d e l of the h u m a n B B B h a s b e e n previously 25  established.  H u m a n brain m i c r o v e s s e l E C isolated from autopsy brains a n d temporal  lobectomy s p e c i m e n s form highly o r g a n i z e d , confluent m o n o l a y e r s that retain important properties of their in vivo counterparts [135].  W h e n grown to c o n f l u e n c e , the cultures  display the following B B B characteristics: 1) tight junctions with high electrical resistance a n d 2) rare pinocytotic v e s i c l e s .  T h e endothelial nature of t h e s e cells is confirmed by  their synthesis of F a c t o r VIII / von Willebrand factor, binding of U l e x e u r o p a e u s agglutinin a n d uptake of acetylated low density lipoprotein ( D i l - A c - L D L ) .  T h i s model has b e e n  reproducibly u s e d to study the r e s p o n s e s of the B B B endothelium under physiological conditions a n d in a n inflammatory milieu a n d the role of brain E C in leukocyte a d h e s i o n a n d migration a c r o s s the B B B [50, 124-126, 129-131].  1.6 1.6.1  O B J E C T I V E A N D S P E C I F I C  A I M S  Hypothesis T h e C N S is c o n s i d e r e d an immunologically privileged site d u e to the p r e s e n c e of  the B B B that normally restricts the entry of white blood cells into the brain and the a b s e n c e of conventional lymphatic drainage.  T h e m e c h a n i s m s that regulate  the  recruitment of inflammatory cells to sites of antigenic c h a l l e n g e in the C N S are not fully understood.  P r o t e a s o m e inhibitors have b e e n s h o w n to h a v e a protective  against the p r o g r e s s i o n of M S through an a s yet unknown m e c h a n i s m .  effect  S i n c e E C are  the first native cells of the C N S to encounter blood-borne leukocytes, it is possible that E C - l e u k o c y t e interactions contribute to antigen presentation e v e n t s a n d the recruitment of inflammatory cells a c r o s s the B B B during C N S inflammation. T h e overall objective of this study is to test the hypothesis that p r o t e a s o m e inhibitors c a n downregulate both the M H C c l a s s I and a d h e s i o n m o l e c u l e e x p r e s s i o n by brain E C , thereby reducing the a d h e s i o n of C D 8 + T lymphocytes to the B B B endothelium during 26  C N S inflammation.  1.6.2 S p e c i f i c  A i m s  T h i s study will utilize a previously well-characterized in vitro m o d e l of the h u m a n blood-brain barrier to a c h i e v e the following specific a i m s : 1. To  determine  the  RNA  and  protein  expression  of  the  proteasome  and  i m m u n o p r o t e a s o m e subunits by h u m a n brain m i c r o v e s s e l endothelial cells ( H B M E C ) and their regulation by cytokines. 2.  To characterize the regulatory role of cytokines a n d the i m m u n o p r o t e a s o m e on M H C  c l a s s I e x p r e s s i o n by H B M E C . 3.  To determine the role of the i m m u n o p r o t e a s o m e in the a d h e s i o n of C D 8 + T cells to  H B M E C in vitro.  28  C H A P T E R 2: M A T E R I A L S A N D M E T H O D S  2.1  E N D O T H E L I A L C E L L  C U L T U R E S  2.1.1 I s o l a t i o n o f H u m a n B r a i n M i c r o v e s s e l E n d o t h e l i a l C e l l s ( H B M E C ) H B M E C w e r e isolated from normal autopsy brains less than 12 hours (h) post mortem.  T h e clinical history w a s reviewed to e n s u r e that the brains w e r e normal.  The  m e n i n g e s w e r e r e m o v e d a n d the cerebral cortex w a s cut into 1-2 m m c u b e s a n d incubated in 0 . 5 % d i s p a s e (Life Technologies Inc.) for 3h in a 37°C s h a k i n g water bath. Digested tissue w a s centrifuged  for  10 minutes  at  1000x g.  T h e pellets  were  r e s u s p e n d e d a n d centrifuged in 1 5 % dextran ( S i g m a C h e m i c a l C o . , St.Louis, M O ) at 5800x g for 10 minutes.  T h e pellets containing m i c r o v e s s e l E C s , b a s e m e n t m e m b r a n e  a n d pericytes, w e r e collected a n d incubated overnight in 0 . 1 % c o l l a g e n a s e / d i s p a s e ( R o c h e D i a g n o s t i c s , L a v a l , Q B ) in a 37°C s h a k i n g water bath to remove b a s e m e n t m e m b r a n e a n d pericytes. serum,  layered  Following w a s h i n g , the pellets w e r e s u s p e n d e d in 5 % horse  over 4 5 %  Percoll gradients  (Percoll, S i g m a C h e m i c a l Co.)  and  centrifuged at 1000x g for 10 minutes in order to s e p a r a t e E C from b a s e m e n t m e m b r a n e , pericytes a n d red blood cells.  T h e layer containing E C w a s aspirated a n d w a s h e d in  1 0 % horse s e r u m in M 1 9 9 .  T h e isolated E C w e r e s u s p e n d e d in complete growth  m e d i a (see section 2.1.3) a n d plated onto fibronectin-coated  tissue culture  plates  (Corning C o s t a r C o r p . , C a m b r i d g e , M A ) .  2.1.2 Isolation of H u m a n Umbilical Vein Endothelial C e l l s ( H U V E C ) Primary cultures of H U V E C w e r e u s e d to m a k e direct c o m p a r i s o n s between the r e s p o n s e s of brain m i c r o v e s s e l a n d extracerebral large v e s s e l endothelium.  HUVEC  were isolated from umbilical cords obtained from term delivery p l a c e n t a s at the B C 29  W o m e n ' s a n d C h i l d r e n ' s Hospital, according to the m e t h o d d e s c r i b e d by Jaffe a n d c o l l e a g u e s [136].  Briefly, cord veins w e r e w a s h e d with H a n k ' s b a l a n c e d salt solution  (Life T e c h n o l o g i e s Inc.) at p H 7.4, infused with 0 . 1 % (1 mg/ml) c o l l a g e n a s e (Worthington Biochemical  Corporation, Lakewood, NJ) and  incubated  at 37°C for  15  minutes.  Following w a s h i n g with w a r m M 1 9 9 containing 2 0 % h o r s e s e r u m E C w e r e collected a n d s u s p e n d e d in c o m p l e t e m e d i a .  2.1.3 Culture  Conditions  H B M E C a n d H U V E C cultures w e r e grown under similar conditions on either fibronectin- a n d c o l l a g e n - c o a t e d plates respectively. complete media) contained M 1 9 9 s u p p l e m e n t e d with  Culture m e d i a (referred to a s 1 0 % horse s e r u m  (Hyclone  Laboratories, L o g a n , U T ) , 100 ug/ml heparin ( S i g m a C h e m i c a l C o . ) , 2 0 u g / m l endothelial growth s u p p l e m e n t ( S i g m a C h e m i c a l C o . ) , 1% antibiotic/antimycotic solution (Invitrogen) and 300 ug/ml glutamine ( S i g m a C h e m i c a l C o . ) .  C u l t u r e s w e r e incubated at 37°C in a  humidified 5 % CC>2/95% air incubator a n d the culture m e d i a w e r e c h a n g e d every s e c o n d day.  C u l t u r e s r e a c h e d c o n f l u e n c e after about 7-10 d a y s .  T h e purity a n d endothelial  nature of the cells w e r e determined by positive staining for F a c t o r V l l l - r e l a t e d antigen and U l e x e u r o p e a u s I lectin binding.  2.2  C Y T O K I N E S A N D P R O T E A S O M E INHIBITOR  T R E A T M E N T  H B M E C a n d H U V E C m o n o l a y e r s were grown to c o n f l u e n c e in replicate wells and incubated with cytokines for 24, 4 8 or 7 2 h to simulate inflammatory conditions. T w o cytokines w e r e u s e d : tumor n e c r o s i s factor a ( T N F a , 1 0 - 1 0 0 U/ml, S i g m a C h e m i c a l Co.) a n d interferon y (IFN-y, 2 0 0 - 5 0 0 U/ml, NIAID, B e t h e s d a , M D ) . Cultures w e r e treated with the p r o t e a s o m e inhibitor lactacystin ( L C , 7.5-90 u M , S i g m a 30  C h e m i c a l C o . ) a l o n e or in combination with IFN-y for 2 4 - 4 8 h to inhibit p r o t e a s o m e activity.  2.3  A N T I B O D I E S For i m m u n o c y t o c h e m i c a l studies the following primary antibodies (Abs) were u s e d :  M o u s e a - h u m a n H L A - A B C antigen: clone W 6 / 3 2 ( D A K O , D e n m a r k ) , m o u s e anti-human C D 4 5 leukocyte c o m m o n antigen ( L C A ) : clone 2B11 + P D 7 / 2 6 ( D A K O ,  Denmark),  m o u s e anti-human C D 4 5 L C A conjugated with H o r s e r a d i s h p e r o x i d a s e : clone 2B11 + P D 7 / 2 6 ( D A K O , D e n m a r k ) , m o u s e anti-human C D 5 4 ( I C A M - 1 ) : c l o n e 8 4 H 1 0 ( B e c k m a n Coulter,  M i s s i s s a u g a , Ontario),  (Beckman  Coulter,  mouse  a-human  M i s s i s s a u g a , Ontario),  mouse  CD106  ( V C A M - 1 ) : clone  monoclonal  anti-human  1G11 CD62E  (E-selectin): c l o n e C L 2 ( B i o S o u r c e Inter. Inc., C a m a r i l l o C A ) , m o u s e anti-human 2 0 S p r o t e a s o m e subunit a 5 (zeta) (Affiniti R e s . P r o . Ltd. C e d a r l a n e , Hornby, O N ) , m o u s e a - h u m a n 2 0 S p r o t e a s o m e subunit B1i ( L M P 2 ) (Affiniti R e s . P r o . Ltd. C e d a r l a n e , Hornby, O N ) , m o u s e a - h u m a n 2 0 S p r o t e a s o m e subunit B5i ( L M P 7 ) (Affiniti R e s . Pro. Ltd. Cedarlane,  Hornby,  ON).  Peroxidase-conjugated  Secondary  AffiniPure  antibodies  goat  used  anti-Mouse  in IgG  this  study (H+L)  included: (Jackson  I m m u n o R e s e a r c h W e s t G r o v e , P A ) , L M G o a t a n t i - m o u s e IgG (H+L) conjugated to 5nm G o l d particles (British Biocell International, Cardiff, U K ) . used  for  flow  cytometry:  (monocyte)  anti-human  T h e following antibodies w e r e  C D 1 4 conjugated  with  P E ; (T  lymphocytes) anti-human C D 4 conjugated with P E , anti-human C D 8 conjugated with F I T C , anti-human C D 3 P e r C P ; (B lymphocytes) anti-human C D 1 9 conjugated with A l l o p h y c o c y a n i n ; ( N K cells) anti-human C D 1 6 + 5 6 conjugated with P E , anti-human C D 3 conjugated with F I T C .  All A b s are m o n o c l o n a l m o u s e A b s from B D B i o s c i e n c e s  ( M i s s i s s a u g a , Ontario). 31  2.4  S E M I - Q U A N T I T A T I V E R E V E R S E T R A N S C R I P T I O N  P C R  Confluent H B M E C a n d H U V E C cultures w e r e grown on 6 0 m m diameter fibronectin or c o l l a g e n - c o a t e d plates.  T h e cultures w e r e c o i n c u b a t e d for 24 hrs with  100 U/ml T N F - a a n d 5 0 0 U / m l IFN-y.  E C w e r e lysed o n the plate using the R N e a s y  Protect Mini Kit ( Q i a g e n Inc.,  M i s s i s s a u g a , O N ) a c c o r d i n g to the  manufacturer's  r e c o m m e n d a t i o n s . 2-5ug of total R N A w a s reverse transcribed using Omniscript R T Kit (Qiagen Inc., M i s s i s s a u g a , O N ) with Oligo d T primers (Invitrogen, Burlington, O N ) for 60 minutes at 37°C.  1-5 y g of reverse-transcribed c D N A w a s amplified by P C R using  specific s e n s e a n d a n t i s e n s e primers for p r o t e a s o m e subunit l o c u s P S M B 6 [61], P S M B 9 [S1i], P S M B 7 [62], P S M B 1 0 [B2i], P S M B 5 [65], P S M B 8 [B5i] a n d h o u s e k e e p i n g g e n e G A P D H (Table 1).  