studies on the mechanism of action of an anti-edema peptide · 2018-12-27 · studies on the...

Studies on the mechanism of action of an

anti-edema peptide

DISSERTATION

zur Erlangung des akademischen Grades eines

Doktors der Naturwissenschaften (Dr. rer. nat.)

an der Universität Konstanz

Mathematisch-naturwissenschaftliche Sektion

Fachbereich Biologie

vorgelegt von

Dominik Wolfram Geiger

Konstanz, September 2006

Tag der mündlichen Prüfung: 27.11.2006

1. Referent: Prof. Dr. Klaus P. Schäfer

2. Referent: Prof. Dr. Albrecht Wendel

http://www.ub.uni-konstanz.de/kops/volltexte/2008/5656/

http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-56564

DANKSAGUNG

Die vorliegende Dissertation wurde in der Abteilung Biotechnologie (RPR/BT) der

ALTANA Pharma AG in Konstanz angefertigt.

An dieser Stelle möchte ich Herrn Prof. Dr. Klaus P. Schäfer recht herzlich für die

Bereitstellung des interessanten Themas und des Arbeitsplatzes in seiner Abteilung

bedanken. Sein Engagement und Enthusiasmus ermöglichten spannendes Forschen.

Herrn Prof. Dr. Albrecht Wendel danke ich für die freundliche Erstellung des

Zweitgutachtens.

Frau Dr. Inge Mühldorfer möchte ich hiermit meinen ganz besonderen Dank aussprechen:

Vielen Dank für die ausgezeichnete Betreuung, für die Unterstützung in allen Belangen,

die ich während der letzten Jahre stets von Dir erhalten habe, und für die Durchsicht des

Manuskriptes dieser Arbeit.

Weiterhin danke ich allen Mitarbeitern der Abteilungen RPR/BT, RPR/PX und RPR/FG für

ihre stete Hilfsbereitschaft und die tolle Stimmung und Arbeitsatmosphäre. Besonders

möchte ich mich bei der ehemaligen „Mibi-Crew“, - Waltraud Burckhardt-Boer, Anja

Buttkewitz, Fatma Kabaoglu, Karsten Keldermann, Thomas Reinberg und Katja Schürer -,

für die freundschaftliche Zusammenarbeit bedanken, sowie bei meinem „Dome Brother“

Aswin Mangerich für die hervorragende Teamarbeit.

Die Mitgliedschaft im Graduiertenkolleg „Biomedizinische Wirkstoffforschung“ ermöglichte

mir die Teilnahme an exzellenten Fortbildungskursen und Seminaren, die mir auch

Einblicke in andere Forschungsgebiete eröffneten. Die Kontakte und Freundschaften im

Graduiertenkolleg haben die Zeit der Promotion sehr bereichert. Deshalb gilt mein

besonderer Dank den Leitern des Graduiertenkollegs Prof. Dr. Albrecht Wendel und Prof.

Dr. Klaus P. Schäfer, sowie PD Dr. Jutta Schlepper-Schäfer für die hervorragende

Koordination innerhalb des Kollegs.

Dr. Jochen Strassner ermöglichte die Biacore-Messungen, Dr. Georg Rast stand immer

mit Rat und Tat bei elektrophysiologischen Fragen zur Verfügung, Dr. Jürgen Paal war bei

den bioinformatischen Analysen behilflich, Silke Müller führte mich in die Mysterien der

Affinitätschromatographie ein und Klaus Hägele trug durch seine Unterstützung bei der

Massenspektrometrie sehr zum Gelingen dieser Arbeit bei. Ein herzliches Dankeschön an

sie alle.

Meinen Mitbewohnern, - das sind Carolina Otero, Jens Lutz und Konrad Bergen -, und all

meinen Freunden danke ich, dass sie immer für mich da waren und für angenehme

Abwechslung während meiner Promotionszeit sorgten.

Mein größter Dank gilt meiner Familie für alle Unterstützung auf meinem bisherigen Weg.

ABBREVIATIONS

i

Abbreviations

aa amino acid(s)

ARDS acute respiratory distress syndrome

bp base pair(s)

BSA bovine serum albumin

CFTR cystic fibrosis transmembrane conductance regulator

CV column volume

EDTA Ethylenediaminetetraacetic acid

ENaC epithelial sodium channel

HBSS Hank’s balanced salt solution

kb kilo base pairs

LLC lung liquid clearance

LY lucifer yellow

Nedd neural precursor cell expressed developmentally down-regulated

NMDG N-methyl-D-glucamin

PCR polymerase chain reaction

SD standard deviation

SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis

SEM standard error of the mean

TEER transepithelial electrical resistance

Tip Tip peptide

TNF-α tumor necrosis factor α

TEMED N,N,N’,N’-Tetramethylethylenediamine

Tris Tris(hydroxymethyl)aminomethane

ABBREVIATIONS

ii

Units SI prefix

A Ampere K Kilo (103)

°C Degree Celsius D Deci (10-1)

Da Dalton C Centi (10-2)

g Gram M Milli (10-3)

h Hour Μ Micro (10-6)

l Liter N Nano (10-9)

M Molar P Pico (10-12)

min Minute

s Second

V Volt

Nucleotides Amino acids (aa)

A Adenine A Ala Alanine M Met Methionine

C Cytosine C Cys Cysteine N Asn Asparagine

G Guanine D Asp Aspartate P Pro Proline

T Thiamine E Glu Glutamate Q Gln Glutamine

F Phe Phenylalanine R Arg Arginine

G Gly Glycine S Ser Serine

H His Histidine T Thr Threonine

I Ile Isoleucine V Val Valine

K Lys Lysine W Trp Tryptophane

L Leu Leucine Y Tyr Tyrosine

TABLE OF CONTENTS

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Table of Contents

1 Introduction............................................................................................ 1

1.1 Structure and Function of the Human Lung ....................................................... 1

1.2 Pulmonary Edema ................................................................................................ 3

1.3 Lung Liquid Clearance and the Resolution of Pulmonary Edema .................... 5

1.3.1 Ion Channels and Transporters Involved in Lung Liquid Clearance ..................... 5

1.4 Tumor Necrosis Factor α and the Tip Peptide.................................................... 7

1.4.1 The Lectin-like Domain of TNF-α and the Tip Peptide .......................................... 8 1.4.2 Role of the Lectin-like Domain of TNF-α and the Tip Peptide in Lung Liquid

Clearance .............................................................................................................. 9

1.5 Aims of the Study ............................................................................................... 11

2 Materials and Methods........................................................................ 12

2.1 Materials.............................................................................................................. 12

2.1.1 Chemicals and Reagents .................................................................................... 12 2.1.2 Laboratory Equipment and Technical Devices.................................................... 13 2.1.3 Kits....................................................................................................................... 13 2.1.4 Peptides............................................................................................................... 14 2.1.5 Antibodies............................................................................................................ 14 2.1.6 Oligonucleotides .................................................................................................. 15 2.1.7 Solutions.............................................................................................................. 16 2.1.8 Cell Culture.......................................................................................................... 16 2.1.9 Computer Software.............................................................................................. 18

2.2 Methods .............................................................................................................. 19

2.2.1 Cell Culture.......................................................................................................... 19 2.2.2 TNF-α Cytotoxicity Activity Assay........................................................................ 19 2.2.3 Molecular Biological Methods.............................................................................. 19 2.2.4 Biochemical Methods .......................................................................................... 21 2.2.5 Immunofluorescence Staining ............................................................................. 28 2.2.6 Microscopy .......................................................................................................... 29 2.2.7 Transepithelial Electrical Resistance (TEER) Assay........................................... 29 2.2.8 Lucifer Yellow Rejection Assay ........................................................................... 30 2.2.9 Dome Assay ........................................................................................................ 31 2.2.10 Statistical Analysis............................................................................................... 32

3 Results ............................................................................................... 33

3.1 Characterization of Human Lung Epithelial Cell Lines .................................... 33

3.1.1 Analysis of A549, H441 and Calu-3 Cells in Regard to Their Expression of Ion Transporters Involved in Lung Liquid Clearance (LLC)....................................... 33

3.1.2 Analysis of A549, H441 and Calu-3 Cells in Regard to Their Expression of the Water Channel Aquaporin-5 Involved in LLC...................................................... 36

TABLE OF CONTENTS

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3.2 Establishment And Validation of In Vitro Test Systems for Pulmonary Edema Resorption.............................................................................................. 37

3.2.1 Establishment of the Transepithelial Electrical Resistance (TEER) Assay......... 37 3.2.2 Establishment of the Dome Assay ...................................................................... 39 3.2.3 Validation of TEER and Dome Assays................................................................ 40

3.3 In Vitro Studies on the Mechanism of Action of the Tip Peptide..................... 45

3.3.1 Influence of the Tip Peptide on Dome Formation and TEER in Calu-3 Cell Monolayers .......................................................................................................... 45

3.3.2 Influence of TNF-α on TEER and Dome Formation in Calu-3 Cell Monolayers . 48 3.3.3 Influence of pH on the Activity of rhTNF-α and the Tip Peptide in the

TEER Assay ........................................................................................................ 50 3.3.4 Investigation of Ions Involved in the Activity of the Tip Peptide .......................... 51 3.3.5 Influence of the Tip Peptide on β-Adrenergic Receptor Activation and

Intracellular cAMP Level...................................................................................... 52 3.3.6 Influence of Oligosaccharides on the Activity of the Tip Peptide ........................ 54 3.3.7 Effect of Acetate on TEER and Dome Formation of Calu-3 Cell Monolayers..... 56 3.3.8 Identification of Potential Interaction Partners of the Tip Peptide ....................... 58

3.4 Screening of Alternative Anti-Pulmonary Edema Peptide Drug Candidates.. 61

3.4.1 Bioinformatic Analysis of the Tip Peptide ............................................................ 61 3.4.2 In Vitro Activity Screening of Potential Anti-Edema Peptides ............................. 61

4 Discussion ........................................................................................... 68

4.1 Lung Epithelial Cells for In Vitro Study on Lung Liquid Clearance................. 69

4.2 In Vitro Test Systems for Pulmonary Edema Resorption ................................ 72

4.3 Mechanism of Action of the Tip Peptide ........................................................... 75

4.4 Identification of Prospective Anti-Pulmonary Edema Peptides ...................... 80

5 Summary .............................................................................................. 81

6 Zusammenfassung.............................................................................. 83

7 References ........................................................................................... 85

INTRODUCTION

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1 Introduction

1.1 Structure and Function of the Human Lung

The main function of the lung is gas exchange, i.e. oxygen uptake and carbon dioxide

excretion. This is accomplished by a well-coordinated interaction of the lung with the

central nervous system as well as circulatory system, diaphragm and chest wall

musculature. The functional structure of the lung can be divided into i) the conducting

airways, comprising the cartilaginous trachea, bronchia, and the membranous

bronchioles, and ii) the gas exchange portion, consisting of respiratory bronchioles,

alveolar ducts, alveolar sacs, and alveoli (Figures 1.1 and 1.2).

Figure 1.1: Schematic diagram of the human respiratory system. Adapted from 1.

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The acinus is the functional respiratory unit of the lung and includes all structures

from the respiratory bronchiole to the alveolus (Figure 1.2). An acinus is about 0.75 mm in

diameter. Each person has about 20,000 acini containing 300 million alveoli surrounded

by the pulmonary capillaries 2.

Figure 1.2: Schematic representations of an acinus and the distal pulmonary epithelium.

(A) The acinus is the functional respiratory unit of the lung and includes all structures from the respiratory bronchiole to the alveolus. Alveolar ducts are small ducts leading from the respiratory bronchioles to the alveolar sacs. Adapted from 1.

(B) The respiratory bronchiole epithelium consists of ciliated cuboidal cells and Clara cells; the alveolar epithelium consists of type I and type II pneumocytes. All of these polarized epithelial cells have the capacity to transport sodium and chloride. Adapted from 3.

The airways and alveoli in the adult human lung constitute the interface between

lung parenchyma and the external environment and are lined by a continuous epithelium.

The distal airway epithelium is composed of terminal respiratory and bronchiolar units with

polarized epithelial cells, including ciliated Clara cells and nonciliated cuboidal cells 4-6.

These cells synthesize, secrete, and recycle surfactant components.

In the healthy lung, there are three mechanisms responsible for keeping the

interstitial tissue and alveoli dry, thus allowing a proper functioning of gas exchange 7:

INTRODUCTION

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(1) The difference between the inwardly directed plasma oncotic pressure

(~ 25 mmHg) and the outwardly directed hydrostatic pressure (7 – 12 mmHg)

within the pulmonary capillaries 8.

(2) The impermeability of connective tissue and cellular barriers to plasma proteins.

(3) Extensive removal of excess fluid from the lung tissue by the lymphatic system.

Pulmonary edema may develop as a result of either malfunction of these three

mechanisms or of overwhelming excess fluid.

1.2 Pulmonary Edema

Pulmonary edema is defined as the excess accumulation of extravascular fluid in the lung

tissue 9. Pulmonary edema is a common medical emergency, which can be life-

threatening. Fluid accumulation in the interstitial tissue and distal airspaces of the lung

results in an impaired gas exchange leading to an unacceptably low oxygen level in the

blood 10. Patients with pulmonary edema show typical symptoms including shortness of

breath, lung-crackling sounds, pink-stained sputum cough, and anxiety. During edema

formation the excessive fluid first accumulates in the interstitial spaces of the lung

(interstitial pulmonary edema), producing only a few clinical symptoms. At a later stage,

flooding of the alveoli results in an alveolar pulmonary edema (Figure 1.3).

Figure 1.3: Formation of pulmonary edema.

(A) The formation of pulmonary edema begins with an increased filtration through the loose junctions of the pulmonary capillaries into the interstitial space of the lungs (= interstitial pulmonary edema).

(B) As the intracapillary pressure increases, the normally impermeable tight junctions between alveolar epithelial cells open, permitting alveolar flooding to occur (= alveolar pulmonary edema). Adapted from 11.

INTRODUCTION

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Three main causes have been described for the development of pulmonary

edema 8,12,13:

(1) In patients with a left-sided insufficiency of the heart, an increased hydrostatic

capillary pressure and subsequent congestion in the lung blood vessels can lead

to cardiogenic pulmonary edema.

(2) In patients with nephritic syndrome or fluid overload of the body (e.g. by infusions),

reduced plasma protein levels (hypo-albuminemia) can cause renal pulmonary

edema.

(3) In patients with lung inflammation (pneumonia) or blood infection (sepsis), and in

patients aspirating gastric contents, direct or indirect lung injuries can damage the

epithelium and increase vascular permeability; the accompanying inflammatory

processes enhance capillary permeability. The clinical manifestations of the

resulting toxic pulmonary edema is in most cases acute respiratory distress

syndrome (ARDS) 14.

Pulmonary edema requires immediate emergency treatment. The goal of

treatment is to reduce the amount of fluid in the lungs, improve gas exchange, and to

correct the underlying disease. Treatment includes 15:

•••• Placing the patient in a sitting position.

•••• Administration of oxygen.

•••• Assisted or mechanical ventilation (in severe cases).

•••• Drug therapy, including morphine, nitroglycerin, diuretics, angiotensin-converting

enzyme (ACE) inhibitors, and vasodilators.

Morphine is very effective in reducing the patient's anxiety, easing breathing, and

improving blood flow. Nitroglycerin reduces pulmonary blood flow and decreases the

volume of fluid entering the overloaded blood vessels. Diuretics, like furosemide (Lasix®),

promote the elimination of fluids through urination, helping to reduce pressure and fluids in

the blood vessels. ACE inhibitors reduce the pressure against which the left ventricle must

expel blood. In patients who have severe hypertension, a vasodilator may be used.

It is noteworthy that none of the currently employed drug therapies specifically

induces the resolution of pulmonary edema in the target organ lung. The limiting capacity

INTRODUCTION

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of existing substances to induce pulmonary edema resorption at the side of action, namely

the lung epithelium, makes it indispensable to identify new effective compounds that

stimulate active transepithelial fluid transport in the lung.

1.3 Lung Liquid Clearance and the Resolution of Pulmonary

Edema

Several studies indicate that active salt transport by pulmonary epithelial cells drives

osmotic water transport in the distal airspaces of the lung, a process known as lung liquid

clearance (LLC, reviewed in 16). This transport accounts for the ability of the lung to

remove water at the time of birth as well as in the mature lung when pathological

conditions lead to the development of pulmonary edema. Active fluid resorption can occur

in all segments of the pulmonary epithelium. However, since alveolar epithelial cells

comprise 99% of the total airway surface, it is likely that the alveolar epithelium plays a

predominant role, although the distal bronchiolar epithelium may contribute 17.

Most experimental studies have attributed a primary role for a vectorial sodium

transport in LLC. Sodium ions enter pulmonary epithelial cells on the apical side through

the amiloride-sensitive sodium channel ENaC and are subsequently transported across

the basolateral membrane by the ouabain-inhibitable Na,K-ATPase 3,18-21. Water follows

passively the generated osmotic force. This vectorial salt and water transport accounts for

the ability of the human lung to remove water at the time of birth as well as in the mature

lung when pathological conditions lead to the development of pulmonary edema

(Figure 1.4).

1.3.1 Ion Channels and Transporters Involved in Lung Liquid Clearance

The epithelial sodium channel ENaC plays an essential role in the regulation of

transepithelial sodium and fluid balance in various tissues and organs including the lung.

Furthermore, ENaC is a key player of the process of LLC and consequently of pulmonary

edema resorption 3,18,19. In the lung, ENaC is a heterotetrameric complex of three

homologous subunits (2α:1β:1γ). Each subunit comprises two transmembrane domains, a

large extracellular loop with numerous N-linked glycosylation sites, and two short

intracellular N- and C-termini 22-26. Expression and function of ENaC are assumed to be

regulated, in part, by gluco- and mineralcorticoids (e.g. dexamethasone and aldosterone,

respectively) 27-29, transmembrane serine proteases 30-33, Nedd4-2 mediated

INTRODUCTION

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ubiquitination 34-36 and association with the cystic fibrosis transmembrane conductance

regulator (CFTR) 37-41. Mutations in ENaC subunits are a cause of severe arterial

hypertension (Liddel’s syndrome) 42-45 and the salt wasting disease

pseudohypoaldosteronism type 1 46,47.

Figure 1.4: Schematic diagram of the process of lung liquid clearance (LLC).

Alveolar type I and type II cells are responsible for an active vectorial transport of sodium ions from the apical to the basolateral surface of the alveolar epithelium. This active transport of sodium appears to provide a major driving force for removal of fluid from the alveolar air space into the lung interstitium, where it leaves into blood vessels or is cleared by lymph drainage. Three ion transporters are critical for LLC: The amiloride-sensitive sodium channel ENaC and the glibenclamide-inhibitable chloride channel CFTR on the apical surface, as well as the ouabain-inhibitable Na,K-ATPase on the basolateral side. Adapted from 18.

Several investigations support the hypothesis that also the glibenclamide-

inhibitable chloride channel CFTR may contribute to LLC 3,48-50. CFTR is a dimeric ATP-

binding cassette (ABC) transporter glycoprotein that functions in transporting chloride ions

across apical membranes of epithelial cells found in the lung, liver, pancreas, digestive

tract, reproductive tract, and skin 51,52. Mutations in the gene that codes for the CFTR

protein can cause two genetic disorders, cystic fibrosis and congenital bilateral absence of

vas deferens (CBAVD) 52,53.

INTRODUCTION

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Na,K-ATPases are basolaterally localized transmembrane proteins consisting of α

and β subunits. The α-subunit binds and cleaves the high-energy phosphate bond of ATP,

whereas the β-subunit is responsible for the assembly and normal function of the enzyme

complex in the plasma membrane 54,55. In LLC, the active transport of sodium and

potassium by Na,K-ATPases in coordination with the apical sodium uptake through ENaC

generates an ionic gradient that drives passive water movements from the airspace to the

lung interstitium 3,18-21.

The second messenger cyclic adenosine monophosphate (cAMP) activates LLC

by influencing the expression and/or activity of all ion channels and transporters

mentioned above. More precisely, cAMP augments the open channel probability of

ENaC 56-58, enhances the activity of CFTR 57,59,60, increases the delivery of ENaC and

Na,K-ATPase subunits to the apical and basolateral membranes, respectively 36,61-63, and

induces the phosphorylation of Na,K-ATPase α-subunits 63,64. Therefore, cell-permeable

cAMP analogues and synthetic β2-adrenergic agonists (e.g. terbutaline, salmeterol or

isoproterenol) that increase the intracellular cAMP level, are used as gold standards for

studies of vectorial transepithelial ion and fluid transport in vitro and in vivo.

1.4 Tumor Necrosis Factor α and the Tip Peptide

The cytokine tumor necrosis factor α (TNF-α) is mainly produced by activated

macrophages and other immune cells such as mast cells, B- and T-lymphocytes.

