1

THE ROLE OF DIRECTED GP130-MEDIATED

SIGNALLING IN BLEOMYCIN-INDUCED

MURINE PULMONARY FIBROSIS

Robert Joseph James O’Donoghue B.Comm., B.Sc. Hons.

This thesis is presented for the degree of Doctor of Philosophy of The University

of Western Australia in the School of Medicine and Pharmacology.

January 2008.

2

ABSTRACT

Fibrosis is a feature of many pulmonary conditions, including idiopathic

pulmonary fibrosis (IPF), which is characterised by the accumulation of

fibroblasts/myofibroblasts and excessive deposition of collagen. IPF is a disease of

unknown aetiology that is unresponsive to current therapy and is typically fatal. The

inflammatory cytokine interleukin (IL)-6 is elevated in patients with IPF and recent

studies have shown that IL-6-induced signalling is altered in lung fibroblasts from

patients with IPF. IL-6 belongs to the gp130 cytokine family, which is a group of ten

structurally related cytokines, that all require the membrane bound glycoprotein gp130

to activate intracellular signalling pathways.

Gp130 activates intracellular signalling through the Shp2-ERK1/2 and STAT1/3

pathways to mediate cellular activities. This thesis tests the hypothesis that gp130-

mediated signalling is dysregulated in the development and progression of pulmonary

fibrosis. To address this hypothesis, I assessed the role of gp130-mediated signalling in

a mouse model of bleomycin-induced lung fibrosis. This thesis utilised two novel gp130

mutant mice strains with directed and enhanced gp130-mediated Shp2-ERK1/2

(gp130ΔSTAT/ΔSTAT) or STAT1/3 (gp130757F/757F) signalling.

I observed complete protection from fibrosis in gp130ΔSTAT/ΔSTAT mice up to 60

days after bleomycin treatment and profound fibrosis in gp130757F/757F mice compared to

wt controls. The enhanced fibrosis observed in gp130757F/757F mice was diminished by

monoallelic deletion of STAT3 (gp130757F/757F;STAT3+/-), identifying gp130-STAT3

signalling as a novel promoter of lung fibrosis. Collagen αI type I mRNA transcription

was enhanced in lung tissue following bleomycin treatment of gp130757F/757F mice

compared to wt and gp130757F/757F;STAT3+/- mice suggesting gp130-STAT3 signalling

enhanced the progression of lung fibrosis. Collagen luciferase gene reporter assays

indicated that IL-6-activated the collagen promoter in murine embryonic fibroblasts

3

through the gp130-STAT3 pathway. Gp130ΔSTAT/ΔSTAT mice showed some airspace

enlargement and degradation of ECM was enhanced by increased MMP-9 and MT-

MMP activity in gp130ΔSTAT/ΔSTAT mice, while collagen αI type I mRNA transcription

was decreased compared to wt mice after bleomycin treatment.

Inflammation within the lung parenchyma was enhanced in gp130757F/757F mice

relative to wt, gp130ΔSTAT/ΔSTAT and gp130757F/757F;STAT3+/- mice three days after

bleomycin treatment. This inflammatory response was largely composed of neutrophils

and lymphocytes, suggesting that inflammatory cells were involved in the progression

of lung fibrosis in gp130757F/757F mice using this model. To determine the role of

directed gp130-mediated signalling within inflammatory cells in vivo in this model,

bone marrow of wt, gp130ΔSTAT/ΔSTAT and gp130757F/757F mice was transferred to whole

body irradiated host mice of a different genotype, creating chimeric mice. These

experiments demonstrated that the profound fibrosis observed in gp130757F/757F mice

was, in part, due to enhanced gp130-STAT1/3 signalling within bone-marrow derived

cells. However, the presence of gp130757F/757F cells in wt host mice treated with

bleomycin did not enhance fibrosis, suggesting that a mutation or anomaly within

resident cells of the lung is required for fibrosis to develop.

In vitro studies showed that gp130-mediated signalling regulated the growth and

phenotype of primary lung fibroblasts. Activation of gp130-STAT1/3 promoted

myofibroblast differentiation and proliferation of primary lung fibroblasts, while others

have shown that gp130-ERK1/2 signalling promoted proto-myofibroblast

differentiation. In addition, IL-6/11 activation of gp130-mediated signalling modulated

transforming growth factor (TGF)-β-induced effects on adult fibroblast proliferation

and myofibroblast differentiation. Interaction between IL-6/11 and TGF-β1 on

fibroblast proliferation was dependent on both the gp130-ERK1/2 and gp130-STAT1/3

pathways. Loss of either pathway abrogated the effects of IL-6 and IL-11 on TGF-β1-

4

induced fibroblast proliferation. However, it was clear that gp130-STAT3 signalling

inhibited TGF-β1-induced myofibroblast differentiation of primary lung fibroblasts. The

inhibition of myofibroblast differentiation was associated with gp130-STAT3

dependent inhibition of TGF-β1-induced Smad3 phosphorylation. These results indicate

that IL-6 and IL-11 promote myofibroblastic differentiation of lung fibroblasts, while

gp130-STAT3 signalling inhibits TGF-β1-induced Smad3 phosphorylation and

myofibroblastic differentiation of lung fibroblasts

While the pathogenesis of IPF is unknown, it is believed that excessive collagen

deposition, aberrant fibroblast behaviour and an inflammatory response are critical to

the progression of this disease. It has been shown here that IL-6 family cytokines

mediate the development and progression of bleomycin-induced lung fibrosis by

increasing collagen synthesis, fibroblast proliferation, myofibroblast differentiation and

inflammation through gp130-STAT3 signalling. This thesis has demonstrated that

differential activation of cytoplasmic signalling pathways by a membrane bound

receptor can have a profound effect on pulmonary responses to injury. Furthermore, this

thesis is the first study to identify the gp130-STAT3 pathway as a therapeutic target in

the treatment of IPF.

5

DECLARATION

The work presented in this thesis was performed solely by the author except

where otherwise acknowledged and has not been submitted previously for any other

degree.

Robert J. J. O’Donoghue.

6

ACKNOWLEDGEMENTS

I would like to acknowledge my mentors and supervisors Associate Professor

Steven Mutsaers and Associate Professor Darryl Knight whose wisdom has given me

insightful knowledge in the field of pulmonary fibrosis, and support has guided me to

completion of this thesis. I thank you for teaching me essential skills and broadening my

scientific knowledge. I also acknowledge the tremendous generosity and support

Associate Professor Matthias Ernst for donating his gp130 mutant mice to this study and

nurtured my interest in IL-6 family cytokines

I am also indebted to my collaborators and mentors Associate Professor Philip

Thompson (University of Western Australia and Lung Institute of Western Australia),

Professor Gary Anderson (University of Melbourne, Victoria, Australia), Dr Hong Jian

Zhu (University of Melbourne, Victoria, Australia) for their critical analysis of my data

and advice.

I would also like to acknowledge the assistance of the following people: Ms

Jessica Jones for performing BAL, culls, tissue collections and teaching me the trans-

nasal method of bleomycin treatment; Dr Steven Bozinovski, Dr Anthony Kicic and Mr

Paul Stevens for assisting me with zymography; Dr Bo Wang for performing Smad3

reporter assays; Ms Dianne Grail and Dr Vance Matthews for their assistance with

animal experiments and collagen reporter assays respectively; Dr Philip Stumbles and

Dr Deborah Strickland for their assistance with bone marrow reconstitution

experiments; Professor Geoffrey Laurent and Dr Chris Scotton for performing HPLC

analysis; Dr Cecilia Prele for assistance with western blots and critical reading of this

manuscript; The staff of PathWest Laboratory Medicine WA for their assistance the

histology

I am very grateful for the friendship and encouragement of past and present

members of Sir Charles Gairdner Hospital Unit, Department of Medicine and the Lung

7

Institute of Western Australia especially; Kathleen Brown, Alison McDonnell, Steve

Broomfield, Sathish Mahendran, Irma Larma, Scott Cornwall, Peter Graham, Amanda

Sherwood, Christine Blundell, Deborah Yeoman, Karen Anderson, Ann Jolghazi,

Ivonne van Bruggen, Ian Dick, Biljana Koloska, Neil Misso, Andrea Mladinovic, Amy

Prosser, Cleo Robinson, Marg Sinmaa, Paula Leigh, Robbert van der Most, Sally

Lansley, Hui Ling Lau, Amelia Scaffidi, Julie Long, Richelle Searles, Rachel Venables,

Vanessa Hawthorn, Bernadette Bradley, Kirrily O’Hara, Kendle Estcourt, Pam

McManus, Conny Bertram, Laura Genovesi, Bernadette Pedersen, Carla Thomas,

Bahareh Badrian, Svetlana Baltic, Shashi Aggarwal, Mirjana Fogel-Petrovic, Rosie

Gulliver, Sylwia Wilkosz and Helen Peake.

Finally, I would like to thank my parents and wife Natalie Kon-yu, your unerring

love, support and encouragement have been a source of strength during my studies.

8

TABLE OF CONTENTS

THE ROLE OF DIRECTED GP130-MEDIATED SIGNALLING IN BLEOMYCIN-INDUCED MURINE PULMONARY FIBROSIS ....................................................... 1

Abstract ......................................................................................................................... 2

Declaration .................................................................................................................. 2

Acknowledgements ................................................................................................... 6

Table of Contents ...................................................................................................... 8

Abbreviations ........................................................................................................... 12

CHAPTER ONE ......................................................................................................... 15

LITERATURE REVIEW ............................................................................................ 15

1.1 Overview ............................................................................................................... 16 1.2 Fibrosis ................................................................................................................. 16 1.3 Collagen Metabolism .......................................................................................... 17 1.4 Epidemiology and Clinical Characteristics of Idiopathic Pulmonary Fibrosis ..................................................................................................................................... 20 1.5 Genetics of IPF .................................................................................................... 21 1.6 Pathogenesis of IPF ............................................................................................ 23

1.6.1 Inflammation and IPF ............................................................................. 24 1.6.2 Wound Healing and IPF ......................................................................... 26 1.6.3 Fibroblast Phenotypes in IPF ................................................................ 28

1.7 Animal Models of IPF .......................................................................................... 30 1.8 Interleukin-6 and IPF .......................................................................................... 32 1.9 The IL-6 Family of Cytokines ............................................................................. 34 1.10 Gp130-Mediated Signalling ............................................................................. 39

1.10.1 SHP-2-ERK Signalling ......................................................................... 42 1.10.2 STAT1/3 Signalling ............................................................................... 43 1.10.3 Inhibition of Gp130-Mediated Signalling ............................................ 45

1.11 IL-6, IL-11 and Pulmonary Physiology ........................................................... 47 1.12 Gp130-Mediated Signalling and Fibrosis ....................................................... 51 1.13 Gp130 Mutant Mice........................................................................................... 54 1.14 Transforming Growth Factor-β ........................................................................ 58

1.14.1 Smad Signalling .................................................................................... 59 1.14.2 Physiological Role of TGF-β ............................................................... 61

1.15 TGF-β and IPF................................................................................................... 63 1.16 Thesis Overview ................................................................................................ 65

CHAPTER TWO ........................................................................................................ 16

9

MATERIALS AND METHODS ................................................................................ 67

Materials .................................................................................................................... 68

2.1 Buffers and Solutions .......................................................................................... 68

Methods ..................................................................................................................... 81

2.2 Bleomycin Administration ................................................................................... 81 2.3 Bone Marrow Reconstitution .............................................................................. 81 2.4 Broncho-alveolar Lavage (BAL) ........................................................................ 83 2.5 Collagen Assays .................................................................................................. 84

