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Role of lipopolysaccharide in pulmonary
inflammation and fibrosis – an in vitro study
Dissertation
zur Erlangung des akademischen Grades
des Doktors der Naturwissenschaften
an der Universität Konstanz (Fachbereich Biologie)
vorgelegt von
Marina I. Borisova
Tag der mündlichen Prüfung: 09.07.2010
Referent: Prof. Dr. Dr. Thomas Hartung
Referent: PD. Dr. Florian Gantner
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For Mom with all my love
На мама с любов
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ACKNOWLEDGEMENT
The work presented in this PhD thesis was carried out between August 2006 and
June 2010 at the Chair of Biochemical Pharmacology at the University of Konstanz
under the supervision of Prof. Dr. Dr. Thomas Hartung and PD Dr. Sonja von Aulock.
First of all, I want to thank Prof. Dr. Dr. Thomas Hartung for his valuable scientific
advice, inspiring discussions during my PhD work and for correcting my manuscripts.
I sincerely appreciate having such a warm and caring teacher. I wish you all the best
in your future career and private life.
I especially want to thank my supervisor PD Dr. Sonja von Aulock for her
unbelievable patience, outstanding support and stimulating ideas even in the time of
being a Mom. I am also grateful for the excellent working facilities and financial
support, for creating a friendly atmosphere and for reading my manuscripts.
Many thanks also go to Dr. Thomas Meergans for his never ending enthusiasm and
interest in my work, for his excellent experimental support and for his friendship.
I am indebted to all “Pulmo-net” members for the incredible time and for organization
of valuable seminars and courses and to the EU Framework 6 for financial support in
this work.
I thank Prof. Dr. Albrecht Wendel for welcoming me into his group and for the
unforgettable party time together.
Sincere thanks go to all current and former colleagues - Dr. Anna Góra, Dr. Christian
Draing, Dr. Christoph Rockel, PD Dr. Corinna Hermann, Dr. Mardas Daneshian, Dr.
Sebastian Bunk, Sonja Erath, Dr. Stefanie Sigel, Dr. Tobias Speicher, Dr. Oliver
Dehus and especially my wonderful students Jenny, Rebecca and Eva for sharing
good and bad times, for creating an excellent working atmosphere and for the
unforgettable time together inside and outside the lab.
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I also thank the team of current and former technicians Teodora Baris, Annette Haas,
Tamara Rupp, Margarete Kreuer-Ullmann and Anne Gnerlich for their technical
assistance.
I would like to thank my Mom and my sister for their never ending love and mental
support during the time I was far away from home.
Last but not at least I thank Christoph for his love and for creating a harmonic and
stress-free atmosphere in the last stage of my PhD work. I am so lucky to have you in
my life!
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TABLE OF CONTENTS
Abbreviations.................................................................................................. 8
I. General introduction ............................................................................... 11
1. INFLAMMATORY LUNG DISEASES ............................................................. 11
1.1. Physiology of the lung.................................................................................. 11
1.2. Inflammatory lung diseases ......................................................................... 12
1.3. Etiology of IPF ............................................................................................. 12
1.4. Treatment of IPF.......................................................................................... 14
1.4.1. Pharmacotherapy ..................................................................................... 14
1.4.2. Non-pharmacological treatment................................................................ 15
1.5. Animal models of IPF................................................................................... 16
2. MECHANISMS OF INFLAMMATION ............................................................. 17
2.1. Innate immunity ........................................................................................... 17
2.2. Lung innate immunity................................................................................... 18
2.3. Toll-like receptors and other pathogen recognition receptors ...................... 18
2.4. Pathogen associated molecular patterns..................................................... 20
2.4.1. Lipopolysaccharide ................................................................................... 20
2.4.2. Peptidoglycan ........................................................................................... 21
2.4.3. Staphyloccocal enterotoxin B.................................................................... 22
2.5. Toll-like receptor signaling ........................................................................... 22
2.6. Inflammatory cytokines ................................................................................ 23
2.6.1. Tumor necrosis factor-α............................................................................ 23
2.6.2. Interleukin-1 family of cytokines................................................................ 23
2.6.3. Interleukin-6 .............................................................................................. 25
2.6.4. Interleukin-8 .............................................................................................. 25
2.6.5. Transforming growth factor-β.................................................................... 25
3. IN VITRO MODELS OF PULMONARY INFLAMMATION AND DISEASE ..... 26
3.1. In vitro models of pulmonary inflammation .................................................. 26
3.1.1. Epithelial cells ........................................................................................... 27
3.1.2. Macrophages............................................................................................ 28
3.1.3. Co-culture models..................................................................................... 29
3.2. In vitro models of fibrosis ............................................................................. 29
3.2.1. Epithelial to mesenchymal transition......................................................... 29
6
3.2.2. Initiators of EMT in vitro ............................................................................ 30
4. AIMS OF THE STUDY.................................................................................... 32
II. Lung epithelial cells constitutively produce an
immunomodulatory factor for cytokine release by mononuclear
cells.................................................................................................33
1. ABSTRACT..................................................................................................... 34
2. INTRODUCTION ............................................................................................ 35
3. RESULTS ....................................................................................................... 37
3.1. A549 cells modulate inflammatory response of PBMC upon LPS, SEB and
PGN stimulation.................................................................................................. 37
3.2. Modulation of cytokine secretion by epithelial cells does not require direct
cell-cell contact ................................................................................................... 38
3.3. αIL-1β, but not αTNF-α neutralizing antibody, inhibits epithelial cell-mediated
amplification of IL-6 and IL-8 release in co-culture ............................................. 38
3.4. LPS-induced inhibition of TNF-α in mixed co-culture is regulated on post-
transcriptional level ............................................................................................. 39
3.5. Conditioned supernatant from resting A549 cells induces down-regulation of
TNF-α protein and mRNA in LPS-stimulated PBMC........................................... 40
3.6. The TNF-α inhibitory compound appears to be a peptide............................ 43
4. DISCUSSION ................................................................................................. 44
III. Conditioned supernatant from lipopolysaccharide stimulated
blood mononuclear cells induces epithelial to mesenchymal
transition in A549 cells: role of IL-1β and TNF-α .............................. 48
1. ABSTRACT..................................................................................................... 49
2. INTRODUCTION ............................................................................................ 50
3. RESULTS ....................................................................................................... 52
3.1. TGF-β1 induces EMT in A549 cells ............................................................. 52
3.2. A549 cells stimulated with LPS do not undergo EMT .................................. 53
3.3. Conditioned supernatant from LPS-stimulated PBMC induces EMT in A549
cells .................................................................................................................... 54
3.4. LPS-induced EMT is not driven by TGF-β1 ................................................. 55
3.5. IL-1β and TNF-α induce EMT in A549 cells ................................................. 56
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3.6. LPS-stimulated PBMC produce IL-1β and TNF-α, which induce EMT in lung
epithelial cells ..................................................................................................... 58
4. DISCUSSION ................................................................................................. 60
5. MATERIAL AND METHODS .......................................................................... 63
5.1. Reagents and antibodies ............................................................................. 63
5.2. Cell line........................................................................................................ 63
5.3. Isolation of human peripheral blood mononuclear cells ............................... 63
5.4. Enzyme-linked immunosorbent assay ......................................................... 64
5.5. A549-PBMC co-cultures .............................................................................. 64
5.6. Neutralization of IL-1β and TNF-α activity.................................................... 65
5.7. Inhibition of TNF-α processing by Brefeldin A.............................................. 65
5.8. LPS stimulation of PBMC in medium or A549 conditioned supernatant ...... 66
5.9. Total RNA extraction and cDNA synthesis................................................... 66
5.10. Quantitative Real-Time PCR...................................................................... 66
5.11. Characterization of TNF-α anti-inflammatory compound ........................... 67
5.12. SDS-PAGE and Western blot .................................................................... 67
5.13. Statistics .................................................................................................... 68
IV. References............................................................................................... 69
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ABBREVIATIONS
AcP IL-1 receptor accessory protein
ALI acute lung injury
ANOVA analysis of variance
ARDS acute respiratory distress syndrome
BAL bronchoalveolar lavage
BCA bicinchronic acid
BSA bovine serum albumin
CD cluster of differentiation
COPD chronic obstructive pulmonary disease
DMEM Dulbecco’s Minimal Essential Medium
DNA deoxyribonucleic acid
ECL enhanced chemiluminescence
EGF epidermal growth factor
ELISA enzyme-linked immunosorbent assay
EMT epithelial to mesenchymal transition
FCS fetal calf serum
FGF fibroblast growth factor
GlcNAc N-acetyl-glucosamine
HGF hepatocyte growth factor
HRP horseradish peroxidise
HSA human serum albumin
HSP heat shock protein
hu human
ICE IL-1β converting enzyme
IgG immunoglobulin G
IL interleukin
IL-1ra interleukin-1 receptor antagonist
IPF idiopathic pulmonary fibrosis
IFN interferon
IRAK IL-1 receptor-associated kinase
IRF interferon regulatory factor
kDa kilo Dalton
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LPS lipopolysaccharide
LTA lipoteichoic acid
MCP macrophage chemotactic protein
MD-2 lymphocyte antigen 96
MIP macrophage inflammatory protein
MyD88 myeloid differentiation protein
min minute
mRNA messenger RNA
mu murine
MurNAc N-acetyl-muramic acid
NaCl sodium chloride
NFκB nuclear factor κB
NOD nucleotide-binding oligomerization domain
NSIP nonspecific interstitial pneumonia
PAGE polyacrylamide gel electrophoresis
PAMP pathogen-associated molecular pattern
PBMC peripheral blood mononuclear cells
PBS phosphate buffered saline
PCR polymerase chain reaction
PDGF platelet derived growth factor
PGN peptidoglycan
PRR pattern recognition receptor
RIPA radioimmunoprecipitation assay buffer
RPMI Roswell Park Memorial Institute medium
R receptor
RT room temperature
SDS sodium dodecyl sulphate
SEB staphylococcal enterotoxin B
SEM standard error of the mean
SP surfactant protein
SV40 simian virus 40
TBS-T Tris-buffered saline with Tween 20
TGF-β transforming growth factor beta
TIR toll-interleukin 1 receptor
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TLR toll-like receptor
TMB 3,3’,5,5’-tetramethylbenzidine
TNF tumor necrosis factor
TRAF TNF receptor-associated family of proteins
TRIF TIR-domain-containing adapter-inducing interferon-β
Tris tris(hydroxymethyl)aminomethane
Tween20 polyoxyethylensorbitan monolaurate
UIP usual interstitial pneumonia
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I. GENERAL INTRODUCTION
1. INFLAMMATORY LUNG DISEASES
1.1. Physiology of the lung
The main function of the respiratory system is gas exchange. The respiratory system
consists of two functionally and structurally distinct regions known as upper and lower
respiratory tract. The upper respiratory tract includes the nose, the nasal cavity,
larynx and trachea and serves to filter, warm and humidify the inhaled air.
Downstream the trachea divides into left and right main bronchi, which enter the lung
parenchymal tissue. Bifurcation of the trachea in 23 generations forms the conductive
zone (main bronchi, small bronchi, bronchioles, terminal bronchioles) and respiratory
zone (respiratory bronchioles, alveolar ducts and the alveoli) of the lower respiratory
tract. The alveoli, also called air sacs, are surrounded by many blood capillaries. This
arrangement ensures optimal gas diffusion on the thin air-blood interface composed
of alveolar epithelial cells and capillary endothelial cells (Ehrhardt et al., 2002).
The total alveolar epithelial surface of an average adult human lung is very large -
140 m2 (Gehr, Bachofen & Weibel, 1978), and is comprised of type I and type II
epithelial cells. Type I squamous epithelial cells, mainly involved in gas exchange,
represent 93% of the epithelial surface area and only 33% the alveolar epithelial cell
number. Alveolar cuboidal type II cells are much smaller and represent the remaining
7% of the surface area and 67% of the epithelial cell number (Crapo et al., 1982).
Type II cells are situated in the corners of the alveoli and their physiological functions
include to serve as progenitors of damaged type I cells (Bhaskaran et al., 2007) and
to produce and secrete pulmonary surfactant. Pulmonary surfactant is composed of
80-90% phospholipids and about 10% surfactant proteins (SP) A, B, C and D and
plays an important role in maintaining physiological conditions during breathing by
reducing the surface tension in the lungs (Fehrenbach, 2001), (Goldmann et al.,
2009), (Johnston et al.). Alveolar epithelial type II cells also participate in ion
transport across the alveolar epithelium (Berthiaume, Lesur & Dagenais, 1999).
The most abundant phagocytic cell population in the lung airspaces is presented by
the alveolar macrophages, also called dust cells. These cells, derived by
differentiation of blood monocytes, account for approximately 95% of the lung
leukocytes (Martin & Frevert, 2005) and have a density of 17 x 103 cells/µl alveolar
12
fluid (Losa Garcia et al., 1999). The other 5% are distributed between lymphocytes,
neutrophils, dendritic cells and eosinophils.
1.2. Inflammatory lung diseases
Inflammation in general can be classified as acute or chronic. Acute lung
inflammation is initiated by resident alveolar macrophages and is characterized by
infiltration of blood monocytes and neutrophils into the lung tissue and in alveolar
spaces (Stockley, 1998), (Ulich et al., 1991). Acute inflammation goes through
phases of initiation, amplification and resolution coordinated by inflammatory
molecules called cytokines. During prolonged or chronic inflammation, resolution
cannot take place, progressive tissue destruction and development of pulmonary
illness occurs.
Inflammation is thought to play an important role in the pathogenesis of lung
diseases such as acute lung injury (ALI), acute respiratory distress syndrome
(ARDS), chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary
fibrosis (IPF). IL-1β, TNF-α and TGF-β1 were shown as common inflammatory
mediators implicated in asthma, COPD and pulmonary fibrosis (Araya & Nishimura).
Anti-inflammatory treatment of patients with low doses of corticosteroids was found
effective for diseases like ALI, ARDS (Lamontagne et al., 2009), (Tang et al., 2009)
and asthma (Rodrigo, 2006), (Cates & Lasserson, 2009), but ineffective for COPD
(Barnes, 2005) and IPF (Mapel, Samet & Coultas, 1996), (Richeldi et al., 2003).
Interestingly, particularly in smokers, COPD and IPF were found to coexist (Gauldie
et al., 2006).