However, endothelial cells, fibroblasts and epithelial cells also produce it. Soluble TNF-α

is a 185 amino acid glycoprotein hormone cleaved from a 212 amino acid-long

transmembrane precursor. Both, transmembrane and soluble TNF-α are biologically

active as homotrimers containing two receptor binding sites (Figure 1.5) 65. TNF-α is

mainly known for its receptor-mediated proinflammatory functions in the systemic

inflammatory response and apoptosis induction 66,67. In these processes TNF-α exerts its

activities by interacting with two distinct TNF receptors with molecular masses of 55 kDa

(TNFR1) and 75 kDa (TNFR2), respectively, which are expressed on cell surfaces of

various cell types in many different tissues 68,69.

TNF-α is an important mediator of innate immunity. Produced at sites of bacterial,

fungal, parasitic or viral invasion, it efficiently recruits and activates defense mechanisms,

e.g. by promoting the production of a wide range of other cytokines, such as IL-1, IL-6, IL-

8 and granulocyte-monocyte colony stimulating factor 70-72. However, high systemic levels

of TNF-α can result in pathologic conditions like sepsis, cerebral malaria, and autoimmune

diseases such as rheumatoid arthritis 73. In addition, the sustained generation of TNF-α is

INTRODUCTION

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associated with multiple organ failure 74, multiple sclerosis 75,76, cardiac dysfunction 77,

arteriosclerosis 78, and inflammatory bowel disease 79.

1.4.1 The Lectin-like Domain of TNF-α and the Tip Peptide

Although cytokines exert their activity by interacting with specific receptors, the discovery

that some cytokines have carbohydrate-binding (lectin) properties makes these molecules

bi-functional and opens new concepts in the understanding of their mechanism of

action 80-82. In particular, TNF-α has been shown to contain a lectin-like domain that is

located at the tip of the TNF-α trimer (tip domain), spatially distinct from the receptor

binding sites (Figure 1.5) 83. This carbohydrate-recognition domain is responsible for the

dula property of TNF-α, participating in innate immune functions by receptor binding and

oligosaccharide binding.

Figure 1.5: Amino acid sequence and structure of human TNF-α and the TNF-α derived Tip peptide.

Human TNF-α possesses three functional domains: two TNF receptor binding-sites and one lectin-like domain. The lectin-like domain extends from Ser100 to Glu116 of the soluble form of human TNF-α (bold letters). Derived from this domain a peptide was synthesized consisting of 17 amino acids (underlined letters). As the lectin-like domain is spatially distinct from the receptor binding domains and is located at the tip of the TNF-α molecule the synthesized peptide is designated Tip peptide. A disulfide bond (orange letters) circularizes the Tip peptide to mimic the loop-like structure of the lectin-like tip domain (yellow circle). Substitution of several amino acids within the lectin-like domain of human TNF-α led to the identification of three amino acids that are critical for the lectin-like activity: Thr105, Glu107, and Glu110 (blue underlined letters). Adapted from 73,83.

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The lectin-like tip domain extends from Ser100 to Glu116 of the soluble form of

human TNF-α and is responsible for the specific binding of oligosaccharides such as

N,N’-diacetylchitobiose and branched tri-mannoses 83-85. In addition, soluble TNF-α has

been shown to bind conserved chitobioseoligomannose (GlcNAc(2)-Man(5-9)) moieties of

the variant surface glycoprotein (VSG) antigen of African trypanosomes by means of its

lectin-like tip domain, resulting in the lysis of these parasites 86,87. Further investigations

revealed that the amino acids Thr105, Glu107, and Glu110 within the tip domain are critical for

the trypanolytic activity of TNF-α (Figure 1.5) 83.

A synthesized 17-amino acid peptide imitating the lectin-like domain of TNF-α has

been shown to be trypanolytic by itself 83. This peptide is called Tip peptide since it is a

derivative of the tip region of TNF-α (Figure 1.5). The Tip peptide is circularized via a

disulfide bond between its terminal cysteine residues in order to mimic its loop structure

within TNF-α.

1.4.2 Role of the Lectin-like Domain of TNF-α and the Tip Peptide in Lung

Liquid Clearance

Another interesting effect of the lectin-like domain of TNF-α and also of the Tip peptide is

the influence on ion channels in several cell types. Patch clamp studies using either the

human lung epithelial cell line A549, murine microvascular endothelial cells, or murine

peritoneal macrophages showed that TNF-α and the Tip peptide induce an amiloride-

sensitive current, indicating the activation of sodium specific ion channels 88,89. Since

several studies specify that active salt transport drives resorption of edema fluid from the

distal airspaces of the lung (see chapter 1.3), it may be inferred that the sodium channel

activating effect of TNF-α and the Tip peptide in lung epithelial cells induces fluid

clearance from the lung. This hypothesis is supported by the fact that intratracheally

administered Tip peptide has been shown to stimulate LLC in isolated perfused rat lungs

as well as in in situ and in vivo rodent models 73,90-92. This effect could be blocked with the

oligosaccharide N,N’-diacetylchitobiose, which specifically binds to the lectin-like domain

of TNF-α and blocks its activity 91,92. Moreover, a peptide mutated in the three amino acids

essential for the lectin-like activity showed no effect on lung liquid clearance. Furthermore,

instillation of TNF-α into the lungs of ventilated rats resulted in an increase of neutrophil

infiltration into the alveolar space, whereas instillation of the Tip peptide did not have any

inflammatory consequences 91,92. Therefore, the Tip peptide was suggested as a new

therapeutic agent for the resolution of pulmonary edema 73.

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Taken together, these findings support the following hypothetic mechanism of

action of the Tip peptide in LLC (Figure 1.6):

Since the Tip peptide consists of only 17 amino acids it is unlikely that Tip forms a

sodium channel by itself as it has previously been described for TNF-α 93,94. Furthermore,

the inhibition of the LLC-stimulating activity with amiloride and specific oligosaccharides

such as N,N’-diacetylchitobiose implies that the Tip peptide activates an endogenous

sodium channel in a direct or indirect manner via a potentially glycosylated receptor at the

surface of lung epithelial cells. The activation of sodium channels such as ENaC may

result in an active ion transport from the alveolar to the interstitial space of the lung with

water following passively to the blood and lymph vessels, finally enhancing the rate of LLC

and contributing to the resolution of pulmonary edema.

Figure 1.6: Schematic diagram of a hypothetic mechanism of action of TNF-α and the Tip peptide in LLC.

Intratracheal administration of TNF-α and the Tip peptide up-regulates LLC 90-92. This activity can be blocked by the sodium specific ion channel inhibitor amiloride and specific oligosaccharides such as N,N’-diacetylchitobiose 91,92. These findings imply that TNF-α and the Tip peptide potentially activate an endogenous sodium channel such as ENaC in a direct or indirect manner via a probably glycosylated receptor at the surface of lung epithelial cells resulting in the induction of a vectorial transepithelial sodium transport that drives LLC. Adapted from 18.

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1.5 Aims of the Study

The aims of this study can be summarized as follows:

(1) The establishment and evaluation of a cell-based in vitro test system that mimics

the human lung epithelium and allows the investigation of anti-pulmonary edema

drug compounds.

(2) The use of this test system for better characterization of the mechanism by which

the Tip peptide stimulates transepithelial ion and fluid transport.

(3) The use of this test system for screening and identification of new effective anti-

edema drug candidates alternative to the Tip peptide.

MATERIALS AND METHODS

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2 Materials and Methods

2.1 Materials

2.1.1 Chemicals and Reagents

All chemicals and reagents were of p.a. quality or of highest available purity.

Table 2.1: Chemicals and reagents.

Chemical/Reagent Manufacturer

4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI) Sigma-Aldrich, Deisenhofen, Germany

Acetate, sodium salt Sigma-Aldrich, Deisenhofen, Germany

Acrylamide/Bisacrylamide Roth, Karslsruhe, Germany

Alamar Blue BioSource International, Camarillo, USA

Amiloride Sigma-Aldrich, Deisenhofen, Germany

Ammoniumpersulfate (APS) Biorad, Munich, Germany

Bovine serum albumine (BSA) Sigma-Aldrich, Deisenhofen, Germany

Butyrate, sodium salt Sigma-Aldrich, Deisenhofen, Germany

Cellobiose Sigma-Aldrich, Deisenhofen, Germany

N,N’-diacetylchitobiose Sigma-Aldrich, Deisenhofen, Germany

Dibuturyl (db) cAMP Sigma-Aldrich, Deisenhofen, Germany

Forskolin Sigma-Aldrich, Deisenhofen, Germany

Glibenclamide Sigma-Aldrich, Deisenhofen, Germany

Tumor necrosis factor α, recombinant, human (rhTNF-α) ALTANA Pharma, Konstanz, Germany

N-Methyl-D-glucamin (NMDG) Sigma-Aldrich, Deisenhofen, Germany

β-Mercaptoethanol Merck, Darmstadt, Germany

Ouabain Sigma-Aldrich, Deisenhofen, Germany

Paraformaldehyde Merck, Darmstadt, Germany

Propranolol Sigma-Aldrich, Deisenhofen, Germany

Sodiumdodecylsulfate (SDS) Roth, Karlsruhe, Germany

Terbutaline Sigma-Aldrich, Deisenhofen, Germany

Tetramethylethylenediamine (TEMED) Merck, Darmstadt, Germany

Triton X-100 Merck, Darmstadt, Germany

TWEEN 20 Merck, Darmstadt, Germany


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2.1.2 Laboratory Equipment and Technical Devices

Table 2.2: Laboratory equipment and technical devices

Equipment/Technical device Manufacturer

Branson sonifier 150 Heinemann, Schwäbisch Gmünd,

Germany

Cell counter Cedex AS20 Innovatis, Bielefeld, Germany

Cell culture incubator Thermo Electron Corp., Waltham, USA

Cell culture material Greiner Bio-One, Frickenhausen, Germany

Nunc GmbH, Wiesbaden, Germany

Clean bench HERASafe Thermo Electron Corp., Waltham, USA

Eppendorf research pipets Eppendorf, Hamburg, Germany

Eppendorf Thermomixer 5436 Eppendorf, Hamburg, Germany

inoLab pH meter VWR International, Darmstadt, Germany

Magnetic stirrer Dunn Labortechnik, Asbach, Germany

Rotary Shaker IKA Werke, Staufen, Germany

Table top centrifuges 5415 D/5417R Eppendorf, Hamburg, Germany

Further laboratory equipment and technical devices are listed in paragraphs of the

corresponding methods.

2.1.3 Kits

Table 2.3: Kits.

Kit Manufacturer

DNA-free Kit Ambion, Austin, USA

Expand Long Template PCR System Roche, Penzberg, Germany

Expand Reverese Transcriptase Kit Roche, Penzberg, Germany

Lumi.LightPLUS Western Blotting Substrate Roche, Penzberg, Germany

Micro BCA Protein Assay Kit Pierce, Rockford, USA

ProteoExtract, Subcellular Proteome Extraction Kit Merck, Darmstadt, Germany

RNeasy Mini Kit Qiagen, Hilden, Germany

TaqMan Gene Expression Assay Applied Biosystems, Foster City, USA


- 14 -

2.1.4 Peptides

Peptides were prepared as acetate salts by fully automated solid-phase peptide synthesis

using the Fmoc/tBu-strategy and a Fmoc-amino acid-TCP-polystyrene resin. Suppliers

were Bachem, Bubendorf, Switzerland and EMC, Tübingen, Germany.

Table 2.4: Peptides

Peptide Manufacturer

Human circular Tip peptide (Tip), mutant Tip peptide

(mTip), scrambled Tip peptide (scTip)

Bachem, Bubendorf, Switzerland

Human circular scrambled-2 Tip peptide (sc2Tip) EMC, Tübingen, Germany

Biotinylated Tip peptide (bTip), mutant Tip peptide

(bmTip), scrambled Tip (bscTip)

EMC, Tübingen, Germany

Other peptides, circular or linear EMC, Tübingen, Germany

2.1.5 Antibodies

Table 2.5: Antibodies.

Primary antibody Manufacturer

Polyclonal rabbit-anti-human α-ENaC Sigma-Aldrich, Deisenhofen, Germany

Monoclonal mouse-anti-human β-ENaC Santa Cruz Biotechnology, Santa Cruz, USA

Polyclonal rabbit-anti-human γ-ENaC Sigma-Aldrich, Deisenhofen, Germany

Monoclonal mouse-anti-human CFTR Santa Cruz Biotechnology, Santa Cruz, USA

Monoclonal mouse-anti-human α-Na,K-ATPase Sigma-Aldrich, Deisenhofen, Germany

Monoclonal mouse-anti-human β1-Na,K-ATPase Sigma-Aldrich, Deisenhofen, Germany

Polyclonal rabbit-anti-human occluding Santa Cruz Biotechnology, Santa Cruz, USA

Polyclonal rabbit-anti-human ZO-1 Santa Cruz Biotechnology, Santa Cruz, USA

Polyclonal goat-anti-human AQP5 Santa Cruz Biotechnology, Santa Cruz, USA

Secondary antibody Manufacturer

Goat-anti-mouse IgG, peroxidase conjugated Dianova, Hamburg, Germany

Goat-anti-rabbit IgG, peroxidase conjugated Dianova, Hamburg, Germany

Donkey-anti-goat IgG, peroxidase conjugated Dianova, Hamburg, Germany

Goat-anti-mouse IgG, Cy2 or Cy3 conjugated Dianova, Hamburg, Germany

Goat-anti-rabbit IgG, Cy2 or Cy3 conjugated Dianova, Hamburg, Germany


- 15 -

2.1.6 Oligonucleotides

Oligonucleotides used for RT-PCR were purchased from Invitrogen, Carlsbad, USA.

Table 2.6: Oligonucleotides.

Primer Name Gene Sequence 5’-3’ Orientation

Mibi 906 α-ENaC GCTAATGAGATTCCTGTCGCTTCCATCCC Forward

Mibi 907 α-ENaC CTCTGCCCCCTTCCTTTGGTCTTCTTCC Reverse

Mibi 908 β-ENaC AGACTACGTCCCCTTCCTTGCGTCCAC Forward

Mibi 909 β-ENaC ATATTGGTGCTTTGGTCCCGCTCCTG Reverse

Mibi 910 γ-ENaC CAGTGCGCCCTCCTCGTCTTCTCCTTC Forward

Mibi 911 γ-ENaC CCCATGCATCGGGTGGTGAAAAAGCGT Reverse

Mibi 912 CFTR CGACAGGGTGAAGCTCTTTC Forward

Mibi 913 CFTR TCTGGCTTGCAAAACACAAG Reverse

Mibi 914 HPRT AATTATGGACAGGACTGAACGTC Forward

Mibi 915 HPRT GTGGGGTCCTTTTCACCAGCAAG Reverse

Mibi 1192 PEPT1 GTTTGTGGCTCTGTGCTACCTGACG Forward

Mibi 1193 PEPT1 TTTGGGAGATGAGCCGCTCAT Reverse

Mibi 1194 PEPT2 CCTTTCCAGAAAAATGAGTCCAAGGA Forward

Mibi 1195 PEPT2 TTGTCCTCCCAGTATTGGTAAGGC Reverse

Mibi 1211 AQP5 CTTCCTCAAGGCCGTGTTC Forward

Mibi 1212 AQP5 GCTGGAAGGTCAGAATCAGC Reverse

Probe sets for quantitative TaqMan gene expression analyses were purchased from

Applied Biosystems, Foster City, USA.

Table 2.7: Probe sets for TaqMan gene expression assays.

Gene Probe Set

α-ENaC Hs00168906_m1

β-ENaC Hs00165722_m1

γ-ENaC Hs00168918_m1

CFTR Hs00357011_m1

α1-Na+/K+ ATPase Hs00167556_m1

β1-Na+/K+ ATPase Hs00426868_g1


- 16 -

As an endogenous control 18S rRNA was amplified using the following primer set:

Table 2.8: Primer set for amplification of 18S rRNA (Applied Biosystems, Foster City, USA).

Primer Name Sequence 5’-3’ Orientation

TQ 18SRNA S CGGCTACCACATCCAAGGAA Forward

TQ 18SRNA A GCTGGAATTACCGCGGCT Reverse

TQ 18SRNA PR (VIC)-TGCTGGCACCAGACTTGCCCTC-(TAMRA) Probe

2.1.7 Solutions

Hank’s balanced salt solution (HBSS; Invitrogen, Carlsbad, USA):

2 mM CaCl2, 5.4 mM KCl, 0.4 mM KH2PO4, 0.5 mM MgCl2, 0.4 mM MgSO4, 137 mM

NaCl, 1.3 mM Na2HPO4, 4 mM NaHCO3 5 mM Glucose, pH 7.4

Dulbecco’s phosphate buffered saline (D-PBS; PAA Laboratories, Pasching, Austria):

137 mM NaCl, 8.1 mM Na2HPO4, 2.7 mM KCl, 1.5 mM KH2PO4, pH 7.4

The composition of further solutions is listed in paragraphs of the corresponding methods.

2.1.8 Cell Culture

2.1.8.1 Cell Lines

Different human lung epithelial cell lines were supplied by American Type Culture

Collection (ATCC), Rockville, USA:

Table 2.9: Characteristics of human lung epithelial cell lines used in this study.

Cell line A549 H441 Calu-3

Organism Homo sapiens Homo sapiens Homo sapiens

Origin Lung carcinoma Lung adenocarcinoma Lung adenocarcinoma

Growth properties Adherent Adherent Adherent

Morphology Epithelial Epithelial Epithelial

Properties of Alveolar type II cells Bronchiolar Clara cells Bronchial ciliated cells

Passages used in experiments

84-92 81-89 21-29


- 17 -

Furthermore, the mouse fibroblast cell line WEHI-13VAR (ATCC, Rockville, USA) was

used as a bioassay system to measure the activity of recombinant human tumor necrosis

factor α.

2.1.8.2 Cell Culture Reagents

2.1.8.2.1 Complete Growth Media

Table 2.10: Complete growth media for cell cultivation.

Medium Kaighn’s F-12K RPMI 1640 Earle’s MEM

Manufacturer Invitrogen, Carlsbad, USA Invitrogen, Carlsbad, USA PAA Laboratories, Pasching, Austria

Supplements 2 mM L-glutamine

1.5 g/l sodium bicarbonate

10% fetal bovine serum

1% penicillin-streptomycin

2 mM L-glutamine

10 mM Hepes, pH 7.4

1 mM sodium pyruvate

4.5 g/l glucose




2 mM L-glutamine

1 mM sodium pyruvate

1 mM nonessential amino acids




Cell line A549 H441, WEHI-13VAR Calu-3

Stock solutions of Hepes buffer (1 M), L-glutamine (200 mM), MEM sodium pyruvate

(100 mM), sodium bicarbonate (7.5%), MEM nonessential amino acids (100x), and

penicillin-streptomycin (10,000 units/ml and 10,000 µg/ml, respectively) were purchased

from Invitrogen, Carlsbad, USA. D-(+)-glucose (45%) and fetal bovine serum were

purchased from Sigma-Aldrich, Taufkirchen, Germany.

2.1.8.2.2 Additional Cell Culture Reagents

Table 2.11: Additional cell culture reagents.

Reagent Manufacturer

Accutase PAA Laboratories, Pasching, Austria

Dimethyl sulfoxide (DMSO) Merck, Darmstadt, Germany

Trypan blue solution (0.4%) Sigma-Aldrich, Taufkirchen, Germany

Trypsin-EDTA (0.05% Trypsin, 0.53 mM EDTA) Invitrogen, Carlsbad, USA


- 18 -

2.1.9 Computer Software

The following software was used for analysis and presentation of data:

Table 2.12: Computer Software

Software Manufacturer

Office 2000 Microsoft Corporation ,Redmond USA

Graph Pad Prism 4.02 GraphPad Software Inc., San Diego, USA

Image Reader LAS 1000 Pro V2.1 Fujifilm, Düsseldorf, Germany

Gene Genius Bio Imaging Syngene, Rockville, USA

Agilent 2100 Bio Sizing Agilent Technologies, Karlsruhe, Germany

AxioVision LE Rel. 4.1 Zeiss, Göttingen, Germany

Advanced Image Data Analyzer 3.12 Raytest GmbH, Straubenhardt, Germany


- 19 -

2.2 Methods

2.2.1 Cell Culture

A549, H441, Calu-3 and WEHI-13VAR cells were grown in different complete growth

media (see chapter 2.1.8.2) with 21% O2, 5% CO2 and balanced N2 at 37°C and 95%

humidity. Medium was replaced every other day. Cultures were passaged once to three

times a week in a subcultivation ratio of 1:3 to 1:10, using Trypsin/EDTA or Accutase.

For cryoconservation trypsinized cells were adjusted to a cell density of 2-4 x 106

cells per ml in complete growth medium supplemented with 5% (v/v) DMSO. One-ml

aliquots were cooled to –80°C in a Nalgene Cryo 1°C freezing container (Nalge Nunc

International, Rochester, USA) and stored in the gas phase of liquid nitrogen at –142°C.

