The role of T-cells in an acute and subchronic animal model of cigarette smoke-induced pulmonary inflammation [Elektronische Ressource] = Die Rolle von T-Zellen in einem akuten und subchronischen Tiermodell der Zigarettenrauch-induzierten pulmonalen Entzündung / vorgelegt von Ewald Benediktus

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The role of T-cells in an acute and subchronic animal model of cigarette smoke-induced pulmonary inflammation — Die Rolle von T-Zellen in einem akuten und subchronischen Tiermodell der Zigarettenrauch-induzierten pulmonalen Entzündung Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades Dr. rer. nat. vorgelegt von Ewald Benediktus aus Marienburg Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 13.10.2010 Vorsitzender der Promotionskommission: Prof. Dr. Rainer Fink Erstberichterstatter: Prof. Dr. Andre Gessner Zweitberichterstatter: Prof. Dr. Lars Nitschke Vorwort Vielen Dank an Herrn Prof. Dr. Andre Gessner für die Betreuung der Arbeit und die Erstellung des Erstgutachtens. Herrn Prof. Dr. Lars Nitschke danke ich für die Erstellung des Zweitgutachtens. Mein ganz besonderer Dank gilt Frau Dr. Birgit Jung und Herrn PD Dr. Florian Gantner dafür, dass sie die Kooperation zur Durchführung der Arbeit ermöglicht haben und mich in jeder Hinsicht unterstützt und gefördert haben. Weiterhin möchte ich mich bei Dr. Klaus Erb, Dr. Franz-Joseph Schneider, Dr. Lutz Wollin und Dr.
Publié le : vendredi 1 janvier 2010
Lecture(s) : 39
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Source : D-NB.INFO/1008557854/34
Nombre de pages : 119
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The role of T-cells in an acute and subchronic animal model of cigarette
smoke-induced pulmonary inflammation



Die Rolle von T-Zellen in einem akuten und subchronischen Tiermodell
der Zigarettenrauch-induzierten pulmonalen Entzündung







Der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur
Erlangung des Doktorgrades Dr. rer. nat.














vorgelegt von
Ewald Benediktus
aus Marienburg





Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg


















Tag der mündlichen Prüfung: 13.10.2010

Vorsitzender der
Promotionskommission: Prof. Dr. Rainer Fink

Erstberichterstatter: Prof. Dr. Andre Gessner

Zweitberichterstatter: Prof. Dr. Lars Nitschke





Vorwort

Vielen Dank an Herrn Prof. Dr. Andre Gessner für die Betreuung der Arbeit und die Erstellung des
Erstgutachtens.
Herrn Prof. Dr. Lars Nitschke danke ich für die Erstellung des Zweitgutachtens.

Mein ganz besonderer Dank gilt Frau Dr. Birgit Jung und Herrn PD Dr. Florian Gantner dafür, dass sie
die Kooperation zur Durchführung der Arbeit ermöglicht haben und mich in jeder Hinsicht unterstützt
und gefördert haben.

Weiterhin möchte ich mich bei Dr. Klaus Erb, Dr. Franz-Joseph Schneider, Dr. Lutz Wollin und Dr. Rolf
Goeggel für ihre stetige Bereitschaft zum wissenschaftlichen Austausch bedanken.

Die unkomplizierte und freundschaftliche Hilfestellung durch die Mitarbeiter des Labors Dr. Jung
(Mathilde Borsch, Madlen Konetzky, Almut Schüle, Kerstin Butscher, Petra Schweighöfer) und des
Labors Dr. Goeggel (Christian Seitz, Helene Lichius, Janine Beier) haben es mir ermöglicht viele neue
Methoden zu erlernen und die großen Versuchsreihen zu bewältigen. Vielen Dank dafür. Darin
einschließen möchte ich auch die zahlreichen anderen Mitarbeiter der Abteilung Atemwegsforschung
mit denen ich ebenfalls äußerst angenehm zusammenarbeiten konnte.

Diese Arbeit wäre kaum möglich gewesen ohne die hervorragende Unterstützung durch Prof. Dr.
Andreas Pahl. Die Zusammenarbeit hat sich nie auf das fachliche beschränkt und hat mir in
schwierigen Phasen immer wieder neue Perspektiven eröffnet.
Die stetige Motivation und der freundschaftliche Umgang haben wesentlich zum erfolgreichen
Abschluss des Projektes beigetragen.

