Transcriptional profile of the mononuclear phagocyte system in the lungs [Elektronische Ressource] / vorgelegt von Zbigniew Jan Zasłona

Zaslona, Zbigniew Jan

Sujets

• Gesundheit

Informations

• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2009
• Nombre de lectures: 7
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

Transcriptional profile of the mononuclear
phagocyte system in the lungs






Inauguraldissertation
zur Erlangung des Grades eines Doktors der Humanbiologie
des Fachbereichs Medizin
der Justus-Liebig-Universität Gießen











vorgelegt von
Zbigniew Jan Zasłona
aus Warschau, Polen




Gießen 2009
Aus dem Zentrum für Innere Medizin
des Klinikums der Justus-Liebig-Universität Gießen
Direktor: Prof. Dr. W. Seeger

























Gutachter: Prof. Dr. Jürgen Lohmeyer


Gutachter: Prof. Dr. Klaus Preissner


Tag der Disputation:
23.03.2010
Table of Contents
 - I -
I. Table of Contents

I. Table of Contents................................................................................................................. I
II. List of Figures .................................................................................................................. V
III. Abbreviations...............................................................................................................VIII
1. Introduction...................................................................................................................... 1
1.1 The mononuclear phagocyte system................................................................................ 1
1.1.1 Monocytes.................................................................................................................. 2
1.1.2 Monocytes are precursors for lung macrophages and lung dendritic cells................ 3
1.1.3 Alveolar macrophages ............................................................................................... 5
1.1.4 Lung dendritic cells.................................................................................................... 6
1.1.5 Lung macrophages and dendritic cells have different functions and location in
the lung....................................................................................................................... 7
1.1.6 Genes involved in the trafficking of mononuclear phagocytes ................................. 8
1.2 Serotonin.......................................................................................................................... 9
1.2.1 Serotonin as a neurotransmitter ................................................................................. 9
1.2.2 Serotonin and the immune system ............................................................................. 9
1.2.3 Serotonin receptors in the lung ................................................................................ 10
1.3 Granzymes ..................................................................................................................... 11
1.3.1 The Granzyme family .............................................................................................. 11
1.3.2 Non-apoptotic roles of granzymes A and B in immune cells .................................. 11
1.4 Aim of the study............................................................................................................. 13
2. Materials and Methods.................................................................................................. 14
2.1 Animals.......................................................................................................................... 14
2.2 Isolation of peripheral blood monocytes, lung macrophages and lung dendritic cells.. 14
2.3 Intratracheal LPS installation......................................................................................... 15
2.4 Pappenheim-stained cytocentrifuge preparations .......................................................... 15
Table of Contents
 - II -
2.5 Gene expression analysis ............................................................................................... 16
2.5.1 RNA isolation .......................................................................................................... 16
2.5.2 cDNA synthesis ....................................................................................................... 16
2.5.3 Real time quantitative PCR...................................................................................... 17
2.5.4 Microarray experiment............................................................................................. 18
2.6 Protein expression analysis............................................................................................ 21
2.6.1 Western blot............................................................................................................. 21
2.6.2 Flow cytometric analysis and flow sorting .............................................................. 22
2.6.3 Enzyme-linked immunosorbent assay ..................................................................... 23
2.6.4 Fluorescent substrate activity assay......................................................................... 24
3. Results ............................................................................................................................. 25
3.1 Immunophenotypic identification and high purity isolation of peripheral blood
monocytes, lung dendritic cells and lung macrophages.............................................. 25
3.2 Differentially expressed genes between peripheral blood monocytes, lung dendritic
cells and lung macrophages ........................................................................................ 28
3.3 Isolation and gene expression profiling of subpopulations of peripheral blood
monocytes and lung macrophages .............................................................................. 38
3.4 Confirmation of selected integrin expression by flow cytometry on subsets of
peripheral blood monocytes, lung macrophages, and lung dendritic cells ................. 43
3.5 Serotonin receptor expression in the lung...................................................................... 45
3.5.1 Alveolar macrophages express the 5-HT receptor ................................................ 45 2C
3.5.2 The serotonin receptors 5-HT are expressed by alveolar epithelial cells.......... 45 2A/2B
3.6 Serotonin action in the lung ........................................................................................... 47
3.6.1 Serotonin increase CCL2 production by alveolar macrophages acting through
the 5-HT receptor.................................................................................................. 47 2C
3.6.2 Microarray analysis reveal transcriptional changes in alveolar macrophages
stimulated with serotonin acting exclusively through the 5-HT receptor............. 48 2C
Table of Contents
 - III -
3.6.3 Validation of candidate genes by real time quantitative PCR ................................. 50
3.6.4 Serotonin increase the production of MIP-2 in alveolar epithelial cells.................. 51
3.6.5 TNF-α production in alveolar macrophages is not modulated by presence of
serotonin................................................................................................................... 52
3.7 Gramd3 as a potential cell surface marker for lung dendritic cells ............................... 54
3.8 Expression of proteases on lung dendritic cells............................................................. 55
3.9 Granzyme B is active in lung dendritic cells ................................................................. 56
4. Discussion........................................................................................................................ 58
4.1 Microarray analysis as a tool for studying the mononuclear phagocyte system in the
lung ............................................................................................................................. 58
4.1.1 Differentially regulated genes between lung macrophages and lung dendritic cells
are essential for cell migration................................................................................. 59
4.1.2 Differentially regulated integrins: validation of microarray results at the protein
level.......................................................................................................................... 60
4.2 Serotonin acts on alveolar macrophages through the 5-HT receptor.......................... 60 2C
4.2.1 Expression of serotonin receptors in the lung.......................................................... 60
4.2.2 Alveolar macrophages increase CCL2 production upon serotonin stimulation ...... 61
4.2.3 Serotonin changes the transcriptional profile of alveolar macrophages .................. 63
4.3 Expression of proteases in lung dendritic cells.............................................................. 66
4.3.1 Differential expression of MMPs among peripheral blood monocytes, lung
dendritic cells and macrophages ............................................................................