2.5.1 Chloramine T Assay ............................................................................... 84 2.5.2 Reverse-phase High Pressure Liquid Chromatography (HPLC) ...... 84 2.5.3 Collagen Luciferase Reporter Assay .................................................... 85

2.6 DNA Isolation from Tissue Biopsy..................................................................... 85 2.7 Enzyme Linked Immuno-Sorbent Assay (ELISA) ........................................... 87 2.8 Eukaryotic Cell Culture ....................................................................................... 88

2.8.1 Cell Isolation ............................................................................................ 88 2.8.2 Propagation of Eukaryotic Cells ............................................................ 88 2.8.3 Immortalisation of Primary Lung Fibroblasts ....................................... 89

2.9 Histology ............................................................................................................... 90 2.9.1 Immunohistochemistry (IHC) ................................................................. 90

2.10 Immunocytochemistry ....................................................................................... 91 2.11 In situ Inflammation ........................................................................................... 92 2.12 Prokaryotic Cell Culture .................................................................................... 93

2.12.1 Preparation of Heat Shock Competent Cells .................................... 93 2.12.2 Heat Shock Transformation of Competent Cells .............................. 93 2.12.3 Plasmid Extraction ................................................................................ 94

2.13 Proliferation Assays .......................................................................................... 94 2.13.1 Brominated Deoxy-Uridine Incorporation (BrdU) Assay .................. 94 2.13.2 Methylene Blue Binding (MBB) Assay ............................................... 95 2.13.3 Methyl-Tetrazolium Reduction (MTT) Assay ..................................... 95

2.14 Reverse-Transcriptase Real-Time Polymerase Chain Reaction ................ 96 2.15 Protein Biochemistry ......................................................................................... 97

2.15.1 Protein Isolation .................................................................................... 97 2.15.2 SDS-Polyacrylamide Gel Electrophoresis ......................................... 98 2.15.3 Zymography .......................................................................................... 99

2.16 Oligonucleotide Primers ................................................................................... 99 2.17 Primary and Secondary Antibodies .............................................................. 102 2.18 Cytokines and Growth Factors ...................................................................... 103 2.19 Plasmids ........................................................................................................... 103 2.20 TGF-β Reporter Assay ................................................................................... 104

CHAPTER THREE .................................................................................................. 105

GP130-STAT3 SIGNALLING REGULATES THE DEVELOPMENT OF BLEOMYCIN-INDUCED LUNG FIBROSIS IN VIVO ......................................... 105

3.1 Introduction ........................................................................................................ 106 3.2 Materials and Methods ..................................................................................... 109 3.3 Results ................................................................................................................ 112

3.3.1 Chloramine T Assessment of Lung Collagen Content ..................... 112

10

3.3.2 Gp130-STAT1/3 Signalling Enhances Bleomycin-Induced Pulmonary Fibrosis ............................................................................................................ 112 3.3.3 Gp130-Mediated STAT3 Signalling Promotes Bleomycin-Induced Lung Fibrosis ................................................................................................... 122 3.3.4 Gp130-Mediated ERK1/2 Signalling Increases MMPs after Bleomycin-Induced Lung Injury. ................................................................... 125 3.3.5 IL-6 Gp130-STAT3 Signalling Promotes Collagen Transcription ... 127

3.4 Discussion .......................................................................................................... 130

CHAPTER FOUR .................................................................................................... 137

GP130-STAT3 SIGNALLING LINKS INFLAMMATION TO THE DEVELOPMENT AND PROGRESSION OF BLEOMYCIN-INDUCED LUNG FIBROSIS IN VIVO ................................................................................................. 137

4.1 Introduction ........................................................................................................ 138 4.2 Materials and Methods ..................................................................................... 141 4.3 Results ................................................................................................................ 144

4.3.1 The Bleomycin-Induced Inflammatory Response ............................. 144 4.3.2 Inflammation and Fibrosis ................................................................... 146 4.3.3 The Effect of Bone Marrow Transfer on Bleomycin-Induced Lung Fibrosis ............................................................................................................ 149 4.3.4 Gp130-Mediated Signalling within the Lung Regulates Bleomycin-Induced Lung Fibrosis .................................................................................... 152

4.4 Discussion .......................................................................................................... 157

CHAPTER FIVE ....................................................................................................... 165

DIRECTED GP130-MEDIATED SIGNALLING REGULATES THE GROWTH AND PHENOTYPE OF MOUSE LUNG FIBROBLASTS .................................. 165

5.1 Introduction ........................................................................................................ 166 5.2 Materials and Methods ..................................................................................... 169 5.3 Results ................................................................................................................ 173

5.3.1 Optimisation of Lung Fibroblast Isolations ........................................ 173 5.3.2 Cytoplasmic Signalling in Primary Lung Fibroblasts from Wt and Gp130 Mutant Mice ........................................................................................ 173 5.3.3 Growth and Phenotype of Primary Lung Fibroblasts from Wt and Gp130 Mutant Mice ........................................................................................ 176 5.3.4 Derivation and Characterisation of Immortalised Lung Fibroblast Cell Lines ................................................................................................................. 176 5.3.5 Assessment of Fibroblast Proliferation Assays ................................. 178 5.3.6 IL-6 and IL-11 Differentially Regulate Fibroblast Proliferation ........ 182 5.3.7 Proliferating Cells are Associated with Fibrotic Lung Tissue .......... 182 5.3.8 Phosphorylated STAT3 and α-SMA Co-localise in Fibrotic Foci .... 182 5.3.9 Gp130-STAT3 Signalling Regulates Fibroblast-Myofibroblast Differentiation .................................................................................................. 185 5.3.10 Directed Gp130-Mediated Signalling Regulates TGF-β-Induced Smad3 Responses ......................................................................................... 188 5.3.11 IL-6 and IL-11 Modulate TGF-β1-Induced Fibroblast Proliferation .......................................................................................................................... 189

11

5.3.12 Gp130 Signalling Mediates TGF-β1-Induced Fibroblast-Myofibroblast Differentiation .......................................................................... 189

5.4 Discussion .......................................................................................................... 195

CHAPTER SIX ......................................................................................................... 201

GENERAL DISCUSSION ....................................................................................... 201

6.1 General Discussion ........................................................................................ 202

6.1.1 Introduction ............................................................................................ 202 6.1.2 Directed Gp130-STAT3 Signalling Regulates Lung Remodelling .. 203 6.1.3 Directed Gp130-STAT3 Links Inflammation to Fibrosis .................. 205 6.1.4 Gp130-Mediated Signalling Regulates Fibroblast Growth .............. 210 6.1.5 Gp130-Mediated Signalling Regulates Fibroblast Phenotype ........ 212 6.1.6 Directed Gp130-STAT3 Inhibits TGF-β-Induced Smad3 Signalling214

6.2 Future Studies ................................................................................................... 216

References .............................................................................................................. 221

12

ABBREVIATIONS

1o primary

2o secondary

3o tertiary

α-SMA alpha smooth muscle actin

μl microlitre

μM micromoles per litre

Ab antibody

ABTS 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)

BAL broncho-alveolar lavage

BALF broncho-alveolar lavage fluid

bp base pair

BrdU brominated deoxy-uridine

BSA bovine serum albumin

C-HLF HLF derived from control patients

DAB di-amino benzidene

ddH2O double distilled water

dH2O distilled water

DMEM Dulbecco’s modified eagles medium

DNA deoxy-ribose nucleic acid

EDTA ethyl-diamine tetra-acetic acid

ELISA enzyme linked immunosorbent assay

Fb fibroblast

GKN glucose potassium sodium buffer

Gy grays

H&E haemotoxylin and eosin

13

HLF human lung fibroblast

HMW high molecular weight solution

ICC immunocytochemistry

IHC immunohistochemistry

IL-6 interleukin-6

IL-11 interleukin-11

IL-6Rα interleukin-6 alpha receptor

i.p. intra peritoneal

IPF idiopathic pulmonary fibrosis

IPF-HLF HLF derived from IPF patient

i.v. intra venous

L litre

LF lung fibroblast

M moles per litre

MBB methylene blue binding assay

ml millilitre

MLF transformed mouse lung fibroblast

MSM minimal serum media

MTT methyl tetrazolium salts

ng nanogram

NGM normal growth media

PBS phosphate buffered saline

PBS-1%BSA 1% w/v BSA supplemented PBS

PBS-T 0.05% v/v Tween 20 supplemented PBS

PCR polymerase chain reaction

PFA paraformaldehyde

14

PLF primary lung fibroblast

Pn1 type 1 pneumocyte

Pn2 type 2 pneumocyte

rpm revolutions per minute

RNA ribose-nucleic acid

SDS sodium dodecyl sulphate

S-HRP streptavidin conjugated horse-radish peroxidase

SV40 simian virus 40

T0 initial timepoint

T1 first timepoint

TBS Tris buffered saline

TBS-0.05T 0.05% v/v Tween 20 supplemented TBS

TBS-0.2Tx 0.2% v/v Triton-X 100 supplemented TBS

TE tris EDTA buffer

TGF-β transforming growth factor beta

Thy1+/- thymocyte antigen 1 positive/negative

TMB 3,3’,5,5’-tetramethyl benzidine

TUNEL terminal deoxy-uridine nick-end labelling

UIP usual interstitial pneumonia

15

CHAPTER ONE

LITERATURE REVIEW

16

1.1 Overview

Idiopathic pulmonary fibrosis (IPF), also know as cryptogenic fibrosing alveolitis

(CFA), is debilitating disease that is always fatal. Current knowledge about this disease

is limited with no effective treatment available to patients with the disease. There is

marginal gender bias towards males, which has lead to many investigators to speculate

there is an occupation related cause to the disease (White et al., 2003, Knight et al.,

2003). While other lung diseases such as pneumoconiosis, asbestosis and anthracosis

have been linked to inhalation of mineral dusts, asbestos and coal dust, no such link

exists with IPF (Mossman and Churg, 1998, White et al., 2003, Knight et al., 2003).

The focus of research into IPF over the last 25 years has been on the link between

inflammatory mediators and the development of fibrosis (Kolb et al., 2001, Keogh and

Crystal, 1982, Haslam et al., 1990, McAnulty et al., 1997, Bonniaud et al., 2005). The

cytokine interleukin-6 (IL-6), is an inflammatory mediator that is enhanced during

periods of inflammation and has been associated with a variety of lung diseases (Olman

et al., 2004, Lesur et al., 1994, Asokananthan et al., 2002, Kuhn et al., 2000). More

recently IL-6, and other members of this cytokine family, have been implicated in the

development and progression of IPF (Pantelidis et al., 2001, Knight et al., 2003,

Moodley et al., 2003b, Moodley et al., 2003a, Scaffidi et al., 2002). This thesis will

examine the role the common intracellular signalling pathways, of the IL-6 family of

cytokines in a mouse model of IPF.

1.2 Fibrosis

Tissue fibrosis can be defined as an excessive deposition of extracellular matrix

(ECM) components that results in the destruction of normal tissue architecture and a

compromise in organ function (Wynn, 2007, Noble, 2003, Border and Noble, 1994). If

17

fibrosis occurs in the major organs such as lung, liver, kidney or heart, it will inevitably

lead to organ failure and death of the individual. Fibrosis is a feature of many

pulmonary disorders including cancer and tuberculosis, where it is secondary to the

primary lesion. However, in asthma and a variety of interstitial pneumoniae, fibrosis is a

significant cause of morbidity and mortality.

The development of fibrosis is thought to occur after a variety of stimuli including

infection, injury or chronic inflammation (Wynn, 2007). Regardless of the initial

aetiology, the common feature of fibrotic diseases is an accumulation of fibroblasts,

tissue remodelling and enhanced interstitial collagen deposition. Clearly, the normal

fine control of cell-cell and cell-ECM interactions is disturbed. Determining where this

control is lost is paramount to finding ways of preventing and treating this condition.