1.3. Etiology of IPF
IPF, also called cryptogenic fibrosing alveolitis, is a chronic inflammatory and fibrotic
lung disease with unknown etiology, characterized by progressive scar formation in
the lung parenchyma. It affects approximately 5 million people worldwide with slightly
greater prevalence in men then in women. IPF is the most lethal interstitial lung
disease with no current cure and a median survival of 5 years from the time of
diagnosis. Different factors have been proposed as etiological factors – infections,
13
cigarette smoking, autoimmune disorders, chemotherapy, allergens and toxins (Kelly
et al., 2003), (Meltzer & Noble, 2008), (Taskar & Coultas, 2006).
The classical pathology of IPF, usual interstitial pneumonia (UIP), does not
demonstrate influx of inflammatory cells and is insensitive to conventional anti-
inflammatory treatment. In addition to UIP pathology, nonspecific interstitial
pneumonia (NSIP) is also diagnosed clinically as IPF. NSIP has more pathologic
inflammation than UIP. However, patients with IPF have been shown to have periods
of acute exacerbation characterized by inflammatory cell infiltration seen in
bronchoalveolar lavage samples and lung biopsies, and numerous inflammatory
mediators were also found in patients with active IPF (Kalluri, 2009), (Maher, Wells &
Laurent, 2007), (Meltzer & Noble, 2008), (Strieter & Mehrad, 2009), (Wilson & Wynn,
2009). However, hints exist that inflammation is a regulator of epithelial-
mesenchymal communication in the pathogenesis of this disease (Selman & Pardo,
2002), (Strieter & Mehrad, 2009), (Willis, duBois & Borok, 2006).
Fig. 1: Current hypothesis of IPF pathogenesis (Wynn, 2007).
14
The current hypothesis shown in Figure 1 suggests that repeating episodes of
unknown stimulus induce epithelial injury and the subsequent activation of
inflammation, platelet activation and blood clot formation. The cytokines produced
further activate epithelial cells to produce chemokines and recruit leukocytes from the
blood circulation. Specific feature of the injured place is the accumulation of cells
called myofibroblasts, which represent the hallmark of the fibrotic lung.
Myofibroblasts are cells of mesenchymal origin that produce extracellular matrix
compounds like collagen and fibronectin. During the normal wound healing process
re-epithelialization and regeneration of damaged tissue occurs. In case of chronic
injury or uncontrolled inflammation persistent myofibroblast activation leads to
excessive matrix deposition and scar formation (Wynn, 2007).
Myofibroblasts, the key mediators of fibrosis in the lung, are believed to have 3
different cellular sources (Ramos et al., 2001), (Selman, King & Pardo, 2001). The
morphological similarity with resident pulmonary fibroblasts has led to the idea that
myofibroblasts derive from these cells. Transdifferentiation of fibroblasts into
myofibroblasts in vitro was induced by stimulation with pro-fibrotic growth factor TGF-
β1 through a smad-dependent mechanism (Phan, 2002). In addition a population of
circulating bone-marrow-derived progenitor cells (fibrocytes) producing extracellular
matrix compounds was found to be an important source of lung myofibroblasts during
IPF (Hashimoto et al., 2004), (Quan et al., 2004). The contribution of alveolar
epithelial cells to myofibroblasts through a process called epithelial to mesenchymal
transition (EMT) is also extensively discussed (Phan, 2002), (Willis et al., 2006).
Based on different models of lung injury the number of cells deriving from resident
fibroblasts is estimated to be 60-80%, from epithelial cells 10-25% and from
fibrocytes 5-25% (Kisseleva & Brenner, 2008a). However, the relative contribution of
each source of myofibroblasts to the progression of pulmonary fibrosis is still under
investigation.
1.4. Treatment of IPF
1.4.1. Pharmacotherapy
There is currently no effective treatment for IPF. Different drugs have been proposed
and different clinical trials have been ongoing for anti-inflammatory, anti-fibrotic, anti-
15
antioxidant and anti-coagulant therapy. The traditional anti-inflammatory treatment
with corticosteroids was shown ineffective and even some data suggest rather
harmful than beneficial effects in treated patients (Flaherty et al., 2001). In addition,
immunosuppressive and cytotoxic agents such as cyclophosphamide (Zisman et al.,
2000), azathioprine (Raghu et al., 1991) and colchicin (Douglas et al., 1998) alone or
in combination with corticosteroids have shown disappointing results. Promising
results have been observed using an anti-fibrotic agent called pirfenidone, TGF-β
antagonist, shown to significantly decrease the number of acute exacerbations in IPF
(Azuma et al., 2005), (Raghu et al., 1999).
Increased amounts of TNF-α cytokine are observed in patients with IPF. Promising
results also point to etanercept, a TNF-α antagonist, which led to a decreased rate of
disease progression (Behr, 2007), (Raghu et al., 2008).
Confusing results were observed in phase II trials using a therapy with interferon-γ
(Meltzer & Noble, 2008). Moreover, clinical phase III trials testing were terminated in
2007 owing to observed side effects of treatment vs. placebo effects.
Another potential therapy for IPF is with N-acetylcysteine, which is a molecular
precursor of the naturally occurring antioxidant glutathione. Glutathione is lacking in
the epithelial lining fluid and intracellularly in BAL cells in the lungs of patients with
IPF (Meltzer & Noble, 2008). N-acetylcysteine, which was also shown to have anti-
inflammatory effects in the lung has more satisfactory results compared to other
current used substances to deteriorate IPF (Cui A., 2009).
1.4.2. Non-pharmacological treatment
Benefits of lung transplantation were demonstrated in patients with end-stage IPF.
The survival post transplantation was 73% at 1 year, 56% at 3 years, 44% at 5 years
and 24% at 10 years (Mason et al., 2007). One likely explanation for the low long
term survival could be the older age of patients undergoing surgery. However,
rejection represents a common problem and therefore transplantation is performed in
only carefully selected patients taking risks and benefits into consideration. In
addition, life-long treatment with immunosuppressants is necessary in order to
prevent lung rejection. Another challenge taking such a decision is the timing of
pulmonary transplantation (Meltzer & Noble, 2008), (Nathan, 2005).
16
As supportive therapy supplemental oxygen was also considered. However, no
improvement of quality of life was of patients receiving oxygen was observed (De
Vries, Kessels & Drent, 2001).
1.5. Animal models of IPF
Involvement of different cells and inflammatory mediators are studied in rodent
models of lung fibrosis. Bleomycin, a cancer chemotherapeutic drug, was found to
induce as a side effect fibrosis with a pathology which closely resembles that of IPF.
Although important differences in the animal pulmonary fibrosis and human IPF
disease were observed, the bleomycin model is still considered a valuable tool in
studying molecular mechanisms of pulmonary fibrosis (Agostini & Gurrieri, 2006),
(Bringardner et al., 2008).
Pulmonary radiation is another known cause of lung injury and fibrosis in humans
(Coggle, Lambert & Moores, 1986), (Movsas et al., 1997). Generation of free radicals
induces DNA damage and apoptosis. In addition, the inflammatory cascade is
activated and increased infiltration of neutrophils and alveolar macrophages occurs.
Irradiation is also a well-characterized and used model of pulmonary fibrosis in mice.
Fibrosis can also be induced by inhaled irritants like silica and asbestos. The
crystalline form of silica is toxic. Inhalation in humans leads to accumulation of
alveolar macrophages, activation of inflammation and may induce fibrosis. Exposure
to larger silica doses induces a massive fibrosis. Inhalation of asbestos fibers leads
to their deposition in the lung epithelium and induces injury. Alveolar macrophages
engulf these fibers and initiate inflammatory cascade that finally results in fibrosis. An
important element in silica and asbestos models is the involvement of the
inflammatory cascade (Barboza et al., 2008), (Robledo & Mossman, 1999).
Another experimental model proposed by Kolb et al. (2001) shows that transient
expression of IL-1β induces ALI and pulmonary fibrosis in mice. This model was used
to study the inflammatory reaction in the lung. The high expression of IL-1β was
accompanied by increased expression of IL-6 and TNF-α and later by pro-fibrotic
cytokines like TGF-β1 and platelet derived growth factor (PDGF) (Kolb et al., 2001).
The role of environmental pollutants like lipopolysaccharide (LPS) in the
pathogenesis of pulmonary fibrosis was long under discussion. Single intratracheal
administration to LPS in mice was shown to induce ALI as pathological features (Liu
17
& Luo, 2006). Phan et al. published that LPS-induced ALI could rather inhibit
bleomycin-induced pulmonary fibrosis (Phan & Fantone, 1984). Current
investigations indicate that chronic, but not acute, administration of LPS may induce
fibrotic lung disorders. Indeed, in 2008 Brass et al. have shown that long-term
exposure to aerosolized endotoxin and bleomycin-induced fibrosis share common
gene activation (Brass et al., 2008). Moreover, chronic LPS-induced ALI and
pulmonary fibrosis in mice was published by two independent research groups last
year, establishing a new pulmonary fibrosis model and indicating LPS as a link
between ALI and pulmonary fibrosis (He, Zhu & Jiang, 2009), (Li et al., 2009). This
model supports the previous postulations that IPF is in part an environmental disease
and indicates a correlation between LPS pollution and the manifestation of this
devastating disease (Taskar & Coultas, 2006).
2. MECHANISMS OF INFLAMMATION
2.1. Innate immunity
Higher vertebrates have developed two interactive protective systems: the innate and
adaptive immune system. The innate immune system is immediately responsible and
ready to recognize and inactivate infectious threats, i.e. bacteria, fungi, viruses, or
destroy cancer cells. It is older and consists of soluble proteins, which bind microbial
products, and phagocytic leukocytes, i.e. macrophages, dendritic cells and
neutrophils, which float through the bloodstream and migrate into tissues at sites of
infection, or reside in tissue waiting for foreign materials (Martin & Frevert, 2005),
(Strieter, Belperio & Keane, 2002). Innate immunity has relatively broad specificities.
It is based on the recognition of common microbial motifs called pathogen-associated
molecular patterns (PAMPs) by pattern recognition receptors (PRRs) expressed on
immune cells and as a result of their activation, cytokines and growth factors further
drive the specialized T- and B-cell responses of the adaptive immune system
(Takeda & Akira, 2005), (Toews, 2001). In contrast, the adaptive immune system,
also called acquired immunity, is characterized by memory and specificity. Together,
both immune systems enable the host to react to an array of microbial and other
products (Martin & Frevert, 2005).
18
2.2. Lung innate immunity
The lung possesses different mechanisms to protect itself from pathogen invasion.
Particles that enter the lungs with sizes between 1 to 5 µm are quickly and easy
removed by the mechanism of cough reflex or the mucociliary escalator. During
mucociliary clearance, ciliated and mucus-producing cells of the conductive airways
trap foreign particles and physically remove them outside the respiratory system by
coordinated upward cilia beating (Diamond, Legarda & Ryan, 2000). Beside the
importance of lung immune cells, the respiratory epithelium has been also shown to
participate in lung innate defenses through expression of PAMPs (Greene &
McElvaney, 2005), and receptors for inflammatory mediators (Bader & Nettesheim,
1996). In addition, phagocytes and airway epithelial cells produce constitutively or
upon bacterial and cytokine activation different anti-microbial agents. Anti-microbial
peptides like α- and β-defensins and cathelicidins (LL-37) act not only as
endogenous antibiotics but were also found to act as chemokines for neutrophils and
to participate in regulation of inflammation in the lung (Diamond et al., 2000),
(Hiemstra, 2006), (Tecle, Tripathi & Hartshorn, in press). An important role of SP-A
and SP-D, produced by alveolar epithelial type II cells in local innate immunity was
also indicated (McCormack & Whitsett, 2002).
A concept for organ-specific regulation of immune responses was proposed by Raz
(2007), which postulated that key inflammatory lung reactions are driven also by non-
immune cells (Raz, 2007).
2.3. Toll-like receptors and other pathogen recognition receptors
The most important PRRs in mammals are Toll-like receptors (TLRs). The first Toll-
protein was identified in the fruit fly Drosophila melanogaster, acting as a key
receptor regulating antifungal defense (Lemaitre et al., 1996). Then, a homologue of
the Toll-protein was found in vertebrates and shown to activate the release of
cytokines in human monocytes (Medzhitov, Preston-Hurlburt & Janeway, 1997). To
date, 10 TLRs have been identified in humans and 13 in mice. They are
transmembrane receptors with highly conserved regions. The extracellular domain
consists of leucine-rich repeats, flanked by cystein-rich regions. The intracellular
19
region contains a Toll-interleukin 1 receptor (TIR) domain, which shows similarities
with the cytoplasmic domain of the mammalian interleukin-1 type 1 receptor (IL1R1).
Most of the receptors are expressed predominantly on the surface of antigen-
presenting cells, except for TLR-3, -7, -8 and -9 which are intracellular (Mogensen,
2009), (Tabeta et al., 2004). TLR-2 has been shown to heterodimerize with TLR-1 or
TLR-6, enabling different PAMP recognition (Takeda & Akira, 2005). Illustration of the
TLR localization on immune cells is shown on Figure 2.
Fig. 2: Cellular localization of TLRs on antigen presenting cells (MacKichan, 2005)
Other PRRs also expressed on lung immune cells are scavenger receptors
(Palecanda et al., 1999), (Resnick et al., 1993), platelet-activating factor receptors
(Stengel et al., 1997), (Thivierge & Rola-Pleszczynski, 1995) or nucleotide
oligomerization domain (NOD) 1 and 2 receptors, found to be expressed
intracellularly (Chamaillard et al., 2003), (Girardin et al., 2003).
Epithelial cells represent a significant part of the lung tissue and therefore the
contribution of these cells to the innate immunity defense is an interesting area of
current research. Indeed, primary tracheal and bronchial epithelial cells were shown
20
to express TLRs 1-6 and 9 (Greene & McElvaney, 2005); (Platz et al., 2004). In
contrast to immune cells, TLR-4 expression in airway epithelial cells and the A549
alveolar epithelial cell line was mainly observed in the intracellular compartment, as
also seen in interstitial epithelial cells (Guillot et al., 2004). Another study has shown
that TLR2 was predominantly expressed on monocytes with only little TLR-2 being
detected in airway epithelium (Armstrong et al., 2004), (Mayer & Dalpke, 2007). In
agreement, Uehara et al. found that various human epithelial cells, including from
lung, express functional TLR-4, NOD1 and NOD2 to produce rather anti-microbial
products like β-defensin 2 than inflammatory mediators (Uehara et al., 2007). All
these data indicate that epithelial cells possess a number of differences in PRRs
expression grade and function, compared to professional immune cells.