P r i m e r s w e r e d e s i g n e d by online program P r i m e r 3 (Whitehead  Institute for B i o m e d i c a l R e s e a r c h ) and p u r c h a s e d from Invitrogen (Burlington, O N ) .  For  the P C R reaction, 1uL of reverse transcribed product w a s mixed with 2 u L of 1 0 u M of e a c h primer sets listed a b o v e along with 1x P C R buffer, 0 . 2 m M d N T P , 2 u M M g C I (all 2  from A p p l i e d B i o s y s t e m s , F o s t e r City, C A ) , 1.25 p o l y m e r a s e (Amplitaq G o l d , Perkin-Elmer).  units of A m p l i T a q G o l d ™ D N A  T h e c y c l e p a r a m e t e r s are: initial hot start  for 2 minutes of 94 °C, 2 5 - 3 5 c y c l e s of 4 5 s e c o n d s of denaturation at 94 °C, 30 s e c o n d s of annealing at temperature listed in Table 1, a n d 30 s e c o n d s of e x t e n s i o n at 72 °C, a n d a final 5 minute e x t e n s i o n period at 72 °C after the c y c l e s .  T h e incubation condition is  as  product  follows:  A  10ul  s a m p l e of e a c h  P C R reaction  was  run  on  6%  polyacrylamide g e l s , stained with ethidium bromide, a n d v i s u a l i z e d by under U V light. Amplification of c D N A with s e n s e and a n t i s e n s e primers of the h o u s e k e e p i n g g e n e G A P D H s e r v e d a s internal control.  F o r negative controls, total cellular R N A without  reverse transcription w a s amplified with all d e s i g n e d primers in order to confirm the 32  a b s e n c e of contaminating g e n o m i c D N A . computer program results.  B a n d intensities w e r e m e a s u r e d using the  ImageJ (NIH) for semi-quantitative  a s s e s s m e n t of the  RT-PCR  P r o t e a s o m e subunit band intensities w e r e divided by the G A P D H  band  intensities to obtain a ratio of band intensity normalized by G A P D H R N A e x p r e s s i o n .  T A B L E 1: P C R PRIMER S E Q U E N C E S GENE  PRODUCT  PRIMER SEQUENCE  SIZE G A P D H sense  5'-GCCAGTAGAGGCAGGGATGATGTTC-3'  G A P D H antisense  5'-CCATGTTCGTCATGGGTGTGAACCA-3'  /35 sense  5'-TCAGTGATGGTCTGAGCCTG-3'  /3 5 antisense  5'-ATGGTGCCTAGCAGGTATGG-3'  /31 sense  5'-CAGAACAACCACTGGGTCCT-3'  /? 1 antisense  5'-TCCCGGTAGGTAGCATCAAC-3'  /32 sense  5'-AAGGGATGGTTGTTGCTGAC-3'  /32 antisense  5'-GATTCTTGGCTTCCTCCTCC-3'  05\ sense  5'-ATGGAGTGATTGCAGCAGTG-3'  y35i antisense  5'-AGCCACAGATCATACTGCCC-3'  /31i sense  5'-CATCATGGCAGTGGAGTTTG-3'  131i antisense  5'-ATGCTGCATCCACATAACCA-3'  /32i sense  5'-GGGCTTCTCCTTCGAGAACT-3'  /32i antisense  5'-AACGCGTGTAGCTCCATCTT-3'  2.5  ANNEALING  (bp)  TEMP. CC)  272  57  208  60  381  60  396  60  282  60  419  60  291  60  C E L L VIABILITY A S S A Y S  2.5.1 MTT (Thiazolyl Blue Tetrazolium Bromide) cytotoxicity assay It w a s important to determine the toxicity of lactacystin o n H B M E C cultures before performing a s s a y s s u c h a s E L I S A , which d e p e n d upon the cell viability of the monolayer. Confluent H B M E C  cultures w e r e treated with titrated concentrations of lactacystin  ( 7 . 5 u M , 1 5 u M , 3 0 u M a n d 6 0 u M ) for 24 and 4 8 h .  E a c h treatment w a s performed in  triplicate wells. T h e cell morphology w a s monitored under the m i c r o s c o p e to evaluate the effect of the drug.  M T T a s s a y s were performed at the e n d of the incubation period 33  to determine the viability of the cells. 1mg/ml of M T T for 2 h .  T h e cultures w e r e w a s h e d a n d incubated in  T h e cells w e r e then w a s h e d with P B S followed by 10-15  minutes isopropanol extraction to d i s s o l v e the f o r m a z a n crystals.  T h e isopropanol  extracted f o r m a z a n w a s transferred to another 96 well plate for spectre-photometric quantification at a w a v e l e n g t h of 5 7 0 n m .  2.5.2 L I V E / D E A D ®  Cell-Mediated Cytotoxicity  A s s a y  Confluent H U V E C a n d H B M E C m o n o l a y e r s w e r e treated with lactacystin (7.5 - 90 MM),  IFN-y (200U/ml), or T N F - a (100U/ml) for 2 4 - 4 8 h .  T h e m o n o l a y e r viability w a s  m e a s u r e d using the L I V E / D E A D ® C e l l - M e d i a t e d Cytotoxicity Kit (Molecular P r o b e s Invitrogen, Burlington, Ontario) following the manufacturer's instructions. m o n o l a y e r s w e r e w a s h e d twice with 37 °C D u l b e c c o ' s P B S .  Briefly, the  C a l c e i n A M (2.0 uM) a n d  Ethidium homodimer-1 ( E t h D - 1 , 4 u M ) w e r e mixed with D u l b e c c o ' s P B S and b e c o m e working solution.  T h e working solution w a s a d d e d to e a c h well of a 96 well plate a n d  incubated for 4 5 minutes at 37 °C.  T h e morphology of cultured cells w a s e x a m i n e d  using a n inverted m i c r o s c o p e with epi-fluorescence a n d a long p a s s blue excitation f l u o r e s c e n c e filter with excitation wavelength of 4 9 5 n m .  Pictures w e r e taken from  e m i s s i o n s of C a l c e i n A M a n d EthD-1 at 5 1 7 n m a n d 6 1 7 n m respectively.  2.6 2.6.1  I M M U N O C Y T O C H E M I S T R Y I m m u n o g o l d Silver Staining (IGSS) for Surface Localization of M H C class I  and E C adhesion  molecules  Untreated, cytokine-treated, and/or lactacystin-treated E C m o n o l a y e r s were w a s h e d briefly with w a r m phosphate-buffered saline ( P B S , p H 7.4) containing 1% bovine s e r u m albumin ( B S A , S i g m a C h e m i c a l C o . ) , 1% normal goat s e r u m ( N G S ) a n d 0 . 0 5 % s o d i u m 34  azide (NaN ). 3  C u l t u r e s w e r e incubated with primary A b s (10ug/ml in carrier buffer  consisting of P B S , 5 % B S A , 4 % N G S and 0 . 0 5 % N a N ) for 6 0 minutes, w a s h e d with 3  w a s h buffer a n d incubated with s e c o n d a r y A b s conjugated to 5 n m gold particles for 60 min.  After  several  additional  washes,  the  cells  were  fixed  with  buffered  f o r m a l d e h y d e / a c e t o n e for 30 s e c o n d s a n d then incubated with silver e n h a n c e m e n t solution ( A m e r s h a m Life S c i e n c e s , B u c k i n g h a m s h i r e , E n g l a n d ) .  T h e precipitation of  metallic silver on the colloid gold particles w a s monitored under a m i c r o s c o p e . nuclei w e r e stained with G i e m s a .  Cell  Controls included m o n o l a y e r s incubated with carrier  buffer, irrelevant primary antibodies (anti-human L C A ) or normal m o u s e IgG.  T h e cells  were e x a m i n e d under a Nikon L a b o p h o t light m i c r o s c o p e .  2.6.2  I m m u n o g o l d  Proteasome  Silver  Staining  (IGSS)  for  Intracellular  Localization o f  Subunits  Confluent m o n o l a y e r s w e r e w a s h e d with P B S a n d p e r m e a b i l i z e d with buffered f o r m a l d e h y d e / a c e t o n e containing 0 . 0 3 % Triton X - 1 0 0 for 10 minutes.  After several  w a s h e s , the culture w e r e incubated with primary A b s (10ug/ml in carrier buffer consisting of PBS,  5 % B S A , a n d 4 % N G S ) for 1h at room temperature followed by incubation with  s e c o n d a r y A b s (10nm gold conjugated goat a n t i - m o u s e A b , A m e r s h a m  International).  Silver e n h a n c e m e n t solution w a s applied and metallic silver precipitation w a s monitored. Nuclei w e r e stained with G i e m s a .  Controls included m o n o l a y e r s incubated with carrier  buffer, irrelevant primary antibody (anti-human L C A ) or normal m o u s e IgG. were e x a m i n e d under a Nikon Labophot light m i c r o s c o p e .  2.7  E N Z Y M E - L I N K E D I M M U N O S O R B E N T A S S A Y  2.7.1 S u r f a c e  Expression 35  (ELISA)  T h e cells  HBMEC untreated  grown to c o n f l u e n c e on triplicate wells of 96-well plates were u s e d  or treated  with  IFN-y (200-500  U/ml)  up to 4 8 h .  Following  cytokine  stimulation, m o n o l a y e r s w e r e fixed in 0 . 0 2 5 % glutaraldehyde a n d then incubated with primary A b (5ug/ml) against h u m a n M H C c l a s s I for 60 minutes at room temperature followed by H R P - c o n j u g a t e d goat anti-mouse IgG for 1 h.  2mg/ml o-phenylenediamine  ( O P D ) w a s a d d e d to allow colour d e v e l o p m e n t a n d the reaction w a s s t o p p e d with 2 M sulfuric a c i d . monolayers  A b s o r b a n c e w a s read on a plate reader at 4 9 0 n m . incubated with carrier buffer, irrelevant  primary  Controls included  antibody  (anti-human  leukocyte c o m m o n antigen) or normal m o u s e IgG.  2.7.2 Intracellular HBMEC  Expression  grown to c o n f l u e n c e on triplicate wells of 96-well plates were u s e d  untreated or treated with IFN-y (200-500 U/ml) for up to 4 8 h .  Following cytokine  stimulation, m o n o l a y e r s w e r e fixed in 1 0 0 % methanol followed by incubation with e n d o g e n o u s p e r o x i d a s e blocking solution (97% methanol a n d 3 % H 0 ) for 30 minutes. 2  2  T h e cultures w e r e then incubated with primary A b s against h u m a n p r o t e a s o m e subunits a 5 , 61 i a n d B5i at 5|jg/ml for 60 minutes at R T followed by p e r o x i d a s e - c o n j u g a t e d goat a - m o u s e IgG ( J a c k s o n I m m u n o R e s e a r c h ) for 1 h.  2mg/ml o-phenylenediamine (OPD)  w a s a d d e d to allow colour d e v e l o p m e n t a n d the reaction w a s s t o p p e d with 2 M sulfuric acid.  A b s o r b a n c e w a s read on a n E L I S A microtitre plate reader at a wavelength of  490nm.  Controls included m o n o l a y e r s incubated with carrier buffer, irrelevant primary  antibody (anti-human L C A ) or normal m o u s e IgG.  2.8  I S O L A T I O N A N D C H A R A C T E R I Z A T I O N O F C D 8 + TL Y M P H O C Y T E S Peripheral blood m o n o n u c l e a r cells ( P B M C ) w e r e isolated from v e n o u s blood of 36  healthy adult volunteers. equal volume  of  B l o o d w a s drawn into S o d i u m heparin tubes, diluted with  P B S , layered  over  centrifuged at 4 0 0 x g for 30 minutes.  Histopaque-1077  (1.077  g/ml,  Sigma)  and  T h e layer containing white blood cells w a s  s u s p e n d e d in P B S a n d centrifuged at 2 5 0 x g for 10 minutes. w a s h e s w e r e carried out to remove platelets.  Additional 2 to 3 P B S  C D 8 + T cells w e r e s e p a r a t e d from P B M C  using H u m a n C D 8 + T cell R e c o v e r y C o l u m n Kits ( C e d a r l a n e , Hornby, O N ) .  