1.3 Collagen Metabolism

The distinguishing feature of fibrosis in all organ systems is the accumulation of

ECM, specifically collagen. Collagen is the most abundant protein found in the ECM

and the vertebrate body (Cutroneo, 2003, Trojanowska et al., 1998). The collagens

comprise of 19 different proteins that are all composed of a triple helical domain

containing three polypeptide chains (Trojanowska et al., 1998). These polypeptide

chains are termed α-chains and consist of repeating amino acid triplet of glycine-X-Y,

where every third X is a proline and every third Y is hydroxyproline (Miller, 1985). The

collagen family is segregated into fibrillar collagens, non-fibrillar collagens, short-chain

collagens and fibril-associated collagens with interrupted triple helices (FACIT) (Shaw

and Olsen, 1991). The fibrillar collagens are the most abundant in the mammalian body

and consist of collagen types I, II, III, V and XI. The primary role of these collagens is

to confer tensile strength to the tissue. In fibrosis the dysregulated accumulation of type

I collagen compromises normal organ function and eventually leads to organ failure.

18

Fibroblasts are the predominant cell type responsible for the synthesis of fibrillar

collagens. Collagen synthesis begins with the transcription of a pro-collagen α chain

gene to form mRNA which is then translated to pre-procollagen. This protein undergoes

co- and post-translational modifications including the hydroxylation of Y positioned

proline and lysine residues. The hydroxylation of prolines in the Y position is essential

for the stability of the triple helix, underhydroxylated procollagen is unable to form

stable triple helices and is susceptible to rapid degradation (Berg and Prockop, 1973,

Steinmann et al., 1981) . Final modifications to the procollagen peptide include

glycosylation of hydroxylysine residues and the transfer of mannose-rich

oligosaccharides onto the C-propeptides of fibrillar collagens. Triple helix formation

occurs through the alignment of three α-chains such that cysteine residues are

juxtaposed in the C-terminal propeptides (Engel and Prockop, 1991). This results in the

formation of intra- and interchain disulphide bonds between opposing lysine and

hyrdoxylysine residues. These initial bonds are strengthened by intra- and inter-

molecular cross-linking of lysine and hyrdoxylysine residues by lysyl oxidase (Siegel et

al., 1970). After this co- and post-translational modification procollagen molecules are

transported from the rough endoplasmic reticulum (RER) to the Golgi apparatus where

they are packaged into secretory vesicles. Following secretion the N- and C-propeptides

are enzymatically cleaved producing mature collagen fibrils (Miyahara et al., 1982,

Njieha et al., 1982).

Collagen undergoes a continuous cycle of synthesis and degradation throughout

life. Animal studies have estimated that at maturity approximately 10% of the total lung

collagen content is being synthesised and degraded each day (McAnulty and Laurent,

1987, Laurent, 1982). Newly synthesised collagen can be rapidly degraded within the

cell by two different pathways termed basal and enhanced degradation (Berg et al.,

1980, Bienkowski, 1984, Bienkowski, 1989). Basal degradation refers to the level of

19

degradation that occurs when conditions are right for optimal hydroxylation of proline

residues. It was recently shown that this is a post-translational event that occurs within

lysosomes (Gotkin et al., 2004). Enhanced degradation is dependent upon cathepsins B,

D and L and occurs when cellular conditions prevent proline hydroxylation and stable

triple helix formation (Berg et al., 1980). For a protein with such an important structural

role it is advantageous to the organism to be able to utilise degradative pathways that

are adaptable, such as basal degradation, and ensure protein quality is maintained, such

as enhanced degradation.

Mature collagen fibrils secreted into the ECM are highly resistant to proteolytic

degradation. In the lung it is believed that mature collagens may not be degraded under

normal circumstances. However, during infection or injury inflammatory cells and

mesenchymal cells are capable of synthesising and releasing a battery of proteinases

that are capable of degrading collagen in the ECM. These proteinases include the matrix

metalloproteinases (MMPs), a family of proteinases that is comprised of interstitial

collagenases, gelatinases and stromelysins. The activity of these enzymes is tightly

regulated by the tissue inhibitors of metalloproteinases (TIMPs). These antiproteinases

are synthesised by most mesenchymal cells and macrophages and form irreversible

covalent complexes with the active forms of MMPs (Cawston, 1996).

Collagen fibrils form vital structural components to all tissues in the vertebrate

body. The formation and maintenance of these fibrils is dependent upon hydroxylation

of proline and lysine residues for the formation of stable triple helices and collagen

fibrils. However, collagen is routinely subject to intra- and extracellular degradation as a

consequence of maintaining tissue structure or due to infection and injury. An

understanding of the processes involved in collagen synthesis and degradation is vital to

improve our knowledge of collagen associated diseases such as fibrosis.

20

1.4 Epidemiology and Clinical Characteristics of Idiopathic Pulmonary Fibrosis

IPF is a one of the idiopathic interstitial pneumoniae (IIP), that are more broadly

recognised as diffuse lung parenchymal diseases (DPLDs) (Maher et al., 2007). The

IIPs are a heterogeneous group of progressive and largely untreatable disorders of

unknown aetiology (ATS, 2000, Maher et al., 2007). The incidence of IPF is

approximately 7 per 100,000 females and 10 per 100,000 males. It typically occurs in

people over the age of 60, with the incidence and death rate increasing with age (ATS,

2000, Baumgartner et al., 2000, Coultas et al., 1994, Knight et al., 2003, Mannino et al.,

1996, Schwartz et al., 1994b). IPF describes a heterogeneous group of disorders

including usual interstitial pneumonia (UIP), non-specific interstitial pneumonia

(NSIP), respiratory bronchiolitis interstitial lung disease (RB-ILD), cryptogenic

organising pneumonia (COP), desquamative interstitial pneumonia (DIP) and lymphoid

interstitial pneumonia (LIP) (White et al., 2003). Of the subtypes of IPF, UIP is

typically associated with extensive and persistent lung fibrosis and has a worse

prognosis. It is for these reasons that all subsequent discussion on IPF will be in

reference to UIP.

Potential risk factors for the development of IPF have been investigated including

cigarette smoking, occupational exposure and viruses (Baumgartner et al., 2000,

Baumgartner et al., 1997, Billings and Howard, 1994, Egan et al., 1997, Vergnon et al.,

1984). Genetic and environmental factors have also been investigated, however links

between any aetiological agent or genetic factor and IPF have only been determined in a

minority of cases (Hubbard and Venn, 2000, Zamo et al., 2005).

The symptoms of IPF, such as dyspnea upon exertion and non-productive cough,

are present, on average, for two years prior to diagnosis (Selman et al., 2001).

Pulmonary function tests reveal inspiratory crackles, restrictive impairment, reduced

diffusing capacity for carbon monoxide and arterial hypoxemia exaggerated or elicited

21

by exercise (ATS, 2000, Guerry-Force et al., 1987).Chest radiography and high

resolution computed tomography (HRCT) show patchy, predominantly peripheral, sub-

pleural, lower lung zone opacities (Guerry-Force et al., 1987, ATS, 2000). HRCT also

shows variable but limited ground glass opacities and sub-pleural honeycombing.

The histologic characteristics of UIP are a heterogenous appearance at low

magnification with alternating areas of normal lung, interstitial inflammation,

fibroblastic foci, dense fibrosis and honeycomb change (Selman et al., 2001). These

features are most severe in the peripheral subpleural parenchyma (ATS, 2000, Selman et

al., 2001).

A definitive diagnosis of IPF-UIP requires the exclusion of known causes of

interstitial lung disease and a surgical lung biopsy showing UIP (Perez et al., 2003). For

accurate diagnosis of UIP it is crucial that histological and radiological assessments

confirm UIP. The median survival of patients with a histologic pattern of UIP and an

indeterminate HRCT pattern is approximately 6 years, yet the prognosis for patients

with concordant histological and HRCT patterns of UIP is worse as their median

survival is only two years (Flaherty et al., 2003).

The end stage of the disease is characterised by abnormal parenchymal tissue

remodelling; increased collagen synthesis and deposition, decreased collagenolytic

activity, aberrant re-epithelialisation and angiogenesis, with consequent progressive

destruction of normal lung tissue and loss of normal lung function (King et al., 2001,

Selman et al., 2001).

1.5 Genetics of IPF

Studies have identified genetic mutations involved in rare sporadic and familial

cases of IPF. However, no causative genetic factors or aetiological agents have been

identified in the majority of disease cases (Scott et al., 1990, Lawson et al., 2004,

22

Hubbard and Venn, 2000). Furthermore, there does not appear to be a sex linkage, since

the male/female ratio is close to 1:1 (Schwartz et al., 1994a, Hubbard and Venn, 2000).

Familial IPF has also been described with the onset of disease occurring during

childhood (Thomas et al., 2002). This disease has been associated with mutations in

SPC that impair pro-SPC processing by the Golgi apparatus and retention of this protein

within the endoplasmic reticulum (Thomas et al., 2002). A number of other familial

disorders are associated with the development of pulmonary fibrosis, for example the

development of pulmonary fibrosis associated with Hermansky-Pudlak syndrome has

been frequently reported (Davies and Tuddenham, 1976, Gahl et al., 1998).

Genetic studies examining the aetiology of pulmonary fibrosis have focused on

known genetic loci with a high degree of polymorphism and those involved in the

inflammatory response (Awad et al., 1998, Pantelidis et al., 2001, Vasakova et al., 2007,

Whyte et al., 2000). Of these, the HLA system located on chromosome 6 has been the

most widely studied (Musk et al., 1986, Falfan-Valencia et al., 2005). However, none of

these studies have been conclusive and the majority have provided conflicting results.

Microsatellite instability and loss of heterozygosity has been demonstrated in DNA

from patients with familial IPF, suggesting that allelic imbalance may occur in this

disease (Vassilakis et al., 2000). The loss of heterozygosity was shown to occur in

microsatellite markers immediately adjacent to putative tumour suppressor genes

(Demopoulos et al., 2002). A recent report has documented several mutations in the

telomere reverse transcriptase (TERT) gene present in IPF patients with a family history

of adult-onset IPF (Tsakiri et al., 2007). These mutations included five missense and

two frame-shift deletions that resulted in the expression of a defective TERT. Further

examination of patients with IPF found they had significantly shorter telomeres than

unaffected family members (Tsakiri et al., 2007). Given that the relative risk of

developing lung cancer is seven times higher if IPF is also diagnosed, these data suggest

23

that DNA damage or dysfunctional repair may be involved in the pathogenesis of the

disease (Demopoulos et al., 2002, Hubbard et al., 2000).

1.6 Pathogenesis of IPF

As the precise mechanisms leading to the development of IPF are unknown,

several different hypotheses on its pathogenesis have been proposed. It is generally

believed that IPF is the result of chronic inflammatory processes or a disturbance in the

normal communication between epithelia and the mesenchyme (figure 1.1) (Selman et

al., 2001, Mason et al., 1999). The inflammation hypothesis for the development of IPF

is driven by initial observations of increased inflammatory cells in the broncho-alveolar

lavage fluid (BALF) of IPF patients relative to normal subjects (Merrill and Reynolds,

1983). This hypothesis proposes sub-types of the disease that show evidence of active

inflammation, such as DIP and NSIP, represent early lesions in the disease’s

progression towards UIP (Keogh and Crystal, 1982). In these forms of IPF, alveolar

septa may be thickened by a sparse inflammatory infiltrate but this lesion rarely

contains fibroblastic foci or the extensive fibrotic pattern observed in UIP (Selman et

al., 2001). Empirical data challenges this hypothesis, as current anti-inflammatory

treatments for UIP, including immunosuppressants and corticosteroids, appear to be

only marginally effective. The survival rate of patients after three years of these

treatments is only 50% (Monaghan et al., 2004). Furthermore, the inflammatory

component of early UIP is usually mild and is focussed around regions of collagen

deposition or honeycomb change. Recent studies have determined that alveolitis and

interstitial inflammation are not predictive of survival. Rather, it is the fibroblastic foci

that are typical of UIP are the best prognostic indicator of patient survival (Monaghan et

al., 2004).

dysregulated wound repair implicating enhanced and persistent inflammation,

apoptosis and impaired re-epithelialisation. Adapted from Selman et al., Ann. Intern.