Bei meiner Familie möchte ich mich für die Unterstützung in den vergangenen Jahren bedanken.



1 INTRODUCTION ...................................................................................................................... 1
1.1 Chronic Obstructive Pulmonary Disease ........................................................................................................ 1
1.1.1 Definition and Classification ....................................... 1
1.1.2 Burden and risk factors ............................................................................................................... 2
1.1.3 Pharmacologic treatment ............................................ 3
1.2 Models of COPD ................................................................................................................................................ 5
1.3 Cellular and molecular pathophysiology of COPD ......................... 7
1.4 The role of the adaptive immune system in COPD ....................................................................................... 11
1.5 Aim of the study .............................................................................. 15
2 MATERIAL AND METHODS ................................................................................................. 16
2.1 Animals ............................................................................................................................ 16
2.2 Cigarette smoke exposure .............................................................. 16
2.3 Bronchoalveolar lavage and tissue collection ................................................................ 17
2.4 Cytokine measurement ................................................................... 18
2.5 Single cell suspensions of lungs and lymph nodes ..................................................................................... 18
2.6 Flow cytometry ................................................................................ 19
2.7 Gene expression analysis in lung tissue ...................................................................... 20
2.8 Histology .......................................................................................................................... 21
2.9 Data analysis.................................................... 21
3 RESULTS .............................................................................................................................. 22
3.1 CD4-/- mice and CD8-/- mice are not protected from cigarette smoke-induced pulmonary
inflammation ................................................................................................................................................... 22
3.1.1 BAL fluid cells ........................... 22
3.1.2 BAL fluid mediators ................................................................................................................................................... 26
3.1.3 Lung tissue cells ........................ 28
3.1.4 Gene expression in lung tissue .................................................................................................................................. 31
3.1.5 Peribronchial collagen deposition ............................ 41
3.1.6 Parathymic lymph nodes ........................................................................................................................................... 43


Doctoral Thesis – Ewald Benediktus I


3.2 Nude mice are not protected from cigarette smoke-induced pulmonary inflammation ............................ 45
3.2.1 BAL fluid cells ........................................................................................................................................................... 45
3.2.2 BAL fluid mediators ................... 48
3.2.3 Gene expression in lung tissue .................................................................................................................................. 51
3.3 Resolution of cigarette smoke-induced inflammation in nude mice and wild type mice depends on
the experimental setup .................................................................................................................................. 59
3.3.1 BAL fluid cells ........................................................... 60
3.3.2 BAL fluid mediators ................................................................................................................... 62
3.4 IFN-γ producing Th1 and Tc1 cells are involved in cigarette smoke-induced pulmonary inflammation . 65
3.4.1 BAL fluid cells ........................................................................................................................................................... 65
3.4.2 Lung tissue cells ........................ 67
3.5 IL-10-/- mice display increased pulmonary inflammation in response to cigarette smoke....................... 69
3.5.1 BAL fluid cells ........................................................................................................................................................... 69
3.5.2 BAL fluid mediators ................... 73
3.5.3 Lung tissue cells ........................................................................................................................................................ 75
3.5.4 Gene expression in lung tissue .................................. 78
4 DISCUSSION ......................................................................................................................... 87
4.1 CD4+ T-cells and CD8+ T-cells are not required for pulmonary inflammation in a mid-term cigarette
smoke model ................................................................................................................................................... 87
4.2 Nude mice indicate that T-cells are dispensable for the onset and persistence of cigarette smoke-
induced pulmonary inflammation ................................................................................................................. 92
4.3 Airway neutrophilia tends to resolve faster in nude mice than in wild type mice dependent on the
specific experimental setup........................................................................................................................... 96
4.4 Th1 and Tc1 cells contribute to cigarette smoke-induced inflammation ................... 98
4.5 Cigarette smoke-induced inflammation is controlled by IL-10 .................................................................... 99
5 SUMMARY ........................................................................................................................... 104
6 ZUSAMMENFASSUNG ....................................................................................................... 106
7 REFERENCES ..................................................................................................................... 108
Doctoral Thesis – Ewald Benediktus II