2.2.2 TNF-α Cytotoxicity Activity Assay

The cytotoxic effect of TNF-α on the murine fibrosarcoma cell line WEHI-13VAR was

measured by the reduction of the tetrazolium dye Alamar Blue (BioScource International,

Camarillo, USA) by viable cells. Herefore, WEHI-13VAR cells were plated into a 96-well

cell culture plate at a density of 2 x 104 cells/well and grown overnight. Consecutively,

serial dilutions of recombinant human TNF-α in RPMI 1640 medium (without phenole red)

+ 25 mM Hepes pH 7.4 + 3% (v/v) FBS were added in the presence of 0.5 µg/ml

actinomycin D (Sigma-Aldrich, Taufkirchen, Germany). Mock treated cells were used to

set the basal level of cytotoxicity (i.e. 0% cytotoxicity), cells lysed with 0.1% Triton X-100

were used to set its maximum level (i.e. 100% cytotoxicity). After incubation of the plate

for 20 h in a cell culture incubator, 1/10 volume of Alamar Blue dye was added to each

well. After a 4 h-incubation at 37°C, Alamar Blue fluorescence was measured with a

Wallac Victor2 Multilabel Counter spectrophotometer (Perkin Elmer, Wellesley, USA) with

an excitation wavelength of 544 nm and an emission filter of 590 nm.

2.2.3 Molecular Biological Methods

2.2.3.1 RNA Isolation

Total RNA was isolated from A549, H441 and Calu-3 cells using the RNeasy kit (Qiagen,

Hilden, Germany). Traces of DNA in RNA samples were removed by on-column DNase

digestion using the RNase-free DNase Set (Qiagen, Hilden Germany). RNA concentration


- 20 -

and purity was determined by measuring optical densities (OD) of RNA samples at

260 nm and 280 nm using an Eppendorf Biophotometer (Eppendorf, Hamburg, Germany).

An OD260 of 1 corresponds with 33 µg/ml of total RNA. Purities of RNA samples were

estimated by calculating the OD260/OD280 ratios. Additionally, RNA sample quality was

assessed using the RNA 6000 Kit with an Agilent 2100 Bioanalyzer (Agilent Technologies,

Karlsruhe, Germany).

2.2.3.2 Reverse Transcription of mRNA

First-strand cDNA was synthesized using 1 µg of total RNA in a 21 µl reverse

transcription (RT) reaction mixture with the Expand Reverse Transcriptase System

(Roche Diagnostics, Penzberg, Germany) with oligo (dT)15 primers according to the

manufacturer’s protocol.

2.2.3.3 RT-PCR

Four µl of the reverse transcription mixture were used for polymerase chain reaction

(PCR). The Expand Long Template PCR System (Roche Diagnostics, Penzberg,

Germany) was used for the amplification of specific DNA fragments with 20 pmol of

forward and reverse primers in a 50 µl reaction mixture containing 1 µl dNTP mix (10 mM

each), 5 µl of 10x PCR buffer, 0.75 µl MgCl2 (50 mM) and 0.75 µl Expand Long Template

enzyme mix. PCR reactions were carried out using a Mastercycler Gradient PCR Machine

(Eppendorf, Hamburg, Germany) with the following cycle programs:

Table 2.13: Parameters of RT-PCRs.

Temperature

Step Number of

cycles ENaC

α/β/γ CFTR PEPT-1 PEPT-2 AQP5 HPRT

Duration

Denaturing 1 95°C 95°C 95°C 95°C 95°C 95°C 1 min

Denaturing 95°C 95°C 95°C 95°C 95°C 95°C 30 s

Annealing 62°C 54°C 62°C 62°C 63°C 62°C 30 s

Elongation

35

72°C 72°c 72°C 72°C 72°C 72°C 1 min

Final elongation

1 72°C 72°C 72°C 72°C 72°C 72°C 5 min


- 21 -

REU = 2∆Ct x10-7; ∆Ct = Ct(mRNA) – Ct(18S rRNA)

The size of amplified DNA fragments was determined using the Agilent 2100 Bioanalyzer

using the DNA 12000 assay kit according to the manufacturer’s protocol (Agilent

Technologies, Karlsruhe, Germany).

2.2.3.4 Real-Time RT-PCR

Quantitative real-time RT-PCR was performed with 10 ng of cDNA using TaqMan Gene

Expression Assays (Applied Biosystems, Foster City, USA) for human ENaC-α, ENaC-β,

ENaC-γ, CFTR, Na,K-ATPase-α1 and Na,K-ATPase-β1 according to the manufacturer’s

instructions in a total volume of 25 µl. As an internal control 18S rRNA was amplified.

Forty cycles of amplification, data acquisition and data analysis were performed in an ABI

Prism 7900 HT Sequence Detection System (Applied Biosystems, Foster City, USA) with

the following parameters:

Table 2.14: Parameters of TaqMan real-time PCRs.

Step Number of cycles Temperature Duration

Enzyme activation 1 95°C 10 min

Denaturing 95°C 20 s

Annealing/elongation 40

60°C 1 min

The mRNA level of each gene of interest was related to that of the 18S rRNA within each

sample and calculated as relative expression units (REU):

with Ct being the value of that PCR cycle at which the fluorescence signal had reached a

specific threshold value indicating the presence of the mRNA of interest.

2.2.4 Biochemical Methods

2.2.4.1 Crude Membrane Preparation

Cells were grown in 94-mm dishes to confluence. Medium was removed and cells were

washed with phosphate-buffered saline (PBS). Cells were scraped and homogenized by

sonication in 1.5 ml of homogenization buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA,


- 22 -

250 mM sucrose, “Complete Protease Inhibitor Cocktail” (Roche Diagnostics, Penzberg,

Germany). The nuclei and cell debris were removed from the homogenate by

centrifugation at 4000 × g for 15 min at 4°C. The resulting supernatant was centrifuged at

100,000 × g for 1 h at 4°C. The membrane pellet was solubilized in buffer (10 mM

Tris-HCl, pH 7.4, 1 mM EDTA, 1% Triton X-100, Complete Protease Inhibitor Cocktail) by

vortexing for a minimum of 1 h at 4°C.

2.2.4.2 Determination of Protein Content

Protein concentration was determined using the BioRad Protein Assay (Biorad, Munich,

Germany) or the Micro BCA Protein Assay Kit (Pierce, Rockford, USA) according to the

manufacturer’s instructions. Bovine serum albumine (Pierce, Rockford, USA) was used as

a standard. Each measurement of protein concentration was performed in duplicates.

2.2.4.3 Quantitative Determination of Cyclic AMP (cAMP)

Intracellular cAMP content of Calu-3 cells was quantitatively assessed with a “Direct

cAMP Enzyme Immunoassay Kit” (Sigma-Aldrich, Taufkirchen, Germany). Cells were

grown in 6-well plates until the reached full confluence and treated with different

compounds (HBSS, 10 µM foskolin, 10 µM terbutaline and 1 mg/ml Tip peptide) for 15

minutes at 37°C. After this incubation lysis of cells was achieved by treatment of cells with

0.1 M HCl + 0.5% Triton X-100 for 20 minutes at room temperature. Samples from

forskolin and terbutaline treated cells were diluted 1:10 with lysis buffer before

measurement of cAMP content. Assessment of cAMP levels was performed according to

the manufacturer’s instructions and normalized to protein contents of the samples.

2.2.4.4 Streptavidin Affinity Chromatography

Calu-3 cells (8 x 107 cells per experiment) were recovered from the cell culture dish by

scraping with 10 ml HBSS + 10 mM Hepes pH 7.4 + “Complete Protease Inhibitor

Cocktail” (Sigma-Aldrich, Taufkirchen, Germany) and lysed by sonication. Cell debris was

removed by centrifugation with 3500 x g at 4°C for 20 min and supernatant was passed

through a 0.45 µm-filter (Millipore, Bedford, USA). Biotinylated human circular Tip peptide

(bTip) was added to a final concentration of 250 µg/ml. After overnight incubation at 4°C

on an overhead shaker, bTip and interacting proteins were purified using a ÄKTAFPLC

liquid chromatography system (Amersham Biosciences, Uppsala, Sweden) with a HiTrap


- 23 -

Streptavidin HP column (1 ml column volume (CV); GE Healthcare, Uppsala, Sweden).

The column was equilibrated with 10 CV running buffer before the bTip-containing cell

lysate was injected. After washing the column with 10 CV running buffer, elution of

interacting proteins was started by subsequent injection of 2 CV elution buffer 1, 2 CV

elution buffer 2 and 5 CV elution buffer 3. The flow rate was 1 ml/min during equilibration

and washing steps whereas sample injection and elution occurred with 0.3 ml/min. Eluted

proteins were collected in 1 ml fractions and subjected to 4 – 20% gradient Tris-Glycine

SDS-polyacrylamide gels (Invitrogen, Carlsbad,USA).

In addition, control experiments with Calu-3 cell lysate alone, with Calu-3 lysate +

D-Biotin (Sigma-Aldrich, Taufkirchen, Germany), with Calu-3 lysate + biotinylated mutant

Tip peptide and with Calu-3 lysate + biotinylated scrambled Tip peptide were performed.

Running buffer:

HBSS + 10 mM Hepes pH 7.4

Elution buffer 1:

500 µM N,N’-diacetylchitobiose in running buffer

Elution buffer 2:

5 mg/ml Tip peptide in running buffer

Elution buffer 3:

50 mM ammonium acetate, 0.5 M NaCl, pH 4.0

2.2.4.5 Surface Plasmon Resonance Analyses

Sugar binding studies were carried out using a Biacore 3000 surface plasmon resonance

(SPR) sensor (Biacore, Uppsala, Sweden) with control software version 4.0 and Sensor

Chip CM5 (carboxymethylated dextran surface). All assays were carried out at 25°C.

Human recombinant TNF-α and Tip peptide were immobilized via amine groups in two

flow cells, one flow cell served as matrix control. The chip surface was first activated

following a standard EDC/NHS protocol95 with HBS-P buffer + 1 mM CaCl2 used as the

running buffer. TNF-α at a concentration of 50 µg/ml and peptides at a concentration of

1 mg/ml in HBS-P buffer were injected followed by the injection of 1 M ethanolamine


- 24 -

(pH 8.5) to inactivate the residual active groups. N,N’-diacetylchitobiose and cellobiose

(100 µM in HBS-P + 1 mM CaCl2) were injected and capture of the analytes was

documented.

In another series of experiments HBS-EP was used as the running buffer to

evaluate the influence of Ca2+ ions on the lectin-like activity of TNF-α and the Tip peptide.

HBS-P buffer (Biacore, Uppsala, Sweden) + 1 mM CaCl2:

10 mM Hepes pH 7.4, 150 mM NaCl, 0.005% v/v Surfactant P20, 1 mM CaCl2

HBS-EP buffer (Biacore, Uppsala, Sweden):

10 mM Hepes pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20

2.2.4.6 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Proteins were separated according to their molecular weight by discontinuous

SDS-PAGE 96. Protein samples were mixed with 2x Laemmli reducing sample buffer

(Biorad, Munich, Germany) and heated for 10 min at 95°C. Membrane protein

preparations were heated at 40°C for 15 min. A 50-µg protein sample and 10 µl of

SeeBlue Pre-stained Standard (Invitrogen, Carlsbad, USA) were separated in

SDS-PAGE. Therefore, SDS-polyacrylamide mini gels of the size of 8.0 × 7.3 × 0.1 cm,

which comprise a 4% stacking gel and a resolving gel of suitable percentage, were

prepared using the Miniprotean 3 gel system (Biorad, Munich, Germany).

Table 2.15: Composition of polyacrylamide gels.

Reagent Stacking gel Resolving gel

4% 7.5% 10% 12.5% 15%

Acrylamide/Bisacrylamide (30:0.8) 0.67 ml 2.5 ml 3.33 ml 4.17 ml 5 ml

0.5 M Tris-HCl pH 6.8 1.25 ml - - - -

1.5 M Tris-HCl pH 8.8 - 2.5 ml 2.5 ml 2.5 ml 2.5 ml

10% (w/v) SDS 0.1 ml 0.1 ml 0.1 ml 0.1 ml 0.1 ml

80% (w/v) glycerin - 1.2 ml 1.2 ml 1.2 ml 1.2 ml

H2O 3 ml 3.7 ml 2.8 ml 2 ml 1.1 ml

TEMED 3.8 µl 7.5 µl 7.5 µl 7.5 µl 7.5 µl

10% (w/v) APS 100 µl 100 µl 75 µl 75 µl 75 µl


- 25 -

2x Laemmli sample buffer (Biorad, Munich, Germany):

62.5 mM Tris-HCl (pH 6.8), 25% (w/v) glycerin, 2% (w/v) SDS, 0.01% (w/v) bromophenol

blue, 8% (v/v) β-mercaptoethanol

Electrophoresis buffer:

25 mM Tris, 192 mM glycine, 0.1% (w/v) SDS

2.2.4.7 Native PAGE

For native (non-denaturing) PAGE the Novex Gel System (Invitrogen, Carlsbad, USA)

was used with 4-20% Tris-Glycine Gels, Tris-Glycine Native Running Buffer and Tris-

Glycine Native Sample Buffer according to the manufacturer’s instructions. During native

electrophoresis, proteins are separated based on their charge to mass ratios.

2.2.4.8 Coomassie Staining of Proteins

Protein bands on polyacrylamide gels were visualized with SimplyBlue SafeStain

(Invitrogen, Carslbad, USA):

After electrophoresis, the gels were washed three times for 10 min in H2O,

incubated in SimplyBule SafeStain reagent for one hour at room temperature and washed

in H2O for one to two hours or overnight. For documentation, images were captured with a

Gene Genius Bio Imaging System (Syngene, Cambridge, United Kingdom).

2.2.4.9 Silver Staining of Proteins

The silver staining procedure according to Heukeshoven and Dernick 97 was performed as

follows:

Gels were fixed for 30 min in fixing buffer 1, for 30 min in fixing buffer 2 and

washed four times for 15 min in H2O. Staining was performed for 30 min in staining

solution. Afterwards, gels were rinsed in H2O and incubated in developer. The reaction

was stopped with stop solution. After 10 min, gels were washed in H2O and stored at 4°C.

Fixing buffer 1:

10% (v/v) acetic acid, 30% (v/v) ethanol


- 26 -

Fixing buffer 2:

30% (v/v) ethanol, 0.5 M sodium acetate, 0.5% (v/v) glutaraldehyde, 0.2% (w/v) sodium-

thiosulfate

Staining solution:

0.1% (w/v) silver nitrate, 0.02% (v/v) formaldehyde

Developer:

2.5% (w/v) sodium carbonate, 0.01% (v/v) formaldehyde

Stop solution:

1% (v/v) acetic acid

2.2.4.10 In Gel Digest of Protein Bands for Mass Spectroscopic Analysis

Gel pieces with protein bands were cut out from silver-stained polyacrylamide gels. In a

silanized Eppendorf cup protein bands were bleached with 15 mM potassium

hexacyanoferrat/50 mM sodium thiosulfate. After washing the gel pieces 3-times for

15 min with H2O, proteins were reduced with 10 mM dithiotreitol (DTT) and alkylated with

55 mM iodoacetamide. Dehydration of gel slices was started by two washing steps with

50% (v/v) acetonitrile (ACN)/50 mM NH4HCO3 for 15 min and one washing step with ACN

for 5 min. After removal of supernatants and desiccation of gel pieces in a SpeedVac

(Eppendorf, Hamburg, Germany) they were covered with 2.5 ng/µL porcine modified

trypsin (Promega) in 50 mM NH4HCO3 and incubated overnight at 37°C. Supernatants

containing tryptic digests were collected. Gel pieces were incubated twice with 50% (v/v)

ACN/1% (v/v) trifluor acetic acid (TFA) and the extracts were pooled with the tryptic digest

samples. Peptides were dried in a SpeedVac. The dried digest was dissolved in 1% TFA.

Two µL thereof were desalted with a C18 µZiptip (Millipore) and washed three times with

10 µL 0.1% TFA within the Ziptip. The sample was eluated from the Ziptip with 0.8 µL

HCCA solution and directly spotted onto the 600/384 MALDI anchor target (Bruker

Daltonics) according to the dried-droplet method. HCCA solution consisted of 0.5 µg

HCCA per milliliter dissolved in a 2:1 mix of MeCN and 0.1% TFA.


- 27 -

2.2.4.11 Mass Spectroscopy

Matrix Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) mass spectra

were measured with an Ultraflex TOF/TOF mass spectrometer in reflector and positive ion

mode (Bruker Daltonik GmbH, Bremen, Germany). Ions were accelerated in an electric

field of 25 kV. With this method the exact and sensitive identification of tryptic fragments

of proteins of interest was possible. The mass spectrometer was externally calibrated with

the peptide standard Pepmix (Bruker Daltonik GmbH, Bremen, Germany). Additionally an

internal calibration was performed using keratin peaks, which further increased the mass

accuracy to 50 ppm. Obtained peptide mass fingerprints, were used for database

searches in order to identify the sample protein. The Mascot search engine was used

together with NCBI (non-redundant) and MSDB databases to identify proteins that fit to

the measured peptide mass fingerprints. For even more precise identification, peptide ions

of single mass peaks were fragmented and fragments were analyzed in MS/MS mode.

The resulting peptide fragment amino acid sequences were again used for Mascot

database searches (Matrix Science Inc., Boston, USA) in order to identify the respective

proteins.

2.2.4.12 Western Blotting

Proteins from SDS-PAGE gels were transferred to Protean BA85 nitrocellulose

membranes (Schleicher & Schuell, Dassel, Germany) using a Hoefer TE 22 Mighty Small

Transphor Tank transfer unit (Amersham Biosciences, Uppsala, Sweden) with Towbin

transfer buffer 98 at a constant current of 950 mA for one hour. After blotting, membranes

were blocked with 5% (w/v) fat-free milk in TBS by slight shaking for 1 h at room

temperature. Afterwards blots were incubated with antibodies against proteins of interest

diluted 1:1000 in TBS + 0.05% (w/v) fat-free milk for 2 h at room temperature or overnight

at 4°C. After four consecutive washing steps with TBS-T for 10 min bound antibody was

detected using a 1:25000 dilution of horseradish peroxidase-conjugated secondary

antibody (Dianova, Hamburg, Germany) for 2 h at room temperature. After three further

washing steps with TBS-T and one washing step with TBS, Western blots were developed

with Lumi-LightPlus Western Blotting Substrate (Roche Diagnostics, Penzberg, Germany)

and photographed with a LAS-1000 Luminescence Image Analyzer using the Image

Reader LAS 1000 Pro V2.1 software (Fujifilm, Düsseldorf, Germany) and analyzed with

the Advanced Image Data Analyzer V3.12 software (Raytest GmbH, Straubenhardt,

Germany). The used antibodies are listed in table 2.3.


- 28 -

Towbin transfer buffer 98:

25 mM Tris, 192 mM glycine, 20% (v/v) methanol, 0.01% (w/v) SDS, pH 8.3

Tris buffered saline (TBS):

4 mM Tris-HCl, 100 mM NaCl, pH 7.5

TBS-T:

0.05% (v/v) TWEEN 20 in TBS

2.2.4.13 Stripping of Western blots

Bound antibodies were removed from Western blot membranes by incubation for 30 min

at room temperature in Restore Western Blot Stripping Buffer (Pierce, Rockford, USA).

After washing the membranes 3-times for 10 min with TBS-T they were ready for another

immunodetection of proteins.

2.2.5 Immunofluorescence Staining

Cells were grown on Lab-Tek Permanox chamber slights (Nalge Nunc, Naperville, USA)

or on microscopically transparent polyester Transwell membranes (Corning, Acton, USA).

Every step of the immunofluorescence staining procedure was performed at room

temperature. Cells were washed 3-times with PBS and subsequently paraformaldehyde

(4% (w/v) in PBS) was added for fixation. After incubation for 15 min cells were washed 3-

times with PBS and incubated for 10 min with 50 mM NH4Cl in PBS. If membrane

permeabilization was required cells were incubated with the 0.2% (w/v) Triton X-100 in

PBS for 5 min and afterwards washed another 3-times with PBS. For blocking, cells were

incubated with 5% (v/v) fetal bovine serum (FBS) in PBS for 30 min in a humid chamber.

Primary antibody solutions were prepared in 1.5% (v/v) FBS in PBS and added to the

preparations for one hour at room temperature or overnight at 4°C. After three washing

steps with PBS, incubation with secondary antibody solutions followed for one to two

hours in a humid chamber protected from light. The counter staining agent 4’,6-Diamidino-

2-phenylindole dihydrochloride (DAPI; Sigma-Aldrich, Deisenhofen, Germany) was added

to secondary antibody solutions to a final concentration of 1 µg/ml. Thereafter, cells were

washed 3-times with PBS and mounted in Vectashield (Vector Lab, Burlingame, USA).


- 29 -

Slide preparations had been dried overnight protected from light, sealed with enamel, and

stored at 4°C until they were used for fluorescence microscopy.