2.4. Pathogen associated molecular patterns
PRRs recognize a large array of bacterial, fungal and viral PAMPs (Takeda & Akira,
2005). In the current study one focus is on characterization of PAMPs of bacterial
origin – lipopolysaccharide (LPS), peptidoglycan (PGN) and Staphylococcal
enterotoxin B (SEB). Lower respiratory tract infections with these bacterial
compounds is known to cause sepsis and death in humans (Knapp, in press).
2.4.1. Lipopolysaccharide
Lipopolysaccharide (LPS), the major immunostimulatory compound of Gram-negative
bacteria, was shown to be recognized by TLR-4 (Akira, Takeda & Kaisho, 2001),
(Takeda & Akira, 2005). LPS, also termed endotoxin, is an amphiphilic molecule and
consists of an outer part made of the O-antigen, a core region and a lipid A anchor
(Figure 3) (Caroff et al., 2002), (Rietschel et al., 1996). The O-antigen is formed by
repeating oligosaccharide sequences, highly variable among the Gram-negative
bacterial strains. The lipid A anchor, also called glycolipid, is highly conserved and is
responsible for the immunostimulatory potency of LPS (Galanos et al., 1985),
(Tanamoto et al., 1984). The recognition of LPS is extensively studied. LPS initially
interacts with a serum protein, LPS-binding protein, which transfers LPS to CD14
receptor, expressed on macrophages. A cell surface protein MD-2 is required for
binding to TLR4. Accordingly, the LPS recognition complex consists of CD14, MD-2
21
and TLR-4 (Toews, 2001). C3H/HeJ mice with non-functional TLR4 display impaired
LPS responses and are highly susceptible to Gram-negative infections (Poltorak et
al., 1998), (Wright et al., 1990).
Fig. 3: Structure of LPS (http://pathmicro.med.sc.edu/fox/lps.jpg).
2.4.2. Peptidoglycan
The bacterial cell wall of both Gram-negative and Gram-positive bacteria contains
peptidoglycan (PGN). PGN is a polymer of repeating units of N-acetyl-muramic acid
(MurNAc) and N-acetyl-glucosamine (GlcNAc). The carboxyl group of MurNAc is
linked to peptide subunits of four to five L- and D-amino acids that are cross-linked
with the peptide chain of the next MurNAc (D-alanine to mesodiaminopimelic acid).
The precise sequence of the peptide chain is species dependent, but it mainly
contains L-alanine, D-glutamine, lysine or diaminopimelic acid. Peptide bridges
between amino acids located in different glycan chains lead to the formation of a
complex three-dimensional network around the cytoplasmatic membrane (Weidel &
Pelzer, 1964). This arrangement of polymeric glycan, cross-linked by peptides, plays
a major role in the determination of cell shape and in maintenance of the physical
integrity of the bacterium. Although PGN has been demonstrated to play an important
role in bacterial recognition, a controversy remains whether PGN is recognized by
22
extracellular TLR-2 (Asong et al., 2009), (Dziarski & Gupta, 2005) or has to be
internalized to activate intracellular signaling via cytosolic Nod1/2 receptors (Girardin
et al., 2003).
2.4.3. Staphyloccocal enterotoxin B
Staphyloccocal enterotoxin B (SEB) is an exotoxin produced by the Gram-positive
bacterium Staphylococcus aureus. It is also termed superantigen because of its
ability to bind to major histocompatibility complex class II proteins on target cells and
stimulate proliferation of specific receptors on T cells (Krakauer, 1999).
After inhalation, clinical features include fever, respiratory complaints (cough,
dyspnea), and gastrointestinal symptoms. Severe intoxication results in pulmonary
edema, shock, and death. SEB can be easily aerosolized and therefore, in some
countries it is produced as a biological weapon (Mattix et al., 1995).
2.5. Toll-like receptor signaling
Binding of specific ligands to their respective TLRs induces receptor dimerization and
subsequent conformational change allows recruitment of cytoplasmic TIR-domain
adaptor proteins. MyD88 was the first adapter molecule identified (Burns et al., 1998)
as playing a major role in the TLR signaling pathway. MyD88 recruits signaling
molecules including IRAK family kinases and TRAF6 allow downstream activation of
NFκB transcription factors and MAP kinases which lead to the expression of
adhesion molecules, chemokines, cytokines and colony-stimulatory factors and
interferon regulatory factors (IRFs), which in turn induce the expression of type I
interferon and interferon-induced antiviral genes (Mizgerd, 2006), (Mogensen, 2009;
Takeda & Akira, 2005). The best studied and related to observed pro-inflammatory
reactions is the NFκB signaling pathway.
MyD88 is recruited to all TLRs except for TLR-3 (O'Neill & Bowie, 2007). TLR-3
signaling is coordinated by TRIF adaptor protein and links to IRF downstream
pathways (Figure 2).
23
2.6. Inflammatory cytokines
Cytokines are low molecular proteins or glycoproteins that mediate processes of
chemotaxis, growth, differentiation and cell death (Toews, 2001). Relevant cytokines
for the investigations in this thesis will be discussed here.
2.6.1. Tumor necrosis factor-α
Tumor necrosis factor-α (TNF-α) is one of the most important early pro-inflammatory
cytokines produced primarily by macrophages/monocytes upon microbial challenge
(Strieter et al., 2002). TNF-α is produced as a 26 kDa precursor protein which is first
displayed on the cell membrane (membrane TNF-α) and then cleaved by a protease
called TNF-α converting enzyme to 17 kDa non-glycosylated TNF-α. The mature
TNF-α protein is biologically active as a trimer and binds to two different TNF-α
membrane receptors - TNF-α receptor 1 (TNFR1, 55 kDa) and TNF-α receptor 2
(TNFR2, 75 kDa), which possess 1 to 6 cysteine-rich domains in the extracellular
part. TNF-R2 is mainly expressed on T-lymphocytes. TNFR1, also called Fas
receptor, is the universal receptor and therefore its signaling has been largely
studied. Upon ligand binding, TNFR1 trimerizes and activates downstream nuclear
factor NFκB, similar to TLR signaling and respectively enhancing transcription of
genes mediating innate immune responses (Idriss & Naismith, 2000), (Tang, Hung &
Klostergaard, 1996), (Toews, 2001), (Verstrepen et al., 2008).
2.6.2. Interleukin-1 family of cytokines
Interleukin-1 (IL-1) cytokine family includes IL-1α and IL-1β isoforms, encoded by 2
distinct genes, and their naturally occurring inhibitor IL-1 receptor antagonist (IL-1ra).
The members of this family are characterized by extracellular immunoglobulin (Ig)-
like structures and the presence of intracellular TIR domain. IL-1α and IL-1β are
synthesized as precursor molecules without leading sequence with a molecular
weight of 31 kDa. Processing of each precursor by a protease called calpain for IL-1α
and IL-1β converting enzyme (ICE) for IL-1β leads to the mature forms of 17 kDa and
16 kDa respectively. The mature IL-1α protein is mainly membrane-associated, while
IL-1β is a secreted protein. Both α and β isoforms bind to two different receptors IL-
1R1 and IL-1R2, but signal transduction is mediated only by IL-1R1. IL-1R2 contains
24
a very short cytoplasmic tail and cannot convey intracellular signals. Upon binding of
IL-1, IL-1R1 interacts with IL-1 receptor accessory protein (AcP) and forms a
functional receptor complex. With the help of adapter molecules downstream IL-1
pathways overlap with TNF-α and TLR signaling (Figure 4). Inflammatory studies on
IL-1 cytokines concentrate on the role of IL-1β as a predominant secreted cytokine
upon bacterial challenge (Dinarello, 1996), (Sims, Giri & Dower, 1994), (Strieter et al.,
2002).
IL-1β and TNF-α cytokines share similar pro-inflammatory functions, but structural
analyses show only a very short amino acid sequence of homology between the two
proteins and no overlap in receptor binding sites. However, computer analysis of the
second structure shows that these proteins have 8 β-strands and no α-helix
structures in common (Larrick & Kunkel, 1988). All nucleated cells possess functional
IL-1 and TNF-α receptors and these cytokines play a key role in initiation and
augmentation of inflammatory responses (Kelley, 1990).
Fig. 4: Receptor recognition of TNF-α, IL-1, and LPS activates overlap in NFκB
signaling pathway (Strieter et al., 2002).
25
2.6.3. Interleukin-6
Interleukin-6 (IL-6) is a cytokine produced by alveolar macrophages and blood
monocytes upon stimulation with LPS, IL-1β and TNF-α. Receptors for IL-6
recognition are expressed on different lung cells including epithelial cells, alveolar
macrophages and T-lymphocytes. Alveolar macrophages produce large amounts
upon endotoxin exposure and lower IL-6 amounts when exposed to IL-1β or TNF-α
(Kotloff, Little & Elias, 1990). IL-6 expression can be also induced by direct IL-1β and
TNF-α stimulation of the lung epithelial adenocarcinoma cell line A549 (Crestani et
al., 1994).
On systemic level IL-6 is the most important pro-inflammatory mediator of acute
phase response and fever. However, studies have also evaluated a possible anti-
inflammatory effect of this cytokine on systemic level and in the lung. Recombinant
IL-6 reduced TNF-α release in mice administrated with LPS intratracheally (Ulich et
al., 1991). Moreover, LPS administration in the lung in IL-6 gene deficient mice
induced increased neutrophils influx and significantly higher amounts of pro-
inflammatory cytokines TNF-α and macrophage inflammatory protein-1α (MIP-1α)
compared to cytokines in wild type mice (Xing et al., 1998).
2.6.4. Interleukin-8
Interleukin-8 (IL-8) belongs to the group of C-X-C chemokines characterized by
conserved cysteine residues (C), separated by another amino acid (X), and regulates
chemotaxis, adhesion and neutrophils activation in the lung (Strieter et al., 2002),
(Toews, 2001). Peripheral blood monocytes and alveolar macrophages are a
significant IL-8 source upon pathogen activation (Porreca et al., 1999), (Strieter et al.,
1990). In vitro studies also show that primary alveolar epithelial cells and cell lines,
including A549 are able to produce IL-8 upon TNF-α and IL-1β activation (Coulter et
al., 1999), (Henriquet et al., 2007), (Standiford et al., 1990).
Better understanding of the role of chemokines in the mechanisms of respiratory
diseases is still needed.
2.6.5. Transforming growth factor-β
Transforming growth factor-β (TGF-β) is growth factor family of cytokines which
exists in 3 isoforms (β1, β2 and β3) with 60 to 80% amino acid homology. In lavage
26
fluid of healthy volunteers TGF-β was about 5 ng/ml (Kelley, 1990). It plays an
important role in different processes of growth arrest, apoptosis, transformation,
proliferation and differentiation (Agostini & Gurrieri, 2006), (Ahmed & Nawshad,
2007). Interestingly, TGF-β may induce completely opposite biological effects in
different effector cells - activation of proliferation in fibroblasts and inhibition of
proliferation in epithelial cells (Eickelberg, 2001). TGF-β is abundant in the lung and
is produced by a large number of cells in a latent form (Kelley, 1990). The mature
latent form is produced as an inactive homodimer together with latency associated
peptide. This latent form must be activated (transient acidification, alkanization or
proteases) before receptor binding. The active form of TGF-β binds to specific type II
receptors and a tetramer complex is formed through recruitment of type I receptor.
An additional type III receptor has been shown to augment TGF-β responses
(Massague & Gomis, 2006). Well studied is the smad signaling, although TGF-β
smad-independent pathways were also described (Derynck & Zhang, 2003),
(Eickelberg, 2001), (Zhang, 2009). Accumulating data suggest that adhesion
molecules integrins control also TGF-β activation, as well as regulate TGF-β
signaling pathway in health and in diseases like COPD, IPF and cancer (Margadant
& Sonnenberg, in press).
3. IN VITRO MODELS OF PULMONARY INFLAMMATION AND DISEASE
3.1. In vitro models of pulmonary inflammation
In vivo inflammatory studies allow observations within the whole lung. However, the
contribution of single lung cell populations cannot be determined in such a complex
system. Thus, in vitro studies are helpful in understanding details of mechanisms of
lung inflammation. In vitro inflammatory models usually consist of isolated pulmonary
cells - alveolar macrophages or type II epithelial cells. However, it should be kept in
mind that neutrophils, endothelial cells, fibroblasts and dendritic cells are also
discussed to play a role in lung inflammation.
27
3.1.1. Epithelial cells
In vitro approaches to model the epithelium of the respiratory tract ranked in order of
diminishing complexity include isolated perfused organs, tissue explants and cell
culture models. The epithelial cell cultures include primary cultures with a limited life
span and immortalized tumor-derived or virus-transformed cell lines. A schematic
overview of the classification is given in figure 5.
Fig. 5: A) In vitro models of the lung epithelium. B) Subdivision of cell culture models.
For inflammatory studies cell culture models are largely used. Primary cells represent
the native epithelium better but their use faces certain limitations. These cells have to
be freshly isolated each time, isolation yield is very low and donor variations are
observed. Useful techniques and protocols for isolation of primary cells of the
respiratory tract were published by Rogers and Donnely (Rogers & Donnelly, 2001).
An advantage of using cell lines is that they are of known origin, and their immortal
nature allows prolonged studies. They give usually reproducible results, they are
easy to maintain and have low costs in comparison with other in vitro epithelial
models.
Transformed cells are usually manufactured by transformation of primary cells with
constructs containing the large T antigen of the SV40 virus or human papilloma virus
(Steimer, Haltner & Lehr, 2005).
Carcinoma cell lines are derived from various lung tumors. The most often used
cancer-derived model of the alveolar epithelium is the A549 cell line, isolated from
lung adenocarcinoma. This cell line shows similarities to the type II alveolar cells like
synthesis of phospholipids, presence of lamellar bodies and microvilli. However, they
isolated perfused organs
isolated tissues
cell culture models
cell culture models
cell lines primary cells
transformed cancer derived
A B
28
are less suited for transport studies where other cell lines like Calu-3, BEAS-2B,
16HBE14o- have been preferably used (Kelley, 1990), (Steimer et al., 2005).
