Involvement of the LFA-1 ligand JAM-A in inflammatory recruitment of mononuclear cells [Elektronische Ressource] : identification of the JAM-A binding domain in LFA-1 / vorgelegt von Line Rebo Fraemohs

Line Fræmohs

Sujets

• Gesundheit

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2007
• Nombre de lectures: 9
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Involvement of the LFA-1 ligand JAM-A in inflammatory
recruitment of mononuclear cells:
Identification of the JAM-A binding domain in LFA-1



Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der Rheinisch-
Westfälischen Technischen Hochschule Aachen zur Erlangung des akademischen Grades
einer Doktorin der Naturwissenschaften genehmigte Dissertation


vorgelegt von


Cand.Scient (M.Sc.)
Line Rebo Fraemohs

aus Aarhus, Dänemark





Berichter: Universitätsprofessor Dr. rer. nat. Rainer Fischer
Universitätsprofessor Dr. med. Christian Weber


Tag der mündlichen Prüfung: 10. August 2007


Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.


The results in this work were in part published in:



Fraemohs, L., Koenen, R. R., Ostermann, G., Heinemann, B. and Weber, C. (2004). The
functional interaction of the β integrin lymphocyte function-associated antigen-1 with 2
junctional adhesion molecule-A is mediated by the I domain. J Immunol 173, 6259-64.

Ostermann, G.*, Fraemohs, L.*, Baltus, T., Schober, A., Lietz, M., Zernecke, A., Liehn, E.
A. and Weber, C. (2005). Involvement of JAM-A in mononuclear cell recruitment on
inflamed or atherosclerotic endothelium: inhibition by soluble JAM-A. Arterioscler Thromb
Vasc Biol 25, 729-35.

Zernecke, A., Liehn, E. A., Fraemohs, L., von Hundelshausen, P., Koenen, R. R., Corada,
M., Dejana, E. and Weber, C. (2006). Importance of junctional adhesion molecule-A for
neointimal lesion formation and infiltration in atherosclerosis-prone mice. Arterioscler
Thromb Vasc Biol 26, e10-3.




*) equal contribution

Table of Contents i

Table of Contents

Table of Contents .......................................................................................................i
Abbreviations ............................................................................................................v

I INTRODUCTION ........................................................................................................................ 1
I.1 Leukocyte adhesion and transendothelial migration ...................................................................... 1
1.2 Chemokines......................... 2
I.3 Integrins............................................................................................................................................... 2
I.3.1 Integrin structure........................................................................................................................... 3
I.3.2 Integrin activation and signaling................................................................................................... 5
I.3.3 I domain........................................................................................................................................ 6
I.3.4 Integrin ligands ............................................................................................................................. 7
I.3.5 LFA-1 ............................................................................................................................................ 8
I.4 Junctional adhesion molecule-A........................................................................................................ 8
I.4.1 JAM-A expression and localization............................................................................................... 9
I.4.2 Structure of JAM-A ....................................................................................................................... 9
I.4.3 Extracellular homophilic interactions in cis and in trans ............................................................ 10
I.4.4 Extracellular heterophilic interactions ......................................................................................... 11
I.4.5 Intracellular heterophilic interactions........ 11
I.4.6 Functions of JAM-A............. 12
I.4.7 Other JAM family members ........................................................................................................ 13
I.5 Inflammation in atherosclerosis ...................................................................................................... 13
I.6 Aim of this study ............................................................................................................................... 14
II MATERIALS AND METHODS ............................................................................................. 17
II.1 General equipment.................. 17
II.2 General solutions..................... 17
II.3 Chemokines, antagonist, and recombinant proteins..................................................................... 18 Table of Contents ii
II.4 Antibodies ......................................................................................................................................... 18
II.4.1 Primary antibodies...................................................................................................................... 18
II.4.2 Secondary antibodies.................................................................................................................. 19
II.4.3 Isotype controls .......................................................................................................................... 20
II.4.4 Antibody purification from hybridoma cell supernatants........................................................... 20
II.5 Mammalian cell culture procedures............................................................................................... 20
II.5.1 General ....................................................................................................................................... 20
II.5.2 Culturing of adherent cell monolayers ....................................................................................... 21
II.5.3 Culturing of cells in suspension ................................................................................................. 21
II.5.4 Freezing and thawing of mammalian cells.................................................................................21
II.6 Mammalian cell lines ....................................................................................................................... 22
II.6.1 Jurkat cells, clone E6-1 .............................................................................................................. 22
II.6.2 J- β .7 cells .................................................................................................................................. 22 2
II.6.3 Mono Mac 6 cells ....................................................................................................................... 22
II.6.4 Chinese hamster ovary cells ....................................................................................................... 22
II.6.5 SVEC.......................................................................................................................................... 23
II.7 Primary cells..................................................................................................................................... 23
II.7.1 Isolation of PBMCs.................................................................................................................... 23
+ +II.7.2 Isolation of CD4 CD45RO memory T cells ............................................................................. 23
II.7.3 HUVEC ...................................................................................................................................... 23
II.7.4 Isolation of mouse aortic endothelial cells.... 24
II.8 Work with E.coli .............................................................................................................................. 24
II.8.1 Bacteria growth medium ............................................................................................................ 24
II.8.2 Preparation of chemically competent E. coli.............................................................................. 25
II.8.3 Heat-shock transformation of competent E. coli........................................................................ 25
II.9 Recombinant DNA techniques........................................................................................................ 25
II.9.1 Restriction endonuclease digestion of DNA 25
II.9.2 Fill-in of 5’-overhangs to generate blunt ends............................................................................ 26
II.9.3 DNA extraction from agarose gels ............................................................................................. 26
II.9.4 Dephosphorylation of linearized plasmid DNA ......................................................................... 26
II.9.5 Precipitation of DNA.................................................................................................................. 27
II.9.6 Ligation of DNA fragments........................................................................................................ 27
II.9.7 Miniprep: Small-scale purification of plasmid DNA ......................................