2.2.6 Microscopy

2.2.6.1 Brightfield Phase Contrast and Fluorescence Microscopy

Light and fluorescence microscopy was performed with a Zeiss Axio Vert 25 microscope

(Zeiss, Göttingen, Germany) equipped with an Axio Cam MR camera and FITC and DAPI

fluorescence filter sets. Microscopic parameters are indicated on corresponding

photomicrographs. Images were edited with AxioVision LE 4.1 analyzing software (Zeiss,

Göttingen, Germany).

2.2.6.2 Confocal Laser Scanning Microscopy

A Leica DMRE microscope was used with a TCS SP2 True confocal scanner (Leica

Microsystems, Wetzlar, Germany) with ultraviolett (351 nm excitation), Argon/Krypton Ion

(458 nm, 476 nm, 488 nm, 514 nm), and He/Ne Ion (543 nm, 633 nm) laser systems for

image acquisition. The Leica TCS Software was used for picture editing.

2.2.7 Transepithelial Electrical Resistance (TEER) Assay

Cells were seeded at a density of 1.5 x 105 cells/cm2 onto Transwell-Clear 24-well cell

culture inserts with 0.4 µm pore size (Corning Costar, Bodenheim, Germany). By

aspirating the apical fluid 48 h after cell seeding an air-liquid interface was created. The

basolateral medium was changed every other day and monolayers were used for

bioelectrical studies between day 7 and 10 post seeding. Formation of tight monolayers

was tested by measuring the transepithelial electrical resistance (TEER) by using an

EVOMX epithelial voltohmmeter (World Precision Instruments (WPI), Sarasota, USA) in

combination with chopstick STX2 electrodes.

For TEER assay measurements cell monolayers were transferred to an ENDOHM-

6 electrode chamber (WPI, Sarasota, USA) and bathed on both sides with Hank’s

balanced salt solution (HBSS) supplemented with 10 mM Hepes, pH 7.4. Once TEER

reached steady state values, measurements were started. Recordings of TEER before

and after addition of compounds to the apical and/or basolateral compartment were


- 30 -

documented with an EVOMX epithelial Voltohmmeter connected to a Rec 111 recorder

(Amersham Pharmacia, Uppsala, Sweden).

For ion-substitution experiments HBSS was replaced by NaCl-, NMDG- Gluconate- or

CsCl-buffer, respectively:

NaCl-buffer:

140 mM NaCl, 5.4 mM KCl, 1.25 mM CaCl2, 1 mM MgSO4, 10 mM Hepes pH 7.4, 5 mM

glucose

NMDG-buffer:

NaCl was replaced by equimolar amounts of N-methyl-D-glucamine (NMDG)

Gluconate-buffer:

NaCl was replaced by equimolar amounts of sodium gluconate

CsCl-buffer:

KCl was replaced by equimolar amounts of CsCl.

The pH of all solutions was adjusted to 7.4.

For sodium titration experiments a dilution series of NaCl-buffer with NMDG-buffer

was prepared to obtain sodium concentrations of 0 (NMDG-buffer), 17.5, 35 70 and

140 mM (NaCl-buffer) in the assay buffer.

2.2.8 Lucifer Yellow Rejection Assay

Lucifer Yellow (LY; MW 521.6) passes cell monolayers only by passive paracellular

diffusion. Therefore, paracellular monolayer integrity and maintenance can be tested by

addition of LY on one side of the monolayer followed by fluorescence readout of apical

and basal solutions after a defined incubation period. Calu-3 cells had been seeded at a

density of 1.5 × 105 cells in 24-well Falcon HTS multiwell inserts (BD Biosiences, Bedford,

USA; Ø 1 µM, PET membrane) 7 days prior to analysis. Cells were grown under air-liquid

interface conditions to full confluent monolayers with TEER of > 300 Ω × cm2. Before


- 31 -

analysis, filter inserts had been washed 3-times with HBSS (pH 7.4) and set up for LY

measurements. HBSS (1 ml with 1% (v/v) DMSO) was aliquoted to each well of a 24-well

Falcon plate (BD Biosiences, Bedford, USA). Subsequently, HTS multiwell inserts were

placed into the 24-well Falcon plate. LY was dissolved in HBSS (1% (v/v) DMSO) and

diluted in series of 1:3 from 60 to 6.7 µM and from 1 µM to 4.1 nM for determination of

high standard (for apical solution) and low standard curve (for basolateral solution),

respectively. Monolayer integrity during compound incubation was tested by adding

300 µL of LY solution (60 µM LY in HBSS) apically to each filter well. The test compound

of interest was added to the apical or basolateral compartment (as indicated). The plates

had been incubated for one hour while shaking (50 rpm) at 37°C and LY fluorescence

readout was performed in solid black 96-well plates (Nalge Nunc International, Rochester,

USA) at 480/530 nm with a Wallac Victor2 Multilabel Counter spectrophotometer (Perkin

Elmer, Wellesley, USA) in triplicates. Percentage of LY passage and permeability

coefficient for LY were calculated using the following equations:

−×=

apical

lbasolatera

RFU

RFU1100rejectionLY%

RFU, relative fluorescence units

t

C

CA

VP

lbasolateraf,

apicali,

lbasolatera

C ××

=

A, area of growth membrane; Cf,basolateral, final basolateral concentration; Ci,apical, initial

apical concentration; PC, permeability coefficient; Vbasolateral, volume on basolateral side,

t, time in seconds

2.2.9 Dome Assay

For dome assay experiments cells were seeded into 96-well plates at a density of 5 x 104

cells per well in a volume of 200 µl and cultured until they reached full confluence and

started to form domes. Prior to the addition of compounds, dome density was documented

by microscopic photography of each well with the optical image analysis system

CellScreen (Innovatis, Bielefeld, Germany) and analyzed with the CS Processing software

package (Innovatis, Bielefeld, Germany). After addition of compounds dome density was

determined every hour for up to 4 h. After completion of an experiment, image data of


- 32 -

each well were analyzed by counting domes of a defined area of 12.5 mm². Data are

presented as percentage of domes compared with the dome density before compound

addition.

In the ion-substitution experiments the culture medium was removed by aspiration

and the cell monolayers were incubated with NMDG-buffer. In control wells medium was

replaced by NaCl-buffer.

NaCl-buffer:

140 mM NaCl, 5.4 mM KCl, 1.25 mM CaCl2, 1 mM MgSO4, 10 mM Hepes pH 7.4, 5 mM

glucose

NMDG-buffer:

NaCl was replaced by equimolar amounts of N-methyl-D-glucamine (NMDG)

2.2.10 Statistical Analysis

Statistics were calculated using the GraphPad Prism 4.02 software (GraphPad Software,

San Diego, USA). For one-grouping variables differences were determined by one-way

analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test (comparison

of „control versus treated groups“) or Bonferroni’s multiple comparison test („all versus all“

comparisons). In case of two-grouping variables two-way ANOVA was performed using

Bonferroni post-tests to compare replicate means by row. Different statistical significant

levels were indicated with asteriks (P value < 0.05: *; P < 0.01: **; P < 0.001: ***).

RESULTS

- 33 -

3 Results

In this work, two test systems with characteristics of the human lung epithelium were

established and validated. These systems were used for in vitro studies on the

mechanism of action of the TNF-α derived anti-edema Tip peptide and for activity testing

of alternative anti-pulmonary edema peptide drug candidates.

In order to find a human lung epithelial cell line suitable for investigations of active

ion and fluid transport, the following three cell lines derived from different parts of the lung

were investigated in regard to ion channel expression and activity, and to active

transepithelial fluid transport capacities:

(1) A549 cells as model system for alveolar type II cells 99

(2) H441 cells with characteristics of distal bronchiolar Clara-like cells 100

(3) Calu-3 cells that represent bronchial ciliated cells 101

3.1 Characterization of Human Lung Epithelial Cell Lines

The epithelial sodium channel ENaC, the chloride-specific ion channel CFTR and the

Na,K-ATPase are involved in the process of lung liquid clearance (LLC) 3,18,49,102.

Therefore, expression of all subunits of these ion channels and transporters in A549,

H441 and Calu-3 cells was examined using real-time RT-PCR and Western blot analyses.

3.1.1 Analysis of A549, H441 and Calu-3 Cells for the Expression of Ion

Channels and Transporters Involved in LLC

Real-time RT-PCR analysis was performed to quantify mRNA levels of ENaC-α, -β and -γ,

CFTR and Na,K-ATPase-α1 and –β1. Transcript levels were calculated as relative

expression units (REU) related to the internal control 18S rRNA of each sample

(Figure 3.1 A-C). Since variation of 18S rRNA levels between different cell lines was less

than 5%, gene expression levels were quantitatively compared (Figure 3.1 D). ENaC-α

mRNA was clearly detected in A549 cells (1.4e-04 REU). ENaC-α mRNA levels were

higher in H441 and Calu-3 cells than in A549 cells (39.4- and 23.5-fold, respectively).

ENaC-β transcript levels were very low in A549 and Calu-3 cells (2.57e-07 REU and

4e-07 REU, respectively), whereas H441 cells contained about 100-fold more ENaC-β

RESULTS

- 34 -

mRNA (2.7e-05 REU). ENaC-γ mRNA was present in H441 and Calu-3 cells in very low

levels (7.7e-07 REU and 1.1e06 REU, respectively), but could not be detected in A549

cells. CFTR mRNA levels were exceedingly higher in H441 and Calu-3 cells compared to

A549 cells (7.1e+05- and 1207-fold, respectively). Levels of Na,K-ATPase-α1 and –β1

mRNAs were rather high in all three investigated cell lines with about 5-fold higher

expression levels in Calu-3 cells compared to A549 and H441 cells.

Figure 3.1: Taqman gene expression analyses of ion channels and transporters in A549, H441 and Calu-3 cells.

ENaC-α, -β, -γ, CFTR, Na,K-ATPase-α1 and -β1 mRNA levels in A549 (A), H441 (B) and Calu-3 cells (C) were quantified by TaqMan analysis. The level of gene expression was related to the expression of the internal control 18S rRNA in each sample and is presented as relative mRNA expression units. Data represent means + SD of three individual samples, each done in triplicate.

(D) Quantitative comparison of ENaC-α, -β, -γ, CFTR, Na,K-ATPase-α1 and –β1 mRNA levels between A549, H441 and Calu-3 cells. The mRNA levels are presented as “x-fold expression” with the lowest expression level set 1.0.

RESULTS

- 35 -

Western blot analyses were performed to detect ion channel and transporter

proteins involved in the process of LLC in membrane preparations of A549, H441 and

Calu-3 cells (Figure 3.2). Glycosylated ENaC-α, -β and –γ subunits were present in

membranes of all three investigated cell lines (87 kDa, 165 kDa and 105 kDa,

respectively). However, in H441 cells ENaC-β was expressed in an unglycosylated form

(75 kDa; Figure 3.2 A-C). Figure 3.2 D shows that glycosylated CFTR (150 kDa) could be

detected in membrane preparations of Calu-3 cells but not of A549 and H441 cells. In

contrast, Na,K-ATPase subunits α and β1 were present in all three lung epithelial cell lines

with both unglycosylated and glycosylated isoforms of the β1-subunit (42 kDa and 52 –

65 kDa, respectively), whereas the α-subunit existed in an unglycosylated form only

(110 kDa; Figure 3.2 E + F).

Figure 3.2: Western blot analyses of ion channel and transporter proteins in membrane fractions of A549, H441 and Calu-3 cells.

Equal amounts of protein (50 µg) in membrane fractions of A549, H441 and Calu-3 cells were subjected to SDS-polyacrylamide gels. Western blot analyses were performed using specific monoclonal (ENaC-β, CFTR, Na,K-ATPases-α and –β1) or polyclonal (ENaC-α and ENaC-γ) antibodies. Arrowheads indicate detection of specific protein bands.

(A) Anti-ENaC-α.

(B) Anti-ENaC-β.

(C) Anti-ENaC-γ.

(D) Anti-CFTR.

(E) Anti-Na,K-ATPase-α.

(F) Anti-Na,K-ATPase-β1.

RESULTS

- 36 -

3.1.2 Analysis of A549, H441 and Calu-3 Cells for the Expression of the

Water Channel Aquaporin-5 Involved in LLC

It had been shown that lung epithelial cells expressing the water channel aquaporin-5

have a high osmotic permeability to water 103. Aquaporin-5 was conferred to be

responsible for the majority of water transport across the lung epithelium 17,104. Hence,

expression of aquaporin-5 in A549, H441 and Calu-3 cells was investigated using

RT-PCR and Western blot analyses. As shown in Figure 3.3, transcripts (398 bp PCR

product) and protein (28 kDa) of aquaporin-5 could be detected in Calu-3 cells but not in

A549 and H441 cells.

Figure 3.3: Expression analyses of aquaporin-5 in A549, H441 and Calu-3 cells.

(A) RT-PCR analysis. Amplified DNA fragments from RT-PCR were visualized with the Agilent Bioanalyzer. The size of the PCR product of aquaporin-5 (AQP5) was expected to be 398 bp. Amplification of the housekeeping gene hypoxanthine phosphoribosyltransferase (HPRT) served as internal control of the PCR; ‘(-) RT’ (control without reverse transcriptase) and ‘H2O Cont’ (H2O control of PCR) served as negative controls. bp, base pairs; L, molecular weight ladder.

(B) Western blot analysis. Fifty micrograms of protein in membrane fractions of A549, H441 and Calu-3 cells were subjected to a SDS-polyacrylamide gel. Western blot analysis was performed using a specific polyclonal antibody against aquaporin 5 (28 kDa). Arrow head indicates detection of a specific protein band.

RESULTS

- 37 -

3.2 Establishment And Validation of In Vitro Test Systems

Reflecting Pulmonary Edema Resorption

Tumor necrosis factor α (TNF-α) and the Tip peptide are assumed to induce ion currents

across the lung epithelium, resulting in an enhanced rate of LLC 88-92. One aim of this

study was to develop and evaluate an in vitro model of the human lung epithelium that

allows the screening of anti-edema drug candidates.

3.2.1 Establishment of the Transepithelial Electrical Resistance (TEER)

Assay

The transepithelial electrical resistance (TEER) assay was established to investigate the

influence of test compounds on bioelectric properties of human lung epithelial cell

monolayers.

Readout of the TEER assay is the alteration of TEER following the application of a

test compound to the apical and/or basolateral side of a monolayer. An increase of TEER

indicates the reduction of transepithelial currents whereas a decrease of TEER indicates

the induction of ion currents across cell monolayers. Prerequisites of this assay are the

integrity and “tightness” (= intact intercellular contacts) of cell monolayers.

To evaluate the suitability of A549, H441 and Calu-3 cell lines for TEER assay

experiments, cells were cultured on semipermeable membranes, and monolayer

formation and tightness were monitored by TEER measurements. Figure 3.4 shows the

time-development of TEER values of A549, H441 and Calu-3 monolayers grown as liquid-

covered cultures (LCC) or air-interfaced cultures (AIC).

Figure 3.4: Time course of TEER development of A549, H441 and Calu-3 cell monolayers.

Cells were cultured as liquid-covered (LCC) or air-interfaced (AIC) cultures. Trans-epithelial electrical resistance (TEER) values were corrected by subtracting the TEER value of a blank filter and normalized to the growth area of the filter. Data are means ± SD, n = 6.

0 2 4 6 8 10 12 14 16 18 20 220

50

100

150

A549 LCC H441 LCC Calu-3 LCC

250

750

1250

Calu-3 AIC

Time(days post seeding)

TE

ER

(ΩΩ ΩΩ x

cm

²)

RESULTS

- 38 -

A549-LCC failed to form tight monolayers (peak TEER values < 50 Ω x cm²).

H441-LCC developed leaky monolayers with moderate TEER values (peak TEER values

155 Ω x cm² on day 11 post seeding). A549 and H441 cells grown as AIC did not produce

an effective barrier to hydrostatically driven medium seepage from the basolateral to

apical compartment. Different variations of A549 and H441 monolayer cultivation (cell

seeding densities, membrane material, pore size, membrane coating with extracellular

matrix components) did not improve monolayer integrity (data not shown). In contrast,

Calu-3 cells formed high resistance monolayers as a function of time with peak TEER

values of 1280 Ω x cm² for LCC and 497 Ω x cm² for AIC at day six and ten post seeding,

respectively. Formation of tight junctions and paracellular integrity of Calu-3 monolayers

were ascertained by immunofluorescence staining of zonula occludens-1 protein (ZO-1)

and the assessment of lucifer yellow (LY) passage across the monolayers, respectively.

Figure 3.5 illustrates the presence of intact tight junctions between adjacent Calu-3 cells

within a monolayer. Passive paracellular passage of LY through Calu-3 cell monolayers

(n = 7) was inhibited to 99.2 ± 0.3 % and the apparent permeability coefficient (Papp) for LY

was calculated to be 6.85 ± 3.25 nm/s. These values are indicative of well-established cell

monolayers. All TEER assay experiments were performed using Calu-3-AIC monolayers

as air-liquid interface growth conditions mimic the physical situation in the lung.

Figure 3.5: Immunfluorescence staining of tight junctional zonula occludens-1 protein in Calu-3 monolayers.

Tight junctions between adjacent cells were stained using a polyclonal rabbit anti-ZO-1 antibody together with a Cy2-conjugated goat-anti-rabbit IgG antibody (green). Cell nuclei were stained with DAPI (red). Original magnification x400.

RESULTS

- 39 -

3.2.2 Establishment of the Dome Assay

The dome assay was established to investigate the influence of test compounds on

transepithelial fluid transport by human lung epithelial cell monolayers. Domes are fluid-

filled hemicysts that result from active vectorial ion transport by confluent cell monolayers

followed by passive water flow (Figure 3.6 A). Therefore, dome formation by lung

epithelial cells simulates the process of LLC in vitro. Readout of the dome assay is the

quantitative analysis of dome formation within lung epithelial cell monolayers induced by

the application of a test compound.

To evaluate the suitability of A549, H441 and Calu-3 cell lines for dome assay

experiments, cells were grown to full confluence and examined in regard to their ability to

form domes. Additionally, influence of gluco- and mineralcorticoid treatment on dome

formation was investigated, since corticoids influence epithelial barriers by the induction of

tight junctions 105-107.

Figure 3.6: Dome formation of A549, H441 and Calu-3 cells.

(A) Light micrographs of three different focus layers of two domes within a confluent Calu-3 cell monolayer. Focus layers are as depicted in the top cartoon (1: focus on confluent cell layer grown on a culture dish; 2: focus on the middle of domes; 3: focus on apical surface of domes).

Top cartoon adapted from 3. White magnification bar: 100 µm.

(B) Spontaneous (untreated) dome formation capacity and the influence of treatment with gluco- (dexamethasone) and mineralcorticoids (aldosterone) on dome formation of A549, H441 and Calu-3 cells.

Dex, dexamethasone; Ald, aldosterone;

(-), no dome formation; (+), dome formation.

RESULTS

- 40 -

Figure 3.6 B illustrates that A549 cells did not form domes, neither spontaneously

nor if cells were exposed to 1 µM dexamethasone for 24 hours. Dome formation in H441

monolayers was induced by exposure of cells to dexamethasone, whereas untreated cells

did not form domes. Treatment of A549 and H441 cells with the mineralcorticoid

aldosterone did not induce dome formation.

Domes appeared spontaneously within Calu-3 monolayers one day after cell

monolayers had reached full confluence. Sizes of domes within Calu-3 monolayers

ranged from < 100 µm up to several hundreds of micrometers, comprising more than 100

cells. Treatment of Calu-3 cells with either dexamethasone or aldosterone did not

influence size and density of domes.

3.2.3 Validation of TEER and Dome Assays

As described above, the human lung epithelial cell line Calu-3 met all requirements for in

vitro analysis of transepithelial ion and fluid transports with respect to expression of ion

channels and transporters involved in LLC, formation of tight monolayers, and

transepithelial fluid transport (dome formation) within confluent monolayers. Therefore,

Calu-3 cells were used to validate the TEER and dome assays as suitable in vitro

measurements to investigate compounds that modulate transepithelial ion and fluid

movements.

Effects of ion transport modulators on bioelectrical properties of Calu-3 cells were

investigated by time-dependent recording of TEER values before and immediately after

compound addition to the apical and/or basolateral side of cell monolayers. TEER was

measured in an ENDOHM tissue resistance measurement chamber connected to an

EVOMX epithelial voltometer, and changes of TEER due to the inhibition or induction of

transepithelial currents by the investigated compounds were continuously recorded (TEER

assay). Ion transport activators, such as db cAMP (2 mM) and the short-acting β2-agonist

terbutaline (100 µM) decreased TEER within seconds to ~75% of initial TEER when

added to the basolateral side (Figure 3.7 A and B), indicating the induction of

transepithelial currents upon compound addition. When terbutaline was applied to the

apical side of the cell monolayer, final values of approx 90% of initial TEER were

measured (Figure 3.7 B). Comparable results were obtained with the long-acting β2-

agonist formoterol (data not shown).