3.1.2. Macrophages
Isolation of alveolar macrophages is relatively simple procedure and only requires
bronchoalveolar lavage in mice or humans. Seeding of the isolated cells allows
selection of the alveolar macrophages, shown to strongly adhere on the cell culture
plate. The major disadvantage using mice is the low yield and a large number of
animals needed to obtain sufficient number of cells. Therefore, alternatives have
been proposed to replace the use of these cells (Knapp, in press).
Human blood monocytes can be isolated from a whole blood together with
lymphocytes in a mixed population called peripheral blood mononuclear cells (PBMC)
and further purified, e.g. by magnetic cell sorting (Radbruch et al., 1994). Both, blood
monocytes and peripheral blood mononuclear cells are commonly used primary cells
in studies of innate immunity (Krakauer, 2002), (Wewers & Herzyk, 1989).
Immunostimulatory effects of different pathogen molecules, especially LPS, were
tested and comparative cytokine/chemokine profiles of blood monocytes and alveolar
macrophages were evaluated in vitro. Alveolar macrophages were shown to have >
20 fold decreased ability to produce IL-1β cytokine compared to blood monocytes,
measured by enzyme linked immunosorbent assay (ELISA). Measurement of total IL-
1β amounts in lysates indicates decreased processing of IL-1β precursor protein in
alveolar macrophages and respectively accumulation intracellularly (Strieter et al.,
1989), (Wewers & Herzyk, 1989). In contrast, alveolar macrophages were shown to
have 6 to 8 fold increase in TNF-α production, in comparison with blood monocytes.
When both cell types were compared for IL-6 expression, it was appreciated that
alveolar macrophages produce significantly more of this cytokine than blood
monocytes in response to endotoxin (Kotloff et al., 1990). Moreover, LPS was shown
to induce similar IL-8 expression in alveolar macrophages and blood monocytes
(Kelley, 1990).
Another alternative to primary cells is the use of human monocytic (THP-1, U937) or
macrophage Mono-Mac-1 cell lines. SV-40 transformed alveolar macrophage cells
line (MH-S) or macrophage-like cell line (RAW 264.7) from mice were also applied in
studies of in vitro inflammation (Knapp, in press), (Mbawuike & Herscowitz, 1989).
29
3.1.3. Co-culture models
An important role of both, macrophages and alveolar epithelial cells in innate immune
responses as mono-cultures has been suggested and discussed in details. However,
it is still unclear how is the expression of macrophage inflammatory cytokines upon
bacterial compounds modulated in the lung by the presence of alveolar epithelial
cells? Is this modulation stimulus-dependent or common for all PAMPs? Is it
dependent on cell-cell contact mechanisms? Even more intriguing to understand, is
which mechanisms modulate cytokine expression in a co-culture system of
macrophages and lung epithelial cells.
3.2. In vitro models of fibrosis
A better understanding the etiology of IPF may suggest new therapeutic targets.
Therefore, in vitro models of pulmonary fibroblasts, fibrocytes and lung epithelial
cells, are applied to characterize the contribution of each cell population in the
pathogenesis of IPF. Primary human lung fibroblasts, HFL-1 (embryonic) and IMR-90
human lung fibroblasts cell lines were used to assess the role of resident fibroblasts
and factors inducing the accumulation of myofibroblasts. Primary lung epithelial cells
and cell lines were used to study the process of EMT.
3.2.1. Epithelial to mesenchymal transition
The new hypothesis of EMT in the pathogenesis of IPF supports that fully
differentiated epithelial cells transform into mesenchymal cells (fibroblasts and
myofibroblasts). This EMT is a form of metaplasia and does not always require cell
division. During EMT epithelial cells lose their typical characteristics like cell-cell
contact, polarity, adhesion (E-cadherin), cytoskeleton proteins (cytokeratins) and
acquire spindle-like shape and mesenchymal markers like vimentin, α-smooth
muscle actin and collagen.
EMT was recently classified into 3 different types. Type 1 is crucial during
embryogenesis and organ development, where epithelial cells are able to give rise to
the mesenchyme. EMT can be also re-activated in adults during wound healing,
tissue reorganization and organ fibrosis (type 2 EMT); cancer progression and
metastasis (type 3 EMT). The opposite process, called mesenchymal to epithelial
30
transition (MET), is also observed during embryonic development and in adults
(Kalluri & Weinberg, 2009), (Xu, Lamouille & Derynck, 2009).
So far, it is still unclear whether alveolar epithelial type I, type II or both of the cells
undergo EMT (Figure 6). The current knowledge suggests that lung injury activates
type II cells (ATII) to undergo EMT and type I cells (ATI) more likely undergo
apoptosis (Kisseleva & Brenner, 2008b), (Willis et al., 2006).
Another type of cell plasticity is the transdifferentiation of ATII cells into ATI as a
normal mechanism of cell regeneration in the alveolar spaces. Studies in vitro show
that ATI cells may also transdifferentiate into type II cells but the occurrence of this
process in vivo is still in doubt (Kim et al., 2006), (Willis et al., 2006).
Fig. 6: Mechanisms of epithelial cell plasticity. Adopted from Willis et al. (Willis et al.,
2006).
3.2.2. Initiators of EMT in vitro
Different growth factors, transforming growth factor family (TGF-β), epidermal growth
factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF)
(Margadant & Sonnenberg, in press), as well as wnt ligands (Konigshoff &
Eickelberg, in press) and matrix metalloproteinases (Selman & Pardo, 2006) have
been shown to induce EMT in vitro. The most potent recognized in vitro (Kasai et al.,
2005), (Liu, 2008), (Willis et al., 2005) as well as in vivo (Kim et al., 2006) EMT
inducer is the TGF-β1. Therefore, ongoing studies evaluate the precise signaling
EMT
MET
injury
injury ? Apoptosis/ necrosis
ATII
ATI
?
Myofibroblast
31
pathways of TGF-β1 induced EMT in lung epithelial cells. Most commonly, smad-
signaling pathway has been activated upon ligand binding. Recruitment of smad 2
and 3 transcriptional factors to the TGF-β1 receptor complex is necessary for
initiation of the downstream cascade. Smad 2 and 3 molecules partner with smad 4
and the complex of smad 2, 3 and 4 transcriptional factors translocates to the
nucleus. As a result of smad-mediated transcriptional regulation, activation of target
genes results in repression of epithelial and activation of mesenchymal marker gene
expression (Willis et al., 2006), (Xu et al., 2009).
The findings that TGF-β is a “master switch” for EMT and fibrosis suggested that IPF
could be managed by anti-TGF-β therapy. Although promising results have been
shown still better characterization of other involved mediators is necessary to
improve IPF treatment.
Indeed, EMT was recognized as the possible link between lung inflammation and
fibrosis. Due to the late discovery of the chronic LPS–induced model of pulmonary
fibrosis, the hypothesis that IPF is in part environmental disease only entered the
discussion recently. Therefore, there are still no reports in vitro or even in vivo about
the role of environmental factors, particularly LPS and involved inflammatory
mediators in the EMT process.
32
4. AIMS OF THE STUDY
Alveolar macrophages, the most abundant immune cells in the alveolar spaces, are
key players in maintaining homeostasis and regulating immune defenses in the lung.
However, the epithelial cells lining the airways are the first point of contact for inhaled
pathogens. In recent years the role of epithelial cells in lung innate immunity has
become a focus of research interest, as it was discovered that airway epithelial cells
possess not only passive barrier function, but also actively contribute to fighting
respiratory pathogens.
• The aim of the first part of the study was to establish an in vitro lung co-culture
model to study the contribution of epithelial cells to inflammation upon stimulation
with bacterial stimuli from Gram-positive and Gram-negative bacteria. In addition
mechanisms of LPS-induced inflammatory modulation were characterized. The co-
culture model was composed of the alveolar epithelial type II like A549 cell line and
peripheral blood mononuclear cells (PBMC).
Recent in vivo investigations suggest that chronic exposure to environmental factors
like LPS induces lung inflammation and subsequent chronic lung diseases, including
pulmonary fibrosis. Myofibroblasts, the main cell population in pulmonary fibrosis, are
central in the pathogenesis of this disease. It is thought that epithelial cells may
differentiate into myofibroblasts through a process called epithelial to mesenchymal
transition (EMT). Recent understandings in the molecular mechanisms of fibrosis
show that EMT is the missing link between inflammation and fibrosis and indicate an
important role of TGF-β1 as master switch for EMT induction. However, the role of
LPS in the process of EMT was not studied so far.
• The aim of the second study was to investigate whether LPS stimulation of PBMC
in vitro induces an inflammatory response that mediates EMT of lung epithelial A549
cells. A subsequent goal was to establish the identity of the cytokine(s) produced by
PBMC which mediated EMT of A549 cells.
33
II.
Lung epithelial cells constitutively produce an immunomodulatory
factor for cytokine release by mononuclear cells
34
1. ABSTRACT
The contribution of airway epithelial cells to the innate immune response in the lung
has been a focus of recent interest. A human mixed co-culture system of lung
epithelial cell line A549 and primary peripheral blood mononuclear cells (PBMC)
allowed us to study modulation of IL-1β, TNF-α, IL-6 and IL-8 expression triggered by
lipopolysaccharide (LPS), staphylococcal enterotoxin B (SEB) and peptidoglycan
(PGN) in comparison to respective monocultures. All immune stimuli induced
significantly less TNF-α and much higher IL-6 release in co-cultures compared to
PBMC alone. IL-1β release was stimulus-dependent: down-regulated upon LPS, up-
regulated upon PGN and not affected upon SEB stimulation. IL-8 amounts were
significantly increased in LPS and PGN-stimulated co-cultures and slightly increased
upon SEB stimulation. Transwell experiments showed that LPS-induced cytokine
modulation in co-culture is triggered by soluble factors. Using neutralizing IL-1β
antibody we demonstrated that PBMC-derived IL-1β mediates augmented IL-6 and
IL-8 production in LPS-stimulated co-cultures. A still unknown soluble factor in
conditioned supernatant of resting A549 cells was shown to down-regulate TNF-α
release by PBMC on mRNA level. Characterization of this anti-inflammatory
compound suggests a peptide nature and opens an interesting area of future
research.
In conclusion, lung epithelial cells modulate the inflammatory response by down-
regulating TNF-α and increasing IL-6 and IL-8 in co-cultures and must therefore be
considered actors in shaping lung inflammation.
35
2. INTRODUCTION
The human lung is exposed to a large number of airborne pathogens as a result of
the daily inhalation of 10 000 liters of air. The observation that respiratory infections
are nevertheless rare is due to the presence of an efficient host defense system in
the lung. The airway epithelium represents a primary site of entrance and deposition
of potentially pathogenic microorganisms into the body, and therefore is equipped
with a variety of mechanisms to avoid infections (Bals & Hiemstra, 2004), (Diamond
et al., 2000). The epithelial lining fluid in the lower respiratory tract contains immune
cells: alveolar macrophages, T- and B-cells, neutrophils, eosinophils, mast cells and
dendritic cells. Alveolar macrophages, derived from blood monocyte differentiation,
account for up to 95% of the cells recovered by bronchoalveolar lavage (Bingisser &
Holt, 2001) and play a critical role in maintaining homeostasis, host defense,
response to foreign substances and tissue remodeling (Losa Garcia et al., 1999). To
combat infection, the phagocytic cells of the innate immune system express pattern
recognition receptors (PRRs), which recognize pathogen-associated molecular
patterns (PAMPs) on the surface of microorganisms. Toll-like receptors (TLRs)
function as major PRRs in mammals. TLRs are membrane-bound molecules
expressed on the surface or within intracellular compartments that participate in the
recognition of different microbial compounds. 10 members of this family (TLR 1-10)
have been identified in humans so far. The most prominent PAMP,
lipopolysaccharide (LPS or endotoxin) from the cell wall of gram-negative bacteria, is
recognized by TLR-4 (Akira et al., 2001), (Takeda & Akira, 2005). Intracellular
recognition of bacteria appears to also involve a TLR-independent system. Recent
studies indicate that nucleotide-binding oligomerization domain (NOD) 1 and 2
proteins recognize peptidoglycan (PGN) present in the cell wall of Gram-positive
bacteria (Mogensen, 2009). Staphyloccocal enterotoxin B (SEB, superantigen) acts
by directly binding to major histocompatibility complex class II molecules on antigen-
presenting cells and is recognized by αβ receptors on T-cells. Therefore, it is involved
in activation of the adaptive immune system response (Choi et al., 1989), (Krakauer,
2001).
A number of studies indicated that TLRs are also expressed in airway epithelial cells
(Gomez & Prince, 2008), (Muir et al., 2004). It was demonstrated that primary
tracheo-bronchial epithelial cells (Becker et al., 2000), alveolar epithelial type II A549
36
and bronchial epithelial BEAS-2B cell lines (Schulz et al., 2002) express mRNA for
TLRs 1-6. Studies on TLR-4 localization in A549 and BEAS-2B cell lines have
described a perinuclear location in association with the Golgi apparatus, rather than
at the cell surface. Despite TLR-4 expression these cell lines were not directly
responsive to LPS in serum-free medium, rather in the presence of serum and
concentrations higher the 1µg/ml (Guillot et al., 2004). Furthermore, it was observed
that primary lung epithelial cells and cell lines, release IL-6 and IL-8 in response to IL-
1β and TNF-α stimulation (Coulter et al., 1999), (Henriquet et al., 2007), (Jiang,
Kunimoto & Patel, 1998). IL-1β and TNF-α, are the most important early responsive
pro-inflammatory cytokines by immune cells in innate immune response (Strieter et
al., 2002). Secretion of IL-8 chemokine during bacterial infections in the lung was a
prerequisite for recruitment of neutrophils into the alveolar space (Reutershan & Ley,
2004). Apart of acute phase reactions, IL-6 was found to participate in modulation of
lung immune responses exerting stimulus-dependent pro- and anti-inflammatory
activities (Xing et al., 1998).
An in vitro co-culture lung model was devised to study the contribution of epithelial
cells to inflammation in the lung. Here we worked with human pulmonary epithelial
type II cells (A549) and primary peripheral blood mononuclear cells (PBMC). Our
findings indicate a function of alveolar epithelial cells in modulating the inflammatory
reaction in the lung.