24

Epithelial Damage and Inflammation

Wound

Fiba

Myofibroblast Foci Formation

Progenitor Cell

ation

Clot

Recruitment

Impaired Re-epithelias

Fibrosis

Epithelial

Figure 1.1. Pathogenesis of Idiopathic Pulmonary Fibrosis.

Postulated mechanisms for the development and progression of IPF have focused on

dysregulated fibroblast growth, persistance of myofibroblasts in discrete foci, epithelial

Med. 2001.

roblast Migration nd Proliferation

Apoptosis

PDGF

IL-6 TNF-α

TGF-β

1.6.1 Inflammation and IPF

The inflammation hypothesis proposes that IPF is the result of a chronic

inflammatory response to an exogenous insult that culminates in a progressive fibrosis.

Numerous studies have found that the synthesis of pro-inflammatory cytokines, such as

tumour necrosis factor (TNF)-α and interleukin-1β, is associated with the development

of IPF and animal models of progressive fibrosis (Riha et al., 2004, Whyte et al., 2000,

Kolb et al., 2001, Fujita et al., 2003, Liu et al., 2001). The success of corticosteroid and

immunosuppressive therapies in treating autoimmune-induced granulomatoses, and the

association of inflammatory cytokines with fibrosis, lead to the implementation of these

treatments for IPF (Noble, 2003).

25

.

l.,

e inflammation hypothesis has been refined to reflect emerging data from IPF

patien tory

arin

A

ravis,

t

three months of age that developed into progressive fibrosis and premature death by five

Animal studies have shown it is possible to dissociate the inflammatory response

from the development and progression of fibrosis. An early study using interleukin-10

(IL-10) deficient mice in a silica-induced model of pulmonary fibrosis demonstrated

enhanced lung inflammation with less fibrosis than wild-type mice (Huaux et al., 1998)

In the bleomycin model of pulmonary fibrosis, IL-10 deficient mice had enhanced

inflammation without any subsequent change in the fibrotic response (Kradin et a

2004). The dissociation of inflammation from fibrosis has been reported in other

studies, the most significant by Sime and colleagues (Sime et al., 1997). By using a

replication deficient adenovirus to deliver active transforming growth factor (TGF)-β1,

Sime et al. were able to induce a progressive fibrosis with only a mild inflammatory

response. Furthermore, ex vivo studies have demonstrated fibrosis can develop and

progress when the lung is maintained in a blood free environment (Adamson and

Bowden, 1974).

Th

ts and animal models of fibrosis and suggests that enhanced Th2 inflamma

cytokine activity drives the development of tissue fibrosis (Ando et al., 1999, Barb

et al., 2005, Wang et al., 2000a, Wang et al., 2000b). The identification of viral DN

within lungs of IPF patients and the presence of fibrosis-resistant and fibrosis-prone

strains of in-bred mice, have contributed to the development of this hypothesis. The

Balb/C mouse strain has been characterised as fibrosis-resistant and are concordantly

“Th2 tolerant”. This characterisation is due to the propensity of Balb/C mice to produce

enhanced levels of Th2 cytokines (IL-4, IL-13) and IgE in response to allergens,

compared to the fibrosis-prone and “Th2 intolerant” C57BL/6 strain (Haston and T

1997, Kolb et al., 2002, Ma et al., 2006). Furthermore, over-expression of an IL-4

transgene in the lungs of C57BL/6 mice produced an unremitting inflammatory lesion a

26

in

, although seven fold less

than C57BL/6 transgenic mice, but there was no progression to fibrosis (Ma et al.,

2006).

Enhancing the Th inflammatory response through over-expression of an

interferon-γ (IFN-γ) transgene has transferred fibrosis-resistance to an otherwise

fibrosis-prone mouse strain. In addition, deletion of downstream IFN-γ signalling

molecules exacerbated bleomycin-induced lung fibrosis in fibrosis-prone strains (Chen

et al., 2001, Giri et al., 1986, Walters et al., 2005). These studies led Ziesche et al.

(1999) to postulate that IPF patients may have an IFN-γ deficiency and so subsequently

investigated the efficacy of IFN-γ treatment for IPF. The preliminary study found that

12 months of IFN-γ treatment significantly improved lung function in IPF patients

(Ziesche et al., 1999). A more comprehensive follow up study involving 330 IPF

patients revealed that IFN-γ treatment over 58 weeks did not significantly improve

survival or pulmonary function (Raghu et al., 2004). However, no studies have

demonstrated any difference in survival between patients given anti-inflammatory

treatments and patients on immunosuppressant treatments.

is of IPF, and UIP in particular, is that the

lesion at

an et al.,

months of age (Ma et al., 2006). However, when the same transgene was expressed

Balb/c mice, an enhanced inflammatory response was noted

1

1.6.2 Wound Healing and IPF

An emerging theory on the pathogenes

develops and progresses as a result of abnormal wound healing. It is thought th

the wound healing response in IPF patients differs from normal wound healing because

of inadequate re-epithelialisation and dysregulated myofibroblast activity (Selm

2001). The epithelium functions as a barrier to injury and invasion by pathogenic

organisms. Perturbation of this barrier exposes the underlying basement membrane and

vasculature to oxidative damage and pathogenic organisms (Noble, 2003). The

association of mutations in the surfactant protein-C (SPC) gene and familial forms of

27

nuded area occurs rapidly. This process is initiated

throu

e not

ed

tzenstein,

me investigators have observed that epithelial

cells derived from IPF lungs produced larger amounts of pro-apoptotic proteins than

cells derived from control lungs (Maeyama et al., 2001). Furthermore, it is known that

lung fibroblasts isolated from IPF patients secrete molecules that induce apoptosis of

respiratory epithelia, particularly TGF-β and angiotensin (Hagimoto et al., 2002, Wang

et al., 1999, Kuwano et al., 2000b).

Examination of patients with familial IPF found that the dysfunctional epithelium

enhances apoptosis of Pn1 and Pn2, and inhibition of Fas-Fas ligand and Caspase

apoptosis pathways abrogates bleomycin-induced lung fibrosis (Kuwano et al., 2000a,

Thomas et al., 2002, Wang et al., 1999). Therefore, the hyperplastic Pn2 may represent

an inherent defect in the pulmonary epithelia, persistent re-injury of the epithelium or

dysregulated epithelial-mesenchymal interactions (Noble, 2003). The impaired

restoration of the epithelium may serve as an activation signal to the adjacent

mesenchyme and initiate the formation of fibroblast and myofibroblastic foci.

IPF illustrate how defects in normal type 1 and type 2 pneumocytes (Pn1 and Pn2

respectively) function progress to fibrosis (Thomas et al., 2002). It is crucial that

complete re-epithelialisation of the de

gh local mechanisms such as epithelial cell migration, proliferation and

differentiation, and activation of the adjacent mesenchyme. If these mechanisms ar

sufficient to resolve the injury, then systemic mechanisms, including the activation of

bone marrow and the recruitment of circulating and tissue derived progenitor cells, are

utilised (Noble, 2003, Selman et al., 2001).

In IPF the local healing mechanisms appear to be impaired, as there is mark

damage to and loss of Pn1 cells and hyperplasia and apoptosis of Pn2 cells (Ka

1985, Kasper et al., 1995, Kawanami et al., 1982, Falfan-Valencia et al., 2005,

Maeyama et al., 2001). Apoptosis of damaged or defective cells is required for the

resolution of normal tissue function and so

28

Alter ir

ar injury

IPF

ous

ra

naghan et al., 2004, Cool et al., 2006).

Monaghan et al., 2004, Cool et al., 2006). These myofibroblasts represent a contractile

cell phenotype and are believed to be responsible for the large increase in collagen

observed in IPF and animal models of lung fibrosis (Kuhn and McDonald, 1991, Zhang

et al., 1996, Zhang et al., 1997). However, it is well understood that fibroblast

populations within the lung are diverse and multi-functional. Fibroblasts expressing the

thymocyte 1 antigen (Thy1 ) constitutively synthesise large amounts of interstitial

collagen, which can be enhanced by cytokines and growth factors, while Thy1

fibroblasts display a more proliferative phenotype in vivo in response to cytokines and

growth factors including TGF-β1 and IL-6 (Derdak et al., 1992, Hagood et al., 2002,

Silvera et al., 1994). The features of Thy1 and Thy1 fibroblasts are frequently

natively, dysregulated mesenchymal responses to re-epithelialisation may impa

the restoration of the alveolar epithelium.

1.6.3 Fibroblast Phenotypes in IPF

A prominent feature of IPF is the accumulation and proliferation of fibroblasts

within distinct fibrotic foci of the lung parenchyma (Katzenstein and Myers, 1998, King

et al., 2001). It is postulated that these fibrotic foci are sites of persistent alveol

and activation associated with evolving fibrosis (Katzenstein and Myers, 1998, Kuhn et

al., 1989, Monaghan et al., 2004, Selman et al., 2001). Interest in these foci has

increased in recent years with the observation that the abundance of these foci in

correlates with poor prognosis (King et al., 2001, Monaghan et al., 2004). Histological

and morphometric analyses of these foci has determined that, while appearing as

distinct and isolated foci, these fibroblastic bodies are composed of a heterogene

population of cells that form a tightly connected reticulum extending from the pleu

into the lung parenchyma (Mo

A large number of fibroblasts within this reticulum express α-smooth muscle

actin (α-SMA), a defining characteristic of myofibroblasts (Zhang et al., 1996,

+

-

+ -

29

re

st

pheno

et al.,

.,

with

of

s within fibrotic foci are derived from the bone marrow

(Hash

observed within fibroblastic foci confounding attempts to identify a proto-typical

fibrotic fibroblast phenotype.

Desmouliere et al. (2003) proposed the existence of three different phenotypes of

fibroblast; the homoeostatic or quiescent fibroblast that served as a progenitor for

subsequent phenotypes depending upon the stimulus, the proto-myofibroblast that

readily proliferated and did not express α-SMA, and the myofibroblast that produced

abundant collagen, expressed α-SMA and possessed contractile properties (Desmoulie

et al., 2003). While our current understanding of the role these different fibrobla

types have in IPF is unclear, there is significant evidence suggesting that lung

fibroblasts in IPF are different to normal lung fibroblasts (McAnulty et al., 1997,

Moodley et al., 2003a, Moodley et al., 2003b, Ramos et al., 2001, Vancheri

2000).

The presence and abundance of myofibroblasts in IPF lungs appears to be

increased compared to normal lungs (Ramos et al., 2001, Moodley et al., 2003a).

Isolation of myofibroblasts from IPF tissue has revealed that these cells are slow

growing and more likely to apoptose, but secrete higher amounts of collagen type I,

TGF-β1, and gelatinase B (MMP-9) compared to normal lung fibroblasts (Ramos et al

2001). Furthermore, it has been shown that myofibroblasts obtained from patients

IPF have a diminished capacity to synthesise and release prostaglandin E2 (PGE2)

(McAnulty et al., 1997). This eicosanoid inhibits fibroblast proliferation and reduces

collagen deposition. Together these findings suggest that the homeostatic balance

fibroblast-myofibroblast populations in the lung is altered in patients with IPF.