Apical addition of the ion channel inhibitors amiloride (100 µM) and glibenclamide

(10 µM) increased TEER to 105% and 120% of initial TEER, respectively, indicating a

reduction of transepithelial conductance upon compound addition (Figure 3.7 C and D).

RESULTS

- 41 -

No change of TEER was observed, when these substances were injected to the

basolateral side of the cell monolayer. The Na,K-ATPase inhibitor ouabain (100 µM –

5 mM) did not effect changes of TEER, neither by application on the apical nor on the

basolateral side of the monolayer (data not shown). Application of the solvent HBSS in

equal volumes as to those used for compound injection served as a negative control and

had no effect on TEER of Calu-3 monolayers.

Figure 3.7: Validation of TEER assay with activators and inhibitors of transepithelial ion currents.

(A) Typical recording of TEER changes after addition of terbutaline (100 µM) to the basolateral side of Calu-3 cell monolayers. (B) Effects of activators of transepithelial ion currents after apical or basolateral application on TEER of Calu-3 cell monolayers. Minimum TEER values were calculated as percentage of TEER before addition of compounds. Data are means ± SD; significant difference with vehicle control (dotted line) for: ** P < 0.01, *** P < 0.001. (C) Typical recording of TEER changes after addition of glibenclamide (10 µM) to the apical side of Calu-3 cell monolayers. (D) Effects of inhibitors of transepithelial ion currents after apical or basolateral application on TEER of Calu-3 cell monolayers. Peak TEER values were calculated as percentage of TEER before addition of compounds. Data are means ± SD; significant difference with vehicle control (dotted line) for: * P < 0.05, *** P < 0.001.

RESULTS

- 42 -

To distinguish TEER changes caused by alterations of either transepithelial ion

currents, or the paracellular integrity of cell monolayers, the influence of test compounds

on paracellular permeability of Calu-3 cell monolayers was assessed by Lucifer yellow

(LY) rejection. LY is a fluorescent marker, which passes cell monolayers only by passive

paracellular diffusion. Percent LY rejection and apparent permeability coefficients (Papp)

for LY in the presence of the investigated ion transport modulators are listed in Table 3.1.

There were no significant differences in LY rejection and Papp values for LY between

untreated Calu-3 monolayers (HBSS control) and monolayers treated with db cAMP

(2 mM), terbutaline (100 µM), amiloride (100 µM), glibenclamide (10 µM), or ouabain

(100 µM).

Table 3.1: Lucifer yellow rejection of Calu-3 cell monolayers and apparent permeability coefficients for LY in the presence of activators or inhibitors of transepithelial ion currents.

Yellow background: activators of transepithelial ion currents; green background: inhibitors of transepithelial ion currents.

ap, apical; bl, basolateral; HBBS, Hank’s balanced salt solution = negative control; LY, Lucifer yellow; Papp, apparent permeability coefficient.

Substance Concentration ap/bl LY rejection ± SD (%) Papp ± SD (nm/s) N

HBSS control --- ap 98.52 ± 0.73 8.23 ± 3.47 7

db cAMP 2 mM bl 99.32 ± 0.07 5.12 ± 1.03 4

Terbutaline 100 µM bl 99.21 ± 0.23 6.69 ± 2.36 4

Amiloride 100 µM ap 98.27 ± 0.79 12.81 ± 5.87 4

Glibenclamide 10 µM ap 99.43 ± 0.18 4.12 ± 1.50 4

Ouabain 100 µM bl 98.30 ± 0.34 12.72 ± 1.7 4

For validation of the dome assay, the influence of ion transport activators and

inhibitors on dome density within confluent Calu-3 monolayers was examined. Figure

3.8 A shows the effect of sodium transport inhibitors on dome densities. Exposure of

dome forming monolayers to the Na+ specific cation channel inhibitor amiloride for four

hours diminished dome density significantly. Addition of the solvent HBSS to monolayers

served as negative control (HBSS control). Domes disappeared completely four hours

RESULTS

- 43 -

after addition of the Na,K-ATPase inhibitor ouabain, while the CFTR-inhibitor

glibenclamide had no effect on dome densities compared to control monolayers (data not

shown). Only about 10% of the initial dome density (i.e., dome density before buffer

exchange) were present within monolayers that were incubated with a Na+-free buffer

(NMDG-buffer) for four hours. Moreover, no new domes occurred within these

monolayers. In control monolayers that had been incubated with NaCl-buffer, domes

diminished after buffer exchange, but only to about 40% of initial dome density. In contrast

to monolayers incubated with NMDG-buffer, dome formation recovered within these

monolayers after four hours to 80% of the initial dome density (Figure 3.8 B).

The effects of terbutaline and db cAMP were also investigated in dome assay

experiments. Exposure of Calu-3 cells to terbutaline (100 µM) for up to six hours did not

increase dome density. However, treatment of cells with terbutaline in combination with

the non-selective phosphodiesterase inhibitor IBMX (40 µM) increased dome formation of

Calu-3 cells by a factor of 2.5, whereas IBMX treatment alone resulted in a 1.7-fold

enhanced dome formation. Figure 3.8 C illustrates these findings one hour after

compound addition. Comparable to these results, addition of membrane-permeable

db cAMP (2 mM) to cell monolayers for four hours resulted in a 4- to 5-fold increase of the

number of domes compared to monolayers treated with the solvent only.

RESULTS

- 44 -

Figure 3.8: Validation of dome assay with activators and inhibitors of transepithelial ion currents.

(A) Effect of Na+ transport inhibitors amiloride and ouabain on dome density in Calu-3 cell monolayers. Dome density before compound addition (time = 0) was set 100%. Data are means ± SEM (n = 6). Significant difference with vehicle control for: * P < 0.05, ** P < 0.01, *** P < 0.001. (B) Influence of Na+-free medium on dome formation. Growth medium was replaced by NaCl- (squares) or NMDG-buffer (triangles), respectively. Dome density before buffer exchange (time = 0) was set 100%. Data are means ± SEM (n = 6). *** indicates significant difference in dome formation of cells incubated with NMDG-buffer compared with NaCl-buffer (P < 0.001). (C) Effect of terbutaline, IBMX and terbutaline + IBMX on dome density in Calu-3 cell monolayers. Dome density before compound addition (time = 0) was set 100%. Data are means + SEM (n = 6) at time = 1 h after compound addition. *** indicates significant difference (P < 0.001); ns, not significant. (D) Effect of db cAMP on dome density in Calu-3 cell monolayers. Dome density before compound addition (time = 0) was set 100%. Data are means ± SEM (n = 6). *** indicates significant difference (P < 0.001).

RESULTS

- 45 -

3.3 In Vitro Studies on the Mechanism of Action of the Tip

Peptide

The TNF-α derived Tip peptide has been shown to stimulate LLC in isolated perfused rat

lungs as well as in in situ and in vivo rodent models and was suggested as a new

therapeutic agent for the resolution of pulmonary edema 73,90-92. The above described

TEER and dome assays were used to characterize the mechanism by which the Tip

peptide stimulates transepithelial ion and fluid transports that are responsible for its anti-

pulmonary edema activity.

3.3.1 Influence of the Tip Peptide on Dome Formation and TEER in Calu-3

Cell Monolayers

Addition of the Tip peptide to dome-forming Calu-3 cell monolayers resulted in a 2-fold

increase of the number of domes within the cell monolayers, demonstrating the influence

of the Tip peptide on active fluid transport by lung epithelial cells in vitro (Figure 3.9 A).

The Tip peptide (1 mg/ml) significantly increased the dome density in Calu-3 cell

monolayers during a four hours incubation period compared with control monolayers that

were treated with the solvent only (Figure 3.9 B). Addition of the Tip peptide in a 2-fold

higher dose (2 mg/ml) amplified this stimulating activity on dome formation by Calu-3 cells

(Figure 3.9 C). In contrast, the mutant form of the Tip peptide (mTip), in which the three

functionally critical amino acids are replaced by alanines, and scrambled Tip (scTip),

which contains the same amino acids as the Tip peptide but in a random order, did not

increase dome density within Calu-3 cell monolayers (Figure 3.9 D).

RESULTS

- 46 -

Figure 3.9: Effect of Tip peptide on dome formation by Calu-3 cells.

(A) Photomicrographs of a section of a dome-forming Calu-3 monolayer before, and four hours after addition of the Tip peptide (1 mg/ml). White arrowheads indicate domes. Original magnification x40. (B) Quantification of the Tip-induced increase of dome density during a four hours period in Calu-3 cell monolayers. Dome density before addition of the Tip peptide (1 mg/ml) or vehicle (time = 0) was set 100%. Data are means ± SEM (n = 6). Significant difference with vehicle control for: ** P < 0.01, *** P < 0.001. (C) Dose dependency of the dome-increasing activity of the Tip peptide. Dome density before addition of the Tip peptide (1 and 2 mg/ml) or vehicle (time = 0) was set 100%. Data are means + SEM (n = 6) at time = 1 h after compound addition. Significant difference with vehicle control for: ** P < 0.01, *** P < 0.001. (D) Effects of Tip, mutant Tip (mTip) and scrambled Tip (scTip) peptides on dome formation by Calu-3 cells. Dome density before addition of peptides (1 mg/ml) or vehicle (time = 0) was set 100%. Data are means ± SEM (n = 6). *** indicates significant difference (P < 0.001) with vehicle control; ns, not significant.

RESULTS

- 47 -

In order to elucidate the underlying mechanism of the Tip peptide’s capability to

induce dome formation, the effect of the Tip peptide on bioelectric properties of polarized

Calu-3 cell monolayers was investigated. Figure 3.10 A depicts a typical recording of

TEER changes in response to successive application of the Tip peptide (1 mg/ml) to the

basolateral and apical sides of a Calu-3 cell monolayer. Only addition of the Tip peptide to

the apical side resulted in a fast increase (within seconds) of TEER up to 140% of initial

values followed by a steady decrease for about 13 minutes until constant TEER values of

about 100 to 110% of initial values were reached (Figures 3.10 A and B).

Figure 3.10: Effect of the Tip peptide on TEER of Calu-3 cell monolayers.

(A) Typical changes of TEER after the sequential addition of the Tip peptide (1 mg/ml) to the basolateral and apical side of Calu-3 cell monolayers grown as air-liquid interface on permeable supports. (B) Quantitative analysis of the effect of the Tip peptide in TEER assay after the addition to the basolateral or apical side of Calu-3 cell monolayers. Peak TEER values were calculated as percentage of TEER values before addition of the Tip peptide (1 mg/ml) or vehicle into the indicated compartment. Data are means + SD (n = 6). (C) Dose dependent change of TEER by the Tip peptide. The Tip peptide (0.1 to 10 mg/ml) was added into the apical compartment of an ENDOHM-6 chamber containing Calu-3 cells. The initial increase of TEER after the addition of the Tip peptide was calculated as percentage of TEER values before addition of the Tip peptide and blotted against the concentration of Tip. Data points are means ± SD (n = 3). (D) Comparison of effects of Tip, mutant Tip (mTip) and scrambled Tip (scTip) peptides on TEER of Calu-3 cells. The initial increase of TEER after the addition of the peptides was calculated as percentage of initial TEER values and normalized to the activity of the Tip peptide (= 100% activity). Data points are means + SD (n = 4 - 6).

RESULTS

- 48 -

Lucifer yellow (LY) rejection experiments demonstrated that changes of TEER

after application of the Tip peptide were not due to changes of paracellular permeability of

Calu-3 cell monolayers, as in the presence of the Tip peptide (1 mg/ml) transepithelial

passage of LY was rejected to 99.37 ± 0.18% and the apparent permeability coefficient for

LY was calculated to be 3.80 ± 0.84 nm/s (n = 4). These values did not differ significantly

from those of monolayers treated with the solvent only (98.52 ± 0.73% LY rejection;

Papp = 8.23 ± 3.47 (n = 7)). The activity of the Tip peptide on transepithelial conductance

was dose dependent with a medium effective concentration (EC50) of 1.66 mg/ml (Figure

3.10 C). Application of the mTip and scTip peptides to the apical side of Calu-3 cell

monolayers resulted in an increase of TEER followed by a steady decrease. Addition of

mTip and scTip peptides to the basolateral side of cell monolayers elicited no changes in

TEER. Compared with the Tip peptide mTip and scTip peptides had 54% and 46% of Tip

peptide’s activity in the TEER assay, respectively (Figure 3.10 D).

3.3.2 Influence of TNF-α on TEER and Dome Formation in Calu-3 Cell

Monolayers

As the Tip peptide is derived from the lectin-like tip domain of TNF-α 83, the impact of

recombinant human TNF-α (rhTNF-α) on dome formation and TEER of Calu-3 cell

monolayers was investigated. The employed rhTNF-α was synthesized in the department

of Biotechnology at ALTANA Pharma using the methylotrophic yeast Pichia pastoris. Initial

studies were performed to characterize the structure and biologic activity of rhTNF-α.

Figure 3.11 A shows polyacrylamide gel electrophoresis (PAGE) analyses of rhTNF-α

under denaturing and native conditions. Purified rhTNF-α migrated in denaturing SDS-

PAGE as a double band with an apparent molecular weight (MW) of ~ 17 kDa, the

expected size for monomeric TNF-α. To evaluate its oligomerization status, rhTNF-α

(isoelectric point (pI) = 7.0) was subjected to native PAGE and its migration was

compared to that of myoglobin, a protein with the same MW and a similar pI

(MW = 17.6 kDa; pI = 7.3 (main component) and 6.8 (minor component)). Under native

conditions rhTNF-α migrated as a triple band slower than the two components of

myoglobin. This finding is indicative for oligomerization of rhTNF-α subunits. Additionally,

cytotoxic activity against the murine fibroblast cell line WEHI-13VAR was measured to

assess the biologic activity of rhTNF-α. As shown in Figure 3.11 B, rhTNF-α exhibited

cytotoxicity against WEHI-13VAR cells with a medium effective dose (EC50) of

62.4 pg/ml.

RESULTS

- 49 -

Figure 3.11: Characterization of recombinant human TNF-α (rhTNF-α).

(A) PAGE analysis of purified rhTNF-α under denaturing and native conditions visualized by Silver staining. (B) Cytotoxic activity of rhTNF-α against the murine fibroblast cell line WEHI-13VAR in the presence of 0.5 µg/ml actinomycin D. After incubation for 20 h with increasing doses of rhTNF-α, viable cells were measured using the Alamar blue assay.

TEER assay experiments were performed to investigate the effect of rhTNF-α on

bioelectric properties of polarized Calu-3 cell monolayers. Figure 3.12 A depicts a typical

recording of TEER changes in response to successive application of rhTNF-α (100 nM) to

the basolateral and apical sides of a Calu-3 cell monolayer. Only addition of rhTNF-α to

the apical side resulted in a decrease of TEER to ~ 94% of initial values within 1.5 minutes

followed by a steady increase for about six minutes until constant TEER values of ~ 98%

of initial values were reached. This activity of rhTNF-α was dose dependent with a

medium effective concentration (EC50) of 71.29 nM (Figure 3.12 B). The influence of

rhTNF-α on paracellular permeability of Calu-3 cell monolayers was assessed by Lucifer

yellow (LY) rejection. In the presence of rhTNF-α (100 nM) transepithelial passage of LY

was rejected to 98.45 ± 0.31% and the apparent permeability coefficient (Papp) for LY was

calculated to be 8.91 ± 0.59 nm/s (n = 4). These values did not differ from those of

monolayers treated with the solvent only (98.52 ± 0.73% LY rejection; Papp = 8.23 ± 3.47

(n = 7)).

RESULTS

- 50 -

Figure 3.12: Effect of recombinant human TNF-α (rhTNF-α) on TEER of Calu-3 cell monolayers.

(A) Typical changes of TEER after the sequential addition of rhTNF-α (100 nM) to the basolateral and apical side of Calu-3 cell monolayers grown as air-liquid interface on permeable supports. (B) Dose dependent change of TEER by rhTNF-α. Recombinant human TNF-α (10 to 1200 nM) was added into the apical compartment of an ENDOHM-6 chamber containing Calu-3 cells. Decrease of TEER was calculated as percentage of TEER values before addition of rhTNF-α and blotted against the concentration of rhTNF-α. Data points are means ± SEM (n = 5 - 6).

Dome assay experiments were performed to investigate the influence of rhTNF-α

on active fluid transport by lung epithelial cells in vitro. Addition of rhTNF-α to dome-

forming Calu-3 cell monolayers did not influence dome density within cell monolayers

(data not shown).

3.3.3 Influence of pH on the Activity of rhTNF-α and the Tip Peptide in the

TEER Assay

It is reported that TNF-α and the Tip peptide evoke an enhanced increase of ion channel

activity in murine lung microvascular endothelial cells and in peritoneal macrophages at

acidic pH compared to pH 7.3 88. Therefore, the influence of acidic conditions on the

activity of rhTNF-α and the Tip peptide was investigated in the TEER assay. Figure 3.13

shows that the effects of both, rhTNF-α and the Tip peptide, on the conductance of Calu-3

cell monolayers were significantly enhanced under acidic conditions compared to neutral

pH. At neutral pH (pH 7.4), apical addition of rhTNF-α (100 nM) resulted in a decrease of

TEER by 50‰. Under acidic conditions (pH 6.0), the TEER-decreasing activity of rhTNF-α

reached values of up to 112‰ (Figure 3.13 A). Similar results were obtained for the

application of the Tip peptide after preincubation of Calu-3 monolayers at pH 6.0. Under

acidic conditions, TEER values reached 178% of initial TEER values after apical addition

RESULTS

- 51 -

of the Tip peptide (1 mg/ml). At pH 7.4, maximum TEER values of 146% were measured

after treatment of Calu-3 monolayers with the Tip peptide (Figure 3.13 B).

Figure 3.13: Influence of pH on the activity of rhTNF-α and the Tip peptide in the TEER assay.

Cell monolayers were preincubated for 30 min with HBSS at pH 7.4 or 6.0 before measurements were started. Tip peptide and rhTNF-α were dissolved in the corresponding buffers. (A) Effect of rhTNF-α on TEER of Calu-3 cell monolayers under neutral and acidic conditions. Minimum TEER values were calculated as percentage of TEER values before apical addition of rhTNF-α (100 nM; n = 7) or vehicle (n = 5). Data are means + SD. *** indicates significant difference (P < 0.001). (B) Effect of the Tip peptide on TEER of Calu-3 cell monolayers under neutral and acidic conditions. Peak TEER values were calculated as percentage of TEER values before apical addition of Tip (1 mg/ml) or vehicle. Data are means + SD (n = 6). *** indicates significant difference (P < 0.001).

3.3.4 Influence of Ions on the Activity of the Tip Peptide

In order to examine which ion currents influence the activity of the Tip peptide, TEER

changes were investigated in response to the addition of the Tip peptide to Calu-3 cell

monolayers in the presence or absence of various ions or impermeable substitutes in the

assay buffer. For instance, Na+ was replaced by equimolar amounts of

N-methyl-D-glucamine (NMDG), Cl- was replaced by gluconate, and K+ was replaced by

Cs+. Control monolayers were incubated with NaCl-buffer. As demonstrated in Figure

3.14 A, only replacement of Na+ ions resulted in a significant inhibition of Tip peptide-

induced changes in conductance compared to control (NaCl-buffer). Replacement of Cl-

and K+ had no effect on TEER changes induced by the peptide. Consequently, the activity

of the Tip peptide on TEER of Calu-3 monolayers was investigated in the presence of

different Na+ concentrations in the assay buffer (0, 17.5, 35, 70 and 140 mM). TEER

changes evoked by the Tip peptide increased with increasing concentrations of Na+

(Figure 3.14 B) in the assay buffer. These results confirmed the activity of the Tip peptide

RESULTS

- 52 -

on Calu-3 monolayer conductance to be Na+ dependent. Furthermore, these findings are

consistent with the results from dome assay experiments, since the process of dome

formation by Calu-3 cells was proven to be dependent on Na+ transport, and the Tip

peptide increased this process (Figures 3.8 and 3.9).

Figure 3.14: Effects of ion substitutions on Tip peptide’s activity in the TEER assay.

(A) Different ions in the assay buffer were replaced by cell impermeable substitutes (Na+ NMDG+, Cl- gluconate, K+ Cs+). The initial increase of TEER after the addition of the Tip peptide (1 mg/ml) was calculated as percentage of TEER values before addition of Tip. Data points are means + SD (n = 6), significant difference with control (NaCl-buffer) for: ** P < 0.01. (B) Activity of the Tip peptide in TEER assay in the presence of different concentrations (0, 17.5, 35, 70 and 140 mM) of Na+ in the assay buffer. The initial increase of TEER after the addition of the Tip peptide (1 mg/ml) was calculated as percentage of TEER values before addition of Tip. Data points are means + SD (n = 3 – 4).