T h e T cell  recovery c o l u m n s w e r e prepared by w a s h i n g with buffer ( P B S + 2 % fetal calf s e r u m ( F C S ) ) before addition of P B M C , followed by incubation with polyclonal goat anti-mouse a n d goat anti-human IgG A b s ( C o l u m n R e a g e n t ) for a minimum of 1h.  P B M C were  treated with m o u s e anti-human C D 4 A b s (Cell R e a g e n t ) in buffer for 30 minutes on ice at 4°C with agitation. centrifugated  After the incubation, the cell s u s p e n s i o n w a s w a s h e d twice a n d  at 2 5 0 x g for 5-10 minutes at 4°C.  P B M C w e r e r e s u s p e n d e d at a  concentration of 5 x 1 0 cells/ml a n d placed on the c o l u m n s . 7  centrifuged  Eluted C D 8 + T cells w e r e  at 2 5 0 * g for 5-10 minutes, s u s p e n d e d in R P M I ,  a n d frozen in 4 5 %  R P M I / 4 5 % h u m a n A B s e r u m / 1 0 % D M S O at a final concentration of 3 - 5 x 1 0 cell/ml. 6  F r o z e n C D 8 + T cells w e r e thawed a n d w a s h e d twice with 1 x H a n k ' s B a l a n c e Salt Solution ( H B S S , G i b c o ) without calcium a n d m a g n e s i u m at 2 5 0 x g for 10 minutes, resuspended  in  complete  medium  consisting  of  RPMI  supplemented  with  2mM  L-glutamine, 1 0 0 U / m l penicillin, 100ug/ml streptomycin, 0.25ug/ml amphotericin B a n d 10%  A B serum and counted.  flow cytometry.  Isolated T cells w e r e a n a l y z e d for C D 8 + T cell purity by  T h e purity of C D 8 + T cells w a s >95% with minimal contamination by  C D 1 6 + C D 5 6 + N K - c e l l s , C D 4 + T lymphocytes, C D 1 9 + B cells a n d C D 1 4 + C D 4 5 + monocytes.  2.9  C D 8 + T L Y M P H O C Y T E A D H E S I O N T O H B M E C 37  M O N O L A Y E R S  H B M E C grown to c o n f l u e n c e in triplicate fibronectin c o a t e d wells of 96-well plates w e r e treated with T N F - a , IFN-y a n d combination of IFN-y a n d lactacystin for 4, 24 or 4 8 h .  In s o m e wells, 10mg/ml a n t i - M H C c l a s s I A b w a s a d d e d 1h prior to the  a d h e s i o n a s s a y to block c l a s s I M H C m o l e c u l e s on H B M E C .  5x10  5  resting C D 8 + T  cells w e r e a d d e d to e a c h well a n d incubated with H B M E C for 1 h at 37°C.  A t the end of  the incubation period non-adherent T cells w e r e r e m o v e d by 3 gentle w a s h e s with M 1 9 9 a n d 1 w a s h with P B S .  M o n o l a y e r s with adherent T cells w e r e fixed with ice cold  acetone:ethanol (50:50) for 7 min.  T cells w e r e stained for L C A using the  i m m u n o p e r o x i d a s e technique a s previously d e s c r i b e d [49]. counter-stained with haemotoxylin.  indirect  T cells a n d E C w e r e  A d h e r e n t C D 8 + T cells w e r e e n u m e r a t e d  by  counting the n u m b e r of L C A positive cells per millimetre s q u a r e (mm ) of the monolayer 2  surface a r e a in four peripheral and o n e central fields with a 2 0 * objective lens using a n ocular grid.  2.10  C o n t r o l s included unstimulated cerebral E C .  D A T A C O L L E C T I O N A N D  STATISTICS  Data from E L I S A experiments w e r e a n a l y z e d by a n a l y s i s of variance ( A N O V A ) . Non-parametric  Students' t tests were a l s o u s e d w h e n differences were found.  data w e r e  M a n n - W h i t n e y U - tests.  analysed  using  Kruska-Wallis A N O V A  on  Ranks  Significant differences b e t w e e n cytokine treated,  and  inhibitor  treated and control groups are s h o w n a s (*) w h e r e p<0.05.  C H A P T E R 3: R E S U L T S  3.1  H U M A N  B R A I N M I C R O V E S S E L E N D O T H E L I A L  C E L L S  ( H B M E C )  Primary cultures of H B M E C form confluent m o n o l a y e r s (Fig. 1a) after 8-11 d a y s in 38  culture.  Strong perinuclear granular staining for F a c t o r V l l / v o n W i l l e b r a n d antigen (Fig.  1b) a n d binding of Ulex E u r o p a e u s agglutinin (Fig. 1c) indicate the endothelial nature of t h e s e cells.  P r e v i o u s studies from this laboratory h a v e s h o w n that this in vitro model  retains important morphological a n d functional characteristics of the B B B in vivo, mainly: 1)  presence  of  tight  junctions  of  high  electrical  resistance  that  transendothelial p a s s a g e of horseradish p e r o x i d a s e , a n d 2) paucity of  restrict  the  pinocytotic  v e s i c l e s [135].  3.2  H U M A N U M B I L I C A L VEIN E N D O T H E L I A L C E L L S  ( H U V E C )  H U V E C cultures in this study w e r e maintained under identical conditions a s H B M E C cultures.  This  allows  direct  extracerebral e n d o t h e l i u m .  comparisons between  brain  m i c r o v e s s e l and  large  T h e purity and endothelial origin of t h e s e cells w a s  determined using the s a m e markers a s for H B M E C .  3.3 3.3.1  P R O T E A S O M E S U B U N I T E X P R E S S I O N B Y R N A expression RNA  detected  by semi-quantitative  H B M E C R T - P C R  extracted from confluent m o n o l a y e r s of H U V E C a n d H B M E C grown on 6 0 m m  collagen  or fibronectin  expression  pattern  of  coated  plates, respectively, w a s  proteasome  subunits.  R N A from  u s e d to both  investigate  unstimulated  the and  cytokine-treated cultures (100 U/ml T N F - a a n d 2 0 0 U/ml IFN-y) w a s extracted after 24h treatment.  P r o t e a s o m e subunit g e n e primers (Table 1) w e r e u s e d to amplify the  specified length of fragments.  R T - P C R analysis of constitutive p r o t e a s o m e subunits in  primary cultures of H B M E C revealed constitutive e x p r e s s i o n of 61 ( P S M B 6 ) , p2 ( P S M B 7 ) and  65 ( P S M B 5 ) subunits in unstimulated cerebral E C (Figure 2a). Following cytokine  treatment, the level of 61 a n d p2 subunit e x p r e s s i o n r e m a i n e d the s a m e , w h e r e a s there 39  was  a slight d e c r e a s e of the (35 subunit.  In contrast, there w a s a strong upregulation of  the inducible subunits (31 i ( P S M B 9 ) , (32i ( P S M B 1 0 ) a n d a slight i n c r e a s e of (35i ( P S M B 8 ) following cytokine stimulation of H B M E C a s c o m p a r e d to the unstimulated cultures. Semi-quantitive a n a l y s i s of the R T - P C R results s h o w s that cytokine stimulated H B M E C exhibited a two-fold i n c r e a s e of the [31 i subunit R N A e x p r e s s i o n , a three-fold i n c r e a s e of the (32i subunit R N A e x p r e s s i o n and a slight upregulation of the B5i subunit R N A a s c o m p a r e d with the unstimulated cultures (Figure 2b). immunoproteasome proinflammatory  subunits  cytokines.  p r o t e a s o m e subunits after  of  HBMEC  HUVEC  T h e results indicate that the  are upregulated  also  showed  cytokine stimulation  at the  upregulation  a n d maintained  mRNA  level  of  inducible  all  by  a similar level of  e x p r e s s i o n of constitutive p r o t e a s o m e subunits in unstimulated a n d stimulated cultures (Figure 2c).  Semi-quantitive analysis of the R T - P C R  results s h o w s that cytokine  stimulated H U V E C d i s p l a y e d a three-fold i n c r e a s e of the (31 i a n d (32i R N A e x p r e s s i o n and  a two-fold i n c r e a s e of (35i R N A e x p r e s s i o n a s c o m p a r e d with the unstimulated  cultures (Figure 2d).  A n a l y s i s of the G A P D H internal control g e n e revealed that similar  amounts of c D N A w e r e a d d e d to the P C R reactions within the s a m p l e s in both cultures.  3.3.2 Intracellular p r o t e a s o m e s u b u n i t protein e x p r e s s i o n b y  H B M E C  Both intracellular i m m u n o g o l d silver staining a n d intracellular E L I S A w a s u s e d to demonstrate p r o t e a s o m e subunit protein e x p r e s s i o n by confluent m o n o l a y e r s of H B M E C following IFN-y (200U/ml) stimulation. for 24h w e r e u s e d for t h e s e studies.  Unstimulated cells a n d cells treated with IFN-y Both studies s h o w e d that the  constitutive  p r o t e a s o m e subunit a 5 is e x p r e s s e d at a b a s a l level by H B M E C without significant upregulation (113%) after stimulation with IFN-y for 2 4 h (Figures 3 a & 3b, panels A & B). The  inducible subunits w e r e constitutively e x p r e s s e d by unstimulated H B M E C . 40  After  incubation with IFN-y for 2 4 h, 61 i w a s significantly upregulated by 1 6 7 % and B5i w a s also significantly upregulated by 1 4 9 % (Figures 3 a & 3b, p a n e l s C - F ) . silver  staining  upregulation  of  of the  HBMEC  monolayers  further  confirmed  inducible subunits a s indicated by the  cytoplasmic staining (Figure 3b).  the  Immunogold  cytokine-induced  i n c r e a s e d intensity  of  T h e r e w a s no difference in staining intensity for the  a 5 subunit after cytokine treatment.  Staining for the inducible subunits 61 i and 65i w a s  mostly perinuclear w h e r e a s the staining for the a 5 subunit w a s uniform throughout the c y t o p l a s m (Figure 3b, panel A & B).  Negative controls for t h e s e experiments, including  normal m o u s e IgG, irrelevant A b ( m o u s e a - h u m a n L C A ) , a n d carrier buffer, s h o w e d no staining (data not shown).  3.4 3.4.1  C E L L VIABILITY A S S A Y S F O R L A C T A C Y S T I N M T T cytotoxicity  T R E A T E D  H B M E C  assay  Mitochondrial d e h y d o g e n a s e conversion of MTT, or the M T T cytotoxicity assay, w a s u s e d to evaluate the viability of the confluent m o n o l a y e r s of H B M E C after lactacystin treatment at different concentration for different lengths of time.  T i m e point experiments  of lactacystin at various concentrations s h o w e d that about 8 0 % of a b s o r b a n c e remained in the culture after 2 4 h treatment with 7 . 5 u M lactacystin (Figure 4a). about 7 0 % after 4 8 h treatment.  T h i s dropped to  Higher concentrations ( 1 5 u M a n d a b o v e ) of lactacystin  for 24h resulted in reduction of a b s o r b a n c e to 5 0 % or l e s s a s c o m p a r e d to untreated cultures. between  E x a m i n a t i o n by light m i c r o s c o p y s h o w e d c o m p a r a b l e numbers of untreated  cultures a n d cultures treated  with 7 . 5 u M  cells  lactacystin for 4 8 h .  Therefore, the optimal concentration w a s set at 7 . 5 u M , s i n c e lower concentration had no significant effect a s s e e n in later experiments (data not shown).  Accordingly, the  optimal concentration a n d incubation time c h o s e n w a s 7.5u.M for 4 8 h for the blocking 41  p r o t e a s o m e e x p e r i m e n t s d e s c r i b e d under A i m s #2 a n d 3.  3.4.2 L I V E / D E A D ® C e l l - M e d i a t e d C y t o t o x i c i t y  A s s a y  C e l l viability w a s further confirmed with the LIVE/DEAD® C e l l - M e d i a t e d Cytotoxicity Kit from M o l e c u l a r P r o b e s .  