De-activation of immune cells through modulation of extracellular matrix metalloproteinase inducer (EMMPRIN) by statins [Elektronische Ressource] / vorgelegt von Venkata Sasidhar Manda

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Informations

• Publié par: heinrich-heine-universitat_dusseldorf
• Publié le: 01 janvier 2007
• Nombre de lectures: 31
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

De-activation of immune cells through modulation of extracellular matrix metalloproteinase
inducer (EMMPRIN) by statins







Inaugural-Dissertation

zur
Erlangung des Doktorgrades der
Mathematisch-Naturwissenschaftlichen Fakultät
der Heinrich-Heine-Universität Düsseldorf





vorlegt von
Venkata Sasidhar Manda
aus Visakhapatnam
Indien
July,2007



Aus dem Institut für Pharmazeutische Biologie und Biotechnologie
der Heinrich-Heine-Universität Düsseldorf
















Gedruckt mit der Genehmigung der
Mathematisch-Naturwissenschaftlichen Fakultät der
Heinrich-Heine-Universität Düsseldorf






Referent : Priv. Doz. Dr. Oliver Neuhaus

Koreferent: Prof. Dr. Peter Proksch

Tag der mündlichen Prüfung: July 6th 2007



TABLE OF CONTENTS I
I TABLE OF CONTENTS
I TABLE OF CONTENTS.............................................................................................................I
II LIST OF FIGURES ..................................................................................................................V
III LIST OF TABLES...VIII
IV LIST OF ABBREVIATIONS...................................................................................................IX
SUMMARY ................................................................................................................................XI
ZUSSAMMENFASSUNG ........................................................................................................XIII
1 INTRODUCTION ...............................................................................................................1
1.1 Immunology of multiple sclerosis ..............................................................................1
1.1.1 MS etiology ...........................................................................................................3
1.1.2 Mechanisms of induction of MS by infectious agents............................................3
1.1.2.1 Molecular mimickry ......................................................................................3
1.1.2.2 Bystander activation.....................................................................................4
1.1.3 Pathogenetic stages in the disease process of MS: lesion pathology...................4
1.1.4 Lessons from therapies.........................................................................................6
1.2 EMMPRIN .................................................................................................................7
1.2.1 EMMPRIN structure..............................................................................................8
1.2.2 Cellular expression ...............................................................................................9
1.2.2.1 EMMPRIN expression in monocytes..........................................................10
1.2.2.2 EMMPRIN expression in the CNS environment .........................................11
1.2.2.3 EMMPRIN expression in T cell lymphomas and T cells .............................11
1.2.3 General EMMPRIN Functions.............................................................................12
1.2.3.1 MMP induction and activation ....................................................................13
1.2.3.2 Chaperone function for monocarboxylate transporters...............................15
1.2.3.3 Lectin activity..............................................................................................16
1.2.3.4 Monocyte-dependent T cell de-activation through EMMPRIN and its associated
CD98...............................................................................................................16
1.2.3.5 Monocyte-independent T cell de-activation through EMMPRIN and its
associated proteins......................................................................................................19
1.2.4 Membrane translocation of EMMPRIN................................................................20
1.2.5 EMMPRIN and signal transduction .....................................................................21
1.2.6 Molecular regulation of EMMPRIN expression....................................................21
1.2.7 EMMPRIN-associated cell surface molecules.....................................................22


TABLE OF CONTENTS II
1.2.8 Therapeutic targeting of EMMPRIN ....................................................................22
1.3 Neuroprotection via de-activated T cells .................................................................23
1.4 Statins .....................................................................................................................24
1.4.1 Mechanism of statin action .................................................................................24
1.4.2 Pathways inhibited due to statin action...............................................................25
1.4.3 Immunosuppression by statins ...........................................................................26
1.4.3.1 Anti-inflammatory actions of statins in atherosclerosis...............................27
1.4.4 Statins in multiple sclerosis and rheumatoid arthritis ..........................................27
2 AIM OF THE STUDY .......................................................................................................29
3 MATERIALS AND METHODS .........................................................................................30
3.1 Materials..................................................................................................................30
3.1.1 General chemicals ..............................................................................................30
3.1.2 Other materials ...................................................................................................31
3.1.3 Cell culture reagents...........................................................................................32
3.1.4 Peptides..............................................................................................................32
3.1.5 Cell lines and media............................................................................................32
3.1.6 Antibodies ...........................................................................................................33
3.2 Methods ..................................................................................................................34
3.2.1 Monocyte activation ............................................................................................34
3.2.1.1 Cell Culture ................................................................................................34
3.2.1.2 Cytofluorometric analysis ...........................................................................34
3.2.1.3 Gelatin zymography ...................................................................................34
3.2.1.4 Western blotting .........................................................................................35
3.2.1.4.1 Protein isolation from cells ....................................................................35
3.2.1.4.2 Protein separation by poly acrylamide gel electrophoresis (PAGE) ......35
3.2.1.4.3 Immunoblot ...........................................................................................36
3.2.1.5 Cell surface biotinylation ............................................................................36
3.2.1.6 Experminetal design - monocyte de-activation...........................................37
3.2.2 T cell activation ...................................................................................................37
3.2.2.1 Isolation of T cells ......................................................................................37
33.2.2.2 T cell proliferation - radio(methyl- H)-thymidineincorporation assay ..........38
3.2.2.3 CFSE - proliferation assay .........................................................................38
3.2.2.4 Lactate estimation38


TABLE OF CONTENTS III
3.2.2.4.1 Intracellular pH and metabolic starvation assay....................................38
3.2.2.4.2 Lactate assay........................................................................................39
3.2.3 Experimental design for T cell activation - costimulatory assay ..........................39
3.2.4 Neuroprotection ..................................................................................................40
3.2.4.1 Human neuronal and astrocyte cell line cultures........................................40
3.2.4.2 Neuronal apoptosis coculture assay ..........................................................40
3.2.4.3 Coculture assay with statin treated T cells .................................................40
3.2.4.4 Antibodies used to attenuate T cell cytotoxcity via monocytes...................40
3.2.4.5 MTT assay .................................................................................................41
3.2.5 Neuroprotection model................................................