3.3.5 Influence of the Tip Peptide on β-Adrenergic Receptor Activation and

Intracellular cAMP Level

The β2-adrenergic agonist terbutaline and the Tip peptide are activators of LLC. To

confirm the hypothesis that these substances activate LLC by different mechanisms of

action 92, the inhibitory effect of the β–blocker propranolol on the activities of terbutaline

and the Tip peptide were assessed using the TEER assay. Preincubation of Calu-3

monolayers with propranolol for five minutes completely inhibited the TEER-decreasing

effect of terbutaline (Figure 3.15 A). In contrast, there was no difference in the activity of

the Tip peptide in the TEER assay in the presence versus the absence of propranolol

(Figure 3.15 B).

RESULTS

- 53 -

In order to investigate whether the Tip peptide could increase intracellular cAMP

levels in a different way than by β–adrenergic receptor activation, cAMP contents were

quantified after treatment of Calu-3 cells with either terbutaline or the Tip peptide. The

cAMP levels of cells treated with the adenylate cyclase agonist forskolin served as the

positive control, cells treated with the solvent served as the negative control.

Figure 3.15 C demonstrates that a 15-min treatment of Calu-3 cells with forskolin and

terbutaline (both 10 µM) resulted in a ~ 7-fold increase of intracellular cAMP. The Tip

peptide (1 mg/ml) did not influence the intracellular cAMP levels of Calu-3 cells.

Figure 3.15: Effects of the Tip peptide on β-adrenergic receptors and intracellular cAMP level.

(A) Effect of the β-blocker propranolol on terbutaline-induced TEER decrease of Calu-3 monolayers. Monolayers were preincubated with propranolol (10 µM) or vehicle for 5 min before addition of terbutaline (10 µM). Decrease of TEER was calculated as percentage of TEER values before addition of terbutaline or vehicle. Data points are means + SD (n = 5). *** indicates significant difference (P < 0.001). Terb + Prop, terbutaline + propranolol.

(B) Effect of the β-blocker propranolol on Tip peptide-induced TEER increase of Calu-3 monolayers. Monolayers were preincubated with propranolol (10 µM) or vehicle for 5 min before addition of the Tip peptide (500 µg/ml). Increase of TEER was calculated as percentage of TEER values before addition of the Tip peptide or vehicle. Data points are means + SD (n = 5). ns, not significant; Tip + Prop, Tip peptide + propranolol.

(C) Effects of forskolin, terbutaline and the Tip peptide on intracellular cAMP level of Calu-3 cells. Calu-3 cells were treated with forskolin (10 µM), terbutaline (100 µM), Tip peptide (1 mg/ml) or vehicle for 15 min. After incubation intracellular cAMP level was determined using an ELISA Kit (Sigma-Aldrich, Deisenhofen, Germany). Data are means + SD (n = 6). Significant difference with vehicle control for: *** P < 0.001; ns, not significant.

RESULTS

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3.3.6 Influence of Oligosaccharides on the Activity of the Tip Peptide

The Tip peptide is derived from the lectin-like tip domain of TNF-α that specifically binds

such as N,N’-diacetylchitobiose but not cellobiose 83. Therefore, the influence of these

oligosaccharides on the activity of the Tip peptide in TEER and dome assays was

investigated.

Apical addition of the Tip peptide (0.5 mg/ml) which had been preincubated with a

~ 10-fold molar excess of N,N’-diacetylchitobiose (1 mg/ml), led to a change in the TEER

response curve compared to addition of the Tip peptide alone: Initial peaks in TEER

following addition of the Tip peptide were completely inhibited by N,N’-diacetylchitobiose

(Figure 3.16 A). Additionally, plateau levels of TEER 10 minutes after addition of Tip +

chitobiose (114.5% ± 1.8%) were higher than those of monolayers treated with the Tip

peptide alone (108% ± 0.4%). The oligosaccharide cellobiose had the same inhibitory

effect on the Tip action in the TEER assay. However, only N,N’-diacetylchitobiose, but not

cellobiose, inhibited the dome-increasing activity of the Tip peptide in the dome assay

(Figure 3.16 B). The sugars alone had no effect on dome formation (data not shown).

Figure 3.16 Influence of oligosaccharides on the activity of the Tip peptide in TEER and dome assays.

(A) Typical changes of TEER after apical application of the Tip peptide (500 µg/ml; black curve) and Tip preincubated with N,N’-diacetylchitobiose (Chi; 1 mg/ml; red curve) or cellobiose (Cel; 1 mg/ml; red curve) for 30 min. (B) Influence of N,N’-diacetylchitobiose and cellobiose on activity of the Tip peptide in dome assay. Tip peptide (1 mg/ml) was preincubated with cellobiose (1 mg/ml; ‘+ Cellobiose’) or N,N’-diacetylchitobiose (1 mg/ml; ‘+ Chitobiose’) for 30 min. Dome density before addition of substances or vehicle (time = 0) was set 100%. Data are means ± SEM (n = 6) at time = 2 h after compound addition. * (P < 0.05) and ** (P < 0.01) indicate significant differences with monolayers treated with the Tip peptide.

RESULTS

- 55 -

In order to elucidate whether the inhibitory effect of N,N’-diacetylchitobiose on the

activity of the Tip peptide may result from a lectin-like activity of the peptide, sugar binding

studies were performed using the surface plasmon resonance technique. Figure 3.17

illustrates qualitative binding of N,N’-diacetylchitobiose and cellobiose to recombinant

human TNF-α (rhTNF-α) and the Tip peptide. An interaction of N,N’-diacetylchitobiose

with immobilized rhTNF-α could clearly be detected (Figure 3.17 A; ∆ = 9.83 RU). This

binding was oligosaccharide specific since cellobiose did not bind to rhTNF-α (Figure

3.17 B; ∆ = 0.67 RU). Moreover, no interaction between N,N’-diacetylchitobiose and the

Tip peptide could be detected (Figure 3.17 C; ∆ = -0.43 RU).

Figure 3.17: Interaction analyses of rhTNF-α and the Tip peptide with specific oligosaccharides by surface plasmon resonance.

Recombinant human TNF-α and the Tip peptide were immobilized as described in flow cells (Fc) 2 and 3 of a Biacore CM5 sensor chip, respectively. Flow cell 1 served as blank control to detect unspecific binding to the chip matrix. Sensogramms represent response differences F2 - F1 for rhTNF-α and F3 - F1 for the Tip peptide. Response differences correspond to specific interaction between rhTNF-α or the Tip peptide and the oligosaccharides N,N’-diacetylchitobiose or cellobiose, respectively, and are presented in response units (RU). ∆ indicates difference between report points (red crosses) before and after injection of the corresponding analyte.

(A) Interaction analysis of rhTNF-α with N,N’-diacetylchitobiose.

(B) Interaction analysis of rhTNF-α with cellobiose.

(C) Interaction analysis of the Tip peptide with N,N’-diacetylchitobiose.

RESULTS

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3.3.7 Effect of Acetate on TEER and Dome Formation of Calu-3 Cell

Monolayers

The peptides used in this work had been produced by chemical synthesis and were

supplied by BACHEM (Bubendorf, Switzerland) as acetate salts. Consequently, the effect

of acetate on TEER and dome formation of Calu-3 cell monolayers was investigated

(Figure 3.18).

A Tip peptide stock solution (100 mg/ml in HBSS) was dialyzed against HBSS to

remove residual acetate ions. The activity of the dialyzed Tip peptide (1 mg/ml) was

compared with not-dialyzed Tip peptide (1 mg/ml) in the TEER assay. Dialysis of the Tip

peptide solution led to a decline in activity by ~ 16% (Figure 3.18 A). Consequently, the

effect of acetate on conductance of Calu-3 cell monolayers was assessed. Figure 3.18 B

shows a typical recording of TEER changes after the subsequent application of 3 mM

sodium acetate to the basolateral and apical side of a Calu-3 cell monolayer. Only

addition of sodium acetate to the apical side resulted in tri-phase TEER changes: TEER

values declined within seconds to ~ 90% of initial TEER directly followed by an increase to

~ 115% of the initial TEER value. Consecutively, TEER decreased continuously until it

reached a constant value of ~ 105% of initial TEER after five to six minutes. The activity of

sodium acetate was dose dependent with a medium effective concentration (EC50) of

42 mM (Figure 3.18 C). Butyrate, another short chain fatty acid, evoked similar effects on

TEER of Calu-3 monolayers (data not shown). The effect of acetate on transepithelial fluid

transport of Calu-3 cells was also investigated using the dome assay. Fifteen minutes

after the addition of sodium acetate (3 mM) to dome-forming Calu-3 monolayers dome

densities within monolayers were significantly increased compared to solvent-treated

control monolayers (Figure 3.18 D). This activity was rather short acting since 1.5 hours

after compound addition dome densities in acetate-treated monolayers did not differ from

those of control monolayers (data not shown).

RESULTS

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Figure 3.18: Effect of acetate on TEER and dome formation of Calu-3 cell monolayers.

(A) Comparison of effects of the Tip peptide on TEER of Calu-3 cells before (B) and after (A) dialysis. Dialysis of Tip peptide stock solution (100 mg/ml in HBSS) was performed against HBSS over night at 4°C to remove residual acetate ions in the peptide solution. The initial increase of TEER after the addition of the dialyzed Tip peptide (1 mg/ml) was calculated as percentage of initial TEER values and normalized to the activity of the Tip peptide before dialysis (= 100% activity). Data points are means + SD (n = 5). *** indicates significant difference (P < 0.001). (B) Typical changes of TEER after the sequential addition of sodium acetate (3 mM) to the basolateral and apical side of Calu-3 cell monolayers grown as air-liquid interface on permeable supports. (C) Dose dependent change of TEER by sodium acetate. Sodium acetate (2 to 200 mM) was added into the apical compartment of an ENDOHM-6 chamber containing Calu-3 cells. Initial decrease of TEER was calculated as percentage of TEER values before addition of sodium acetate and blotted against the concentration of sodium acetate. Data points are means ± SD (n = 3). (D) Effect of sodium acetate on dome formation by Calu-3 cells. Dome density before addition of sodium acetate (3 mM) or vehicle (time = 0) was set 100%. Data are means ± SEM (n = 6). *** indicates significant difference (P < 0.001) with vehicle control.

RESULTS

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3.3.8 Identification of Potential Interaction Partners of the Tip Peptide

Different methods were employed to discover protein interaction partners of the Tip

peptide that potentially mediate its anti-edema activity. These methods included Far

Western analyses with lung epithelial cell proteins and biotinylated Tip peptide under

denaturing and native conditions, surface plasmon resonance (Biacore) analysis with

immobilized Tip peptide and lung epithelial cell fractions as analytes, a small scale pull-

down assay with biotinylated Tip peptide and Calu-3 cell proteins using strepatavidin

beads, and affinity chromatography analysis with biotinylated Tip peptide to capture

possible binding partners on a streptavidin column. Only using the affinity chromatography

approach, the generally high unspecific binding of proteins was minimized, and several

potentially specific protein binding partners of the Tip peptide were isolated. In brief, whole

cell lysate of 8 x 107 Calu-3 cells was incubated with 250 µg/ml biotinylated Tip peptide

(Figure 3.19 A), 250 µg/ml biotinylated scrambled Tip peptide (Figure 3.19 B), equimolar

amounts of D-biotin (Figure 3.19 C) or vehicle (Figure 3.19 D). Afterwards, lysate was

applied to a streptavidin column. Flowthrough and wash fractions were collected. Elution

of interacting proteins was carried out in three consecutive steps: (1) N,N’-

diacetylchitobiose (500 µM), (2) Tip peptide (5 mg/ml) and (3) pH downshift (50 mM

ammonium acetate, 0.5 M NaCl, pH 4.0). Additionally, streptavidin beads were boiled in

SDS-sample buffer to obtain interacting proteins that were not dissociated with the

eluents. Collected fractions of affinity chromatography runs were subjected to 4-20% Tris-

Glycine polyacrylamide gradient gels. Only elution with pH 4.0 of chromatography runs

with biotinylated Tip and scrambled Tip peptides yielded eight and three interacting

human proteins, respectively. Protein bands in elution fractions were cut out and identified

by mass spectroscopy (MALDI-TOF and MS/MS analyses) followed by Mascot search

against MSDB database. Identified proteins and their functions according to Swiss-

Prot/TrEMBL databases are presented in Table 3.2. Chromatography runs with D-biotin

and vehicle did not yield interacting proteins indicating absence of unspecific binding to

biotin and the streptavidin column matrix, respectively.

RESULTS

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Figure 3.19: Protein interaction analysis of biotinylated Tip peptide with Calu-3 cell proteins using streptavidin column affinity chromatography.

Silver-stained 4-20% Tris-Glycine polyacrylamide gels with fractions of affinity chromatography analyses to discover protein interaction partners of the Tip peptide. Elution of interaction partners was carried out with (1) N,N’-diacetylchitobiose (500 µM), (2) Tip peptide (5 mg/ml), (3) pH downshift with 50 mM sodium acetate, 0.5 M NaCl, pH 4.0 and (4) by boiling column material in SDS-PAGE sample buffer.

Framed and numbered protein bands were cut out and further analyzed by mass spectroscopy (MALDI-TOF and MS/MS analyses followed by Mascot search against the MSDB database).

(A) Fractions from streptavidin affinity chromatography run with biotinylated Tip peptide.

(B) Fractions from streptavidin affinity chromatography run with biotinylated scrambled Tip peptide.

(C) Fractions from streptavidin affinity chromatography run with D-biotin.

(D) Fractions from streptavidin affinity chromatography run with vehicle.

kDa, kilodalton; M, SeeBlue Pre-Stained Standard (Invitogen); FT, flowthrough; SB, streptavidin beads; ?, MS signal intensity too low for significant protein identification.

RESULTS

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Table 3.2 Results from MALDI-TOF and MS/MS analyses followed by Mascot database search to identify eluted proteins from streptavidin affinity chromatography runs.

Identification probability (Mascot search): P < 0.05; MS, mass spectroscopy; MALDI-TOF, matrix-assisted laser desorption/ionization time-of–flight.

Band No.

Identified protein Primary

accession No.

MW (kDa)

Subcellular location

Function

1 Protein-tyrosin kinase erbB2 precursor

P04626 141 Cell membrane

Tyrosine kinase-type cell surface receptor

2 UV-damaged DNA binding factor

Q16531 127 Nucleus DNA repair

3 Nucleolin (protein C23)

P19338 76 Nucleus, nucleolus

Chromatin decondensation

4 T-complex protein 1, delta / theta subunit

P50991 / P50990

58 / 60 Cytoplasm Molecular chaperone

5 Elongation factor 1-alpha 1

P68104 50 Cytoplasm Promotion of protein biosynthesis

Diphosphomevalonate decarboxylase,

P53602 44 ? Isoprene biosynthesis 6

Aktin, cytoplasmic 1

P60709 42 Cytoplasm Cell motility

Ribose-phosphate pyrophosphokinase I / II,

P60891 /

P11908

35

? Metabolic intermediate biosynthesis

7

Annexin A2

P07355 39 Basement membrane

Calcium-regulated membrane-binding protein

Annexin A4, P09525 36 ? Calcium/phospholipid-binding protein, membrane fusion

8

Annexin A5

P08758 36 ? Calcium/phospholipid-binding protein, anticoagulant protein

9 Protein disulfide-isomerase A4 precursor

P13667 73 Endoplasmic reticulum lumen

Rearrangement of disulfide bonds in proteins

10 Streptavidin, fragment

(Streptomyces avidinii)

P22629 19

(full length)

Secreted protein

Biotin binding

RESULTS

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3.4 Screening of Alternative Anti-Pulmonary Edema Peptide Drug

Candidates

The described studies on the mechanism of action of the Tip peptide proofed the TEER

and dome assays to be suitable for in vitro activity testing of substances that regulate

transepithelial ion and fluid transports across an epithelial cell monolayer. Consequently,

the TEER and dome assays with the human lung epithelial cell line Calu-3 were used as

in vitro screening tools for the identification of new effective anti-pulmonary edema peptide

drug candidates.

3.4.1 Bioinformatic Analysis of the Tip Peptide

A comprehensive bioinformatic analysis of the Tip peptide was performed in the

department of chemo- and bioinformatics at ALTANA Pharma AG, Konstanz, Germany, in

order to suggest alternative anti-pulmonary peptide drug candidates. Searches for

alternative peptides were carried out using SPASM substructure, Sybyl/Biopolymer loop,

Relibase, Blastp for short nearly exact matches, and JPRED secondary structure

prediction databases. Accordingly, 40 alternative peptides were investigated for their in

vitro activities on transepithelial ion and fluid transports using the TEER and dome assays.

3.4.2 In Vitro Activity Screening of Potential Anti-Edema Peptides

The activities of peptides on conductivity of lung epithelial cell layers was assessed by the

TEER assay using at least three individual Calu-3 cell monolayers and compared to the

activity of the Tip peptide (= positive control/gold standard). Peptides were added to both,

the apical and basolateral sides of cell monolayers in a concentration of 250 µM. Peptides

with activities comparable or higher than that of the Tip peptide were further investigated

for their abilities to induce transepithelial fluid transport using the dome assay. The

screening results are presented in Tables 3.3 to 3.5.

RESULTS

- 62 -

Table 3.3: TEER assay analysis of peptides with the same amino acid composition as the Tip peptide (white background), peptides derived from Tip-related domains of coelomic cytolytic factor-1 and lymphotoxin-α (light grey background), and primary sequence-based homologues of the Tip peptide (dark grey background).

The initial increase of TEER after the addition of the peptides was calculated as percentage of initial TEER values and normalized to the activity of the Tip peptide (= 100% activity; orange background). Blue letters indicate amino acids identical to those in Tip peptide; underline points to central TPEGAE-motif critical for Tip peptide’s anti-edema activity.

Activity in TEER assay

(c = 250 µM)

[% of Tip ; Tip = 100%] Peptide ID

linear

or

cyclic

Amino acid sequence

Mean SD N

Tip

= positive control /

Gold standard

cyclic CGQRETPEGAEAKPWYC 100,0 13,1 39

1 cyclic CYWPKAEAGEPTERQGC 15,7 12,1 3

2 cyclic CGTKPIELGPDEPKAVC 130,1 4,6 8

3 cyclic CANPWWVDFWEWGKPWGC 0,0 0,0 3

4 cyclic CNWKWTWDDEGDNNAMGC 0,0 0,0 3

5 cyclic CQKMVYPGLQEPWLHSC 0,0 0,0 3

6 cyclic CSGKAYSPKAPSSPLYC 0,0 0,0 3

7 cyclic CGVVTTPEGAEGMYLRC 2,0 13,9 3

8 cyclic CGEWETPEGCEQVLTGC 20,6 25,1 3

9 cyclic CGSKETPEPEEVDPWDC 47,1 16,2 3

10 cyclic CGRGVTSEGAEARWYSC 10,5 17,1 3

11 cyclic CGEREYPEGSEAYNFYC 34,3 11,5 3

12 cyclic CGHTESPEQAEAKPWYC 19,9 4,3 3

13 cyclic CGQKTVKGAREASPWYC 17,6 8,4 3

14 cyclic CGRKAAEDDGEPSAWYC 8,4 5,0 3

15 cyclic CGQRETPRGAKTNPWYC 13,0 8,7 3

16 cyclic CGQRETPQGAKTKPWYC 18,6 9,8 3

In summary, a peptide with the same amino acids as the Tip peptide but in

reversed order (peptide 1, reversed Tip) did not elicit TEER changes of Calu-3 cell

monolayers to the same extent as Tip. In contrast, a peptide with the same amino acids

as the Tip peptide but in a random order (peptide 2, scrambled-2 Tip) showed a higher

activity compared to the Tip peptide (130.1% compared to Tip). This superior activity was

confirmed in the dome assay, were the scrambled-2 Tip peptide enhanced dome densities

within Calu-3 cell monolayers 2.7-fold more than the Tip peptide after a two-hour

incubation (data not shown).

A set of peptides derived from tip domain homologue structures of coelomic

cytolytic factor-1 (CCF-1; peptides 3 and 4), a lectin-like defense molecule of the

RESULTS

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earthworm Eisenia foetida, and of lymphotoxin-α (LT; peptides 5 and 6), a member of the

tumor necrosis factor ligand superfamily, did not influence TEER of Calu-3 cell

monolayers. Furthermore, ten peptides (peptides 7 to 16) with primary sequence-based

homologies to the Tip peptide were compared to Tip’s activity in the TEER assay. None of

these peptides elicited TEER changes of Calu-3 cell monolayers in the same manner as

the Tip peptide.

Amino acid sequences and TEER assay activities of the above-described peptides

are presented in Table 3.3.

RESULTS

- 64 -

Table 3.4: TEER assay analysis of variants of the Tip peptide (white background) and mutants of the Tip peptide (light grey background).