T h i s test u s e s both calcein a n d E t h D - 1 .  C a l c e i n is a green  fluorescent d y e converted by live cell e s t e r a s e , w h e r e a s EthD-1 enters cells with damaged  membranes and  binds to  nucleic a c i d s ,  resulting  in red f l u o r e s c e n c e .  Untreated H B M E C a n d cells treated with both IFN-y s h o w e d g r e e n c y t o p l a s m i c staining (Figure 4 b . p a n e l A - B ) .  C u l t u r e s treated with 7 . 5 u M lactacystin d i s p l a y e d an overall  green f l u o r e s c e n c e after 2 4 - 4 8 h of treatment (Figure 4 b . p a n e l C ) .  In cultures treated  with higher concentrations (15-90uM) of lactacystin for 4 8 h most cells s h o w e d red nuclear f l u o r e s c e n c e indicating d e c r e a s e in viability (Figure 4 b . panel D-E). confirmed the optimal concentration of 7.5 u M .  T h i s further  Control included cells treated with 7 0 %  methanol that s h o w e d 1 0 0 % red nuclear f l u o r e s c e n c e (Figure 4 b . panel F).  3.5  S U R F A C E E X P R E S S I O N O F M H C C L A S S IM O L E C U L E S B Y  H B M E C  S u r f a c e E L I S A a n d surface immunogold silver staining w e r e u s e d to determine the level of M H C c l a s s I m o l e c u l e s e x p r e s s e d by confluent H B M E C m o n o l a y e r s before a n d after treatment with IFN-y for 2 4 - 7 2 h , a n d a combination of IFN-y with lactacystin for 24-72h.  T h e r e w a s a b a s a l e x p r e s s i o n of M H C c l a s s I in the unstimulted cultures  (Figure 5, panel A ) .  Treatment of H B M E C cultures with IFN-y (200U/ml) for 24-72h  induced upregulation of M H C c l a s s I e x p r e s s i o n that w a s time-dependent with m a x i m a l levels at 7 2 h (Figure 5). Immunogold silver staining confirmed a n overall increase of staining intensity for M H C c l a s s I after 4 8 h treatment with IFN-y in a time-dependent m a n n e r (Figure 5, panel B).  Co-incubation with IFN-y (200U/ml) a n d lactacystin for 4 8 h 42  downregulated M H C c l a s s I e x p r e s s i o n to a level similar to that of unstimulated cultures in a time-dependent m a n n e r (Figure 5).  This w a s confirmed by a moderate d e c r e a s e in  staining intensity by the immunogold silver staining (Figure 5, panel C ) .  Controls  incubated with irrelevant A b s a n d carrier buffer had no noticeable staining (data not shown).  3.6  S U R F A C E E X P R E S S I O N  O F A D H E S I O N M O L E C U L E S  B Y  H B M E C  S u r f a c e E L I S A a n d surface immunogold silver staining w e r e u s e d to determine the levels of I C A M - 1 , V C A M - 1 a n d E-selectin e x p r e s s i o n by confluent H B M E C monolayers before a n d after treatment with T N F - a for 2 4 h , a n d a combination of T N F - a with lactacystin for 2 4 h .  R e s u l t s from both surface E L I S A a n d I G S S s h o w e d that treatment  with T N F - a resulted in significant upregulation of E - s e l e c t i n , I C A M - 1 a n d V C A M - 1 in primary  HBMEC  cultures.  Co-incubation of T N F - a  with  lactacystin  significantly  downregulated the T N F - a - induced upregulation of E-selectin a n d V C A M - 1 , w h e r e a s downregulation of I C A M - 1 w a s less p r o n o u n c e d . results.  I G S S results supported the E L I S A  T h u s , treatment with T N F - a resulted in a significant i n c r e a s e in surface staining  for I C A M - 1 , V C A M - 1 a n d E-selectin (Figure 6b, p a n e l A - B , D - E , G - H ) .  Co-incubation  with T N F - a a n d lactacystin r e d u c e d the intensity of staining significantly (Figure 6b, panel C , F, I).  Immunostaining of unstimulated H B M E C for I C A M - 1 w a s significantly  more intense in about 4 0 % of cells c o m p a r e d with only scattered cells positive for V C A M - 1 , w h e r e a s E-selectin staining s h o w e d only rare positive cells.  After T N F - a  stimulation for a m a x i m a l time period, 1 0 0 % of H B M E C s h o w e d strong upregulation of ICAM-1 a s s h o w n by the intense staining of positive cells. upregulation  of V C A M - 1  and  heterogeneity b e t w e e n cells.  E-selectin, but  to  a  T h e r e w a s a l s o significant  lesser  extent,  with  apparent  Co-incubation with lactacystin resulted in an overall 43  d e c r e a s e in intensity of staining for I C A M - 1 .  V C A M - 1 a n d E-selectin w e r e significantly  downregulated a s s h o w n by E L I S A data a n d by the d e c r e a s e in the number of cells e x p r e s s i n g t h e s e m o l e c u l e s a n d the reduced intensity of staining of the positive cells.  3.7  C D 8  +  TL Y M P H O C Y T E A D H E S I O N T O  H B M E C  C D 8 + T cell a d h e s i o n to resting H B M E C w a s minimal (Figure 7 a , panel A ) . A d h e r e n t T cells localized mostly around the nuclear a r e a of E C , indicating that the C D 8 + T C a d h e r e d to the E C in a non-random m a n n e r (Figure 7 a , panel A - D ) . Incubation with IFN-y for 4 8 h upregulated C D 8 + T cell a d h e s i o n by 2 0 % (Figure 7 a a n d panel A & B ) .  C o i n c u b a t i o n with lactacystin completely inhibited this  (Figure 7 a a n d p a n e l C ) .  upregulation  M o n o c l o n a l A b blocking of M H C c l a s s I o n H B M E C 30  minutes prior to the a d h e s i o n a s s a y resulted in significant d e c r e a s e in a d h e s i o n e v e n after IFN-y stimulation for 2 4 h (Figure 7 a a n d panel D).  Stimulation with T N F - a  (100U/ml) for 2 4 h a n d 18h upregulated C D 8 + T cell a d h e s i o n significantly w h e r e a s 4 h treatment s h o w e d minimal upregulation of a d h e s i o n (Figure 7 c ) . 100U/ml T N F - a  a n d lactacystin significantly  downregulated  C o i n c u b a t i o n with  the T N F - a  - induced  upregulation of C D 8 + T cell a d h e s i o n after 2 4 h incubation (Figure 7c).  C H A P T E R 4 : DISCUSSION  4.1  H B M E C A S A N in vitro M O D E L O F T H E A  B B B  wide range of neurological disorders are c h a r a c t e r i z e d by the migration of  circulating white blood cells a c r o s s the B B B that normally restricts their entry into the CNS.  In vitro m o d e l s of the B B B offer a unique opportunity to study brain E C - l e u k o c y t e  interactions without the complexity of factors involved in vivo. 44  O u r laboratory h a s  d e v e l o p e d a n d fully c h a r a c t e r i z e d a n in vitro m o d e l of the h u m a n B B B that consists of primary cultures of H B M E C that retain E C properties a s well a s important morphological and  functional characteristics of the h u m a n B B B in situ.  T h e s e include e x p r e s s i o n of  Factor VIIIR: A n t i g e n , binding of the Ulex e u r o p a e u s lectin, p r e s e n c e of tight junctional complexes  between  adjacent  E C that  restrict  t h e paracellular  movement  of  m a c r o m o l e c u l e s , paucity of c y t o p l a s m i c v e s i c l e s a n d a b s e n c e of a vesicular transport system.  T h i s in vitro s y s t e m h a s b e e n u s e d reproducibly to study the r e s p o n s e s of  t h e s e E C to cytokine activation a n d their role in the trafficking of leukocytes a c r o s s the BBB  in C N S inflammation.  4.2  E X P R E S S I O N O F P R O T E A S O M E S U B U N I T S B Y The  H B M E C  present studies demonstrate that recombinant h u m a n IFN-y is c a p a b l e of  inducing upregulation of the i m m u n o p r o t e a s o m e subunits by H B M E C in primary culture. At the m R N A level, unstimulated H B M E C grown under standard conditions e x p r e s s b a s a l level of the consititutive p r o t e a s o m e subunits (31, (32 a n d (35 a n d the inducible subunits (31 i, (32i a n d (35i.  After treatment with T N F - a a n d IFN-y, t h e 6 1 , 6 2 and (35  subunits s h o w e d no significant upregulation. B2i,  E x p r e s s i o n of the inducible subunits (31 i,  and 65i w a s significantly upregulated after cytokine treatment although the (35i had a  slightly higher b a s a l level than other inducible subunits.  T h e e x p r e s s i o n profiles of the  inducible subunits w e r e similar between H B M E C a n d H U V E C . However, differences w e r e found b e t w e e n t h e b a s a l level of subunit e x p r e s s i o n .  T h e results suggest that  both large v e s s e l E C a n d cerebral m i c r o v e s s e l E C regulate p r o t e a s o m e subunit m R N A expression  similarly  in r e s p o n s e to cytokine  e x p r e s s i o n w a s d e m o n s t r a t e d with intracellular  stimulation.  Intracellular  E L I S A a n d confirmed with  protein IGSS.  Stimulated H B M E C cultures s h o w e d a trend towards upregulation of the a 5 subunits by 45  ELISA,  however,  the  IGSS  showed  a  unstimulated and IFN-y treated cultures.  comparable  staining  intensity  between  S i n c e a 5 exists in both constitutive  and  inducible p r o t e a s o m e s , results from both I G S S and E L I S A indicate that the total population of p r o t e a s o m e remained constant after IFN-y stimulation.  O n the other h a n d ,  both (31 i and (35i subunits w e r e upregulated in IFN-y stimulated cultures a s s h o w n by E L I S A and I G S S .  S i n c e the population of the p r o t e a s o m e after IFN-y  treatment  remains constant, the i n c r e a s e d e x p r e s s i o n of the inducible subunits s u g g e s t s a shift of the intracellular p r o t e a s o m e population from the constitutive to the inducible form. P r o t e a s o m e studies in the C N S are extensive but quite diverse in focus.  T h e y vary  from effective markers for d i s e a s e s s u c h a s Huntington's d i s e a s e and P a r k i n s o n ' s d i s e a s e , to apoptotic m e c h a n i s m s of neurodegerative d i s e a s e s a n d inhibition of the ubiquitin-proteasome pathway against stroke.  In addition, the p r o t e a s o m e h a s b e e n  described a s a n autoantigen targeted by humoral immunity that c a n serve a s a marker for d i s e a s e progression in multiple s c l e r o s i s [137].  A t present, the involvement of the  proteasome in C N S a u t o i m m u n e d i s e a s e s is poorly understood.  A l t h o u g h the BBB has  been the f o c u s of studies into the pathogenesis of neuroinflammatory d i s e a s e s , the e x p r e s s i o n of the p r o t e a s o m e at the blood-brain barrier, its regulation by cytokines and its role in C N S inflammation  has not b e e n previously investigated.  