37
3. RESULTS
3.1. A549 cells modulate inflammatory response of PBMC upon LPS, SEB
and PGN stimulation
A549 cells directly stimulated with 100 ng/ml LPS, 100 ng/ml SEB or 1 µg/ml PGN
did not release IL-1β, TNF-α, IL-6 or IL-8 (data not shown). However, the presence of
epithelial cells significantly down-regulated TNF-α and IL-1β expression of LPS-
stimulated PBMC (Figure 1A). In contrast, LPS exposure amplified IL-6 and IL-8
production in mixed co-cultures, compared to PBMC mono-cultures (Figure 1B).
A B
con LPS con LPS0
2
4
6TNF-αααα IL-1 ββββ
***
***
[ng
/ml]
con LPS con LPS0
50
100
150
200 IL-6 IL-8
******
[ng
/ml]
C D
con PGN SEB con PGN SEB0
5
10
15
20 TNF-αααα IL-1 ββββ
*** ***
***
ns
[ng
/ml]
con PGN SEB con PGN SEB0
50
100
150
200
250 IL-6 IL-8
***
***
***
ns
[ng
/ml]
Fig. 1: A549 cells modulate inflammatory response of PBMC alone upon LPS, SEB and
PGN stimulation. Mixed co-culture of A549 and PBMC (white bars) or PBMC alone (black
bars) were stimulated with 100 ng/ml LPS, 100 ng/ml SEB, 1 µg/ml PGN or PBS (con). After
24 h cell-free supernatants were assayed for TNF-α and IL-1β (A); IL-6 and IL-8 (B) by
ELISA. Data are means ± SEM from 11 PBMC donors in 3 independent experiments. TNF-α
and IL-1β (C); IL-6 and IL-8 (D) release upon stimulation with PGN and SEB were measured
by ELISA. Data are means ± SEM, 8 PBMC donors in 2 independent experiments (Repeated
measures ANOVA followed by Bonferroni’s multiple comparison test).
38
We investigated whether cytokine modulation is similar upon stimulation with other
PAMPs. The presence of A549 cells inhibited TNF-α expression also dramatically in
PGN- and SEB-activated PBMC. PBMC produced very low IL-1β amounts upon SEB
stimulation and cytokine amounts were not affected in co-cultures (Figure 1C). In
contrast, PGN activated PBMC to produce IL-1β and its amounts were increased in
co-cultures. SEB-stimulated PBMC in mono-culture did not produce IL-6, but a
massive IL-6 amplification was present in PGN- and SEB-stimulated co-cultures. We
also observed that PGN significantly up-regulated and superantigen tended to
increase IL-8 expression in co-culture compared to PBMC alone (Figure 1D).
3.2. Modulation of cytokine secretion by epithelial cells does not require
direct cell-cell contact
To find out whether LPS-driven inflammatory effects in the co-culture system are
modulated by cell contact mechanisms cells in mono-culture and co-culture were
seeded on 24 well normal or transwell plates with 0.4 µm pore size of the membrane
inserts (Figure 2). Cytokine release of IL-1β, TNF-α, IL-6 and IL-8 in both settings
had similar patterns arguing that the modulation is mediated by soluble factors, not
cell-cell contact.
3.3. αIL-1β, but not αTNF-α neutralizing antibody, inhibits epithelial cell-
mediated amplification of IL-6 and IL-8 release in co-culture
It was previously observed that IL-1β and TNF-α induce IL-6 and IL-8 release in A549
cells (Coulter et al., 1999), (Henriquet et al., 2007), (Jiang et al., 1998). We confirmed
these results (data not shown) and hypothesized that IL-1β and TNF-α from PBMC
mediate the amplification of IL-6 and IL-8 release in LPS-activated co-cultures.
Antibodies against IL-1β and TNF-α were used to neutralize cytokine effects upon
LPS stimulation. The neutralizing activity of the αTNF-α and αIL-1β antibodies was
demonstrated by inhibited IL-6 and IL-8 production in A549 cells stimulated with TNF-
α and IL-1β in quantities released by LPS-stimulated PBMC (data not shown).
Antibodies alone did not induce IL-6 and IL-8 production in non-stimulated cells.
αTNF-α antibody did not affect IL-6 and IL-8 production in LPS-stimulated co-cultures
39
and PBMC. Addition of αIL-1β to LPS-activated cells resulted in no significant
difference in IL-6 and IL-8 amounts between co-cultures and PBMC (Figure 3).
con LPS LPS0
1
2
3
4
Direct contact Transwells
***
***
TNF-αααα
TN
F- αα αα
[n
g/m
l]
con LPS LPS0.0
2.5
5.0
7.5
Direct contact Transwells
***
***IL-1ββββ
IL-1
ββ ββ [
ng
/ml]
con LPS LPS0
10
20
30
40
50
60
70
80
Direct contact Transwells
***
***
IL-6
IL-6
[n
g/m
l]
con LPS LPS0
10
20
30
40
****
Direct contact Transwells
IL-8
IL-8
[n
g/m
l]
Fig. 2: Comparison of cytokine induction in co-culture vs. PBMC upon LPS stimulation
in direct cell-cell contact and on transwells. Cells in mixed co-culture (white bars) or
PBMC (black bars) were seeded on 24 well plates in direct contact or were physically
separated by transwells (polycarbonate membrane, 0.4 µm pore size). After 3 h cells were
stimulated with LPS (100 ng/ml) or vehicle (PBS), supernatants were harvested after 24 h
and analyzed by ELISA. Data are means ± SEM from 6 PBMC donors in 2 independent
experiments (Repeated-measures ANOVA with Bonferroni’s multiple comparison test).
3.4. LPS-induced inhibition of TNF-α in mixed co-culture is regulated on
post-transcriptional level
In figure 4 we show that TNF-α down-regulation in LPS-stimulated co-cultures occurs
on post-transcriptional level. 3 h LPS stimulation of cells in co-culture or PBMC alone
in the presence of 5 µg/ml Brefeldin A blocked extracellular release of TNF-α, as
expected. This allowed the observation of a significant down-regulation of
intracellular pro-TNF-α in co-culture compared to amounts detected in PBMC.
40
con
LPS αααα
TNF-
αααα
αααα
TNF-
αααα
LPS +
ββββ
IL-1
αααα
ββββ
IL-1
αααα
LPS +
0
50
100
150
200
250***
***IL-8
ns
IL-8
[n
g/m
l]
con
LPS αααα
TNF-
αααα
αααα
TNF-
αααα
LPS +
ββββ
IL-1
αααα
ββββ
IL-1
αααα
LPS +
0
25
50
75
100
125
150
******IL-6
nsIL-6
[n
g/m
l]
Fig. 3: Effects of αIL-1β and αTNF-α neutralizing antibody on IL-6 and IL-8 production
in co-culture vs. PBMC. Cells in co-culture (white bars) or PBMC mono-culture (black bars)
were seeded in direct cell-cell contact settings on 48 well plates. In addition, neutralizing
TNF-α and IL-1β antibodies (1 µg/ml) were added and after 1 h cells were stimulated with
vehicle (con) or 100 ng/ml LPS for 24 h. Supernatants were collected and analyzed for IL-8
and IL-6 expression by ELISA. Data are means ± SEM and represent 2 independent
experiments, 8 PBMC donors (Repeated-measures ANOVA with Bonferroni’s multiple
comparison test).
3.5. Conditioned supernatant from resting A549 cells induces down-
regulation of TNF-α protein and mRNA in LPS-stimulated PBMC
We next tested whether the inhibitory effect on TNF-α release is exerted also by
conditioned supernatant from non-stimulated A549 cells. A549 conditioned medium
from different time points (2 h, 4 h, 8 h and 24 h) was added to PBMC and after 3 h
cells were stimulated with 100 ng/ml LPS for additional 24 h. We observed down-
regulated TNF-α secretion by LPS-stimulated PBMC in a time dependent manner
41
consistent with accumulation of an inhibitory factor in the supernatant of the resting
A549 cells (Figure 5).
con Brefeldin A LPS LPS + Brefeldin A0.0
0.5
1.0
1.5
2.0
2.5A - extracellular
***
TN
F-αα αα
[n
g/m
l]
con Brefeldin A LPS LPS + Brefeldin A0.0
0.2
0.4
0.6
0.8B - intracellular
***
TN
F-αα αα
[n
g/m
l]
Fig. 4: Regulation of TNF-α expression upon LPS stimulation occurs on post-
transcriptional level, not at release stage. Cells in mixed co-cultures (white bars) vs.
PBMC (black bars) were seeded on 48 well plates and after 3 h were stimulated with vehicle
(con) or 100 ng/ml LPS in the presence or absence of 5 µg/ml Brefeldin A. After 3 h
supernatants were collected for TNF-α ELISA measurement (A). Cell pellet was resuspended
in PBS and 3 freeze-thaw cycles were performed. Cell-free supernatants were harvested and
intracellular pro-TNF-α was analyzed by ELISA (B). Data are means ± SEM (8 PBMC donors
in 2 independent experiments). Statistical differences are analyzed by repeated-measures
ANOVA followed by Bonferroni’s multiple comparison test.
42
2 4 8 24 con0
1
2
3
***
time [h]
****** ***
TN
F-αα αα
[n
g/m
l]
Fig. 5: Conditioned supernatant from resting A549 cells induces down-regulation of
TNF-α in LPS-stimulated PBMC. PBMC were seeded in conditioned supernatant (76%)
from A549 (white bars) or medium (black bars) collected after different conditioning times
(2h, 4h, 8h and 24h). After 3 h PBMC were stimulated with vehicle (con) or 100 ng/ml LPS
for 24 h. Cell-free supernatants were analyzed for TNF-α by ELISA. Data represent mean ±
SEM from 3 independent experiments, 9 PBMC donors (Repeated measures ANOVA with
Bonferonni’s multiple comparison test).
The fact that the inhibitory factor was produced by resting A549 cells simplified the
investigation of whether its effect is already found on mRNA level. Stimulation of
PBMC with LPS in the presence of 24 h epithelial-derived supernatant showed a
significantly decreased TNF-α mRNA levels (Figure 6).
A549 SN Medium0
25
50
75
100
125
150
**
TN
F- αα αα
mR
NA
no
rmali
zed
to
GA
PD
H
Fig. 6: Effect of conditioned supernatant from A549 cells on TNF-α mRNA regulation in
LPS-activated PBMC. PBMC were seeded in 76% 24 h conditioned supernatant from A549
cells (white bar) or medium (black bar). After 3 h, PBMC were incubated with PBS or
stimulated with 100 ng/ml LPS. Expression of TNF-α mRNA was examined by quantitative
RT-PCR. Transcript expression was normalized to the housekeeping gene GAPDH. Control
values were subtracted from LPS values. Results are means ± SEM from 7 PBMC donors in
duplicates in 3 independent experiments (Mann-Whitney Test).
43
3.6. The TNF-α inhibitory compound appears to be a peptide
We aimed to characterize the TNF-α anti-inflammatory compound in the conditioned
supernatant of resting A549 cells. We worked with 76% and 30% supernatants. Our
results indicate that TNF-α inhibitory factor is heat and freeze (Figure 7A) stable.
However, 30% conditioned supernatant treatment with 0.5 mg/ml pronase reversed
TNF-α levels to those observed in the absence of conditioning. Experiments
identifying the size, using a filter with cut-off 10 kDa, have shown that eluted 76%
conditioned medium still possessed strong TNF-α suppressing activity (Figure 7B),
similar to observed without filtering and that filtrate of 30% conditioned medium
partially lost its activity (figure 7C). These data point towards a peptide as the anti-
inflammatory agent.
Fig. 7: TNF-α inhibitory compound
appears to be a peptide. A) 30%
conditioned supernatant from A549 cells
(A549 30%, white bars) or medium (black
bars) was not-treated; heated at 95°C for 15
min; digested with 0.5 mg/ml pronase and 2 x
freeze at -80°C. B) 76% (A549 76%) and C)
30% (A549 30%) conditioned supernatant
from A549 (white bars) or medium (black
bars) was filtrated through a filter with cut-off
10 kDa and eluted fractions were used for
experiment. 70µl PBMC were added to 380
µl supernatants and after 3 h were stimulated
with vehicle or 100 ng/ml LPS. After 24 h
cell-free supernatants were analyzed by
TNF-α ELISA. Data are means ± SEM (6
PBMC donors in 2 independent
experiments). Repeated measures ANOVA
with Bonferroni’s multiple comparison test
was applied for statistical differences.
con LPS con LPS con LPS con LPS0
1
2
3
4
5
95°C/15 min pronase freeze-thaw
A A549 30 %medium
******
***ns
TN
F- αα αα
[n
g/m
l]
con LPS con LPS0
1
2
3
4
5
eluted fraction
Bmedium
A549 76%
*** ***
TN
F- αα αα
[n
g/m
l]
con LPS con LPS0
1
2
3
4
5 C
eluted fraction
A549 30%
medium
******
TN
F- αα αα
[n
g/m
l]
44
4. DISCUSSION
Until recently the focus of research into the innate immune response of the lung has
been on the role of resident alveolar macrophages. In 2007, a study has shown that
various human epithelial cells, including from lung, express functional TLRs, NOD1
and NOD2 to produce anti-microbial products like β-defensin 2 rather than
inflammatory mediators (Uehara et al., 2007). In agreement we found that stimulation
of A549 cells in mono-culture with LPS, SEB or PGN did not activate these cells to
produce IL-1β, TNF-α, IL-6 or IL-8. In addition, Schulz et al. have shown that LPS
activates A549 cells to produce IL-6 and IL-8 only in µg doses and in the presence of
serum (Schulz et al., 2002). These results indicate that during infection, direct
stimulation of airway epithelial cells with bacterial compounds is not a significant
source of inflammatory mediators.
To better mimic the in vivo situation in the lung we designed an in vitro co-culture
system of A549 cells and PBMC and studied regulation of inflammation upon immune
stimulation. Here we show that the presence of lung epithelial cells modulate the
immune response of stimulated PBMC. All of the 3 chosen stimuli (LPS, PGN and
SEB) induced significantly lower TNF-α, and much higher IL-6 expression in
stimulated co-cultures compared to cytokine release in respective stimulated PBMC
mono-cultures. Early produced TNF-α cytokine from alveolar macrophages was
found to play an important role during initiation and augmentation phases of
inflammation (Kelley, 1990), (Strieter et al., 2002). IL-6 was shown as pro-
inflammatory regulator of acute phase responses and fever. However, in an LPS-
induced acute lung inflammation model IL-6 was described rather as anti-
inflammatory cytokine (Xing et al., 1998). Leemans et al. postulated differential roles
of IL-6 in lung inflammation by LTA (anti-inflammatory) and PGN (pro-inflammatory
effect) both from Staphylococcus aureus (Leemans et al., 2002). These results
indicate that indeed epithelial cells may create an anti-inflammatory milieu in the lung
through modulation of TNF-α and IL-6 produced from alveolar macrophages.
