Role of lung dendritic cells in acute pulmonary inflammation [Elektronische Ressource] / by Werner von Wulffen

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• Gesundheit

Informations

• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2008
• Nombre de lectures: 10
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Role of lung dendritic cells in acute pulmonary inflammation




Inaugural Dissertation
submitted to the
Faculty of Medicine
in partial fulfillment of the requirements
for the PhD-Degree
of the Faculties of Veterinary Medicine and Medicine
of the Justus Liebig University Giessen


by
Werner von Wulffen
of
Hannover
Giessen 2008




















From the Department of Internal Medicine II
Director / Chairman: Prof. Dr. W. Seeger
of the Faculty of Medicine of the Justus Liebig University Giessen




First Supervisor and Committee Member: Prof. Dr. J. Lohmeyer, Giessen
Second Supervisor and Committee Member: Prof. Dr. Th. Tschernig, Hannover
Committee Members: Prof. Dr. H.-J. Thiel and PD Dr. K. Mayer, Giessen
thDate of Doctoral Defense: August 6 , 2008
Table of Contents I
Table of Contents
Table of Contents................................................................................................I
Abbreviations ................................................................................................... IV
List of Figures VI
List of Tables VII

1 Introduction ..................................................................................................1
1.1 Dendritic cells ..........................................................................................1
1.2 Dendritic cells in the lung.........................................................................6
1.3 Fms-like tyrosine kinase 3-ligand (Flt3L) .................................................7
1.4 Pneumonia, acute lung injury (ALI), and adult respiratory distress
syndrome (ARDS) ...................................................................................9
1.5 Klebsiella pneumoniae...........................................................................11
1.6 Aims of the study12

2 Materials and Methods...............................................................................13
2.1 Mice.......................................................................................................13
2.2 Reagents ...............................................................................................13
2.3 Monoclonal antibodies15
2.4 Treatment protocols17
2.4.1 Treatment with Flt3L and in vivo application of mAb .......................17
2.4.2 Intratracheal instillation of LPS........................................................17
2.4.3 Infection experiments with Klebsiella pneumoniae..........................18
2.4.4 In vivo depletion of circulating neutrophils.......................................18
2.4.5 Peritoneal LPS injection ..................................................................18
2.5 Analysis of mice.....................................................................................19
2.5.1 Recovery of blood, spleens, bronchoalveloar lavage fluid, and
mediastinal lymph nodes19
2.5.2 Isolation and identification of lung DC and lung M φ.........................20
2.5.3 Flow cytometry and cell sorting.......................................................21
2.5.4 Immunohistochemistry ....................................................................22
2.5.5 ELISA and protein concentration measurement..............................22
2.5.6 SDS-PAGE of BAL fluid ..................................................................23 Table of Contents II
2.5.7 FITC albumin leakage assay...........................................................23
2.6. Functional characterization of lung DC .................................................24
2.6.1 Allogenic mixed lymphocyte reaction and MTT test .........................24
2.6.2 Sorting of DC and in vitro stimulation ..............................................25
2.6.3 RNA isolation, cDNA synthesis, and PCR.......................................25
2.7 Statistics ................................................................................................29

3 Results ......................................................................................................30
3.1 Identification and characterization of lung DC........................................30
3.1.1 Flow cytometric characteriziation .....................................................30
3.1.2 Morphology of flow-sorted lung DC and M φ .....................................31
3.1.3 Stimulatory properties in an
allogenic mixed lymphocyte reaction (MLR)32
3.2 Effect of systemic Flt3L application........................................................32
3.2.1 Effect of Flt3L application on the accumulation of lung DC .............32
3.2.2 Time course of Flt3L-elicited DC accumulation in
peripheral blood, spleen, and mediastinal lymph nodes...................35
3.3 Expression patterns of adhesion molecules ...........................................36
3.4. Blockade of Flt3L elicited DC recruitment to the lung by
monoclonal antibodies against adhesion molecules..............................39
3.5 Effect of Flt3L induced lung DC accumulation on the
lung inflammatory response to LPS.......................................................41
3.5.1 Cells in lung homogenate................................................................41
3.5.2 Cells in BAL fluid .............................................................................43
3.5.3 Proinflammatory cytokines in BAL fluid ...........................................44
3.5.4 Assessment of lung leakage ............................................................45
3.6 Klebsiella pneumoniae infection in Flt3L pretreated mice......................47
3.7 LPS-induced peritonitis in Flt3L pretreated mice ...................................50
3.8 Expression of proinflammatory cytokines by lung DC............................51
3.8.1 In vivo expression of proinflammatory cytokines in lung DC
after LPS instillation .........................................................................51
3.8.2 Effect of Flt3L on LPS-induced cytokine
gene expression in lung DC .............................................................52 Table of Contents III
4 Discussion ..................................................................................................53
4.1 Identification and characterization of lung DC........................................54
4.2 Treatment with Flt3L..............................................................................55
4.3 Adhesion molecules and recruitment of lung DC...................................56
4.4 Aggravated lung inflammation to LPS in Flt3L pretreated mice .............60
4.5 Klebsiella pneumoniae infection in Flt3L pretreated mice......................63
4.6 Conclusion and future perspectives.......................................................64

5 Summary.....................................................................................................67
5.1 Summary ...............................................................................................67
5.2 Zusammenfassung ................................................................................69

6 References..................................................................................................71

7 Appendix.....................................................................................................87
7.1 Publications ...........................................................................................87
7.2 Declaration ............................................................................................88
7.3 Acknowlegdments .................................................................................89
7.4 Curriculum vitae.....................................................................................90


Abbreviations IV
Abbreviations
A Absorption at 550/620 nm 550/620
ALI acute lung injury
APC allophycocyanin
ARDS adult respiratory distress syndrome
BAL bronchoalveolar lavage
bp base pairs
CD cluster of differentiation
CFU colony-forming units
DC dendritic cell(s)
FCS fetal calf serum
FITC fluorescein-iso-thiocyanate
Flt3L Fms-like tyrosine kinase-3 ligand
FSC forward scatter
HBSS Hank’s balanced salt solution
HEPES 2-(4-(2-Hydroxyethyl)- 1-piperacinyl)-ethansulfonic
acid
HSA human serum albumin
ICAM-1 intercellular adhesion molecule-1
IgG immunoglobulin G
IL-12 Interleukin-12
JAM-c junctional adhesion molecule-c
LPS lipopolysaccharide
M φ macrophage(s)
mAb monoclonal antibody
MHC major histocompatibility complex
MIP-2 macrophage inflammatory protein-2
MLR mixed lymphocyte reaction
MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5
diphenyltetrazoliumbromide
rAM resident alveolar macrophage
SSC side scatter Abbreviations V
SD s