Furthermore,  studies on p r o t e a s o m e subunit e x p r e s s i o n by large v e s s e l E C are limited. proteasome extracerebral  is well-known tissues.  a s a m e m b e r of the antigen Studies  in  vivo s h o w  that  presentation  The  pathway  dysregulation  of  in the  i m m u n o p r o t e a s o m e is a risk factor of autoimmune d i s e a s e s s u c h a s type I diabetes [138].  A similar p r o t e a s o m e - p r o c e s s i n g error in the activation of the NFKB pathway w a s  found in both (31 i knock-out mice and n o n - o b e s e diabetic m i c e ( N O D ) , the murine model of type I diabetes [139].  A h u m a n renal c a r c i n o m a cell line s h o w e d upregulation of both 46  m R N A a n d protein levels of the inducible subunits by IFN-y without additional effect on constitutive subunit e x p r e s s i o n [140].  In the murine s y s t e m , the i m m u n o p r o t e a s o m e is  nearly a b s e n t in m o u s e brain extracts, while in the s p l e e n e x p r e s s i o n is high, supporting the postulate that the brain is an immune-privileged site [141].  G e n e t i c studies s h o w  that the murine B2i g e n e c a n a l s o be regulated by IFN-y reciprocally with high similarity at the promoter region with the h u m a n p r o t e a s o m e [142].  T h i s indicates that IFN-y is  the main cytokine r e s p o n s i b l e for upregulation of i m m u n o p r o t e a s o m e subunit g e n e s in both h u m a n s a n d m i c e .  T h e e x p r e s s i o n of p r o t e a s o m e subunits by E C has not b e e n  fully investigated.  F o s s et al. studied the effect of a transfected promotional factor  protein  IFN-y signalling pathway that c a n simulate the effect of IFN-y  under the  upregulation of the B5i p r o t e a s o m e subunit in a h u m a n E C line [143].  A b a s a l level of  m R N A e x p r e s s i o n of the a 1 , a 2 , a 3 , a 4 a n d B2 p r o t e a s o m e subunits w a s found in H U V E C [144, 145], suggesting that large v e s s e l E C e x p r e s s the constitutive p r o t e a s o m e under normal conditions.  A limited number of studies s u g g e s t that the proteasome  m R N A e x p r e s s i o n profile in r e s p o n s e to IFN-y stimulation in E C h a s similarities a m o n g different cell types in both h u m a n a n d murine s y s t e m s .  A t the protein level, B1 i, B2i a n d  B5i upregulation w a s found in h u m a n c a r c i n o m a cells treated with IFN-y for 10 d a y s [146].  It h a s b e e n s u g g e s t e d that i m m u n o p r o t e a s o m e e x p r e s s i o n is important  i m m u n e system-related cells.  for  T h u s non-immune cell lines, s u c h a s c a n c e r a n d E C  lines, s h o w low constitutive B2i e x p r e s s i o n , while cell lines derived from m o n o c y t e s and B cells have a high consititutive level of B2i e x p r e s s i o n [147]. reported in murine studies showing greater constitutive subunits by thymic a n d s p l e e n i c cells.  Similar results w e r e  e x p r e s s i o n of 61 i and 65i  T h e high e x p r e s s i o n of 62i constitutively  by  immune-related cells m a y reflect functional differences b e t w e e n various cell types.  In  large v e s s e l E C s u c h a s H U V E C , treatment with IFN-y a l o n e upregulates the 65i subunit 47  most significantly [148].  T h e role of D C in the M H C c l a s s I presentation pathway h a s  b e e n well c h a r a c t e r i z e d particularly regarding the p r o t e a s o m e subunit e x p r e s s i o n . have b e e n s h o w n to upregulate the inducible subunits a s a result of maturation.  DC  Mature  D C usually h a v e i n c r e a s e d M H C c l a s s I e x p r e s s i o n that e n a b l e s them to present antigens for C D 8 + lymphocyte priming.  P r e v i o u s studies s h o w e d that the inducible  p r o t e a s o m e subunit (31 i is downregulated during the time w h e n M H C c l a s s I is highly expressed.  T h i s s u g g e s t s that the i m m u n o p r o t e a s o m e shift is n e c e s s a r y only during  the stage of peptide digestion, thus confirming its importance in M H C c l a s s I generation a n d e x p r e s s i o n [107]. after 4 8 h [106].  L P S stimulated L a n g e r h a n s D C upregulate (31 i a n d (35i m R N A  Following IFN-y or L P S stimulation mature D C have a n overall higher  e x p r e s s i o n of inducible subunit m R N A while the constitutive subunits remain mostly the s a m e [149]. proteasome  At the protein level, mature subunits  after  48h  of  D C s h o w upregulation  L P S treatment  [106].  of the It  inducible  appears  that  i m m u n o p r o t e a s o m e upregulation is most significant in D C d u e to their primary antigen presentation function.  I m m u n o p r o t e a s o m e s are mainly e n r i c h e d around the E R , while  the constitutive p r o t e a s o m e s are present in the n u c l e u s a n d throughout the cytoplasm [109].  It h a s b e e n s u g g e s t e d that E R accumulation of the i m m u n o p r o t e a s o m e  related to its c l o s e binding to the T A P transporter [109]. rat liver a n d h u m a n H e L a cells.  is  A similar localization is found in  T h i s indicates that IFN-y stimulation a n d D C maturation  leads to upregulation of the inducible subunits. T h e present studies s h o w that H B M E C e x p r e s s inducible subunits  after  IFN-y  stimulation.  Upregulation  characterizes D C w h i c h are professional A P C s .  of  inducible  immunoproteasome subunit  expression  T h u s , upregulation of the inducible  subunits in H B M E C by IFN-y m a y r e s e m b l e the ability of D C to produce M H C c l a s s l-peptide c o m p l e x e s .  In addition, perinuclear staining for the inducible subunits in 48  H B M E C in this study s u g g e s t s that stimulated H B M E C utilize the i m m u n o p r o t e a s o m e for peptide generation.  4.3  MHC C L A S S I M O L E C U L E E X P R E S S I O N Increased e x p r e s s i o n of M H C c l a s s I is believed to i n c r e a s e the probability for  cognate T C R - M H C  interaction.  B e n h a m et  al. s h o w e d  that the  IFN-y-induced  p r o t e a s o m e subunits are the rate-determining factor for M H C c l a s s I e x p r e s s i o n [102]. T h e y a l s o s h o w that M H C c l a s s I e x p r e s s i o n by IFN-y treated cells is downregulated by lactacystin, s u g g e s t i n g a d e c r e a s e in the stability of M H C c l a s s I e x p r e s s e d on the surface.  In addition, C D 8 + T lymphocytes c a n only initiate their effector function upon  encounter of the correct M H C c l a s s l-peptide c o m p l e x . p r o t e a s o m e on the  Studies on the effect  of  interactions between C T L a n d M H C c l a s s I e x p r e s s i n g cells  concentrate mainly on C T L cytotoxicity.  C e r u n d o l o et al. d e m o n s t r a t e d that lysis of  h u m a n c a r c i n o m a cell lines by influenza matrix epitope specific cytotoxic C T L is d e c r e a s e d w h e n the cell lines are treated with lactacystin [97].  IFN-y is required for  upregulating m e l a n o m a - a s s o c i a t e d antigen ( M A G E ) specific M H C c l a s s I e x p r e s s i o n in h u m a n tumor cells transfected with the precursor peptide s e q u e n c e M A G E - 3 [150]. This observation correlates with the upregulation a n d e x c h a n g e b e t w e e n p r o t e a s o m e a n d i m m u n o p r o t e a s o m e subunits by IFN-y s i n c e /3 5\ subunit substitution alone is sufficient to digest M A G E - 3 . peptide  from  mouse  K u c k e l k o r n et al s h o w e d that cells incubated with digested  small  intestine,  which  are  expressing  relatively  more  i m m u n o p r o t e a s o m e , i n c r e a s e the lysis by C T L specific for H S P 6 0 peptide  [151].  Respiratory epithelial cells a l s o upregulate (31 i a n d 35i subunits following infection with the respiratory syncytial virus a n d this upregulation l e a d s to the upregulation of M H C c l a s s I, which c a n be further downregulated by lactacystin [152]. 49  Immunoproteasome  e x p r e s s i o n affects the digestion of intracellular proteins, resulting in upregulation of M H C c l a s s I d u e to the i n c r e a s e of available peptides. In the h u m a n brain, i m m u n o h i s t o c h e m i c a l studies s h o w e d that E C are the only cell type e x p r e s s i n g M H C c l a s s I m o l e c u l e s consititutively [153]. lesions  displayed  a  moderate  to  strong  immunostaining  A c t i v e demyelinating M S for  the M H C - s p e c i f i c  transcription factors a n d a l s o high e x p r e s s i o n of M H C m o l e c u l e s by virtually every cell type within M S l e s i o n s [154, 155].  However, recent i m m u n o h i s t o c h e m i c a l studies  utilizing a more specific M H C c l a s s I A b (targeted on a-chain) demonstrated a difference in e x p r e s s i o n b e t w e e n  cell types.  Specifically, astrocytes a n d  oligodendrocytes  displayed lower e x p r e s s i o n , w h e r e a s inflammatory cells s u c h a s T cells, B cells a n d m a r c r o p h a g e s a l o n g with E C s stained intensely for M H C c l a s s I [26].  However, the  underlying m e c h a n i s m for the upregulation and the p r o t e a s o m a l effect on M H C c l a s s I e x p r e s s i o n in H B M E C r e m a i n s largely unknown.  In this study, w e found that M H C  c l a s s I e x p r e s s i o n in H B M E C is upregulated significantly by IFN-y after 4 8 h but not after 24h stimulation.  T h i s s u g g e s t s that n e w e x p r e s s i o n of M H C c l a s s I o c c u r s between  24h  IFN-y  to  48h  of  stimulation.  In  addition,  the  expression  is  not  entirely  time-dependent s i n c e there is no further significant i n c r e a s e in e x p r e s s i o n after 4 8 h a n d up to 7 2 h .  D e c r e a s e in M H C c l a s s I e x p r e s s i o n by lactacystin a l s o r e a c h e d significant  levels only after 4 8 h .  However, the inhibitory effect is slower, unlike studies with  lymphoblastoid cell lines in w h i c h the inhibitory effect started after 4 h [102].  The slow  downregulation m a y be related to the nature of H B M E C , s i n c e the B B B endothelium is highly selective for hydrophilic c h e m i c a l intake.  T h e results of the present studies  indicate that i m m u n o p r o t e a s o m e subunit upregulation by IFN-y in H B M E C results in upregulation of M H C c l a s s I e x p r e s s i o n .  