Peptide ID linear or cyclic Amino acid sequence


(c = 250 µM)

[% of Tip ; Tip = 100%]

Mean SD N

Tip


Gold standard


17 cyclic CQRETPEGAEAKPWYC 96,7 12,0 3

18 cyclic CQRETPEGAEAKC 92,3 6,3 3

19 cyclic

(by A E peptide bond)

AKPAYGGQRETPEGAE 22,9 4,9 3

20 cyclic CGQRESPEGAEAKPWYC 120,9 13,5 3

21 cyclic CGQRESPDGAEAKPWYC 88,8 17,4 3

22 cyclic CGQRESPEGADAKPWYC 119,4 7,2 3

23 cyclic CGQRESPDGADAKPWYC 127,7 17,5 3

24 cyclic CGQRETPDGAEAKPWYC 130,4 7,7 3

25 cyclic CGQRETPEGADAKPWYC 139,8 16,0 3

26 cyclic CGQRETPDGADAKPWYC 172,4 12,6 3

27 cyclic CGQRETPEGGEAKPWYC 108,7 14,9 3

28 cyclic CGQRESPDGGDAKPWYC 123,6 11,6 5

Table 3.4 summarizes the results from TEER assay experiments with variants and

mutant forms of the Tip peptide. Peptides 17 and 18 represent short variants of the Tip

peptide with one or four amino acids omitted, respectively. The activities of these peptides

in the TEER assay were comparable to that of the Tip peptide (96.7 and 92.3% of Tip’s

activity, respectively). Remodeling and circularization of the Tip peptide by a peptide bond

between terminal amino acids alanine and glutamic acid instead of circularization by a

disulfide bond between terminal cysteine residues diminished the activity to 22.9%

(peptide 19).

In a set of nine mutant Tip peptides (peptides 20 to 28) amino acids threonine

(Thr6), glutamic acid (Glu8 and/or Glu11) and alanine (Ala10) of Tip’s central TPEGAE-motif

were replaced by amino acids with similar physical features, namely serine, aspartic acid

and glycine, respectively. Exchange of these amino acids in different variations did not

RESULTS

- 65 -

alter the activity of the Tip peptide. The activating effect on transepithelial ion and fluid

transport of a peptide with four amino acid exchanges (peptide 28) was confirmed in the

dome assay where it significantly enhanced dome densities within Calu-3 monolayers

after a one-hour incubation (data not shown).

RESULTS

- 66 -

Table 3.5: Analysis of linear and cyclic peptides with oligomeric amino acid motifs.


Peptide ID linear or cyclic Amino acid sequence


(c = 250 µM)

[% of Tip ; Tip = 100%]

Mean SD N

Tip


Gold standard


29

(triple Tip) linear

GGSPSQRETPEGAEAKPWYGGSPSQRETPEGAEAKPWYGGSPSQRETPEGAEAKPWY

539,0 20,2 6

30 linear TPEGAE 35,5 4,5 3

31 linear TPEGAETPEGAETPEGAE 57,9 9,5 3

32 linear TPEGAEGGGGGTPEGAEGGGGGTPEGAE

80,0 7,0 3

33 linear TPEGAETPEGAETPEGAETPEGAETPEGAE

93,0 2,3 3

34 cyclic CGTPEGAETPEGAETPEGAEGC 139,9 7,3 5

35 cyclic CGGGGGTPEGAEGGGGGTPEGAEGGGGGTPEGAEGC

156,5 10,6 5

36 cyclic CGTPEGAETPEGAETPEGAETPEGAETPEGAEGC

146,6 11,8 5

37 linear SPEGGEGGGGGSPEGGEGGGGGSPEGGE

87,1 4,8 3

38 cyclic CGSPEGGEGGGGGSPEGGEGGGGGSPEGGEGC

131,7 5,4 5

39 linear SPEGGDGGGGGSPEGGDGGGGGSPEGGD

90,7 14,1 3

40 cyclic CGSPEGGDGGGGGSPEGGDGGGGGSPEGGDGC

146,0 13,7 5

Since the tip domain of TNF-α consists of three identical protein loops, three

successive tip peptide sequences have been combined in a linear artificial monomer.

Spacers of five amino acids have been inserted between the individual tip sequences to

mimic the distances between the tip loops within TNF-α (peptide 29). An equimolar

amount (250 µM) of this triple Tip peptide evoked a 5.4-times higher activity in the TEER

assay compared to the monomeric Tip peptide (Table 3.5). Further investigations of the

triple Tip peptide using the TEER assay revealed a well-fitting dose response relationship

(R² = 0.9563) with a medium effective dose (EC50) of 73 µM. Complementing dome

RESULTS

- 67 -

assay analysis revealed a 2.3-fold higher dome-increasing activity of the triple Tip peptide

than that of the Tip peptide (data not shown).

The effect of oligomerization of amino acid sequences on the in vitro activities of

peptides was further examined with a set of seven peptides (peptides 30 to 36). A linear

hexapeptide (peptide 30) comprising the central TPEGAE-motif critical for the Tip

peptide’s anti-edema activity elicited 35.5% of Tip’s activity in the TEER assay. Peptide

31, which consists of three TPEGAE-sequences, exhibited an elevated activity (57.9%).

Insertion of flexible polyglycine linkers between TPEGAE-motifs resulted in an activity of

80% (peptide 32). Replacement of polyglycine linkers by TPEGAE-sequences

(peptide 33) did not result in a significant increase of activity (80 ± 7% and 93 ± 2%,

respectively). Peptides 34 to 36 represent cyclic forms of peptides 31 to 33. Circularization

by disulfide bonds of terminal cysteine residues further increased the activities of the

corresponding peptides in the TEER assay (57.9% to 139.9% (peptides 31 and 34,

respectively), 80.0% to 156.5% (peptides 32 and 35, respectively), and 93.0% to 146.6%

of Tip’s activity (peptides 33 and 36, respectively)).

According to these results and the results from “conservative” amino acid

exchanges (Table 3.4), peptides 37 to 40 were examined for their activities in the TEER

assay. Analogous to peptides 32 and 35, these peptides contain three TPEGAE-motifs

with conservative mutations that are separated by flexible poly-glycine linkers. Both, linear

and cyclic variants were investigated. According to the above-mentioned findings,

replacement of amino acids within the TPEGAE-motif by amino acids with similar physical

properties did not alter the activity. Furthermore, circular forms of the peptides had a

higher activity than the linear forms, being consistent with the activity patterns of peptides

31 to 36. Activities of peptides 37 to 40 on transepithelial ion and fluid transport were

confirmed in dome assay experiments (data not shown).

Amino acid sequences and TEER assay activities of the above-described peptides

are presented in Table 3.5.

DISCUSSION

- 68 -

4 Discussion

Pulmonary edema is a life-threatening pathological condition caused by excessive

extravascular fluid accumulation in the lung tissue 7,12,18,108-110. The pulmonary epithelium

that actively transports ions, which drives osmotic water transport from the airspaces to

the lung interstitium, is the site of action for the removal of pulmonary edema from the

airspaces of the lung 3,16,17,21,111,112. Excess fluid in the pulmonary interstial spaces can

subsequently be cleared by lung lymphatics and the lung microcirculatio 110.

Consequently, substances that directly act on lung epithelial cells and stimulate active

fluid clearance are attractive pharmaceutical drug candidates against pulmonary edema.

To identify and develop new drugs that stimulate LLC at the site of action, a lung epithelial

cell-based assay is useful for in vitro screening of such drug candidates.

In the present study, examinations on the mechanism of action of the TNF-α

derived Tip peptide were performed using two human lung epithelial cell-based in vitro

systems: the transepithelial electrical resistance (TEER) assay and the dome assay. The

anti-pulmonary edema capacity of the Tip peptide had previously been demonstrated ex

vivo, in situ and in vivo in 11 lung edema animal models (ALTANA Pharma AG, Konstanz,

Germany; confidential data; 90-92). Furthermore, Hribar and co-workers had shown that

treatment of murine microvascular endothelial cells and peritoneal macrophages with the

Tip peptide increased membrane conductance in whole cell voltage-clamped patch clamp

studies, indicating the influence of the Tip peptide on amiloride-sensitive ion channels

and/or transporters 88. In addition, the activity of the Tip peptide had been inhibitable by

the lectin-binding sugar N,N’-diacetylchitobiose, proposing that the Tip peptide features a

lecting-like property that is involved of in its edema clearance activity. Prior to this thesis

no in vitro system based on pathologically relevant human lung epithelial cells was

available to explore the underlying mechanism of action of the Tip peptide, and to screen

for alternative anti-pulmonary edema peptide drug candidates. Consequently, an aim of

this thesis was to establish an in vitro activity assay that reflects the Tip peptide’s anti-

pulmonary edema action.

DISCUSSION

- 69 -

4.1 Lung Epithelial Cells for In Vitro Study on Lung Liquid

Clearance

Three different human lung epithelial cell lines were examined in regard to their

expression of ion channels and transporters involved in the process of lung liquid

clearance (LLC). Since active fluid reabsorption can occur in all segments of the

pulmonary epithelium 17, cell lines derived from different parts of the human lung were

chosen: (i) the A549 cell line, which possesses characteristic functions of alveolar type II

(ATII) cells, (ii) the H441 cell line, which serves as a cell culture model of bronchiolar

Clara cells, and (iii) the Calu-3 cell line that mimics features of serous ciliated cells from

the bronchi 99-101.

The epithelial sodium channel ENaC, the chloride specific ion channel CFTR, the

active ion transporter Na,K-ATPase, and the water channel aquaporin-5 play key roles in

the process of pulmonary edema resorption 17,48-50,60,102-104,113. Results from different rodent

models led to the current hypothesis that ENaC and CFTR may participate in the

mechanism of the Tip peptide’s anti-edema action 90-92.

In order to examine whether A549, H441, and Calu-3 cells are suitable for

investigations of active transepithelial ion and fluid transports, expression of ENaC

subunits α, β and γ, CFTR, Na,K-ATPase-α1 and –β1, and aquaporin-5 were assessed by

mRNA quantification and Western blot analysis.

It was shown that A549 cells contained mRNAs of the α-, and β-ENaC subunits.

However, the β-subunit mRNA was only present at a very low level. The finding that the γ-

ENaC transcript could not be detected by real-time RT-PCR, but was occasionally

observed in qualitative RT-PCR experiments (data not shown), corresponds with

investigations of Lazrak et al. (who used qualitative RT-PCR for mRNA detection) 114.

Since they also found γ-ENaC mRNA to be irregularly present, it appears that the

transcription of γ-ENaC mRNA occurs only temporarily during cultivation of A549 cells.

H441 cells contained transcripts of all three ENaC subunits. The expression pattern of

ENaC subunit mRNAs in H441 cells was found to be similar to that of A549 cells

(α > β > γ), but quantities of transcripts were notably higher than in A549 cells. This is

consistent with previous reports by Itani et al. and Ramminger et al. 115,116. Transcripts of

all three ENaC subunits were detected in Calu-3 cells in the order α > γ ≥ β. Western blot

analysis revealed that all three investigated cell lines expressed ENaC subunits in

glycosylated forms, except for ENaC-β that was exclusively present in H441 cells in an

unglycosylated form. In this context it is noteworthy that only channels composed of all

three ENaC subunits are fully functional with regard to Na+ selectivity and conductance.

DISCUSSION

- 70 -

Channels formed by the α-subunit alone or in combination with one of the additional

subunits β and γ also exist, however, they produce smaller and less selective Na+

currents 22,28. Nevertheless, the α-ENaC subunit is essential for channel formation, and

studies of α-ENaC-deficient mice demonstrated its pivotal role in the process of LLC 22,117.

Thus, fully functional forms of ENaC may be present in A549, H441 and Calu-3 cells,

since these cells contained transcripts and/or proteins of all three ENaC subunits.

Although the chloride channel CFTR is not required for basal fluid clearance in the

lung, there is evidence arising that it plays a major role in stimulated LLC 3,48-50,60,118.

Furthermore, experiments with a rat in situ model had shown that the anti-edema activity

of the Tip peptide can be blocked by the CFTR inhibitor glibenclamide 92. Therefore,

expression of CFTR by the investigated cell lines was deemed critical for the realization of

in vitro studies with the Tip peptide. Transcripts of CFTR were detected in all three cell

lines. Kulaksiz et al. 119 showed that the Clara cell line H441 contains high quantities of

CFTR mRNA, which corresponds with the data of the present thesis. In H441 and Calu-3

cells, CFTR mRNA levels were considerably higher than in A549 cells. These findings are

consistent with the fact that Clara cells and ciliated cells from the bronchioles and the

bronchus, respectively, function as secretory cells. In contrast to expression studies by

real-time PCR, Western blot analysis revealed that CFTR was only present in membrane

fractions of Calu-3 cells.

The Na,K-ATPase which is located on the basolateral side of the lung epithelium,

is assumed to be the essential driving force for LLC 3,20,120. The Na,K-ATPase-α1 subunit

gene was transcribed at a constantly high level in A549, H441 and Calu-3 cells, according

to the nature of a housekeeping gene. Similarly, Na,K-ATPase-β1 transcripts were

present in all cell lines at higher levels compared to α1 subunit mRNAs. Accordingly,

Na,K-ATPase subunits α and β1 were detected with specific antibodies in membrane

fractions of A549, H441 and Calu-3 cells. The β1-subunit was found in unglycosylated and

glycosylated isoforms, whereas the α-subunit was found in an unglycosylated form only.

It is known that lung epithelial cells expressing the water channel aquaporin-5

have a high osmotic permeability to water 103. Aquaporin-5 was conferred to be

responsible for the majority of water transport across the lung epithelium 17,104,121,122.

Hence, expression of aquaporin-5 in A549, H441 and Calu-3 cells was investigated using

RT-PCR and Western blot analysis. Aquaporin-5 mRNA and protein was detected in

Calu-3 cells but not in A549 and H441 cells.

In summary, the results from expression analysis using RT-PCR and Western

blots indicate the presence of functional ion channels and transporters as well as water

channels that are crucial for active transepithelial ion and water transport in Calu-3 cells.

DISCUSSION

- 71 -

In contrast, only transcripts of CFTR were present in A549 and H441 cells, whereas no

CFTR proteins were detected in these cells. Furthermore, A549 and H441 cells did not

express the water channel aquaporin-5.

The pathologically relevant human lung epithelial cell line Calu-3 met all

requirements for in vitro analysis of transepithelial ion and fluid transports in regard to

expression of ion channels and transporters involved in LLC, and of the water channel

aquaporin-5. Therefore, the TEER and dome assays, two in vitro test systems for

substances that influence the process of LLC, were established and validated using the

human lung epithelial cell line Calu-3.

DISCUSSION

- 72 -

4.2 In Vitro Test Systems for Pulmonary Edema Resorption

To identify and develop new drugs that stimulate LLC at the site of action, i.e. the lung

epithelium, a lung epithelial cell-based assay should be used for screening of candidates

in vitro. In this thesis, the human lung epithelial cell lines A549, H441 and Calu-3 were

used to establish two complementary in vitro assays for pulmonary edema resorption:

(1) Bioelectrical properties of polarized lung epithelial cell monolayers were studied in

a transepithelial electrical resistance (TEER) assay.

(2) The ability of lung epithelial cells for spontaneous dome formation within confluent

monolayers allowed quantitative examination of transepithelial fluid transport

(“dome assay”).

The TEER assay was established and validated to investigate the influence of

putative anti-lung-edema drug compounds on bioelectric properties of human lung

epithelial cell monolayers. It allows time-dependent measurements of the electrical

resistance across an epithelial cell monolayer. Readout of this assay is the alteration of

TEER due to the application of a compound to the apical and/or basolateral side of a

monolayer. Prerequisites of this assay are the integrity and “tightness” (= intact

intercellular contacts) of cell monolayers. From the three investigated human lung

epithelial cell lines A549, H441 and Calu-3 only Calu-3 cells formed high resistance cell

monolayers (TEER values up to 1280 Ω x cm²) allowing TEER assay measurements. Cell

monolayers that develop TEER values greater than 300 Ω x cm² provide representative

models of epithelial barriers and are convenient for bioelectric studies 101,123,124. To mimic

the microenvironment of the lung as accurately as possible, Calu-3 cells were grown at an

air-liquid interface on permeable filter membranes. The exposure of the apical surface of

respiratory epithelial cells to air is the determinant for their differentiation and

polarization 125-127. Formation of tight junctions, an indication for epithelial polarity 128,129,

and paracellular integrity of Calu-3 monolayers were ascertained by immunofluorescence

staining of the tight junction zonula occludens-1 protein and the assessment of lucifer

yellow passage across the monolayers, respectively. Validation of the TEER assay using

known stimulants and inhibitors of transepithelial ion currents proved this test system

suitable for screening of such modulators of transepithelial currents. As expected, the

CFTR inhibitor glibenclamide and the ENaC inhibitor amiloride increased TEER of Calu-3

monolayers. Activators of transepithelial ion currents, such as db cAMP, terbutaline and

DISCUSSION

- 73 -

formoterol, decreased TEER of Calu-3 monolayers. These results are consistent with the

findings of others 17,21,102,124 and are in agreement with the current theory of the molecular

mechanism of LLC that implies a vectorial transepithelial ion current from apical to

basolateral as driving force. Importantly, the above decribed compounds did not affect the

paracellular integrity of Calu-3 monolayers, indicating that the observed TEER changes

were due to alterations of transepithelial ion currents. Furthermore, these experiments

demonstrated high functional polarity of the cell monolayers, since amiloride and

glibenclamide increased TEER only after their addition to the apical side of the cell

monolayers, while the β2-agonist terbutaline exerted a 3-fold higher activity when added to

the basolateral side.

The formation of fluid filled hemicysts within an epithelial cell monolayer,

designated domes, has been reported for different epithelial cell types that actively

transport fluid, including primary alveolar type II cells 130-132. The presence of domes

indicates an intact active transport function of the cells within the monolayer, which may

reflect an important mechanism for the maintenance of fluid-free air spaces and normal

lung fluid balance in mammalian lungs in vivo. This thesis shows that Calu-3 cells but not

A549 and H441 cells are capable to spontaneously form domes when grown to full

confluence on non-porous substrates. Corticoids influence epithelial barriers by the

induction of tight junctions 105-107. A549 cells did not form domes, neither spontaneously

nor if cells were exposed to the glucocorticoid dexamethasone. In agreement with data

from Shylonsky and co-workers 133, H441 cells only formed domes after treatment with

dexamethasone. However, an in vitro model based on dexamethasone-treated H441 cells

would be inappropriate, as dexamethasone influences ENaC expression and activity in

H441 cells affecting transepithelial fluid transport itself 27,115,116,133,134.

As a result from these findings, Calu-3 cells were used to establish and validate

the dome assay for quantitative examination of transepithelial fluid transport. Readout of

this assay is the quantitative analysis of dome formation within lung epithelial cell

monolayers induced by the application of a drug test compound.

Experiments with ion transport modulators were performed to confirm the dome

assay with Calu-3 cells to be an appropriate model for studies on LLC in vitro. Terbutaline

increased dome formation by Calu-3 cells in the presence of the non-selective

phosphodiesterase inhibitor IBMX. An increase of dome densities was also obtained after

treatment of dome forming Calu-3 monolayers with db cAMP. The present study also

demonstrates that dome formation of Calu-3 cells can be blocked by the Na+ transport

inhibitors amiloride and ouabain and by the replacement of Na+ ions in the assay buffer by

the cell impermeable substitute NMDG+. Consistent with these findings it had been shown

DISCUSSION

- 74 -

that dome formation of lung epithelial cells is dependent on Na+ transport and that dome-

forming lung epithelial cells absorb Na+ ions through amiloride-sensitive channels 131-133.

In conclusion, the dome assay allows the quantitative analysis of substances that

modulate transepithelial fluid transport in a 96-well format, making it suitable as an in vitro

screening system for compounds that stimulate LLC. Since dome formation as well as

LLC depends on vectorial Na+ and fluid transport from the apical to the basolateral side of

an epithelial cell monolayer, quantitative analysis of dome formation by lung epithelial

cells may provide a valuable tool for in vitro studies on the process of LLC.

In summary, dome formation and the electrical properties of polarized monolayers

indicate that Calu-3 cells actively transport electrolytes and fluid, simulating the process of

LLC in vitro. As previously discussed, these cells express ion channels and transporters

that mediate LLC. Consequently, the TEER and dome assays using the human lung

epithelial cell line Calu-3 are suitable systems for in vitro activity testing of anti-pulmonary

edema compounds and are useful for studies on their mechanism of action.

DISCUSSION

- 75 -

4.3 Mechanism of Action of the Tip Peptide

The Tip peptide has been shown to stimulate LLC in isolated perfused rat lungs as well as

in in situ and in vivo rodent models and was suggested as a new therapeutic agent for the

resolution of pulmonary edema 73,90,91. This 17 aa peptide is derived from the lectin-like tip

domain of TNF-α that extends from Ser100 to Glu116 of the soluble form of human TNF-α

and is responsible for the specific binding of oligosaccharides, such as

N,N’-diacetylchitobiose and branched tri-mannoses 83-85. In order to mimic its loop

structure within TNF-α, the Tip peptide was circularized via a disulfide bond between its

terminal cysteine residues. The amino acids Thr105, Glu107, and Glu110 within the tip

domain of human TNF-α were shown to be critical for the lectin-like activity 83.