IL-8 expression in LPS- and PGN-activated co-cultures was also significantly
enhanced and tended towards an increase in SEB-stimulated co-cultures. It is known
that IL-8 is a very potent chemoatractant for neutrophils in the alveolar space (Coulter
et al., 1999), (Reutershan & Ley, 2004), therefore chemokine amounts increase as a
prerequisite for resolution of infection.
45
Modulation of IL-1β expression was stimulus-dependent: lower upon LPS, higher
upon PGN and unchanged upon SEB stimulation of co-cultures compared to
stimulated PBMC. Although this latter observation was very interesting, regulatory
mechanisms for IL-1β production were not further investigated in details.
To study whether LPS-induced inflammatory responses in co-culture require direct
cell-cell contact or are driven by soluble mediators, cells were seeded in parallel in
mixed and transwell co-cultures. Our results indicated no requirement for cell-cell
contact between lung epithelial cells and blood mononuclear cells for all modulated
co-culture inflammatory mediators. A critical point of using transwell plates was the
choice of the material for membrane inserts. Here polycarbonate material was used,
which produced reproducible and reliable results. Polyester membrane inserts from
the same company in contrast resulted in confusing and non-reproducible results in
comparative experiments. This indicates that the choice of membrane material is
crucial and has to be carefully selected according to the application and cell type.
We further investigated soluble mediators involved in LPS-induced mechanisms of
IL-6 and IL-8 modulation in co-culture. Interestingly, amplification of LPS-induced IL-6
and IL-8 release has also been reported in A549-neutrophil co-cultures (Grandel et
al., 2009). Indeed, stimulation of lung epithelial cells or lung adenocarcinoma cell
lines with IL-1β and TNF-α induces expression of IL-6 and IL-8 (Coulter et al., 1999),
(Henriquet et al., 2007), (Jiang et al., 1998). A549 cells were also found to express
IL-1 type I receptor (Coulter et al., 1999) and TNF-receptor I (Burvall, Palmberg &
Larsson, 2005). Here we show that only neutralizing antibody against IL-1β, but not
TNF-α, returns IL-6 and IL-8 expression in LPS-stimulated co-cultures to levels
comparable with those in LPS-stimulated PBMC mono-cultures. These results
indicate an important role of IL-1β in augmenting the IL-6 and IL-8 response to LPS
in the lung. The importance of IL-1β, but not TNF-α, in enhancing IL-8 expression in
silica-treated lung cell co-cultures of A549 and THP-1 monocyte-derived cell line was
also described (Herseth et al., 2008). One year later, the same group showed that IL-
1β is responsible also for increased IL-6 release in silica activated lung cell co-
cultures (Herseth et al., 2009).
We further characterized LPS-induced inhibition of TNF-α in co-culture. Brefeldin A
blocks protein transport from the endoplasmic reticulum to the Golgi apparatus,
allowing the determination of unprocessed cytokines. We demonstrated that pro-
TNF-α in LPS-stimulated PBMC was already significantly down-regulated by the
46
presence of A549 cells. As we had observed that already conditioned supernatant
from resting A549 cells accumulated the anti-inflammatory activity over time, we
could use this to investigate whether the unknown factor acts on mRNA level.
Therefore, avoiding direct contact between the two cell populations and resulting
difficulties with normalization of the mRNA data, we studied transcriptional TNF-α
regulation of PBMC using 24h conditioned supernatant from A549 cells. TNF-α cDNA
data were also consistent with observed extracellular and intracellular TNF-α
inhibition in co-cultures. In agreement with our observations, it was published recently
that the bronchial epithelial BEAS-2B cell line inhibited secretion of TNF-α by human
monocytes and monocyte-derived macrophages on transcriptional level (Mayer et al.,
2008). However, this group tried to identify anti-inflammatory mediator(s) specifically
produced by lung epithelial cells. In our hands, substitution of A549 cells with
epithelial cervix HeLa cell line and embryonic kidney HEK293T cell lines in mixed co-
cultures with PBMC also resulted in down-regulated TNF-α production (unpublished
observations). These results indicate that inhibition of TNF-α release in co-culture is
not specific to the A549 cell line or epithelial cells of the lung.
Finally, initial characterization of the anti-inflammatory factor in A549-derived
conditioned medium showed that it is heat and freeze stable. This compound filters
through a 10 kDa cut-off filter membrane, but is inactivated by a protease called
pronase. These data suggest a peptide as a possible candidate but further
experiments are needed to prove its nature. Indeed, A549, HEK293T and HeLa cells
were shown to constitutively produce abundant amounts of peptide called beta
defensins 1 (Pazgier et al., 2006), (Valore et al., 1998), (Zucht et al., 1998), (Jang et
al., 2004), (Hegedus et al., 2008). Beta defensin 1 is a member of the defensins
family which was shown to be heat stable (Tunzi et al., 2000) and to have a size of
47 amino acids residues (5 kDa). In addition, defensins were recognized to act not
only as anti-microbial peptides but are also described as anti-inflammatory mediators
in innate immune responses (Semple et al.), (Tecle, Tripathi & Hartshorn).
Taken together, lung epithelial cells in LPS-stimulated co-cultures induce modulatory
effects shown by amplification of IL-6 and IL-8 in co-culture, induced by IL-1β and
down-regulation of TNF-α on protein and mRNA level, caused by a still unknown
soluble factor. The identification and characterization of a possibly novel anti-
inflammatory peptide would greatly improve our understanding of the regulation of
47
inflammation and treatment of inflammatory diseases. Further co-culture studies are
necessary in better understanding the intimate inflammatory mechanisms in the lung.
48
III.
Conditioned supernatant from lipopolysaccharide stimulated blood
mononuclear cells induces epithelial to mesenchymal transition in
A549 cells: role of IL-1β and TNF-α
49
1. ABSTRACT
Recent investigations postulate that lung inflammation due to environmental
pollutants, including lipopolysaccharide (LPS) plays an important role in the
development and progression of different chronic lung diseases, including idiopathic
pulmonary fibrosis. Advances in understanding the molecular mechanisms of this
disease point to epithelial to mesenchymal transition (EMT) as a link between
inflammation and fibrosis, but so far the role of LPS in the process of EMT has not
been investigated. Therefore, we hypothesized that conditioned supernatant from
LPS stimulated peripheral blood mononuclear cells (PBMC) induces an inflammatory
cascade and subsequent EMT in lung epithelial type II A549 cell line. EMT in
epithelial cells was evidenced after 3 days by alteration in cell morphology assessed
by phase contrast microscopy and analysis of Western blot marker expression. A549
cells alone stimulated with LPS did not undergo EMT. Conditioned supernatant from
LPS stimulated PBMC activated A549 cells to undergo EMT, evidenced by
acquisition of mesenchymal morphology, down-regulation of epithelial cytokeratin
and up-regulation of mesenchymal vimentin expression. Direct stimulation with IL-1β
and TNF-α, but not IL-6, IL-8, MCP-1, MIP-1α and IL-10, shown to be produced from
PBMC upon LPS-stimulation, induced fibroblast-like morphology, decreased
cytokeratin and increased vimentin expression in A549 cells. Using neutralizing
antibodies for IL-1β, TNF-α and IL-1 receptor antagonist added to conditioned
supernatants from LPS-stimulated PBMC showed that IL-1β and TNF-α cytokines are
responsible for the observed EMT effect. Thus, our findings indicate a central role of
IL-1β and TNF-α in the in vitro LPS-driven EMT process in A549 cells.
50
2. INTRODUCTION
Idiopathic pulmonary fibrosis (IPF) is a progressive and relentless inflammatory and
fibrotic lung disease. It has been recognized as the most lethal interstitial lung
disease and lung transplantation represents the only option for cure. IPF is
characterized by excessive matrix deposition and destruction of the lung architecture
(Agostini & Gurrieri, 2006), (Selman & Pardo, 2006).
The hallmark of pulmonary fibrosis is the abundance of myofibroblasts. It is
considered that these cells are derived from three different sources: resident lung
fibroblasts, blood-borne cells called fibrocytes, and from epithelial cells through
epithelial to mesenchymal transition (Scotton & Chambers, 2007). EMT is recognized
as a physiological mechanism occurring during embryonic development (Acloque et
al., 2009), and this process may be reactivated in adults during wound healing, tissue
regeneration, organ fibrosis and cancer progression (Kalluri, 2009). EMT is
characterized by loss of cell polarity and a change from cuboidal to fibroblastoid cell
shape. In addition, reorganization of the cytoskeleton system, down-regulation of
epithelial and acquisition of mesenchymal characteristics occurs (Kalluri & Neilson,
2003), (Xu et al., 2009), (Zavadil & Bottinger, 2005).
An experimental animal study has identified epithelial-derived mesenchymal cells in
TGF-β1 driven fibrosis (Kim et al., 2006). Wu et al. observed airway EMT in
bleomycin-induced peribronchial fibrosis mice (Wu et al., 2007). EMT can also be
observed in vitro in a variety of normal epithelial cells and epithelial-derived
carcinoma cell lines by stimulation with soluble growth factors, i.e. transforming
growth factor-β family (TGF-β), fibroblast growth factor (FGF), epidermal growth
factor (EGF) or hepatocyte growth factor (HGF) (Kasai et al., 2005), (Margadant &
Sonnenberg, in press), (Zhang, Dong & Yang, 2006).
In the inflammatory milieu of chronic lung diseases, the major cytokines present are
IL-1β and TNF-α (Araya & Nishimura, 2010). In an in vivo lung injury model, transient
over-expression of IL-1β in rat airway epithelial cells resulted in increased expression
of TNF-α and IL-6, followed by an increase in bronchoalveolar lavage of platelet
derived growth factor (PDGF) and TGF-β1 (Kolb et al., 2001). In a bleomycin-induced
animal model of pulmonary fibrosis soluble TNF-α was found as a prerequisite for the
development of this disease (Oikonomou et al., 2006), suggesting a relation between
IL-1β and TNF-α inflammatory cytokines and the conversion from inflammation into
51
fibrosis. As a common link between inflammation and fibrosis recent findings point
EMT. This understanding was supported by a report that a cocktail of IL-1β, TNF-α
and IFN-γ augments TGF-β1 induced EMT in lung epithelial A549 cells (Liu, 2008).
The role of environmental factors such as pyrogenic bacterial or fungal components
in the pathogenesis of IPF is a subject of current investigations. LPS (endotoxin) is
the predominant pyrogen derived from Gram-negative bacteria. In 1984, Phan et al.
(Phan & Fantone, 1984) reported that a single intraperitoneal endotoxin injection in
mice inhibits bleomycin-caused pulmonary fibrosis. In contrast, Brass et al. (2008)
have shown that ongoing but not single LPS challenge and bleomycin-induced
fibrosis share common patterns of gene expression. It was also reported that LPS
three-hit regiment induces acute lung injury (ALI) and rapid pulmonary fibrosis in
mice (Li et al., 2009). In addition, He et al. found that 72 h LPS administration
induced ALI and caused pulmonary fibrosis (He et al., 2009). All these data taken
together indicate that chronic but not acute exposure to LPS induces pulmonary
fibrosis and represents a new paradigm in understanding the etiology of this disease.
Based on these results, we hypothesized that chronic exposure to environmental
pyrogens in the lung may cause EMT and subsequent fibrosis via the inflammatory
cytokine milieu. To test this hypothesis in vitro, we asked whether LPS stimulation of
peripheral blood mononuclear cells (PBMC) is able to induce an inflammatory
response that can mediate EMT of lung epithelial type II cells (A549). Here we show
that indeed exposure of PBMC to endotoxin activates secretion of soluble mediators
that induce EMT of A549 cells and that IL-1β and TNF-α play a central role in
mediating the EMT process. EMT in epithelial cells was evidenced by acquisition of
fibroblast-like morphology, significant decrease in cytokeratin and increase in
vimentin marker expression.
52
3. RESULTS
3.1. TGF-β1 induces EMT in A549 cells
In agreement with other investigators (Kasai et al., 2005), (Liu, 2008) our study
demonstrated that TGF-β1 induces EMT in A549 cells. The lung epithelial cells
cultured in the presence of the growth factor adopted fibroblast-like morphology and
reduced their cell-cell contacts (Figure 1A). EMT was also evidenced by decrease in
E-cadherin and cytokeratin epithelial markers and increase in vimentin mesenchymal
marker expression (Figure 1B).
Fig. 1: TGF-β1 induces EMT in A549 cells. A549 cells (5 x 104 in 1ml) were plated on 24
well plate in DMEM + 10% FCS. After 16 h, culture medium was replaced by serum-free
RPMI-1640 and cells were stimulated with PBS (control) or with 5 ng/ml TGF-β1 for 72 h. A)
Phase contrast microphotographs of A549 cells show changes in cell morphology upon
treatment (magnification 100 x). B) Cell lysates were immunoblotted for E-cadherin, pan-
cytokeratin, vimentin and β-actin. 20 µg total proteins were loaded on each lane.
Representative blots are shown from 2 independent experiments.
A B
0 5
TGF-β1 [ng/ml]
E-cadherin
cytokeratin
vimentin
β-actin
Control
TGF-β1
53
3.2. A549 cells stimulated with LPS do not undergo EMT
Although A549 cells have been shown to express TLR-4, predominantly in the
intracellular compartment (Guillot et al., 2004), even 10 µg/ml endotoxin exposure
was not able to induce IL-1β, TNF-α, IL-6 and IL-8 secretion in serum-free conditions
(data not shown). Stimulation of epithelial cells with 10 ng/ml LPS for 72 h did not
alter cell morphology and the expression of the molecular markers examined was not
affected (Figure 2).
Fig. 2: LPS does not induce EMT in lung epithelial cells. A549 cells (5 x 104 in 1ml) were
plated on 24 well plate in DMEM + 10% FCS. After 16 h, culture medium was replaced by
serum-free RPMI-1640 and cells were stimulated with PBS (control) or with 10 ng/ml LPS for
72 h. (A) Cell morphology was assessed under phase contrast light microscopy
(magnification 100 x). (B) Equal amount of total proteins (20 µg) were loaded in each lane
and E-cadherin, vimentin and hsp 90 α/β marker expressions were analyzed by Western blot.