T h e m R N A a n d protein e x p r e s s i o n patterns of  i m m u n o p r o t e a s o m e in H B M E C along with the M H C c l a s s I downregulation by lactacystin,  50  resemble similar regulatory p h e n o m e n a in professional A P C a n d further suggest a n important role for the cerebral endothelium in i m m u n e reactions in the C N S .  4.4  A D H E S I O N M O L E C U L E E X P R E S S I O N AT T H E B B B It is now well e s t a b l i s h e d that E C facilitate interactions with circulating leukocytes  leading to t h e initiation of inflammation, although t h e exact m e c h a n i s m s are not fully understood.  A large body of e v i d e n c e on extracerebral E C indicates that recruitment of  leukocytes to a n inflammatory site is at least in part mediated by d e novo e x p r e s s i o n or upregulation of certain E C t r a n s m e m b r a n e glycoproteins that bind to corresponding ligands o n leukocytes.  T h u s , P - , E-selectin mediate t h e initial rolling of leukocytes  along the endothelium, w h e r e a s I C A M - 1 a n d V C A M - 1 are important for firm a d h e s i o n to endothelium. adhesion  I C A M - 1 is a l s o important in transendothelial migration [49].  molecule  - ligand  interactions  a r e important  In addition,  for t h e formation  of t h e  immunological s y n a p s e that is n e c e s s a r y for antigen presentation. S i n c e a d h e s i o n m o l e c u l e s are de novo e x p r e s s e d a n d upregulated by cytokines at inflammatory sites, t h e present studies w e r e undertaken to investigate the effects of p r o t e a s o m e inhibitors o n the cytokine-induced e x p r e s s i o n a n d upregulation of I C A M - 1 , V C A M - 1 a n d E-selectin in a n in vitro model of the h u m a n B B B under both resting a n d inflammatory  conditions.  Previous  studies  showed  that  treatment  with  TNF-a  maximally upregulates I C A M - 1 , V C A M - 1 a n d E-selectin at 2 4 h , 18h, a n d 4 h respectively [50, 124, 125].  In the present study, surface e x p r e s s i o n of I C A M - 1 , V C A M - 1 a n d  E-selectin w a s quantitated by E L I S A a n d v i s u a l i z e d by I G S S .  Unstimulated H B M E C  s h o w e d m o d e r a t e I C A M - 1 e x p r e s s i o n a n d w e a k b a s a l e x p r e s s i o n of V C A M - 1 but not E-selectin,  by both  ELISA and IGSS,  with significantly  i n c r e a s e d staining  upon  stimulation with T N F - a (100 U/ml) for 2 4 h ( I C A M - 1 ) , 18h ( V C A M - 1 ) , a n d 4 h (E-selectin). 51  Coincubation  with  lactacystin  downregulated  the  TNF-a-induced  upregulation  of  V C A M - 1 a n d E-selectin a n d , to a l e s s e r extent, I C A M - 1 . T h e g e n e a n d protein e x p r e s s i o n of I C A M - 1 h a s b e e n studied extensively in extracerebral E C under inflammatory conditions.  A study o n H U V E C s h o w e d that the  g e n e s for I C A M - 1 a n d E-selectin are upregulated in bacterial infections along with activation  of the  transcription  factor  NFKB  [156].  A d h e s i o n m o l e c u l e s are  regulated by s h e a r s t r e s s [157], T N F - a [158] a n d HIV infection [159]. studies involve the N F K B pathway.  also  All of the a b o v e  P r o t e a s o m e inhibition of N F K B h a s b e e n widely  d o c u m e n t e d a n d this b l o c k a d e results in inhibition of a d h e s i o n m o l e c u l e e x p r e s s i o n in three recent E C studies [160-162].  Lactacystin, a s well a s other p r o t e a s o m e inhibitors,  are able to inhibit a d h e s i o n m o l e c u l e e x p r e s s i o n by blocking N F K B .  M o r i s e et al.  s h o w e d that gastric m u c o s a l E C e x p r e s s i o n of I C A M - 1 i n d u c e d by T N F - a is blocked by lactacystin [160].  W a r t o n et al. a l s o s h o w e d that E-selectin g e n e e x p r e s s i o n induced by  IL-16 through the N F K B pathway is b l o c k e d by lactacystin [161].  T h i s s u g g e s t s that the  p r o t e a s o m e is involved in cytokine activation of E C through  upregulation  resulting in a d h e s i o n m o l e c u l e e x p r e s s i o n .  of  NFKB  E C in the C N S a l s o upregulate I C A M - 1  after T N F - a stimulation a n d following reoxygenation after h y p o x i a [163], IL-1B[164], a n d c a d m i u m stimulation [165] through the N F K B pathway. IL-1,  or L P S stimulation  is a l s o d e p e n d e n t  demonstrated in murine cerebral E C [166].  on the  V C A M - 1 e x p r e s s i o n after TNF-a, NFKB  pathway  as  previously  T h e results of the present study are in  a g r e e m e n t with p r e v i o u s reports of E-selectin e x p r e s s i o n by cerebral E C following stimulation with IL-B, T N F - a a n d L P S [125, 167].  M i n i m a l consititutive e x p r e s s i o n by  h u m a n cerebral E C w a s found and upregulation o c c u r r e d 4 h following stimulation with  TNF-a, L P S a n d IL-B.  T h e upregulation w a s further inhibited by a N F K B blocking agent.  At present, there a r e no studies on p r o t e a s o m e inhibition of the N F K B pathway in h u m a n 52  cerebral E C . O u r results s h o w that the T N F - a induced e x p r e s s i o n of the a d h e s i o n m o l e c u l e s ICAM-1,  VCAM-1  and  lactacystin in H B M E C .  E-selectin  is downregulated  by  the  proteasome  inhibitor  T h e y a l s o indicate that p r o t e a s o m e inhibition d e c r e a s e s the  e x p r e s s i o n of both key m o l e c u l e s for antigen presentation ( M H C c l a s s I molecules) and m o l e c u l e s important for leukocyte a d h e s i o n to E C .  4.5  C D 8 + T L Y M P H O C Y T E  A D H E S I O N  It is now d o c u m e n t e d that e x p r e s s i o n of E C a d h e s i o n m o l e c u l e s mediates the recruitment of leukocyte s u b s e t s to inflammatory sites [49]. that migrated  In view of recent reports  resting C D 8 + T cells clonally e x p a n d in the C N S , it is important to  investigate the role of a d h e s i o n molecule e x p r e s s i o n by cerebral E C on the recruitment of resting C D 8 + T cells. Firm a d h e s i o n to the endothelium is a prerequisite for leukocyte migration.  The  current  proteasome  inhibition  on  studies resting  experiments w e r e performed  were  undertaken  CD8+  T  cell  to  investigate  adhesion  to  transendothelial the  cerebral  effects EC.  of The  using a n allogeneic s y s t e m s i n c e C D 8 + T cells and  H B M E C w e r e isolated from 2 different h u m a n hosts.  A d h e s i o n of C D 8 + T cells to  unstimulated  HBMEC  HBMEC  was  minimal.  Stimulation  of  i n c r e a s e d the a d h e s i o n of C D 8 + T cells significantly  by 4 0 % .  with The  IFN-y for  48h  IFN-y-induced  upregulation of C D 8 + T cell a d h e s i o n w a s significantly downregulated by lactacystin. Interestingly, the inhibitory effect of M H C c l a s s I blocking on a d h e s i o n w a s c o m p a r a b l e to that of lactacystin treatment.  T h i s indicates that M H C c l a s s I m o l e c u l e s play a role in  C D 8 + T cell a d h e s i o n to H B M E C . Following H B M E C stimulation with T N F - a , C D 8 + T cell a d h e s i o n w a s i n c r e a s e d 53  significantly a s c o m p a r e d to the unstimulated controls. 24h treatment  with TNF-a,  which significantly  A d h e s i o n w a s maximal after  upregulated  E-selectin a s previously d e s c r i b e d [50, 124, 125].  ICAM-1, VCAM-1  and  C o i n c u b a t i o n with T N F - a  and  lactacystin d e c r e a s e d C D 8 + T C a d h e s i o n to H B M E C m o n o l a y e r s significantly.  The  TNF-a-induced upregulation of C D 8 + T C a d h e s i o n to H B M E C is most likely d u e to the upregulation of a d h e s i o n m o l e c u l e s on H B M E C .  S i n c e the upregulation of a d h e s i o n  m o l e c u l e s by T N F - a w a s prevented by lactacystin (section 3.6), it is possible that the d e c r e a s e d a d h e s i o n of C D 8 + T C is d u e to downregulation of a d h e s i o n m o l e c u l e s at different  time  points.  IFN-y stimulation  upregulates the  e x p r e s s i o n of a d h e s i o n  m o l e c u l e s to a significantly l e s s e r d e g r e e [50, 124, 125] a n d it is p o s s i b l e that the T N F - a induced i n c r e a s e of C D 8 + T cell a d h e s i o n is mainly m e d i a t e d by the  upregulation  I C A M - 1 , V C A M - 1 a n d E-selectin. T h e p r o t e a s o m e regulates T cell a d h e s i o n and transendothelial migration  on  extracerebral E C , possibly through the downregulation of a d h e s i o n m o l e c u l e e x p r e s s i o n . R e a d et a l . s h o w e d that p r o t e a s o m e inhibitors r e d u c e a d h e s i o n and transmigration a c r o s s T N F - a treated H U V E C in an in vitro flow c h a m b e r s y s t e m , through the blocking of a d h e s i o n m o l e c u l e e x p r e s s i o n via NFKB pathway [51].  Further, the s a m e group  s h o w e d that p r o t e a s o m e inhibition is involved in altering lateral junctional structures between E C , w h i c h affect the transmigration of T cells [168].  Pre-treatment of primary  H U V E C cultures with lactacystin for 1h ablated the T N F - a induced e x p r e s s i o n of I C A M - 1 , VCAM-1,  E-selectin a n d polymorphonuclear neutrophil  a d h e s i o n [162].  However,  additional studies indicate that antigen recognition between T cells and E C is also important for T cell a d h e s i o n .  R e c e n t studies e m p h a s i z e that antigen presentation by  E C induces T cell a d h e s i o n and transendothelial migration [53, 169].  Marelli-Berg et al.  demonstrated that i n c r e a s e d e x p r e s s i o n of specific M H C c l a s s l-peptide (H2) c o m p l e x e s 54  by IFN-y stimulated E C i n c r e a s e s antigen-specific T cell transmigration a c r o s s E C m o n o l a y e r s [53].  Similar observations have b e e n reported on C D 4 + T C a d h e s i o n a n d  transendothelial migration a c r o s s IFN-y-stimulated E C , w h i c h e x p r e s s M H C c l a s s II m o l e c u l e s [169].  It is n o w well established that the trafficking of resting T lymphocytes  a c r o s s the B B B requires binding of a d h e s i o n m o l e c u l e s o n E C to corresponding ligands on T cells[49, 170].  However, a study s u g g e s t s that lymphocyte trafficking a c r o s s the  B B B is antigen-specific [171], implicating drainage of brain antigen to the periphery. Therefore, it is p o s s i b l e that cerebral E C play a role a s A P C that assist a d h e s i o n a n d trafficking of antigen specific lymphocytes a c r o s s B B B . O u r results s h o w that the inflammatory cytokines IFN-y a n d T N F - a c a n i n c r e a s e allogeneic a d h e s i o n of C D 8 + T cells to H B M E C through upregulation of both M H C c l a s s I molecules and  E C adhesion molecules.  