Recent animal studies using different fibrotic stimuli have found that 80-95% of

the collagen producing cell

imoto et al., 2004, Epperly et al., 2003). It is unlcear if these cells are

myofibroblasts as some reports describe them as α-SMA positive (Epperly et al., 2003,

30

T+)

lly

humans with chronic asthma and bronchiolitis obliterans. Fibroblast

progenitor cells home to injury sites in the airways of chronic asthma patients and then

ducing fibroblasts; chimerism has been identified within

mese

ated

F-β1

l.,

ve therapies for human diseases is limited

by ou

ot”

Broekema et al., 2006) while another report has described them as α-SMA negative

(Hashimoto et al., 2004). These cells expressed telomere reverse transcriptase (TER

(Hashimoto et al., 2004, Lama and Phan, 2006, Phillips et al., 2004), which is typica

a feature of proliferating cells and a common feature of cells within the fibroblastic foci

(Nozaki et al., 2000, Liu et al., 2007). Cells of extra-pulmonary origin have been

observed in

differentiate into collagen pro

nchymal cells of lung transplant recipients from gender mismatched donors

(Schmidt et al., 2003, Suratt et al., 2003, Brocker et al., 2006). Recently, elev

numbers of circulating fibroblast progenitors have also been identified in patients with

IPF (Mehrad et al., 2007)

While these TERT+ fibroblasts did not constitutively express α-SMA,

differentiation to a myofibroblast phenotype was induced by IL-4. Interestingly TG

stimulation of TERT+ fibroblasts could not induce this differentiation (Nozaki et a

2000, Liu et al., 2002). It is not clear whether these different fibroblast phenotypes

represent discrete phases in the linear progression from a quiescent fibroblast to an

“activated” myofibroblast or if they are different endpoints of fibroblast differentiation.

More importantly, the role of these different fibroblasts phenotypes in IPF is still

uncertain.

1.7 Animal Models of IPF

Progress in the development of effecti

r ability to assess the continuum from normal tissue to diseased tissue.

Researchers typically assess this progression histopathologically providing a “snapsh

of the disease, or as isolated cells in 2-dimensional tissue culture systems. The use of

31

us

lung

the

e acute pulmonary lesions that

resolv

fect. In particular, viral-mediated transfer of TGF-β or IL-1β

has p

g

lower mammalian models of human disease has given medical science a tremendo

insight into the pathogenesis of cancer, genetic and infectious diseases. In order to

understand the pathogenesis of a disease such as IPF it is necessary to use model

systems that mimic the cellular heterogeneity and structural features of the human

and its responses to a variety of stimuli. Animal models of IPF need to reproduce

key features of this disease such as inflammation, fibroblast proliferation, formation of

myofibroblast foci, accumulation of fibrillar collagens and tissue remodelling. A variety

of methods have been used to induce these key features of IPF including

pharmacological agents (bleomycin and fluorescein isothiocyanate), inorganic particles

(silica and asbestos), gene transfer and over-expression (TGF-β, IL-1β and GM-CSF),

and gamma irradiation (Chua et al., 2005).

These methods in general are better at emulating the fibrosis seen in idiopathic

interstitial pneumonia than IPF per se, as they produc

e over time, unlike the chronic and unremitting characteristics of IPF. Inorganic

particles such as asbestos and silica are more appropriate models of occupational

diseases such as asbestosis and silicosis. Gamma irradiation of the rodent thorax

produces a lesion more akin to pneumonia seen in patients undergoing radiation therapy

for the treatment of thoracic tumours. More recently, gene transfer and gene over-

expression methods have been used to induce pulmonary fibrosis and mimic the

features of IPF to great ef

roduced pulmonary lesions that are very similar to UIP and emulate the chronic

features of IPF (Sime et al., 1997, Kolb et al., 2001). However, the dissemination of

these methods is limited, so commercially available pharmacological agents, includin

bleomycin and fluorescein isothiocyanate (FITC), have proven to be useful methods for

modelling the fibrosis observed in UIP. The lesions induced by these drugs are not

32

e

ion,

t

er

tment is the dose-dependent induction of pulmonary

fibrosis which causes death in 3% of affected patients (Chen and Stubbe, 2005,

.

nary

d in

1.8 In

ry

completely comparable to those seen in IPF, as they develop over a relatively short tim

and are resolved over time without any intervention.

While bleomycin-induced rodent pulmonary fibrosis is an acute resolving les

it has been successfully used to elucidate the mechanisms involved in the developmen

and progression of pulmonary fibrosis and IPF. Bleomycin is a potent anti-cancer drug

that is used for the treatment of testicular cancer and some types of lymphoma; howev

a frequent complication of this trea

Horowitz et al., 1973).

Bleomycin was first used experimentally to induce pulmonary fibrosis in 1970 by

a Japanese group, with the first English language report of experimental bleomycin-

induced pulmonary fibrosis in 1971 (Okamoto et al., 1970, Fleischman et al., 1971)

Adamson et al. (1974) were the first to report the pathogenesis of bleomycin-induce

pulmonary fibrosis in mice after i.v. delivery (Adamson and Bowden, 1974). In recent

years, researchers have adopted endotracheal and trans-nasal delivery methods of

bleomycin to model a pneumo-centric mechanism for the development of pulmo

fibrosis. Bleomycin-induced murine pulmonary fibrosis has been used alone an

combination with transgenic and knock-in/knock-out mice to elucidate the role of

inflammatory mediators such as IL-1β, TNF-α, IFN-γ and IL-6, and growth factors

including PDGF, GM-CSF and TGF-β in the pathogenesis of IPF (Cavarra et al., 2004,

Fujita et al., 2003, Zhao et al., 2002, Adamson and Bowden, 1974, Chen et al., 2001,

Giri et al., 1986, Liu et al., 2001).

terleukin-6 and IPF

IL-6 is a cytokine produced by a variety of cells including epithelia, inflammato

cells and fibroblasts. Clinical studies have reported that IL-6 levels are elevated in the

33

y

volun

IL-6

udies have suggested that the normal actions of IL-6 are altered in

patients with IPF (Moodley et al., 2003a, Moodley et al., 2003b). The proliferation of

) was inhibited by IL-6 but the proliferation of

HLFs

as

tients

sera of patients with pulmonary fibrosis, and specifically IPF compared to normal

individuals (Tsantes et al., 2003, Scala et al., 2004). Lesur et al. (1994) have reported

that levels of IL-6 are increased in the BALF of coal workers with symptoms of lung

disease (Lesur et al., 1994). More rigorous examination of these patients found that

those with IPF had enhanced levels of IL-6 in their BALF compared to health

teers and patients with pneumoconiosis. The increase in BALF IL-6 was

determined to be due to increased secretion of this cytokine into the alveolar epithelial

lining fluid and correlated with increased alveolar cellularity and neutrophil numbers

(Lesur et al., 1994). Genetic analysis has found that a polymorphism within in the

gene, intron 4A/G, is associated with IPF and is independently linked to reduced lung

carbon monoxide diffusion capacity; a physiological indicator of IPF (Pantelidis et al.,

2001).

IL-6 is released by cells upon inflammatory stimuli, such as TNF-α and IL-1α/β,

and mitogenic stimuli, such as platelet-derived growth factor (PDGF) (Olman et al.,

2004, Olman et al., 2002, Kohase et al., 1987). There is also emerging evidence on the

ability of IL-6 to alter fibroblast phenotype and actions (Olman et al., 2004, Moodley et

al., 2003a, Moodley et al., 2003b). Thy1+ and Thy1- fibroblasts express the IL-6 α-

receptor (IL-6Rα) and, while IL-6 is not directly mitogenic for human lung fibroblasts

(HLF), blocking IL-6-mediated actions with function blocking antibodies, ablates the

mitogenic effects of IL-1β on HLFs (Olman et al., 2004, Fries et al., 1994).

Recent st

HLFs from control patients (C-HLFs

from patients with IPF (IPF-HLFs) was increased by IL-6 (Moodley et al.,

2003a). Similarly, Fas-ligand-induced apoptosis of C-HLFs from control patients w

enhanced by IL-6 and Fas-ligand-induced apoptosis was reduced in IPF-HLFs pa

34

of these

/2

n

s cyclin

rylation of retinoblastoma associated protein (pRb) (Moodley

et al.,

y

),

r

T-1,

(Moodley et al., 2003b). While there was no difference in the expression of the IL-6 co-

receptor glycoprotein-130 (gp130) between C-HLFs and IPF-HLFs, the ability

cells to activate IL-6-induced signalling pathways was altered. Upon stimulation of IPF-

HLFs with IL-6 there was prolonged activation of the extracellular regulated kinase 1

(ERK1/2) cytoplasmic signalling pathway (Moodley et al., 2003a). Prolonged activatio

of this pathway was associated with the expression of the pro-mitotic molecule

D1, cyclin E1and phospho

2003a). A corollary of the enhanced expression of pro-mitosis molecules was an

increase in anti-apoptosis protein Bcl-2 (B-cell leukaemia/lymphoma protein) (Moodle

et al., 2003b).

1.9 The IL-6 Family of Cytokines

The IL-6 family of cytokines is a group of cytokines that are produced by a

variety of cells in response to inflammatory stimuli and cell differentiation. These

cytokines are related in both structure and function and include IL-6, leukaemia

inhibitory factor (LIF), oncostatin M (OSM), IL-11, ciliary neurotrophic factor (CNTF

cardiotrophin-1 (CT-1), cardiotrophin like cytokine (CLC), IL-27, IL-31 and

neuropoietin (NPN) (Rose-John et al., 2006, Heinrich et al., 2003, Knight et al., 2003).

These seemingly diverse cytokines share a weak structural homology, however thei

effects on target cells are mediated through binding to a specific receptor and forming

complexes with the ubiquitous signal transducer gp130 (Heinrich et al., 1998,

Kishimoto et al., 1995).

The receptors of the IL-6 family cytokines can be divided into non-signalling α-

receptors (IL-6-Rα, IL-11Rα, CNTF-R, WSX-1 and CT-1R) and signal transducing β-

receptors (LIFR, OSMR and IL-31R) (figure 1.2) (Heinrich et al., 1998, Heinrich et al.,

2003, Pflanz et al., 2002, Colgan and Rothman, 2006). IL-6, IL-11, CNTF, NPN, C

35

30 or

o

ctivate

signal

ines to

IL-

d

during

embr

et

CLC and IL-27 bind to specific α-receptors and induce homo-dimerisation of gp1

hetero-dimerisation of gp130 with a β-receptor (IL-27 recruits the IL-12 β-receptor) t

activate cytoplasmic signalling pathways (Rose-John et al., 2006, Heinrich et al., 1998).

LIF and OSM bind their β-receptors directly, which bind to gp130 and a

ling pathways, however IL-31 binds to its own β-receptor which dimerises with

the OSMR to activate intracellular signalling (Heinrich et al., 2003, Rose-John et al.,

2006). The specific cytokine and receptor interactions allow this family of cytok

elicit specific responses from target cells. However, the shared use of the common

signal transducer gp130 explains the overlapping and compensatory properties of IL-6

cytokine family members (Knight et al., 2003, Tang et al., 1996, DiCosmo et al., 1994,

Rose-John et al., 2006).