Representative blots are shown from 2 different experiments.
0 10
LPS [ng/ml]
E-cadherin
vimentin
hsp 90 α/β
A B
Control
LPS
54
3.3. Conditioned supernatant from LPS-stimulated PBMC induces EMT in
A549 cells
Transfer of supernatant from LPS-stimulated PBMC to A549 cells resulted in EMT.
This event has been evidenced by changes in cell morphology and marker
expression (Figure 3). The typical epithelial morphology was replaced by fibroblast-
like and the cells dramatically decreased their cytokeratin expression and acquisition
of vimentin mesenchymal marker expression was observed.
Control LPS
Control LPS0.0
0.5
1.0
***
cyto
kera
tin
/ h
sp
90
αα αα/ ββ ββ
Control LPS0.0
2.5
5.0
7.5
***
vim
en
tin
/ / / /h
sp
90
αα αα/ ββ ββ
Fig. 3: Effect of conditioned supernatant from LPS-stimulated PBMC on EMT in A549.
1 x 106 in 1ml PBMC were seeded on 24 well plates. After 2 h mononuclear cells were not
stimulated with PBS (control) or stimulated with 10 ng/ml LPS for 20 h at 37°C. A549 cells (5
x 104 in 1ml) were plated on 24 well plate in DMEM + 10% FCS. After 16 h epithelial culture
medium was replaced by cell-free supernatants from untreated or LPS-stimulated PBMC and
A549 cells were incubated for 72 h at 37°C. A and B panels represent the morphology of
A549 cells, incubated in supernatant from (A) untreated PBMC or (B) LPS-treated PBMC
(magnification 10 x). C and D panels show respectively the Western blot analysis for
A B
C D
55
cytokeratin and vimentin. Results are based on densitometric analysis of the ratio of each
marker protein to hsp 90 α/β. For each donor control values were set to 1. Data are pooled
from 3 independent experiments (9 different PBMC donors) and represent a mean ± SEM
(Wilcoxon matched pairs test).
3.4. LPS-induced EMT is not driven by TGF-β1
We tested whether TGF-β1 plays a role in PBMC mediated LPS-induced EMT in
A549 cells. TGF-β1 production was analyzed in the supernatant of control and LPS-
stimulated PBMC after 20 h and in supernatants after incubation on epithelial cells for
3 days. Neither PBMC nor epithelial cells secreted TGF-β1 (data not shown),
although 50 ng/ml IFN-γ was shown to activate TGF-β1 secretion in PBMC (control
(94.7 ± 24.3) vs. IFN-γ (1323.3 ± 145.2) pg/ml production of 6 different PBMC
donors).
To investigate when factor(s) inducing EMT in A549 cells were produced PBMC were
stimulated for shorter times - 2 h, 4 h, 6 h, 8 h, 10 h and 20 h before transfer to
epithelial cells (Figure 4). Microscopy showed that 2 h LPS treatment of the PBMC
already induced weak but detectable mesenchymal-like morphology in A549 after
transfer of the supernatant. This alteration from epithelial into mesenchymal
morphology became statistically significant after 4 h conditioning, accumulated over
20 h time of PBMC stimulation with LPS. These results were consistent with a
decrease in cytokeratin and acquisition of vimentin marker expression and indicate
an important role of early pro-inflammatory mediator(s), accumulating with time in the
medium of LPS-stimulated immune cells.
A
Control 2 h LPS 4 h LPS
56
B
con 2 4 6 8 10 200.0
0.5
1.0
1.5
time [h]
**
cyto
kera
tin
/ h
sp
90
αα αα/ ββ ββ
con 2 4 6 8 10 200.0
2.5
5.0
7.5
10.0
vim
en
tin
/ h
sp
90
αα αα/ ββ ββ
time [h]
**
* *
**
Fig. 4: Early produced inflammatory cytokine(s) mediate EMT in lung epithelial
cells. 1 x 106 (1 ml) PBMC were seeded and after 2 h were stimulated with PBS or with 10
ng/ml LPS for 2 h, 4 h, 6 h, 8 h, 10 h and 20 h at 37°C. A549 cells (5 x 104 in 1ml) were
plated on 24 well plate in DMEM + 10% FCS. After overnight culture epithelial cells were
incubated with supernatants from untreated or LPS-stimulated PBMC after the different
incubation times for 72 h. Panel A: Morphology of A549 (magnification 50 x) incubated with
conditioned medium from stimulated with vehicle PBMC (control); PBMC stimulated for 2h
with LPS (2 h LPS); PBMC stimulated for 4h with LPS (4 h LPS). Panel B: Western blot
analysis for cytokeratin and vimentin. Results are based on densitometric analysis of the
ratio of each marker protein to hsp 90 α/β. Control values were set to 1. Data are pooled from
2 independent experiments (4 different PBMC donors) and represent a mean ± SEM
(Repeated Measures ANOVA and Dunnett’s post test on paired row data).
3.5. IL-1β and TNF-α induce EMT in A549 cells
The major cytokines present in the inflammatory milieu of chronic lung diseases are
IL-1β and TNF-α (Lappalainen et al., 2005), which are also known to be the first
cytokines secreted upon stimulation of monocytes with LPS (Jansky, Reymanova &
Kopecky, 2003). Therefore, A549 cells were incubated with TNF-α (3 ng/ml) and IL-
1β (4 ng/ml) for 72 h and effects on cell morphology and marker expression were
investigated (Figure 5). Both pro-inflammatory cytokines induced a change to a
fibroblast-like shape in epithelial cells, reduced their cytokeratin and increased their
vimentin expression. We have also observed in agreement with other investigators
(Jansky et al., 2003) that LPS stimulation of PBMC induces production of IL-6, IL-8,
macrophage chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1α
57
(MIP-1α) and IL-10, shown also to be elevated in IPF and experimental models of
fibrosis (Agostini & Gurrieri, 2006), (Gauldie, Jordana & Cox, 1993; Kelly et al.,
2003). Direct stimulation of A549 cells with these inflammatory mediators did not alter
cell morphology and investigated marker expression (data not shown).
A
B
Control IL-1β TNF-α IL-1β + TNF-α
Fig. 5: IL-1β and TNF-α induce EMT in A549. A549 (5 x 104 /ml) were stimulated with
vehicle (control), 4 ng/ml IL-1β, 3 ng/ml TNF-α and combination of both cytokines for 72 h in
serum-free medium. A) Phase contrast microphotographs are taken with 100 x magnification.
Control IL-1β (4 ng/ml)
TNF-α (3 ng/ml) IL-1β (4 ng/ml) + TNF-α (3 ng/ml)
cytokeratin
vimentin
hsp 90 α/β
58
B). 20 µg of total proteins were loaded on each lane and Western blotting for vimentin and
cytokeratin expression was performed. Data represent one of 3 independent experiments.
3.6. LPS-stimulated PBMC produce IL-1β and TNF-α, which induce EMT
in lung epithelial cells
To investigate the role of these two pro-inflammatory cytokines in the observed EMT
effect of epithelial cells upon conditioning we used neutralizing antibodies against IL-
1β and TNF-α. PBMC-conditioned supernatants diluted to 75% were pre-incubated in
the absence or presence of 1 µg/ml of αIL-1β and αTNF-α, and transferred onto A549
cells for an additional 3 days. In the presence of the antibodies cytokeratin
expression was increased and vimentin expression was decreased, compared to the
marker expression observed in the absence of neutralizing antibodies (Figure 6).
Fig. 6: Neutralization of IL-1β and TNF-α in conditioned supernatant from PBMC with
αIL-1β and αTNF-α. 1 x 106 in 1ml PBMC were seeded on 24 well plates. After 2 h
mononuclear cells were not stimulated or stimulated with 10 ng/ml LPS for 20 h at 37°C.
A549 cells (5 x 104/ml) were plated on 24 well plate in DMEM + 10% FCS. Cell-free
supernatants from PBS- and LPS-stimulated PBMC (75%) were pre-incubated for 2 h with
and without 2 µg/ml of αIL-1β, αTNF-α or a combination of both antibodies at 37°C. Then
conditioned medium was transferred onto A549 cells for 72 h. Results are based on
densitometric analysis of the ratio of each marker protein to hsp 90 α/β. Control values are
set to 1. Western blot data are pooled from 3 independent experiments (9 different PBMC
donors) and represent a mean ± SEM. *p<0.05; **p<0.01, ***p<0.001 all versus LPS values
(Friedman test followed by Dunn’s post test on paired row data).
0
1
2
3
control
LPS
LPS + αααα IL-1ββββ
LPS + ααααTNF-αααα
LPS + α α α α IL-1β + αβ + αβ + αβ + α TNF-αααα
**
ns
5
10
**
vim
en
tin
/ h
sp
90
αα αα/ ββ ββ
0.0
0.5
1.0
control
LPS
LPS + αααα IL-1ββββ
LPS + ααααTNF-αααα
LPS + α α α α IL-1β + αβ + αβ + αβ + αTNF-αααα5
10
***
ns*
cyto
kera
tin
/ h
sp
90
αα αα/ ββ ββ
59
These data were consistent with results using IL-1ra and αmuTNF-α (Figure 7). The
ability of αmuTNF-α to neutralize human TNF-α activity was shown by inhibition of IL-
8 and IL-6 production in TNF-α stimulated A549 cells (data not shown).
0.0
2.5
5.0
control
LPS
LPS + IL1ra + ααααmuTNF-αααα
5
10
**
ns
vim
en
tin
/ h
sp
90
αα αα/ββ ββ
0
1
control
LPS
LPS + IL1ra + ααααmuTNF-αααα
5
10
cyto
kera
tin
/ h
sp
90
αα αα/ββ ββ
**
ns
Fig. 7: Inhibition of IL-1β and TNF-α in conditioned SN from PBMC with IL-1ra and
αmuTNF-α. 1 x 106 in 1ml PBMC were seeded on 24 well plates. After 2 h mononuclear cells
were not stimulated (con) or stimulated with 10 ng/ml LPS for 20 h at 37°C. A549 cells (5 x
104/ml) were plated on 24 well plate in DMEM + 10% FCS. Cell-free supernatants from PBS-
and LPS-stimulated PBMC (75%) were pre-incubated for 2 h in the presence or absence of a
combination of 10 µg/ml and αmuTNF-α (500 µg/ml total protein) at 37°C. Then conditioned
medium was transferred onto A549 cells for 72 h. Results are based on densitometric
analysis of the ratio of each marker protein to hsp 90 α/β. Control values are set to 1.
Western blot data are pooled from 3 independent experiments (4 different PBMC donors)
and represent a mean ± SEM. *p<0.05; **p<0.01, ***p<0.001 all versus LPS values
(Friedman test followed by Dunn’s post test on paired row data).
60
4. DISCUSSION
Inflammation is believed to play an important role in the pathogenesis of chronic lung
diseases such as asthma, chronic obstructive pulmonary disease (COPD) and
idiopathic pulmonary fibrosis (IPF) (Araya & Nishimura, 2010). IPF is a devastating
disease with unknown etiology and a prognosis worse than that of many cancers.
Increasing numbers of studies show that environmental pollutants and oxidative
stress may cause IPF (Maher et al., 2007), (Rahman et al., 1999), (Taskar & Coultas,
2006).
Myofibroblasts, the main cell population that constitutes fibroblastic foci in IPF, are
considered central in the pathogenesis of this disease. Emerging evidence suggests
not only interstitial fibroblasts and blood-borne fibrocytes but also epithelial cells can
differentiate into myofibroblasts through EMT as source (Scotton & Chambers, 2007).
In vitro EMT has been induced in various epithelial cell lines in response to TGF-β1
stimulation (Kasai et al., 2005), (Willis et al., 2005), (Zhang et al., 2006). Kasai et al.
for a first time reported that A549 cells undergo EMT upon stimulation with 5 ng/ml
TGF-β1 and observed up-regulation of fibronectin, down-regulation of E-cadherin,
slight decrease in cytokeratin 19 and no change in vimentin expression (Kasai et al.,
2005). In our studies we confirmed that these cells undergo EMT upon treatment with
TGF-β1 and that A549 cells adopted fibroblast-like morphology, lost expression of
epithelial phenotypic markers (E-cadherin and cytokeratin) and accumulated strongly
mesenchymal vimentin expression. As a difference Kasai et al. maintained A549 cells
in serum-free DMEM containing 0.1% bovine serum albumin (BSA) for 24 h prior
stimulation with cytokines. From our own observations 0.1% BSA in DMEM medium
induced time-dependent cell death in resting A549 cells and probably a loss of
surface cell marker expression.
So far the role of LPS, the principal endotoxin of Gram-negative bacteria, in the
process of EMT has not been investigated. Recent papers (Li et al., 2009), (He et al.,
2009) indicate that repeated LPS administration induces ALI and pulmonary fibrosis
in mice. Few studies also suggested that inflammation links to fibrosis through the
process of EMT (Liu, 2008), (Lopez-Novoa & Nieto, 2009). Therefore we
hypothesized that in vitro LPS stimulation of immune cells, i.e. PBMC, may induce an
inflammatory cascade and result in EMT in A549 cells. In the current study we
demonstrate that 20 h conditioned supernatant from LPS-stimulated PBMC induces
61
EMT in A549 cells. This was evidenced by alteration in cell shape from epithelial into
elongated mesenchymal morphology, significant down-regulation of cytokeratin and
significant up-regulation in vimentin expression. By measuring TGF-β1 content in
supernatant from control and LPS stimulated PBMC and in supernatants incubated
on A549 cells for 3 days we determined that EMT was not driven by TGF-β1 in this
setting. However, it could be shown that recombinant IFN-γ induces significant TGF-
β1 expression in PBMC. In agreement, Olas et al. published that in vitro LPS-
stimulated blood monocytes and PBMC are not a significant source of TGF-β1 (Olas
et al., 2005). Moreover, it was observed here that endotoxin activated PBMC do not
release IFN-γ, as also previously seen by Krakauer (Krakauer, 2002).