Consequently, replacement of these amino acids by alanines resulted in an inactive

mutant form of Tip (mTip) 83,88. The mutant Tip peptide mTip and a peptide consisting of

the same amino acids as Tip in a random order (scrambled Tip; scTip) served as negative

controls in the experiments performed in this study.

Application of the Tip peptide to dome forming Calu-3 cells significantly increased

dome formation within cell monolayers in a dose dependent manner, indicating stimulation

of transepithelial fluid transport. The Tip peptide lost its capacity to stimulate dome

formation upon the incubation of the peptide with the lectin-binding sugar

N,N’-diacetylchitobiose. In conclusion of these findings, the in vitro activity of the Tip

peptide in the dome assay is reflective of Tip’s anti-pulmonary edema activity in situ and in

vivo 91,92.

The impact of the Tip peptide on bioelectric properties of polarized Calu-3 cell

monolayers cultivated at the air-liquid interface on porous membranes was examined. The

results obtained from TEER assay experiments clearly demonstrate that the Tip peptide

influences transepithelial conductance of polarized lung epithelial cell monolayers only

when added to the apical side of the monolayers without affecting the paracellular integrity

of the monolayers. In the lung, the apical sides of lung epithelial cells face the airspace

whereas the basolateral sides are adjacent to the interstitial space and the lung

capillaries. Therefore, the polarized mode of action of the Tip peptide in the TEER assay

may explain the finding of Dr C. Braun 92, who had demonstrated that the intra-venous

application in contrast to the intra-tracheal instillation of the Tip peptide had not shown any

effect on LLC in an in vivo flooded rat lung model.

The Tip peptide induced a biphasic change of Calu-3 monolayer TEER: application

of the Tip peptide resulted in a fast increase (within seconds) of TEER followed by a

steady decrease until constant TEER values were reached. The initial peak in TEER

DISCUSSION

- 76 -

values may represent sustained inhibition of an ion current followed by the activation of a

different ion transport pathway, which may contribute to edema clearance in vivo. These

findings are in agreement with previous observations by others 135-138, who described

biphasic transepithelial ion current responses induced by the treatment of epithelial cell

monolayers with pharmacological active substances.

The activity of the Tip peptide on bioelectric properties of Calu-3 cells was shown

to be dose dependent with a medium effective dose of 1.66 mg/ml and required Na+ ions

in the assay buffer. These findings are consistent with results from patch clamp analyses

using TNF-α and the Tip peptide 88,89. They also reflect the activities of these molecules on

LLC in different animal models that demonstrated the activation of sodium-specific ion

currents 88-90. It had been proposed that the β2-agonist terbutaline and the Tip peptide

activate LLC by different mechanisms of action 92. This study demonstrates that the action

of the Tip peptide in the TEER assay does not depend on β-receptor activation since

experiments in the presence of the unselective β-receptor antagonist propranolol exhibited

no change of the activity of the Tip peptide. The Tip peptide did not up-regulate

intracellular cAMP-levels in Calu-3 cells. Therefore, the results from in vitro studies using

the human lung epithelial cell line Calu-3 confirm the hypothesis that terbutaline and the

Tip peptide activate transepithelial ion and fluid transport by different mechanisms of

action.

The effective doses of the Tip peptide in the TEER and dome assays determined

in this thesis were significantly higher than those determined in previously reported whole

cell patch clamp and in vitro trypanolysis assay experiments 83,88. However,

concentrations used in this study were in the range of magnitude employed in

experiments with rodent models of lung edema (ALTANA Pharma, unpublished data; 90-

92). In a rat ex vivo permeability lung edema model, the medium effective dose (ED50) of

the Tip peptide was estimated to be 1 mg/lung. This corresponds to an instillation of a

~523 µM Tip peptide solution into the lung, which is consistent with the ED50 of Tip

determined in the TEER assay (1.66 mg/ml = 863.2 µM). In general, because of

bioavailability, pharmacologically active substances should be more effective in vitro than

in vivo. In this respect, the above mentioned lung edema animal models may represent a

special case since, similar to in vitro systems, substances are delivered directly to their

target cells without passing the blood stream.

In contrast to the Tip peptide, the mutant and scrambled Tip peptides did not

increase dome densities within Calu-3 cell monolayers. However, the mutant and

scrambled Tip peptides elicited 54.7% and 46.8% of the Tip peptide’s activity in the TEER

assay, respectively. Since the peptides used in this work were synthesized as acetate

DISCUSSION

- 77 -

salts, it was investigated whether the residual activities of the mutant and scrambled Tip

peptides in the TEER assay might be due to acetate ions in the peptide preparations.

Consequently, the effect of acetate on TEER and dome formation of Calu-3 cell

monolayers was investigated. Sodium acetate influenced transepithelial conductance of

polarized Calu-3 monolayers only when added to the apical side of the monolayers

without affecting the paracellular integrity of the monolayers. Moreover, sodium acetate

significantly increased dome density within Calu-3 monolayers in the dome assay.

Consistent with the data from this work, it has been reported that short chain fatty acids,

such as acetate and butyrate, influence transepithelial ion currents in epithelial cells,

including Calu-3 cells 134,139-142, and increase dome densities in lung and kidney epithelial

cells 132,134,143. However, it can be excluded that the observed effects of the Tip peptide

and its variants are solely caused by acetate ions in the peptide preparations, as (i) the

removal of acetate ions from a Tip peptide solution by dialysis resulted in an activity

decrease of only 16%, and (ii), according to the manufacturer’s product analysis sheet

(Bachem, Bubendorf, Switzerland), acetate concentration in a 1 mg/ml Tip peptide

solution was calculated to be 0.5 mM. At this concentration, sodium acetate barely

induced TEER changes of Calu-3 cell monolayers. Nevertheless, the results from TEER

and dome assay experiments with sodium acetate suggest the possibility that the activity

of the Tip peptide on TEER and dome formation of Calu-3 cells may partly be caused by

acetate ions in the peptide preparation.

TNF-α had previously been demonstrated to increase LLC in the lungs of

anaesthetized and ventilated rats 89,144,145. Conversely, the findings of Elia et al. as well as

Braun et al. were unclear regarding TNF-α’s lung edema resorption capacity 90-92. TNF-α

had also been shown to promote lung edema formation most likely by receptor-mediated

neutrophil activation and infiltration 146-148. Moreover, two recent publications revealed that

TNF-α down-regulates the expression and activity of ENaC in type II alveolar epithelial

cells by ~70%, and thus could inhibit lung edema reabsorption 149,150. The results from

in vitro studies with recombinant human TNF-α in this thesis reflect the dual role of TNF-α

in LLC: On one hand, TNF-α decreased TEER of Calu-3 monolayers, indicating a

stimulating effect on transepithelial ion currents. On the other hand, TNF-α failed to induce

dome formation in the dome assay. The effect of TNF-α in the TEER assay was dose

dependent with a medium effective dose of 71.29 nM. It was observed only upon apical

addition and was not due to the loss of paracellular integrity, leading to the assumption

that TNF-α triggered ion fluxes by interaction with an apical localized receptor protein and

not by formation of an ion channel by itself, as reported by Kagan et al. 93.

DISCUSSION

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Interestingly, the effects of both, rhTNF-α and the Tip peptide on the conductance

of Calu-3 cell monolayers in the TEER assay were significantly enhanced under acidic

conditions (pH 6.0). These findings reflect the activities of these molecules on whole cell

currents of murine microvascular endothelial cells and peritoneal macrophages as

published by Hribar et al. 88. A potential physiological role of these observations is

conceivable: It has been reported that the vicinity of alveolar epithelial cells presents an

acidic microenvironment, and that the pH of the alveolar lining fluid can be depressed by

disease or inflammation 151,152. In this context, pulmonary edema associated with the acute

respiratory distress syndrome (ARDS) may be a medical indication for the treatment with

the Tip peptide 14,153.

As described above, the Tip peptide lost its capacity to stimulate transepithelial

fluid transport in the dome assay upon the incubation of the peptide with the lectin-binding

sugar N,N’-diacetylchitobiose. The lectin-like specificity of TNF-α for this oligosaccharide

had previously been elucidated and mapped to the tip domain of TNF-α 83-85. However, a

lectin-like activity of the TNF-α derived Tip peptide had not been demonstrated. The

interaction studies performed in this thesis, using the surface plasmon resonance

technology, do not provide evidence for a lectin-like activity of the Tip peptide since only

rhTNF-α, but not the Tip peptide, specifically bound to N,N’-diacetylchitobiose.

Accordingly, NMR binding studies performed in the laboratory of Prof Dr H. Kessler at the

Munich University of Technology failed to detect an interaction of the Tip peptide with

N,N’-diacetylchitobiose (I. Varnay, unpublished data).

In this thesis, nine potential protein interaction partners that specifically bind the

Tip peptide are presented. Among them, only one transmembrane receptor protein, the

tyrosine kinase-type cell surface receptor ErbB2 (Her-2), was identified. ErbB receptors

are well known to activate the Ras/Mitogen-activated protein kinase (MAPK) signal

transduction pathway (reviewed in 154). The activation of ENaC by the Ras/MAPK-

pathway 155-157 suggests a possible involvement of this pathway in the regulation of LLC.

According to a recent publication 158, TNF-α may be a potent activator of ErbB2 signaling

independent of its receptor-dependent biologic activity. In this context, using an in vivo rat

model of lung edema, Folkesson et al. found that the cytokine transforming growth factor

α (TGF-α) increased lung liquid clearance by an amiloride-sensitive, tyrosine kinase-

dependent mechanism of action, which was independent of cAMP 159.

In conclusion, this study provides evidence that complementary dome assay and

electrophysiological measurements, such as Ussing chamber or the TEER assay, using

Calu-3 cells are valuable in vitro test systems for the screening of anti-pulmonary edema

DISCUSSION

- 79 -

compounds that directly act on the lung epithelium and stimulate active fluid resorption.

The results obtained in this thesis and by others 88-92 suggest that the Tip peptide activates

LLC by the direct or indirect activation of apical localized sodium channels of pulmonary

epithelial cells. This thesis also suggests that

(1) a superimposed effect of acetate ions might contribute to the activity of a Tip

peptide solution on LLC.

(2) ErbB2 receptors and the Ras/MAPK signaling pathway may be involved in the

action of the Tip peptide.

Further investigations should explore the possible involvement of ErbB2 signaling

and the Ras/MAPK pathway in the mechanism of action of the anti-edema Tip peptide.

Consequently, the influence of tyrosine-kinase inhibitors or ErbB2 blockers, such as

Herceptin®, should be subject of further investigations.

DISCUSSION

- 80 -

4.4 Identification of Prospective Anti-Pulmonary Edema Peptides

Initiated by the Tip peptide’s anti-pulmonary edema capacity, 40 peptides were in silico

constructed by the ALTANA Pharma AG. Their in vitro activities in the TEER and dome

assays using the human lung epithelial cell line Calu-3 were compared with that of the Tip

peptide. As described above, these in vitro test systems are reflective of the Tip peptide´s

anti-lung edema activity in vivo. It became obvious that

(1) the activity of the Tip peptide was maintained with „conservative“ amino acid

exchanges within the TPEGAE core motif (T → S, E → D, A → G).circularization

of the peptides enhanced their activity. This observation is consistent with findings

of Dr C. Braun, who investigated the action of variants of the Tip peptide using an

ex vivo rat lung model 92.

(3) trimerization of the TPEGAE core motif (or variants thereof) with flexible poly-

glycine linker sequences increased the activity.

Several of the measured peptides showed activities in the range or above those

elicited by the Tip peptide. The activity of one of these prospective anti-pulmonary edema

peptide drug candidates (peptide 2) has been tested in a murine model of pulmonary

edema. This peptide revealed an enhanced capacity to induce LLC compared with the Tip

peptide (ALTANA Pharma AG, unpublished data). These results further emphasize that

the TEER and dome assays are suitable in vitro tools to screen for anti-edema drug

candidates.

SUMMARY

- 81 -

5 Summary

Pulmonary edema is a life-threatening pathological condition caused by excessive

extravascular fluid accumulation in the lung tissue. In 11 different animal models the Tip

peptide, a cyclic peptide derived from the lectin-like tip domain of the human tumor

necrosis factor α (TNF-α), was shown to up-regulate the rate of lung liquid clearance

(LLC), contributing to the resolution of pulmonary edema (ALTANA Pharma, unpublished

data; 90-92).

One aim of this thesis was to establish an in vitro activity assay that reflects the Tip

peptide’s anti-pulmonary edema action and is useful for studies on its mechanism of

action. For this purpose, the three human lung epithelial cell lines A549, H441 and Calu-3

were characterized with respect to their suitability for in vitro studies on anti-edema drug

candidates. The Calu-3 cell line met all requirements for in vitro analysis of transepithelial

ion and fluid transports in regard to expression of ion channels and transporters involved

in LLC (e.g. the epithelial sodium channel ENaC, the chloride channel CFTR and the

Na,K-ATPase), expression of the water channel aquaporin-5, formation of tight and

polarized monolayers, and the capability of transepithelial fluid transport (dome formation)

within confluent monolayers. Therefore, Calu-3 cells were used to establish two

complementary in vitro assays for pulmonary edema resorption:

(1) Bioelectrical properties of polarized cell monolayers were studied by using the

Transepithelial Electrical Resistance (TEER) technology (“TEER assay”).

(2) The ability of Calu-3 cells for spontaneous dome formation within confluent

monolayers allowed the quantitative examination of transepithelial fluid

transport (“dome assay”).

TEER assay experiments proved that the Tip peptide influences transepithelial

conductance of Calu-3 cell monolayers in a polarized and dose dependent manner. This

activity requires sodium ions in the assay buffer. Dome assay experiments confirmed that

dome formation by lung epithelial cells is a sodium dependent process and that it is

induced by the Tip peptide. The effect of the Tip peptide in TEER and dome assays was

(partly) inhibitable by the oligosaccharide N,N’-diacetylchitobiose. However, a lectin-like

binding of this sugar by the Tip peptide could not be detected. Further investigations

revealed that the action of the Tip peptide on TEER and dome formation of Calu-3 cells

was independent of β-adrenergic receptor activation and cAMP up-regulation. Effects on

SUMMARY

- 82 -

TEER and dome formation may partly be caused by acetate ions in the peptide

preparation. Protein interaction studies led to the identification of nine potential receptor

proteins for the Tip peptide, one of which is the transmembrane receptor tyrosine kinase

ErbB2. Testing of 40 potential anti-pulmonary edema peptides in the TEER and dome

assays revealed that

(1) the activity of the Tip peptide was maintained with “conservative” amino acid

exchanges within the TPEGAE core motif.

(2) circularization of active peptides enhanced their activity.

(3) trimerization of the TPEGAE core motif (or variants thereof) with flexible poly-

glycine linker sequences increased the activity.

In conclusion, this thesis reveals that complementary dome assay and TEER

assay experiments with Calu-3 cells are valuable in vitro test systems for the screening of

anti-pulmonary edema compounds that directly act on the lung epithelium and stimulate

active fluid resorption. It supports the hypothesis that the Tip peptide activates LLC by the

direct or indirect activation of apical localized sodium channels of pulmonary epithelial

cells and suggests that acetate ions and/or ErbB2 signaling may contribute to the Tip

peptide’s activity.

ZUSAMMENFASSUNG

- 83 -

6 Zusammenfassung

Unter einem Lungenödem versteht man die übermäßige Ansammlung von

extravaskulärer Flüssigkeit im Lungengewebe. Es wurde in insgesamt 11 Tiermodellen

gezeigt, dass das Tip-Peptid,- ein zyklisches Peptid, welches die Lektin-artige Domäne an

der Spitze des humanen Tumornekrosefaktors-α imitiert -, eine Aktivität gegen

Lungenödeme besitzt (ALTANA Pharma, nicht-puplizierte Daten; 90-92).

Ein Ziel dieser Arbeit war es, einen In-vitro-Aktivitätstest zu etablieren, der die

Ödemresorptionsaktivität des Tip-Peptids reflektiert. Dieses Testsystem sollte dazu

dienen, Untersuchungen zum Wirkmechanismus des Tip-Peptids durchzuführen. Zu

diesem Zweck wurden die drei menschlichen Lungenepithelzelllinien A549, H441 und

Calu-3 hinsichtlich ihrer Eignung für In-vitro-Analysen mit antiödematösen Substanzen

untersucht. Die Zelllinie Calu-3 entsprach allen Anforderungen für In-vitro-

Untersuchungen zu transepithelialen Ionen- und Flüssigkeittransport in bezug auf die

Expression von Ionenkanälen und -transportern, die am Prozess der

Lungenödemresorption beteiligt sind (z.B. des epithelialen Natriumkanals ENaC, des

Chloridkanals CFTR und der Natrium-Kalium-Pumpe (Na,K-ATPase)), sowie bezüglich

der Expression des Wasserkanals Aquaporin-5. Des weiteren eignen sich Calu-3 Zellen

zur Züchtung dichter, einschichtiger und polarisierter Zellrasen und besitzen die Fähigkeit,

Flüssigkeit zu transportieren (Bildung flüssigkeitsgefüllter Zellkuppeln innerhalb eines

dichten Zellrasens = „Domes“). Demzufolge wurden mit Calu-3 Zellen zwei sich

ergänzende In-vitro-Testsysteme aufgebaut, um die antiödematöse Wirkung des Tip-

Peptids zu untersuchen:

(1) Unter Verwendung des Transepithelialen-Elektrischen-Widerstand-Messsystems

(„TEER-Assay“) wurden die bioelektrischen Eigenschften von polarisierten,

einschichtigen Calu-3 Zellrasen untersucht.

(2) Die Fähigkeit von Calu-3 Zellen innerhalb eines konfluenten Zellrasens spontan

„Domes“ auszubilden ermöglichte die quantitative Untersuchung des

transepithelialen Flüssigkeitstransports („Dome-Assay“).

TEER-Assay-Experimente belegten, dass das Tip-Peptid die Ionenleitfähigkeit von Calu-3

Zellschichten nur nach apikaler Zugabe in einer dosisabhängigen Weise beeinflusst.

Diese Aktivität des Tip-Peptids erfordert Natriumionen im Probenpuffer. Die Ergebnisse

ZUSAMMENFASSUNG

- 84 -

von Dome-Assay-Experimenten bestätigten, dass die Dome-Bildung von

Lungenepithelzellen ein Natriumionen-abhängiger Prozess ist, und dass das Tip-Peptid

diesen Prozess stimuliert. Die Effekte des Tip-Peptids in den TEER- und Dome-Assays

waren (teilweise) durch das Oligosaccharid N,N'-Diacetylchitobiose hemmbar. Eine

Lektin-artige Interaktion dieses Zuckers mit dem Tip-Peptid konnte jedoch nicht

festgestellt werden. Weitere Untersuchungen deckten auf, dass die Wirkungen des Tip-

Peptids von Calu-3 Zellen unabhängig von einer Stimulation β-adrenerger Rezeptoren

und einer Hochregulation des intrazellulären cAMP-Spiegels sind. Die Effekte des Tip-

Peptids auf den transepithelialen Widerstand und die Dome-Bildung könnten teilweise von

Acetationen in der Peptidlösung ausgelöst werden. Proteininteraktionsstudien führten zur

Identifikation von neun möglichen Rezeptorproteinen des Tip-Peptids, darunter die

transmembrane Rezeptortyrosinkinase ErbB2. Die Aktivitätstestung von 40 alternativen

antiödematösen Peptiden zeigte, dass

(1) die Aktivität des Tip-Peptids beim Austausch von Aminosäuren gegen

Aminosäuren mit ähnlichen Eigenschaften erhalten blieb.

(2) die Zirkularisierung von wirksamen Peptiden deren Aktivität erhöhte.

(3) die Aneinanderreihung von drei TPEGAE-Motiven (oder Varianten davon) mit

flexiblen Polyglycin-Verbindungssequenzen zu einer Aktivitätssteigerung führte.

Folglich macht diese Arbeit deutlich, dass sich ergänzende Experimente mit dem Dome-

Assay und einem elektrophysiologischen Messsystem wie dem TEER-Assay mit Calu-3

Zellen hilfreich für die In-vitro-Testung von Substanzen sind, die direkt am Lungenepithel

einen aktiven Flüssigkeitstransport anregen und somit eine anti-lungenödematöse

Wirkung haben. Sie unterstützt die Hypothese, dass das Tip-Peptid durch die direkte oder

indirekte Aktivierung von apikal lokalisierten Natriumkanälen von Lungenepithelzellen die

Resorption von Flüssigkeit in der Lunge bewirkt und gibt Hinweise, dass Acetationen

und/oder die ErbB2 Signalkaskade zum Wirkmechanismus des Tip-Peptids beitragen

könnten.

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