The  proteasome  inhibitor  lactacystin  downregulates C D 8 + T cell a d h e s i o n by downregulating both the M H C c l a s s I and possibly through a d h e s i o n m o l e c u l e e x p r e s s i o n by H B M E C .  T h e s e studies suggest an  important immunoregulatory role of the p r o t e a s o m e at the B B B in inflammatory C N S diseases.  C H A P T E R 5: C O N C L U S I O N S  5.1  S U M M A R Y A N D The  aim of this  S I G N I F I C A N C E  study w a s to  investigate the  differential  e x p r e s s i o n of  the  p r o t e a s o m e a n d i m m u n o p r o t e a s o m e at the B B B a n d their regulatory role on the e x p r e s s i o n of M H C c l a s s I m o l e c u l e s by h u m a n cerebral E C in primary culture.  A  s e c o n d aim w a s to determine the effect of p r o t e a s o m e inhibition on the e x p r e s s i o n of the a d h e s i o n m o l e c u l e s I C A M - 1 , V C A M - 1 and E-selectin by H B M E C a n d the a d h e s i o n of 55  C D 8 + T C to the B B B endothelium.  O u r initial hypothesis p r o p o s e d that treatment with  lactacystin could downregulate the proinflammatory c y t o k i n e - i n d u c e d M H C c l a s s I a n d a d h e s i o n m o l e c u l e e x p r e s s i o n , thereby reducing the C D 8 + a d h e s i o n to H B M E C . results  s h o w that  immunoproteasome  proinflammatory cytokines.  expression  by  HBMEC  The  is upregulated  by  Inhibition of the p r o t e a s o m e a n d i m m u n o p r o t e a s o m e by  specific inhibitors d o w n r e g u l a t e s both M H C c l a s s I a n d a d h e s i o n m o l e c u l e e x p r e s s i o n , resulting in r e d u c e d a d h e s i o n of C D 8 + T C to cerebral endothelium. T h e experiments d o c u m e n t e d here s h o w that H B M E C a n d H U V E C in vitro are c a p a b l e of e x p r e s s i n g constitutive p r o t e a s o m e subunit R N A under both resting a n d cytokine-treated conditions a n d that i m m u n o p r o t e a s o m e subunits c a n be upregulated by inflammatory cytokines.  In both H B M E C a n d H U V E C , the d e g r e e of upregulation of the  i m m u n o p r o t e a s o m e R N A w a s relatively similar, with noticeable upregulation of B1i, B2i a n d B5i after treatment with 100 U/ml T N F - a a n d 2 0 0 U/ml IFN-y for 2 4 h . Intracellular localization of the p r o t e a s o m e a n d i m m u n o p r o t e a s o m e subunits, a s a s s a y e d by E L I S A a n d i m m u n o g o l d silver staining, s h o w e d that constitutive e x p r e s s i o n of the  a5  subunit  Immunoproteasome  remains subunits  constant  before  61 i a n d  and  B5i, on  after  the  IFN-y  other  treatment for  hand,  are  24h.  significantly  upregulated by nearly two-fold after treatment with IFN-y for the s a m e period of time. M H C c l a s s I e x p r e s s i o n w a s upregulated incubation.  Co-incubation  with  lactacystin  by IFN-y only after a 4 8 - 7 2 h and  IFN-y  for  48-72h  resulted  long in  downregulation of M H C c l a s s I e x p r e s s i o n . S u r f a c e e x p r e s s i o n of I C A M - 1 by H B M E C w a s upregulated by T N F - a treatment a n d slightly downregulated by lactacystin.  Similarly, T N F - a treatment upregulated V C A M - 1  e x p r e s s i o n a n d co-incubation with lactacystin nearly ablated the upregulation.  Unlike,  I C A M - 1 a n d V C A M - 1 , E-selectin displayed no constitutive e x p r e s s i o n by H B M E C but its 56  e x p r e s s i o n w a s i n d u c e d after 4 h treatment with T N F - a .  Lactacystin downregulated  E-selectin e x p r e s s i o n slightly but significantly. A d h e s i o n of resting C D 8 + T C to unstimulated H B M E C w a s minimal. with IFN-y for 4 8 h upregulated C D 8 + T cell a d h e s i o n by nearly two fold.  Treatment  Treatment of  H B M E C with lactacystin downregulated C D 8 + T C a d h e s i o n thus d e c r e a s i n g the surface e x p r e s s i o n of M H C c l a s s I on H B M E C .  T N F - a treatment of the m o n o l a y e r s resulted in  nearly 2 fold i n c r e a s e of C D 8 + T cell a d h e s i o n a n d the effect w a s downregulated by lactacystin, possibly through  downregulation  of T N F - a - i n d u c e d a d h e s i o n  molecule  e x p r e s s i o n by H B M E C . T h e s e findings along with previous observations on murine p r o t e a s o m e e x p r e s s i o n in r e s p o n s e to cytokines a n d the functional significance of M H C c l a s s I presentation pathway,  indicate  that  the  BBB  endothelium  has  the  ability  to  express  i m m u n o p r o t e a s o m e subunits after stimulation with IFN-y, the cytokine responsible for initiating the signalling pathway for M H C c l a s s II locus transcription.  A t the protein level,  there are no studies on E C showing that upregulation of i m m u n o p r o t e a s o m e subunits leads to r e p l a c e m e n t of constitutive subunits intracellularly.  Studies with murine a n d  h u m a n cell lines on the formation of i m m u n o p r o t e a s o m e support this point of view. Upregulation of i m m u n o p r o t e a s o m e subunits l e a d s to either c h a n g e in the repertoire of peptides presented by M H C c l a s s I or the number of e x p r e s s e d M H C c l a s s I m o l e c u l e s . In vivo studies in C N S inflammatory d i s e a s e s have f o c u s e d on the cell types responsible for e x p r e s s i o n of M H C c l a s s I m o l e c u l e s but rarely o n the pathway of M H C c l a s s I production.  With increasing e v i d e n c e that autoantigen specific C D 8 + T cells reside a n d  e x p a n d clonally within the C N S , the B B B m a y play a role in presenting brain antigens to leukocytes that are specific to the autoantigen. The  signalling  pathway  for  adhesion 57  molecule  induction/upregulation  is  well-established recruitment.  and  s e v e r a l blocking  studies  have  proven  in  inhibiting  A l t h o u g h m a n y s u c h blockers h a v e a n inhibitory effect on the  pathway, p r o t e a s o m e inhibition h a s b e e n u s e d in E C s t u d i e s . adhesion  effective  molecules  are  important  for  CD8+  T  cell  NFKB  O u r results s u g g e s t that  adhesion  induction/upregulation c a n be regulated via the p r o t e a s o m e pathway.  and  that  their  In addition, in  keeping with the diversity of p r o t e a s o m e functions, w e a l s o s h o w that the p r o t e a s o m e r e d u c e s T cell a d h e s i o n to H B M E C possibly through the d e c r e a s e of M H C c l a s s I molecule e x p r e s s i o n .  T h i s observation is interesting c o n s i d e r i n g that the c o n c e p t of the  immunological s y n a p s e originated from observations of antigen presentation by dendritic cells to T cells.  A l o n g with s e v e r a l studies in H U V E C s h o w i n g that i n c r e a s e d antigen  presentation by M H C c l a s s I results in i n c r e a s e d C D 8 + T cell a d h e s i o n a n d migration, our o b s e r v a t i o n s s u g g e s t a n e w m e c h a n i s m for the entry of antigen specific C D 8 + T cells into the brain a c r o s s the B B B in C N S inflammation.  5.2  F U T U R E  D I R E C T I O N S  T h i s study o p e n s a n u m b e r of potential lines of investigation pertaining to the role of the brain e n d o t h e l i u m in C N S inflammation.  S e v e r a l q u e s t i o n s h a v e b e e n raised from  this study, a n s w e r s to w h i c h m a y s e r v e to further clarify the significance of antigen presentation by E C to inflammatory cells in the initiation of inflammatory reactions in the CNS. First of all, e x p r e s s i o n of M H C c l a s s I in this in vitro m o d e l of B B B c a n be regulated by s e v e r a l intracellular proteins. b l o c k e d with transfection  T A P transporters located at the E R m e m b r a n e c a n be  of a T A P protein  e x p r e s s e d by H e r p e s s i m p l e x virus.  inhibitor  ICP47,  a cytoplasmic  protein  T h i s would further clarify w h e t h e r all the M H C  c l a s s I specific peptides are delivered from cytosol through the T A P transporter to the E R 58  a s in the A P C .  T h e results will further define the function of the endothelium a s a n  antigen presentating cell w h e n comparing the turnover time of M H C c l a s s I e x p r e s s i o n between the inhibitor treated H B M E C a n d the professional A P C s . S e c o n d l y , a s a n increasing body of e v i d e n c e s u g g e s t s that the M H C c l a s s I pathway c a n present e x o g e n o u s antigens, it would be interesting to determine the ability of the B B B endothelium to uptake a n d c r o s s - p r e s e n t e x o g e n o u s peptides.  Evaluation of the  M H C c l a s s I e x p r e s s i o n in the in vitro model of the B B B after treatment with the e n d o s o m a l acidification inhibitor bafilomycin c o m b i n e d with e x o g e n o u s peptide will further elucidate the cross-presentation ability of the brain e n d o t h e l i u m . T h e third study w o u l d be to look at the possibility of initiation of T cell r e s p o n s e s by HBMEC.  A l l o g e n e i c T cells c a n be incubated with the cytokine stimulated  EC  monolayers a n d T cell r e s p o n s e s c a n be monitored by T-cell C D R 3 spectratyping, a P C R - b a s e d detection of s o m a t i c recombination of T cells that undergo clonal e x p a n s i o n . C h a n g e s detected with this m e t h o d , in theory, would provide direct e v i d e n c e for the antigen presentation capability of the B B B . T h e s e experimental a p p r o a c h e s would provide a better understanding of M H C c l a s s I antigen presentation by H B M E C .  Studies presented here provide initial support for the  role of H B M E C a s A P C for C D 8 + T lymphocytes, a n d lay a foundation for future investigations a i m e d at fully characterizing the ability of c e r e b r a l E C to function a s A P C during C N S inflammation.  59  REFERENCES 1.  J a n e w a y , C . 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Eur J Immunol, 2 0 0 5 . 35(4): p. 1 0 7 6 - 8 5 .  