The nature of the inflammatory mediator(s) involved in the LPS-driven EMT process
was studied. We tested the effect of conditioned supernatants from PBMC stimulated
with LPS for shorter times. We were surprised to see initiation of cell elongation of
epithelial cells incubated with supernatant after only 2 h conditioning. 4 h conditioned
supernatant induced a clear fibroblast-like morphology. Western blot results were
consistent with cell morphology data and indicated time-dependent down-regulation
of cytokeratin and up-regulation in vimentin expression. This directed us to
investigate the early inflammatory cytokines IL-1β and TNF-α as possible mediators.
These cytokines have been suggested to play a role in IPF (Allen & Spiteri, 2002).
Liu et al., 2008 (Liu, 2008) have shown that a mixture of inflammatory cytokines (IL-
1β, TNF-α, IFN-γ) augments EMT in A549 induced by TGF-β1. Vesey et al. reported
that IL-1β induces EMT in kidney epithelial cells (Vesey et al., 2002).
Here, we were able to show that IL-1β and TNF-α, but not IL-6, IL-8, MCP-1, MIP-1α,
IL-10, convert A549 cell morphology, decrease cytokeratin and increase vimentin
marker expression. Surprisingly, no synergistic effect on cell morphology and marker
expression was observed when A549 cells were incubated with both cytokines.
The role of IL-1β and TNF-α in LPS-activated mononuclear cell supernatants on the
EMT in A549 cells was further examined using neutralizing antibodies or IL-1ra. In
these experimental settings, 75% of conditioned supernatants from PBS-treated and
LPS-treated PBMC were incubated in the presence or absence of 2 µg/ml αIL-1β,
αTNF-α or a combination of both neutralizing antibodies. Inhibition of IL-1β and TNF-
α activity was also achieved using 10 µg/ml IL-1ra, 500 µg/ml total protein of
αmuTNF-α or a combination of both. Addition of antibodies or IL-1ra to control PBMC
supernatants did not alter cell morphology or marker expression. The effect of
62
neutralizing antibodies or neutralizing antibody and IL-1ra showed greater results
when applied in combination both on cell morphology, cytokeratin and vimentin
expression. Thus, our results indicate a central role of IL-1β and TNF-α in the LPS-
driven EMT process in A549 cells. Interestingly, clinical trials to prevent IPF with
etanercept, a TNF-α antagonist, recently reported a decreased rate of IPF disease
progression and good tolerance (Raghu et al., 2008). However, there have been no
clinical trials with anakinra, IL-1 receptor antagonist effective in reducing the signs
and symptoms of rheumatoid arthritis but leading to increased risk of serious
infections for the treatment of IPF (Salliot, Dougados & Gossec, 2009), (Agostini &
Gurrieri, 2006).
Taking these results further, it would be highly interesting to test weather chronic LPS
administration in vivo causes EMT and weather this process can be attenuated or
inhibited in the presence of neutralizing αIL-1β and αTNF-α antibodies or IL-1ra.
Taken together, our findings indicate that in vitro LPS stimulation of PBMC induces
production of pro-inflammatory cytokines IL-1β and TNF-α able to drive the process
of EMT in the epithelial cell line A549 in the absence of TGF-β1.
63
5. MATERIAL AND METHODS
5.1. Reagents and antibodies
LPS from Salmonella abortus equi, SEB from Staphylococcus aureus, PGN from
Staphylococcus aureus and Brefeldin A were purchased from Sigma (Deisenhofen,
Germany). Further substances were IL-1β and TNF-α from NIBSC (Hertfordshire,
UK) and IL-1 receptor antagonist (IL-1ra) (Kineret® 100 mg of Anakinra in a syringe,
Biovitrum, Sweden). Dulbecco’s PBS, DMEM medium with high glucose and fetal calf
serum (FCS) were from PAA Laboratories (Pasching, Austria) and RPMI-1640 cell
culture medium was purchased from Bio Whittaker (Verviers, Belgium). Recombinant
human TGF-β1 and polyclonal goat anti-human antibodies against TNF-α (αTNF-α)
and IL-1β (αIL-1β) were purchased from R&D Systems (Minneapolis, MN). Polyclonal
sheep anti-mouse TNF-α antibody (αmuTNF-α) was produced in-house (Sommer et
al., 2001). Mouse monoclonal anti-human antibodies against pan-cytokeratin and
vimentin were from Sigma; polyclonal rabbit anti-human hsp 90 α/β antibody was
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); polyclonal anti-human
β-actin antibody was from Cell Signaling Technology, Inc. (Danvers, USA). Goat anti-
rabbit immunoglobulin-HRP and goat anti-mouse immunoglobulin-HRP were supplied
by Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA).
5.2. Cell line
Human lung adenocarcinoma epithelial cell line A549 was supplied from ATCC/LGC
Promochem (Wesel, Deutschland). Cells were maintained in high glucose DMEM
medium supplemented with 10% heat-inactivated FCS at 37°C in a humidified 5%
CO2 atmosphere. A549 cells were harvested every 2 to 3 days after reaching 90 to
95% confluence up to passage 20.
5.3. Isolation of human peripheral blood mononuclear cells
PBMC from healthy volunteers were prepared with CPTTM Cell Preparation Tubes
(BD Biosciences, Heidelberg, Germany) according to the manufacturer instructions.
After centrifugation (1600 x g for 20 min), the white layer above the gel containing the
64
PBMC was removed and the cells were washed 3 times (300 x g for 7 minutes) with
RPMI-1640 containing 2.5 IU/ml heparin (Ratiopharm, Ulm, Germany). Cell counts
were determined with a Pentra 60 apparatus (ABX Diagnostics, Montpellier, France).
5.4. Enzyme-linked immunosorbent assay
Cytokine release was determined by in-house sandwich enzyme-linked
immunosorbent assay (ELISA) based on commercial antibody pairs against human
TNF-α, IL-8 (both Thermo Scientific, Germany), IL-1β and IL-6 (both R&D Systems,
Minneapolis, USA). Recombinant standards for IL-1β, IL-6 and TNF-α were obtained
from NIBSC, Herts, UK, for IL-8 from PeproTech. For TGF-β1 measurements
commercial DuoSet kit was purchased from R&D Systems (Minneapolis, MN).
Assays were carried out in flat-bottom, ultrasorbent 96 well plates (MaxiSorp, NUNC,
Wiesbaden, Germany). Binding of biotinylated secondary antibody was quantified
using streptavidin-conjugated horseradish peroxidase (Jackson Immuno Research,
West Grove, PA, USA) and the substrate TMB (3,3’,5,5’-tetramethylbenzidine,
Sigma). The reaction was stopped by adding 50 µl of 1 M H2SO4 and absorption was
measured at λ = 450/690 nm in an ELISA reader (Rainbow, Tecan, Crailsheim,
Germany).
5.5. A549-PBMC co-cultures
Co-culture experiments were performed in direct contact between epithelial cells and
PBMC (mixed co-cultures) and on transwell plates (transwell co-cultures). In mixed
co-cultures 2 x 105 A549 cells in 500 µl growth medium were seeded on 48 well
plates or 24 well plates and incubated at 37°C overnight for 16 h. Next day PBMC
were prepared as described above. Supernatant of the A549 culture was discarded
and 450 µl RPMI-1640 with heparin was added. 450 µl 5 x 105 cells PBMC were
added to the epithelial cells or seeded on the same plate (PBMC mono-culture). After
3 h, cells were stimulated with vehicle (PBS) or 100 ng/ml LPS, 100 ng/ml SEB or 1
µg/ml PGN. Cell-free supernatants were collected after 24 h and stored at -80°C until
ELISA measurements.
65
In transwell experiments 500 µl 2 x 105 A549 cells were seeded in growth medium in
the lower compartment of 24 well plates (Corning, NY, USA). The transwell inserts
material had a polycarbonate membrane with 0.4 µm pore size (# 3413). After
overnight incubation, medium was removed and 200 µl RPMI-1640 with heparin was
added into each well of the lower compartment. 250 µl RPMI-1640 with heparin was
added into the upper compartment of the transwells when A549 were incubated
alone. For transwell co-cultures and PBMC mono-cultures 250 µl 5 x 105 PBMC were
added to the upper compartment. After 3 h cells in transwell plates were stimulated
with vehicle (PBS) or 100 ng/ml LPS (into the upper compartment) in a final volume
of 500 µl. Cell-free supernatants were collected after 24 h and stored at -80°C until
ELISA measurements.
5.6. Neutralization of IL-1β and TNF-α activity
Cell-free supernatants from stimulated with vehicle or LPS-stimulated PBMC were
diluted to 75% (v/v) in RPMI-1640 with heparin and incubated for 2 h at 37°C in the
presence or absence of 1) 2 µg/ml αIL-1β, 2 µg/ml αTNF-α or a combination of both
neutralizing antibodies; 2) neutralizing αmuTNF-α (500 µg/ml total protein
concentration) antibody, 10 µg/ml IL-1 receptor antagonist (IL-1ra) or a combination
of αmuTNF-α and IL-1ra, before being transferred onto A549 cells, which were then
cultured for 3 additional days.
5.7. Inhibition of TNF-α processing by Brefeldin A
A549 and PBMC mono-cultures and mixed co-culture were seeded on 48 well plates
and after 3 h were left untreated (PBS) or stimulated with 100 ng/ml LPS, in the
presence or absence of 5 µg/ml Brefeldin A (500 µl final volume). After 3 h
stimulation, plates were centrifuged; supernatants were collected and stored at -80°C
until TNF-α ELISA measurement. The cell pellets were resuspended in 500 µl PBS
and 3 x freeze-thaw cycles were performed. Cell-free supernatants were harvested
and intracellular pro-TNF-α was assessed by ELISA.
66
5.8. LPS stimulation of PBMC in medium or A549 conditioned
supernatant
A549 cells (2 x 105 in 500 µl) were seeded in growth medium. After 16 h culture
medium was replaced by 500 µl RPMI-1640 with heparin. In parallel, 500 µl RPMI-
1640 with heparin was also added on the 48 well plates. After 2 h, 4 h, 8 h and 24 h
supernatants from both conditions were harvested and stored at -80°C. PBMC were
prepared according to the protocol described above. 70 µl 5 x 105 cells were added
to 380 µl of collected supernatants (76% v/v) and after 3 h stimulated with vehicle
(PBS) or 100 ng/ml LPS in a final volume of 500 µl. After 24 h TNF-α cytokine was
assessed by ELISA.
5.9. Total RNA extraction and cDNA synthesis
70 µl 5 x 105 PBMC were added to 380 µl 24 h cell-free conditioned supernatant from
A549 cells or RPMI-1640 with heparin, and after 3 h PBMC were stimulated with
vehicle (PBS) or 100 ng/ml LPS. After 3 h plates were centrifuged and supernatants
were removed. Cell pellet was lysed and RNA was extracted using QIAamp RNA
Blood Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer instructions.
RNA samples were diluted in RNAase free water and the DNA was digested. 150 ng
of total RNA was reverse transcribed using oligo (dT)16 primers (Thermo Hybaid,
Ulm, Germany) and M-MuLV Reverse Transcriptase (New England Biolabs, Ipswich,
USA).
5.10. Quantitative Real-Time PCR
cDNA was quantified by Real-Time PCR via the LightCycler system (Roche) with
LightCycler FastStart DNA Master SYBR Green (Roche) using specific primers from
ThermoScientific (ThermoScientific, Hamburg, Germany) for human TNF-α: forward
5´-AGG CCC CAG TTT GAA TTC TT -3´and reverse: 5´-TCC TTC AGA CAC CCT
CAA CC -3´; for human GAPDH: forward 5´-GAA GGT GAA GGT CGG AGT C-3´and
reverse: 5´-GAA GAT GGT GAT GGG ATT TC-3´. The specificity of the PCR
67
products was confirmed by melting curve analysis. The expression levels were
normalized to the housekeeper GAPDH.
5.11. Characterization of TNF-α anti-inflammatory compound
RPMI-1640 with heparin medium, incubated at 37°C for 24 h in the presence or
absence of A549 cells, was collected and treated as described:
a) Boiling at 95°C for 15 min;
b) 2 x freeze-thaw cycles;
c) Protein digestion with 0.5 mg/ml pronase (Roche, Mannheim, Germany) (37°C, 1
h) and subsequent enzyme inactivation (95°C, 15 min). Cell pellet was discarded
by centrifugation (3000 x g, 3 min);
d) Centrifuge filtration (molecular weight cut-off 10 kDa; 2000 x g) in Amicon Ultra-15
centrifugal filter unit (Millipore, Schwalbach, Germany).
70 µl 5 x 105 cells were added to 380 µl of supernatants (76% or 30% v/v) and after 3
h stimulated with vehicle or 100 ng/ml LPS in a final volume of 500 µl. After 24 h
TNF-α cytokine was assessed by ELISA.
5.12. SDS-PAGE and Western blot
A549 cells were washed once with Dulbecco’s PBS and lysed on ice with RIPA buffer
(50 mM Tris-HCl, pH 8, 150 mM NaCl, 1% TritonX 100, 0.5% Sodium Deoxycholate,
0.1% SDS) containing protease inhibitor cocktail (Sigma Aldrich, St Louis, USA).
After scraping, cell suspensions were collected and cleared by centrifugation at
13,000 x g for 30 min at 4°C. Total protein concentration was measured using the
BCA protein assay kit (Pierce) with bovine serum albumin (BSA) as standard protein
according to the manufacturer. Equal amounts of protein (20 µg) were loaded for
each lane on 12% Novex® Tris-Glycine gels (Invitrogen, Carlsbad, CA), followed by
electrophoresis and protein transfers to BioTraceTM NT nitrocellulose membrane (Pall
Corporation, Pensacola, FL) by semi-dry blotting. After the transfer, membranes were
blocked for 1 h at RT with 5% non-fat dry milk in TBS-T buffer (50 mM Tris-base, 150
mM NaCl, 0.1% Tween20, pH 7.5) and then probed with appropriate primary
68
antibody, diluted in TBST with 5% BSA overnight at 4°C. After washing, the
membranes were probed for 1 h at RT with appropriate peroxidase-conjugated
secondary antibodies. After further extensive washing the immunoblots were
visualized by ECL substrate (2.5 mM Luminol, 0.4 mM para- coumaric acid in 10 mM
Tris, pH 8.5) and using LAS-3000 imaging system (Fuji).
5.13. Statistics
Statistical analyses and graphs were performed using the Graph Pad Prism software
(Graph Pad Software, San Diego, USA). All data are given as means ± SEM. P
values less then 0.05 were considered significant (*p ≤ 0.05, **p ≤ 0.01 and ***p ≤
0.001).
69
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