List of abbreviations


AAT α-1 antitrypsin
AP activator protein
APC antigen-presenting cell
BAL bronchoalveolar lavage
BSA bovine serum albumin
CD cluster of differentiation

cDNA complementary deoxyribonucleic acid
COPD Chronic Obstructive Pulmonary Disease

C threshold cycle T
CXCR chemokine (C-X-C motif) receptor
EGFR epidermal growth factor receptor

ELISA enzyme-linked immunosorbent assay
eYFP enhanced yellow-fluorescent protein

FACS fluorescence-activated cell sorting
FCS fetal calf serum
FEV1 forced expiratory volume at 1 second

FVC forced vital capacity
GM-CSF granulocyte macrophage colony-stimulating factor

GOLD Global Initiative for Chronic Obstructive Lung Disease
HBSS Hank’s balanced salt solution
HDAC histone deacetylase

IFN interferon
IL interleukin

IP-10 interferon-γ-inducible protein-10
LABA long-acting β2-agonist
LAMA long-acting muscarinic antagonist

LPS lipopolysaccharide
MAPK mitogen-activated protein kinase

MCP monocyte chemotactic protein
MIP macrophage inflammatory protein
MMP matrix-metallo-proteinase

mRNA messenger ribonucleic acid
NF-κB nuclear factor-kappa-B

PCR polymerase chain reaction
PDE4 phosphodiesterase-4
Doctoral Thesis – Ewald Benediktus III


RANTES regulated upon activation, normal T-cell expressed and secreted
ROS reactive oxygen species
SCID severe combined immuno-deficient
Tc cytotoxic effector T-cells
TGF transforming growth factor
Th0 cells T helper cells type 0
Th1 cells T helper cells type 1
Th17 cells T helper cells type 17
Th2 cells T helper cells type 2
TIMP tissue inhibitor of metallo-proteinases
TNF tumour necrosis factor
Treg regulatory T-cell
yeti yellow-enhanced transcript for IFN-γ
Doctoral Thesis – Ewald Benediktus IV

1 Introduction

1 Introduction
1.1 Chronic Obstructive Pulmonary Disease
1.1.1 Definition and Classification
The Global Initiative for Chronic Obstructive Lung Disease (GOLD) defines Chronic Obstructive
Pulmonary Disease (COPD) as a preventable and treatable disease with some significant extra-
pulmonary effects that may contribute to the severity in individual patients. Its pulmonary component is
characterized by airflow limitation that is not fully reversible. The airflow limitation is usually progressive
and associated with an abnormal inflammatory response of the lung to noxious particles or gases [1].
The progressive loss of lung function is caused by a mixture of small airway obstruction, broncholitis
and destruction of parenchymal tissue (emphysema). In the past years there was increasing evidence
that COPD is accompanied by extra-pulmonary co-morbidities such as osteoporosis or pulmonary
hypertension. Therefore COPD could be considered as a systemic disease rather than a disease only
affecting the lungs [2].
Airflow limitation is characterized by a reduced forced expiratory volume at 1 second (FEV1) and
reduced forced vital capacity (FVC). Both parameters are easy to measure during the diagnosis and
serve for classification of disease severity. Based on spirometry, the severity of disease is classified
into 4 stages [1].
In stage I (mild COPD with FEV1/FVC < 0.70 and FEV1 > 80% predicted) people are usually not aware
of their beginning lung problems because clinical symptoms are rare.
In stage II (moderate COPD with FEV1/FVC < 0.70 and 50% < FEV1< 80% predicted) people may be
commonly driven to the physician for the first time as they may suffer from shortness of breath, cough
and sputum production. In stage III (severe COPD with FEV1/FVC < 0.70 and 30% < FEV1 < 50%
predicted) symptoms are getting worse and more frequent, particularly under exertion. Additionally,
tiredness and reduced exercise capacity occur. Repeated exacerbations have a distinct impact on the
quality of patients´ life. Stage IV is classified as very severe COPD with FEV1/FVC < 0.70 and FEV1 <
30% predicted or FEV1 < 50% predicted plus the presence of chronic respiratory failure. Patients
whose disease is classified as stage IV suffer from dramatic airflow limitation which then may result in
Doctoral Thesis – Ewald Benediktus 1

1 Introduction

chronic respiratory failure. Additionally, effects on the cardiovascular system are seen (cor pulmonale,
pulmonary hypertension). COPD patients may be more susceptible to respiratory infections. Acute
exacerbations can be life-threatening and lead to an accelerated progression of the disease.
Furthermore, severe COPD may be associated with emphysema, weight loss, development of
depression and anxiety.
In general, the progression of COPD may finally lead to respiratory insufficiency and death [3].