IL-6 and IL-11 are unique because they are the only members of this family that

signal exclusively through gp130 homo-dimers and are therefore completely dependent

on the intracellular domains of gp130 to initiate intracellular signalling events (Heinrich

et al., 2003, Heinrich et al., 1998). The gp130 binding sites of IL-6/IL-6Rα and

11/IL-11Rα complexes, while distinctive, overlap and activate qualitatively an

quantitatively identical signalling events in vitro (Kurth et al., 1999, Moodley et al.,

2003a). This similarity in biophysical and biochemical properties explains some of the

functional redundancy observed between these two cytokines.

Gene knockout studies have identified some distinct roles for these cytokines in

vivo (Robb et al., 1998, Nandurkar et al., 1997, Xing et al., 1998, Xing et al., 1994,

Kopf et al., 1994). While large amounts of these cytokines are released

yogenesis, IL-6-/-, IL-11αR-/- and LIF-/- embryos develop normally and reach

maturity without any overt abnormalities. Conversely, OSMR-/- and LIFR-/- embryos fail

to develop beyond 6.5 days post coitum (Kopf et al., 1994, Stewart et al., 1992, Robb

al., 1998). Interestingly, LIF-/-, OSM-/- and IL-11Rα-/- dams are infertile with embryos of

36

GP130IL-11R

GP130

IL-1

1

GP130IL-6R

GP130

IL-6

GP130

OSMR

OS

M

GP130

LIFR

LIF

GP130

LIFR

OS

M

GP130CT-1R

LIFR

CT

-1

GP130CNTFR

GP130

CN

TF

GP130CNTFR

LIFR

CN

TF

GP130CT-1R

LIFR

CLC

GP130CNTFR

LIFR

NP

N

IL-31R

OSMR

IL-3

1

GP130WSX-1

IL-12R

I

Gp1

-

-R

/ L-27

Gp1

30O

SM

R-

X

30G

p130

G

p130

LIF

Fig

.n-

6 C

kia

.

The

ly

yne

s re

en

ecr

ac

tce

llul

6 a

ab

soly

n

gp13

0 c

ang

pLC

, LIF

M b

R

hete

wit

to a

ctiv

nl

sca

lsnd

O

SMR

c h

er

30 t

prom

oti

eal

ling.

Ind

s aα

p-1

2Rer

otrim

ova

intr

lll

-bi

nd’s

sf

c w

hich

form

ei

wo

van

thw

ays.

Ada

pte

fr H

einr

ic a

m.

J.

ure

12.

The

Inte

rleu

ki

IL-6

fam

iof

cto

kilu

tede

pend

ent u

poro

dim

ers

h gp

130

e nt

rac

llula

r sig

ns i

tpe

ciic

reep

tor

yto

ne F

mily

quire

the

mm

bra

e sp

anni

ng c

o-r

epto

gp1

30 to

tiva

e in

trato

ativ

ate

sign

lliat

hway

s, w

hile

CN

TF, N

PN, C

T-1,

Cat

e i

trace

lula

rig

nalli

ng. O

SM

n a

o bi

the

gp13

0-lik

e L-

27 b

i sp

ecifc

-r

ece

tor a

nd fo

rms a

gp1

30-I

L h

ets a

hte

rod

mer

ith

the

OSM

R t

act

ite

sign

alli

g pa

ar si

gnal

ling

path

way

s. IL

-nd

11

are

and

OS

ind

to th

eLI

F a

nd fo

rm

whi

h fo

rms a

tero

dim

ew

ith g

p1o

er t

act

ite

ac

eul

ar si

gnal

ing

and

IL31

d

omh

etl.

(199

8) B

ioch

e

ly

prolif

red by

P) and serum amyloid A (SAA) levels in

sera after challenge with turpentine, Listeria monocytogenes, or LPS. Levels of pro-

GM-CSF and IFN-γ were increased in sera over

24 ho

ble to

)

37

the former failing to implant into the uterus and the latter embryos degenerate rapid

after implantation due to defective decidua formation (Stewart et al., 1992, Robb et al.,

1998). IL-6-/- and IL-11Rα-/- mice are born in the expected Mendelian ratio and yet

upon maturation IL-6-/- mice display deficiencies in circulating T-lymphocytes and

thymocytes, without any change in the proportion of T-cell populations and circulating

polymorphonuclear leukocytes (Nandurkar et al., 1997, Xing et al., 1998, Kopf et al.,

1994). The reduction in circulating T-cells in IL-6-/- mice corresponds with lower

numbers of T-cell progenitors and stem cells in the bone marrow and reduced

eration of the bone marrow stroma (Rodriguez Mdel et al., 2004). In contrast, IL-

11Rα-/- mice undergo normal haematopoiesis with no significant pathology of major

organs described to date (Nandurkar et al., 1997).

Predictably, IL-6-/- mice have an impaired acute phase response as measu

haptoglobin (HP), α1-acid glycoprotein (α-AG

inflammatory cytokines TNFα, MIP-2,

urs after intraperitoneal injection of lipo-polysaccharide (LPS), and anti-

inflammatory IL-10 was decreased compared to wt controls (Xing et al., 1998). Upon

immunological challenge, IL-6-/- mice were unable to generate a response compara

IL-6+/+ controls. The generation and activity of cytotoxic T-cells in vaccinia virus (VV

challenged IL-6-/- mice was up to 10 fold lower than wild-type (wt) controls, while viral

titres were approximately 1000 fold higher in the lungs. Macrophage and T-cell

interactions were assessed in response to L.monocytogenes infection, and similar to

vaccinia virus, listerial titres were up to 1000 fold higher in the spleen and liver of IL-6-

/- mice. There was no significant reduction in the bactericidal activity of IL-6-/-

macrophages as assessed by nitrite release.

38

ody secreting plasma cells

(Uytt bers of

f

ctions

to

invol at

ified a

When IL-6 was first identified and characterised, it was found to induce the

proliferation of B-cells and induce their maturation into antib

enhove et al., 1988, Nemunaitis et al., 1989). Interestingly, baseline num

plasma cells and natural serum antibody levels were not affected by systemic deletion o

IL-6 but IL-6-/- mice were defective in mucosal IgA responses. T and B cell intera

in IL-6-/- mice were assessed after infection with vesicular stomatitis virus (VSV),

examine T-cell independent IgM production, and VV to examine T-cell dependent IgG

production. There was a 10 fold decrease in IgG production over the course of VV

infection in IL-6-/- mice compared to wt, yet early IgM levels were comparable between

genotypes (Kopf et al., 1994, Xing et al., 1998). This suggests IL-6 secretion by T-cells

is required for the maturation of B-cells into IgG producing plasma cells and the

maintenance of a humoral immunity.

While these initial studies suggested that deficiencies in IL-6 production may be

ved in the exacerbation of lymphocytoses, more recent studies have suggested th

IL-6 production may be involved in the pathogenesis of diseases associated with acute

and chronic injury (Ezure et al., 2000, Johnston et al., 2005, Brandt et al., 1990). In a

model of obstructive hepatic biliary disease, IL-6-/- mice had a significantly higher

mortality rate than wt littermates (51% versus 23% respectively) after bile duct ligation

(Ezure et al., 2000). IL-6-/- mice also had a liver morphology that was distinct from

littermate controls and characterised by a relative decrease in the number of hepatocytes

compared to mesenchymal and biliary epithelial cells. These changes corresponded to a

decrease in total liver weight and liver to bodyweight ratio. Interestingly, IL-6-/- mice

had significant leakage of bile duct contents, specifically conjugated bilirubin, into the

circulation suggestive of significant hepatocyte death (Ezure et al., 2000).

While initial investigation of the IL-6 family of proteins suggested their role was

the activation of the acute inflammatory response, subsequent studies have ident

39

se

l Transducer and Activator of

Trans

e

of

ll

en et

alling

e residues within the cytoplasmic tail of human gp130 are

phosp d

ed

variety of activities regulated by these proteins. These proteins are now known to

mediated uterine implantation of mammalian blastocysts, placenta formation,

lymphopoiesis and wound healing responses.

1.10 Gp130-Mediated Signalling

Three intracellular signalling molecules have been found to bind homo-dimerised

gp130; the first being the protein tyrosine phosphatase Src-Homology 2 Phosphata

(SHP-2), the second is the transcription factor Signa

cription (STAT) 1 and STAT3 is the third (figure 1.3). The first stage of gp130-

mediated intracellular signalling is the phosphorylation of membrane proximal tyrosin

at position 683 (Tyr683) and the activation of a signal transducing complex composed

Janus Kinase (JAK) 1, JAK2 and Tyrosine Kinase (TYK) 2 anchored to the internal ce

membrane (Stahl et al., 1994, Lutticken et al., 1994). The JAK1-JAK2-TYK2 complex

becomes phosphorylated upon homo/hetero-dimerisation of gp130 and induces

phosphorylation of tyrosine residues within the cytoplasmic tail of gp130 (Luttick

al., 1994, Stahl et al., 1994). JAK1 is the key regulator of gp130-mediated sign

events, as deletion of this protein impairs the activation of downstream signalling

promoted by IL-6 (Rodig et al., 1998, Guschin et al., 1995).

Five tyrosin

horylated by the JAK1-JAK2-TYK2 complex (Tyr759, Tyr767, Tyr814, Tyr905 an

Tyr915) and induce downstream signalling through the recruitment of SHP-2 and

STAT1/3 (figure 1.3) (Stahl et al., 1995). SHP-2 is phosphorylated by the second

tyrosine residue proximal to the cell membrane, which is part of a four amino acid motif

Tyr-X-X-Valine (YxxV), and interacts with the adaptor proteins Grb-SOS to activate

the Ras-Extracellular Regulated Kinase (ERK) 1/2 cascade. STAT1/3 is phosphorylat

by the remaining four distally located tyrosine residues that are part of the four amino

40

Figure 1.3. IL-6 and IL-11-mediated intracellular signalling.

IL-6 and IL-11 bind to their specific α-receptor and induce homodimerisation of gp130. Homodimerisation of gp130 induces phosphorylation of the membrane bound JAK1/2-TYK2 heterotrimer, activating tyrosine kinase motifs (pY) in the cytoplastail of gp130. Specific pYs phosphorylate SHP2 and STAT1activates the ERK1/2 signalling cascade via the membrane b

mic /3. Phosphorylated SHP2 ound adaptor proteins Sos-

Grb2, Raf and Ras, and the cytoplasmic kinase MEK1/2. Phosphorylated STAT1/3 mers, inducing phosphorylation of serine residues before

leus. ERK1/2 and STAT1/3 bind to IL-6/11 response elemprom of

forms either hetero or homodiit is transported into the nuc

ents (IL-6/11RE) and acute phase resonse elements (APRE) encoded in the oters of target genes. The pathways are inhibited ( ) by dephosphorylation

STAT1/3 by SHP2, PIAS1/3 inhibition of STAT1/3 nuclear translocation and DNAbinding, and inhibition of JAK1/2-TYK2 and pY actions by SOCS1/3.

ERK1/2

MEK1/2

Ras Raf Sos-Grb2

SHP2

STAT1/3

P Tyr

Gp1

30

Gp1

30

IL-6/11Rα

IL-6/11

PIAS1/3

Ser STAT1/3

PTyr P

Ser STAT1/3

PTyrP

SOCS1/3

APRE TARGET GENESIL-6/11RE

-759-

-767-

-812-

-904-

-914-

JAK1/2-TYK2

JAK1/2-TYK2

pY

pY

pY

pY

pY

pY

pY

pY

pY

pY

PIAS1/3

ptional

r

tokine

rotein family consists of eight members.

SOCS

ain

ond

ding

130,

ibition of

e

/2

41

acid motif Tyr-X-X-Glutamine (YxxQ). Phosphorylated STAT1/3 is translocated to the

nucleus upon homo/hetero-dimerisation and induces transcription via the transcri

coactivator CBP/p300 (Stahl et al., 1995, Baumann et al., 1994).