72  F i g u r e 1: Primary cultures of h u m a n brain m i c r o v e s s e l endothelial cells, a n in vitro m o d e l of the h u m a n blood-brain barrier. P a n e l (A) represents a confluent monolayer of H B M E C under p h a s e contrast m i c r o s c o p y . P a n e l (B) shows binding of the Ulex e u r o p e a u s lectin by H B M E C . (C) Strong positive perinuclear staining for Factor VIM/vonWillebrand antigen. Both markers indicate that endothelial characteristics a r e maintained in t h e s e primary cultures. Magnification: (A) 180x; (B, C ) 360x  73  RNA Expression of proteasome subunits by HBMEC Constitutive subunits M  Inducible subunits  U  M PSMB6 [01] (25)  PSMB7 [/32] (28)  PSMB5 [05] (25)  U  500" 400-  PSMB9 [jSli] (30)  300-  PSMB10 [02i] (25)  200-  PSMB8 [05i] (25)  300200-  GAPDH (22)  300200M : mol. weight m a r k e r s  U: unstimulated  S : stimulated  C : - R T negative control  N o . in parenthesis: # of P C R c y c l e  F i g u r e 2a: R N A e x p r e s s i o n of both consitutive a n d cytokine inducible p r o t e a s o m e subunits w a s determined by semi-quantitative R T - P C R . R N A extracted from 8-10 d a y s old confluent H B M E C cultures stimulated (S) with cytokines (100U/ml T N F - a a n d 2 0 0 U / m l IFN-y) for 24 hours and unstimulated cultures (U). c D N A products from the R T experiment w e r e amplified with c y c l e n u m b e r s that optimally s h o w visual differences between stimulated a n d unstimulated conditions. Controls involved amplifying the product from experiment without the r e v e r s e transcriptase. After amplification with g e n e specific primers, all p r o t e a s o m e subunits a n d G A P D H s a m p l e s w e r e run on 6 % polyacrylamide gels a n d v i s u a l i z e d by staining with ethidium bromide under U V light.  74  Semi-quantitative Assessment of Protesome Subunit Expression by HBMEC by PCR  AW F i g u r e 2b: T h e band intensity of p r o t e a s o m e subunit R N A e x p r e s s i o n by H B M E C w a s normalized with G A P D H R N A e x p r e s s i o n a n d d i s p l a y e d a s ratios. T h e inducible subunits (31 i, 32i, B5i) s h o w upregulation by T N F - a a n d IFN-y stimulated cultures w h e r e a s constitutive subunits (31, 32, 35) did not i n c r e a s e substantially after cytokine stimulation. i n  75  RNA Expression of proteasome subunits by HUVEC Constitutive subunits  M  Inducible subunits  U  M PSMB6  400"  PSMB9 [/31i] (30)  500 400  [/SI]  (25)  300-  U  PSMB7  400"  PSMB10  [02i]  (28)  300-  (25)  PSMB5 300"  PSMB8  [05]  [05i]  (25)  (25)  200-  GAPDH (22)  300" 200M : mol. weight markers  U: unstimulated  S : stimulated  C : - R T negative control  N o . in parenthesis: # of P C R c y c l e  F i g u r e 2c: R N A e x p r e s s i o n of both consitutive a n d cytokine inducible p r o t e a s o m e subunits w a s determined by semi-quantitative R T - P C R . R N A extracted from 8-10 d a y old confluent H U V E C cultures stimulated (S) with cytokines (100U/ml T N F - a a n d 2 0 0 U / m l IFN-y) for 2 4 hours a n d unstimulated cultures (U). c D N A products from the reverse transcription (RT) experiment w e r e amplified with c y c l e n u m b e r s that optimally s h o w visual differences between stimulated a n d unstimulated conditions. Controls involved amplifying the product without the reverse transcriptase (-RT). After amplification with g e n e - s p e c i f i c primers, all p r o t e a s o m e subunits a n d G A P D H s a m p l e s w e r e run on on 6 % polyacrylamide gels and v i s u a l i z e d by staining with ethidium bromide under U V light.  76  Semi-quantitative Assessment of Protesome Subunit Expression by HUVEC by PCR  A# F i g u r e 2d: T h e band intensity of proteasome subunit R N A expression by H U V E C was normalized with G A P D H R N A expression and displayed as ratios. T h e inducible subunits (P1i, P2i, P5i) show upregulation by T N F - a and IFN-y in stimulated cultures whereas constitutive subunits (P1, P2, P5) did not increase substantially after cytokine stimulation.  77  Intracellular ELISA for proteasome subunits in HBMEC ns  1.0D=i  F i g u r e 3 a : Intracellular localization of the constitutive (a5) a n d inducible (,/31i /35i) p r o t e a s o m e subunits in H B M E C . Treatments are d e s c r i b e d under e a c h bar a n d e a c h color c o r r e s p o n d s to e a c h p r o t e a s o m e subunit e x p r e s s i o n profile (legend). V a l u e s represent m e a n e x p r e s s i o n ± S E M (n=3); * a b o v e bars = p<0.05 a s c o m p a r e d to unstimulated culture.  78  F i g u r e 3 b : Intracellular localization of p r o t e a s o m e subunits in H B M E C . E x p r e s s i o n of the constitutive ( a 5 ) a n d inducible (/3Y\ & /35i) p r o t e a s o m e subunits by I G S S (A-F). Unstimulated cells ( A , C , E ) d i s p l a y e d a more oval s h a p e than the s p i n d l y - s h a p e d cells stimulated with I F N - j ( 2 0 0 U / m l , 24 hours) (B, D, F). E x p r e s s i o n w a s determined by the fine, granular staining s e e n in all of the a b o v e p a n e l s . T h e cell nuclei w e r e counterstained with G i e m s a . B a r = 0.1 m m  79  MTT Viability Assay for HBMEC treated with lactacystin 0.3-,  J?*  f <>° Jr r>*  &  # f  4?  5^  ^  + -  v °v C?  tjfr  -  A  L C : lactacystin  r>*  \?rTreatments  F i g u r e 4 a : M T T Viability a s s a y performed o n H B M E C . F o r m a z a n crystal formation transforms into a b s o r b a n c e (O.D) on y axis, this a l s o represents viability of the culture. T r e a t m e n t s are d e s c r i b e d under e a c h bar. V a l u e s represent m e a n e x p r e s s i o n ± S E M (n=3). A N O V A p<0.005; * a b o v e e a c h bar = p<0.05 a s c o m p a r e d to unstimulated culture.  80  Cell Viability assay for H B M E C with LIVE/DEAD® Cell-Mediated Cytotoxicity Kit  • %  # %•  #  #l  0  1  •  H  %w l  F i g u r e 4 b : L I V E / D E A D ® C e l l - M e d i a t e d Cytotoxicity A s s a y performed on H B M E C . C e l l Viability of H B M E C w a s determined using a cytotoxicity kit p u r c h a s e d from (Molecular P r o b e s ) . C e l l s with intact cell m e m b r a n e s are indicated by green f l u o r e s c e n c e (Calcein). T h e nuclei of dying cells s h o w red f l u o r e s c e n c e (EthD-1). T h e cultures w e r e (A) untreated, or treated with (B) I F N - j (200U/ml for 4 8 hours); (C) 7.5 // M lactacystin for 4 8 hours; (D) 1 5 / / M lactacystin for 4 8 hours; (E) 9 0 / / M lactacystin for 4 8 hours. C o n c e n t r a t i o n s of 7.5 // M to 90 // M are increasingly toxic a s indicated by the number of nuclei with red f l u o r e s c e n c e . Untreated a n d IFN-vtreated cultures had recovered no visiable red f l u o r e s c e n c e . (F) Control cultures treated with 7 0 % methanol for 15 minutes s h o w nuclei uniformly stained red indicating cell death. B a r in ( A - C , E - F ) = 0.1 m m ; B a r in (D) = 0.04 m m  81  Q u a n t i t a t i o n of M H C c l a s s I e x p r e s s i o n b y s u r f a c e E L I S A in H B M E C cultures 2.5-,  Unstimulated  4 8 h IFN-y(200U/ml)  4 8 h I F N - 7 (200U/ml) & lactacystin  F i g u r e 5 : E x p r e s s i o n of M H C c l a s s I by H B M E C after IFN-y stimulation w a s quantitated by surface E L I S A . Treatments are d e s c r i b e d under e a c h group of bars a n d e a c h color c o r r e s p o n d to different incubation time periods (legend). V a l u e s represent m e a n e x p r e s s i o n ± S E M (n=3). A N O V A p<0.005; * a b o v e bars = p<0.05 a s c o m p a r e d b e t w e e n two treatments. T h e fine granular silver staining by I G S S confirms the E L I S A findings. T h e cells w e r e counterstained with G i e m s a to visualize their nuclei. Treatments a n d time are s h o w n below e a c h p a n e l . B a r = 0.1 m m 82  Quantitation of adhesion molecules by ELISA in HBMEC ICAM-1 VCAM-1 E-selectin  J > ef  i  f  rfP  ^  A  o L C = lactacystin 7 . 5 u M  F i g u r e 6 a : A d h e s i o n m o l e c u l e e x p r e s s i o n by H B M E C after T N F - a stimulation determined by s u r f a c e E L I S A . Treatments a r e d e s c r i b e d under e a c h bar a n d e a c h color c o r r e s p o n d to e a c h a d h e s i o n m o l e c u l e profile (legend). V a l u e s represent m e a n e x p r e s s i o n ± S E M (n=3). A N O V A p<0.001; * a b o v e bars = p<0.05, ** p<0.01, not significant (ns).  83  F i g u r e 6 b : S u r f a c e e x p r e s s i o n of a d h e s i o n m o l e c u l e s in H B M E C . E x p r e s s i o n of the I C A M - 1 ( A - C ) , V C A M - 1 (D-F), a n d E-selectin (G-I) by unstimulated H B M E C cultures ( A , D , G ) , and following treatment with T N F - a for 24hrs to maximally upregulate I C A M - 1 (B), 18hrs T N F - a to maximally upregulate V C A M - 1 (E), 4 h r s T N F - a to maximally upregulate E-selectin (H), a n d 2 4 h r s coincubation with T N F a +lactacystin (C), 18hrs T N F - a + l a c t a c y s t i n (F), and 4 h r s T N F - a + l a c t a c y s t i n (I). E x p r e s s i o n w a s determined by the fine, granular staining s e e n in all of the a b o v e panels. T h e cells nuclei w e r e counterstain with G i e m s a . B a r = 0.1 m m 84  Quantitation of CD8+ T cell Adhesion to HBMEC  ^ °  L C = lactacystin 7 . 5 u M  Quantitation of CD8+ T cell adhesion to HBMEC (actual slides)  A  B  "'  1  i t  Unstimulated  \  \  '\£  IFN-y+ L C  IFN-Y'200U/rrfl  f  *&Jt  * *  %  IFN-y+blocking A b  F i g u r e 7 a : Quantitation of C D 8 + T lymphocyte a d h e s i o n to H B M E C . Adhered C D 8 + T C s w e r e stained with the i m m u n o p e r o x i d a s e technique for L C A a n d a r e s h o w n a s dark dots in the p a n e l s . Treatment a n d incubation times of the m o n o l a y e r s before incubation with of C D 8 + T C a r e s h o w n under the bar. V a l u e s represent m e a n counts of five fields of cells ± S E M (n=3). A N O V A p<0.005; * o n top of e a c h bar = p<0.05 a s c o m p a r e d to unstimulated culture. T h e cell nuclei w e r e counterstain with G i e m s a . B a r in actual slides = 0.1 m m 85  Quantitation of CD8 T cell Adhesion to HBMEC +  F i g u r e 7 b : Quantitation of C D 8 + T lymphocyte a d h e s i o n to H B M E C (cont.). A d h e r e d C D 8 + T C s w e r e stained with i m m u n o p e r o x i d a s e technique for L C A . P r e treatment incubation times of the m o n o l a y e r s before the incubation with C D 8 + T C are s h o w n on x-axis. V a l u e s represent m e a n counts of five fields of cells ± S E M (n=3). A N O V A p<0.005; * at d a t a points: p<0.05 a s c o m p a r e d between two treatments.  86  

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