1.1.2 Burden and risk factors
COPD is a major cause of morbidity and mortality in the world. In 2001, 2.6 million people died of
COPD. While being the sixth commonest cause of death in 1990, COPD is predicted to become the
third leading cause of death by 2020 [4]. In contrast to the decreasing mortality of several other
common chronic disorders like coronary heart disease or infectious diseases, the death rate of COPD is
the only one that has increased dramatically in the past 40 years and will further do [4]. This disorder
represents an enormous economic and social burden, which has been greatly underestimated during
the past years and will probably lead to escalating healthcare costs.
Hospitalizations due to COPD and especially acute COPD exacerbations are increasing all over the
world. Meanwhile, COPD exceeded the costs of asthma by 3-fold [5].
Cigarette smoking is the main risk factor for COPD. In the majority of patients, the disease is provoked
by long-term tobacco smoking. As smoking behaviour intensified during the past, passive smoking of
side stream smoke by originally non-smokers became an increasing problem. Governments throughout
Europe passed laws to ban smoking in many public areas. The fact that only 10% of smokers develop
clinically significant COPD suggests that genetic predisposition has a distinct impact on the individual
risk [6, 7]. Among several genes which are suspected to predispose for COPD, genetic association
studies reliably revealed α-1 antitrypsin (AAT) deficiency as a rare genetic predisposition in COPD
patients. AAT is a serine protease inhibitor which inhibits neutrophil elastase. Patients suffering from a
AAT deficiency develop early onset emphysema [8]. The field of genetics of COPD has to be
investigated intensively in the future, to enable the identification of patients at risk for the disease.
Doctoral Thesis – Ewald Benediktus 2

1 Introduction

Occupational causes such exposure to fumes, chemical substances, and dusts have also been shown
to be relevant for COPD-like symptoms in non-smokers and therefore promote the onset of disease
upon chronic disposition [9]. Air pollution, especially in urban regions, is suspected to play an increasing
role as a risk factor for developing COPD, although its role remains controversial [10].
Inconsistent findings were made concerning gender [11]. The prevalence of disease seems to be equal
in men and women, even though there is a trend towards increasing smoking among women, especially
in developing countries. This may lead to an increased number of female patients suffering from COPD
in the future [11, 12].

1.1.3 Pharmacologic treatment
No current existing pharmacological treatment can stop progression of the disease. Smoking cessation
is the most effective therapy even if the quit rate only rarely exceeds 15%. Nevertheless, despite
cessation of smoking, the disease may continue as inflammation in the airways persists. However,
smoking cessation strategies can nowadays be supported by drugs against nicotine addiction [1].
Current pharmacologic treatment is mainly intended to prevent and control symptoms. Bronchodilators
are the main class of drugs used. They comprise β2-agonists and anti-cholinergics and are given via
the inhaled route to reduce systemic exposure and side effects. Both classes of compounds can be
combined to maximize efficacy. Long-acting β2-agonists like salmeterol and formoterol or the long-
acting anti-cholinergic tiotropium bromide are preferred instead of short-acting compounds, because a
once-daily administration is more convenient for the patient and enhances compliance. Furthermore, a
regular use of bronchodilators is considered more useful rather than the treatment on an as-needed
basis. Long-term treatment with combinations of long-acting β2-agonists (LABAs) and long-acting
muscarinic antagonists (LAMAs) was shown to go beyond bronchodilation by increasing exercise
capacity, improving health status and reducing the rate of acute exacerbations in COPD patients [1, 13].
However, these drugs do not stop progression of the disease.
Despite the great success of inhaled steroids in asthma treatment and the integration of steroids into
standard treatment regimes for asthmatic patients, these drugs do not modify the inflammatory
processes which are ongoing in the lungs of COPD patients [14]. The efficacy of steroids seems to be
Doctoral Thesis – Ewald Benediktus 3

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