The termination of gp130 signal transduction can occur through 3 independent

mechanisms that involve either protein phosphatases, inhibition of activated proteins o

negative feedback. The canonical negative feedback regulators of gp130-mediated

signalling are the SOCS proteins. This family of proteins have been termed the cy

inducible SH2 proteins (CIS), STAT-induced STAT inhibitors (SSI) and the suppressor

of cytokine signalling (SOCS). The SOCS p

1 and SOCS3 are induced by IL-6 and terminate gp130-mediated signals through

inhibiting the phosphorylation of JAK proteins and the actions of STAT1 and STAT3

respectively (figure 1.3) (Naka et al., 1997, Starr et al., 1997). SOCS1/3 actions require

unique effector sites in the JAK-gp130 complex. SOCS1 binds to the activation dom

of JAKs via its Src-Homology 2 (SH2) domain to inhibit its kinase activity. A sec

SOCS1 motif, the kinase inhibitory region (KIR), engages with the substrate bin

site of JAK to further inhibit the catalytic properties of the JAK kinase domain

(Yasukawa et al., 1999). Similar to SHP-2, SOCS3 binds directly to pTyr759 of gp

yet SOCS3 has a higher binding affinity for gp130 and can therefore dislodge SHP-2

from gp130 (De Souza et al., 2002). However, the mechanism of SOCS3 inh

gp130 signal transduction is not clear as it has also been reported to bind JAK2 through

its SH2 domain (Sasaki et al., 1999, Sasaki et al., 2000). In addition to their inhibitory

effects on JAKs, the SOCS proteins have a role in the proteosomal degradation of th

ligand-receptor-gp130 complex through interaction with E3-ubiquitin ligases and

specifically elongins B and C (Zhang et al., 1999).

While SHP-2 is thought to activate signal transduction through the Ras-ERK1

pathway, the inherent phosphatase activity of SHP-2 has implicated it in the negative

42

A family of proteins known as protein inhibitors of activated STATs (PIAS)

consists of five members that are critical regulators of the JAK-STAT pathway. These

proteins appear to inhibit the dimerisation of STAT proteins and their subsequent

nuclear translocation by preventing phosphorylation of Ser727 (Chung et al., 1997, Liu et

al., 1998). It appears that each PIAS protein inhibits the effects of specific STAT family

proteins. Thus PIAS1 inhibits STAT1 and PIAS3 inhibits STAT3 DNA binding and

gene expression (Chung et al., 1997, Liu et al., 1998). While initial reports suggested

that PIAS-mediated actions were located in the cytoplasm, recent studies are suggesting

that at least some members of this family are located in nuclear bodies and exert their

effects after nuclear translocation of dimerised STATs (Tan et al., 2002, Sachdev et al.,

2001, Liu and Shuai, 2001).

ing

regulator of gp130-mediated signalling (Sengupta et al., 1998). Mutation of tyrosine

759 of human gp130 to phenylalanine leads to enhanced IL-6, OSM and LIF-induced

signal transduction and expression of a dominant-negative SHP-2 promotes enhanced

receptor, JAK and STAT phosphorylation (Symes et al., 1997, Kim et al., 1998,

Schaper et al., 1998, Lehmann et al., 2003). The identification of SHP-2-STAT3

complexes and the characterisation of SHP-2 as a phosphatase of pTyr701 and

phosphorylated serine 727 (pSer727) of STAT1 suggests that dephosphorylation of

STAT proteins may occur through interaction with SHP-2 (Wu et al., 2002, Gunaje and

Bhat, 2001).

1.10.1 SHP-2-ERK Signalling

Tyrosine phosphatases have a crucial role in reversing the phosphorylation of

tyrosine residues of membrane bound receptors and intracellular signalling molecules.

However, tyrosine phosphatases can also stimulate intracellular signalling by remov

inhibitory phosphorylation events (Saxton et al., 1997, Noguchi et al., 1994). One

particular protein is the ubiquitous non-transmembrane tyrosine phosphatase SHP-2, an

43

e

an

though

os showed signs of retarded growth compared to wt littermates (Saxton et

al., 1

1.10.2 STAT1/3 Signalling

IL-6 family cytokines activate STAT proteins through their common signal

transducer gp130. Dimerisation of gp130 with either an IL-6 family β-receptor or

another gp130 molecule induces phosphorylation of STAT1 and STAT3. STAT1 and

STAT3 form homo or hetero-dimers that translocate to the nucleus and activate

transcription of specific target genes. STAT3 is unique amongst the STAT family of

proteins as genetic depletion of this molecule is embryonically lethal at E7.0. Embryos

lacking STAT3 are phenotypically normal up to E6.0 but begin to degenerate at E6.5

with no sign of mesoderm formation (Takeda et al., 1997). In order to elucidate the

function of STAT3, in vivo over-expression methods and tissue-specific deletion

techniques have been used (Takeda et al., 1999, Takeda et al., 1998, Hokuto et al., 2004,

Maritano et al., 2004, Alonzi et al., 2001).

One such study induced the deletion of STAT3 specifically in liver once the mice

had matured. Liver specific deletion of STAT3 markedly reduced the transcription of

abbreviation of Src homology-2 phosphatase, which binds directly to transmembrane

receptors linking cytokine/growth factor activities to the Ras-ERK signalling cascad

(Bennett et al., 1996, Li et al., 1994). SHP-2 is an absolute requirement for mammali

embryonic development, with knockout mouse embryos surviving up to E9.5, al

these embry

997). Further characterisation of SHP-2 null embryos indicated these mice had a

defect in gastrulation and were unable to form functional mesoderm structures such as

the notochord. In vitro assays have demonstrated that SHP-2 signalling modulates cell

morphology through organisation of the cytoskeleton (Inagaki et al., 2000). Over-

expression of a catalytically inactive form of SHP-2 in rat fibroblasts dramatically

increased filamentous actin expression and cell-ECM adhesion but impaired their

migration towards ECM components (Inagaki et al., 2000).

44

e anti-

inflam

survi

d

n of

se

c

sts

l

.

acute phase inflammatory proteins such as haptoglobin, α1-acid glycoprotein, the serum

amyloids A (SAA) and P (SAP), and α-2 macroglobin promoted by LPS and IL-6

(Alonzi et al., 2001). However, the absence of STAT3 in the liver caused LPS to

increase the release of inflammatory cytokines IL-6, TNFα, IL-1β and th

matory cytokine IL-10 (Alonzi et al., 2001). STAT3 exists as two different

isoforms, STAT3α and STAT3β, as the result of alternative splicing of the gene

encoding STAT3 during transcription. Early studies identified STAT3β as a dominant

negative form of STAT3α (Schaefer et al., 1995, Caldenhoven et al., 1996).

Investigation into the physiological actions of these, apparently disparate, isoforms by

genetic disruption in the mouse, demonstrated that both proteins have distinctive roles

(Maritano et al., 2004). During pre-natal development, lack of one isoform can be

compensated by the presence of the other as both STAT3α−/− mice and STAT3β-/-

develop normally and survive birth. However, these isoforms appear to have distinctive

roles in post-natal development and survival as STAT3α-/- mice die 16 hours post-

partum with symptoms of respiratory distress and cyanosis, while STAT3β-/- mice

ve birth, are fertile and have a normal life expectancy. STAT3β-/- mice had an

enhanced inflammatory response to LPS, characterised by increased IL-6 and TNF, an

reduced IL-10 secretion by peritoneal macrophages and inflammatory cell infiltratio

tissues, compared to wt mice. LPS treatment produced thickened alveolar septa, inten

congestion and inflammatory infiltration of the lungs. Maritano et al. (2004) further

demonstrated that STAT3α was required to elicit an IL-6 type response from embryoni

fibroblasts. Gene-array analysis of IL-6 stimulated STAT3α-/- embryonic fibrobla

demonstrated that a number of IFN-γ target genes were upregulated, rather than typica

IL-6 target genes (Maritano et al., 2004)

45

tigations into the physiological role of the inhibitors of

this pathway have found that SOCS1/3 and PIAS1 proteins are required for embryonic

3 signalling are inhibited by the

PIAS

ined to

N-γ

effects and

produ et al.,

et al.,

a and

induced by gp130-mediated

signal

001).

lects its induction by several

1.10.3 Inhibition of Gp130-Mediated Signalling

Tight regulation of gp130-mediated signalling is imperative for normal

mammalian development. Inves

development. Gp130-STAT1 and gp130-STAT

1/SOCS1 and PIAS3/SOCS3 proteins respectively.

Mice homozygous for deletion mutations of SOCS1 (SOCS1-/-) die early in

neonatal life with fatty degeneration and hepatic necrosis, lymphocytic and

polymorphonuclear infiltrates of the lung, liver, skin, pancreas, and gut (Starr et al.,

1997, Marine et al., 1999b, Starr et al., 1998). These characteristics were determ

be IFN-γ dependent as treatment of SOCS1-/- mice with neutralising antibodies to IF

and breeding of SOCS1-/- and IFN-γ null mice (IFNγ-/-) ameliorated these

ced healthy adult mice (Marine et al., 1999b, Alexander et al., 1999, Bullen

2001). These features appear to be due to the loss of SOCS1 in bone-marrow-derived

inflammatory cells. Chimeric mice were generated by transplanting SOCS1-/- bone

marrow into SOCS1+/+ animals, and inflammatory foci composed of lymphocytes and

polymorphonuclear cells were observed in the lung, liver and kidney (Metcalf

2003). Interestingly, recipients of SOCS1-/- bone marrow developed pneumoni

alveolar thickening in addition to cuffing of bronchi and bile ducts with fibroblasts

(Metcalf et al., 2003). While the synthesis of SOCS1 is

ling its role in vivo appears to be the regulation of IFN-γ induced STAT1

signalling.

Normal physiologic levels of SOCS3 are an absolute requirement for embryonic

development, as neither SOCS3-/- nor transgenic SOCS3TG mouse embryos are viable

(Takahashi et al., 2003, Marine et al., 1999a, Sasaki et al., 2000, Roberts et al., 2

The fine balance of SOCS3 required for development ref

46

horm

tor

8).

when

with IL-6

g

5, and

y

ependent activities of other growth factors and cytokines.

-/- mice

ones, growth factors and cytokines required for homeostasis; SOCS3 negatively

regulates erythropoietin (EPO), growth hormone (GH), leptin, insulin-like growth fac

(IGF) 1, IL-1 and IL-6 (Sasaki et al., 2000, Paul et al., 2000, Bjorbaek et al., 199

Macrophages derived from foetal livers of SOCS3-/- mice have enhanced pSTAT3

stimulated with IL-6, yet other STAT3 activators such as IL-10 do not produce

enhanced pSTAT3 in SOCS3-/- cells. This indicates that SOCS3 inhibition of STAT3

activation is specific to IL-6 (Lang et al., 2003). Targeted deletion of SOCS3 from

murine liver (SOCS3-/ΔHep) and macrophages (SOCS3-/ΔMac) has been achieved, by loxP-

Cre-induced homologous recombination, to evaluate the physiological significance of

SOCS3 deficiency (Croker et al., 2003). Upon intra venous (i.v.) stimulation

SOCS3-/ΔHep and SOCS3-/ΔMac mice have prolonged and enhanced STAT3

phosphorylation (pSTAT3) (Croker et al., 2003).

Elevated levels of SOCS 3 have been observed in allergic diseases and increasin

levels of SOCS3 correlate with disease severity (Seki et al., 2003). Expression of a

SOCS3 transgene in T-lymphocytes (SOCS3TL-TG), biased Th cell differentiation

towards a Th2 phenotype, characterised by enhanced antigen induced IL-4 and IL-

decreased IFN-γ production (Seki et al., 2003). These characteristics of transgenic T-

lymphocytes were reversed by T-lymphocyte expression of a dominant negative SOCS3

(Seki et al., 2003). This evidence outlines a role for SOCS3 in modulating inflammator

responses through inhibiting the pro-inflammatory effects of IL-6 without interrupting

the STAT3 d

In vivo studies assessing the physiological role of PIAS1 found that PIAS1

are not born at the expected Mendelian ratio (12.9%) from PIAS1+/- parents, however

examination of E17.5 embryos found that approximately 25% of pups were PIAS1-/-

(Liu et al., 2004). This suggests that the interaction of PIAS1 with other loci is critical

for perinatal survival. Cells isolated from PIAS1-/- mice exhibited an increase in mRNA

47

emokine

ination of

of

ely than “strong” STAT1 DNA binding sites such as those in the IRF1 promoter.

t

s.

to

expression of STAT1 target genes such as T-lymphocyte chemokines CXC ch

ligand 9 (CXCL9) and CXCL10, but there was no effect on the proto-typical STAT1

target gene interferon regulatory factor 1 (Irf1) (Liu et al., 2004). Further exam

this observation revealed that PIAS1 was capable of inhibiting STAT1 DNA binding

so-called “weak” STAT1 binding sites located in the CXCL9/10 promoters more

effectiv

At present there is little data on the biochemical role of PIAS3 and no data on the

physiological role of this protein. However it is clear that PIAS3 specifically inhibits

DNA binding of STAT3 and has been implicated in the progression of malignancy

(Chung et al., 1997, Junicho et al., 2000). A recent report has found that PIAS3 can

engage promoters containing Smad binding elements suggesting that PIAS3 induces the

transcription of Smad target genes (Long et al., 2004). Further characterisation of this

result found that PIAS3 bound to the C-terminal of Smad3 and formed a ternary

complex with the transcriptional coactivator CBP/p300, which could be enhanced by

TGF-β stimulation (Long et al., 2004).

The necessity for homeostatic levels of SOCS1/3 during embryonic developmen

has hindered progress into understanding the physiological role of these protein

However, targeted deletion and expression of these molecules indicates there is

similarity between the phenotypes of SOCS1/3 deficient mice and mice that over-

express IL-6 family cytokines.

1.11 IL-6, IL-11 and Pulmonary Physiology

Studies manipulating the global expression level of genes and their translation

protein provide useful insights into systemic physiology. Yet, these approaches do not

address the tissue specific manner in which many proteins are able to modulate organ

function and local responses to injury, inflammation and infection. The role of IL-6

48

se the unique characteristics of lung biology.

ither

-

lls were

arkers of T-

lymp

al

n,

6). These subtle differences

in airway structure elicited significant differences in airways hyper-responsiveness upon

family proteins and downstream gp130-mediated signalling events have been

investigated in vivo through the use of lung specific gene delivery systems or gene

targeting strategies that utili

While IL- 6 family proteins promote the synthesis and release of proteins

associated with the acute inflammatory response, this cytokine appears to modulate

immune-mediated responses to infectious particles and tissue repair mechanisms (Wang

et al., 2000a, Johnston et al., 2005, Kida et al., 2005). Constitutive expression of e

IL-6 (IL-6CC10-TG) or IL-11 (IL-11CC10-TG) in lung epithelium induces similar

morphological alterations in transgenic mice including airspace enlargement,

peribronchial mononuclear aggregates and enlargement of the bronchiolar lumen (Tang

et al., 1996, DiCosmo et al., 1994, Kuhn et al., 2000).

Characterisation of peribronchial aggregates found them to be composed of

lymphocytic lineage cells, while there was little reactivity for macrophage lineage

markers such as Mac-1. The majority of cells within the aggregates were positive for B

lymphocyte markers MHC-II and B220, while a significant proportion of ce

identified as positive for the markers CD3, CD4 and CD8, typical m

hocytes (Tang et al., 1996, DiCosmo et al., 1994).

In IL-6CC10-TG mice, the increase in bronchiolar lumen diameter was proportion

to increases in thickness of the bronchiolar wall (Kuhn et al., 2000). In IL-11CC10-TG

mice the increase in bronchiolar wall thickness was greater than the increase in

bronchiolar lumen diameter (Kuhn et al., 2000). This increase in bronchiolar wall

density was characterised by enhanced subepithelial and adventitial collagen depositio

particularly collagen type III (Tang et al., 1996, Kuhn et al., 2000). Furthermore, the

airways of IL-11CC10-TG mice showed enhance reactivity for the smooth muscle and

myofibroblast markers desmin and α-SMA (Tang et al., 199

49

change in airways hyper-

respo

in

s of IL-6-/- mice compared to control mice. This reduction in airway

sTNF ein

n et

n

methacholine stimulation; IL-6CC10-TG mice had no

nsiveness while there was a significant increase in IL-11CC10-TG mice upon

methacholine challenge compared to wt control mice (Kuhn et al., 2000).

Oxidative damage is a significant cause of tissue injury, evoking apoptosis and

inflammation. After 3 hours of ozone (O3) exposure there is a rapid reduction in the

amount of soluble tumour necrosis factor (TNF)-α receptors 1 and 2 (sTNFR1/2) with

the airway

R1/2 was enhanced after three days of exposure to O3 and although the prot

content of BALF was increased, it was still three times less than wt levels (Johnston et

al., 2005). This reduction in inflammatory proteins corresponded with a decrease in

neutrophil infiltration of the airways of IL-6-/- mice relative to control mice (Johnsto

al., 2005).

An alternative system has utilised the “tet-on-tet-off” system to express a

constitutively active form of STAT3 (STAT3C) in the mouse lung (Lian et al., 2005).

Upon activation of STAT3, oxidative damage was induced by exposure to hyperoxic

conditions (95% O2), and the tissue response was characterised by assessing changes i

gene expression, airways inflammation and tissue remodelling. STAT3C transgenic

mice had increased maximum survival relative to wt mice; 9 days and 7 days

respectively. Pulmonary changes due to hyperoxic conditions were assessed after 4

days. Wt mice had evidence of alveolar haemorrhage with increased capillary

permeability and damage of the endothelial-epithelial interstitial structure along the

alveolar surface. These structural changes were not evident in STAT3C expressing

mice.

LPS is a constituent of bacterial cell membranes such as E.coli and induces a Th1

type immune response. Administration of aerosolised LPS to IL-6-/- mice induced an

enhanced inflammatory response in the lungs compared to wt controls. Over a period of

50

. The

0a).

e

g

10-TG mice and was found to increase in wt animals

and w brosis

and

3

ays

three days IL-6-/- airways secreted approximately 2 fold more TNF-α and MIP-2 and

induced neutrophil infiltration into the airways (Xing et al., 1998). When a Th2 type

inflammatory response was induced by ovalbumin (OVA) sensitisation and challenge in

mice, IL-6 appeared to inhibit this process (Wang et al., 2000a). When challenged with

OVA, IL-6-/- mice had enhanced BALF cellularity with significantly more neutrophils,

eosinophils and lymphocytes compared to wt (Wang et al., 2000a, Qiu et al., 2004)

increase in the airways inflammation was also evident within the lung parenchyma of

IL-6-/- mice. In contrast IL-6CC10-TG mice had markedly less airways and tissue

inflammation and no detectable eosinophilia after OVA challenge (Wang et al., 200

The release of Th2 type cytokines IL-4 and IL-13 were up to 17 times higher in th

BALF of IL-6-/- mice compared to controls. The eosinophil chemokines IL-5 and

eotaxin, and MCP-1, were similarly up regulated in the airways of IL-6-/- animals

(Wang et al., 2000a, Qiu et al., 2004), and were lower in airways of IL-6CC10-TG mice

compared to wt after OVA challenge. This appeared to be a direct effect of IL-6 on

cytokine synthesis as mRNA levels had the same kinetic pattern as the proteins (Wan

et al., 2000a). Interestingly, the anti-inflammatory and pro-fibrotic molecule TGF-β1

was lowest in the BALF of IL-6CC

as highest in IL-6-/- mice (Wang et al., 2000a). However, the subepithelial fi

that is produced by chronic exposure to OVA was reduced in IL-6-/- mice compared to

controls (Qiu et al., 2004).

Studies have found higher levels of SOCS3 in peripheral T-lymphocytes of

patients with asthma and increasing levels of SOCS3 correlate with disease severity

serum IgE levels (Seki et al., 2003). Mechanistic studies using mice carrying a SOCS

transgene specifically in T-lymphocytes (SOCS3TL-TG) confirmed that enhanced

symptoms of OVA-induced atopic asthma correlated with SOCS3 levels (Seki et al.,

2003). Transgenic mice were four fold more sensitive to acetylcholine-induced airw

51

tion

ed

et

at are

ture B-lymphocytes to immunoglobulin secreting plasma-

cells, indicates that this cytokine is vital for a Th2 reaction.

1.12

02,

00).

the

the li

ice

hyper-responsiveness and had a two-fold increase in total inflammatory cell infiltra

of the airways. Th2 cytokines IL-4, IL-13, IL-5 and OVA-specific IgE were elevated in

the BALF of SOCS3TL-TG relative to wt littermates after OVA sensitisation (Seki et al.,

2003). The constitutive expression of SOCS3 in this system suggests the OVA-induc

phenotype of SOCS3TL-TG mice is independent of IL-6-STAT3-induced actions. In an

IL-6 dependent system of SOCS3 synthesis, such as SOCS3+/- mice, these

characteristics of OVA sensitivity were reduced (Seki et al., 2003).

These studies suggest that IL-6 decreases the Th2 type inflammatory reaction y

IL-6 does not simply promote an immunological inflammatory response and inhibit

injury-induced inflammation. Experiments enhancing IL-6 expression, through viral-

induced transient over-expression and constitutive expression of IL-6 in rodent lungs,

induce the formation of peribronchial and perivascular lymphoid aggregates th

primarily composed of B lymphocytes and CD4+/CD8+ T lymphocytes (DiCosmo et

al., 1994, Xing et al., 1994). The initial observation that IL-6 induces the proliferation

and differentiation of imma

Gp130-Mediated Signalling and Fibrosis

Clinical data and animal modelling suggests IL-6 family cytokines are involved in

the development of IPF and pulmonary fibrosis (Tang et al., 1996, Scaffidi et al., 20

O'Hara et al., 2003, Moodley et al., 2003a, Moodley et al., 2003b, Kuhn et al., 20

However, emerging data suggests that an IL-6/gp130 mechanism is involved in

development and progression of fibrotic diseases in other organ systems, particularly

ver.

The role of IL-6 in the development of biliary cirrhosis, a fibrotic disorder

associated with bile duct and gall bladder obstructions, was investigated in IL-6-/- m

52

in acute

ed an

es and

t

tant

lfpCre-flox) and from

other

ent

f

C

sies from

tive to controls

ensitive

al over

mice had collagen accumulation in the liver,

following bile duct ligation (BDL) (Ezure et al., 2000). There was no difference

liver injury after BDL between wt and IL-6-/- mice although there was significantly

higher mortality in IL-6-/- mice during the 12 week experimental period. BDL caus

increase in the mesenchymal cell volume of the liver compared to hepatocyt

biliary epithelial cells (Ezure et al., 2000). Similar results were observed by Streetz e

al. (2003). In this study, plasma levels of IL-6 were elevated in patients with acute and

chronic liver disease. They assessed the role of IL-6 in vivo by using a gp130 mu

mouse with gp130 deleted specifically from hepatocytes (gp130A

liver parenchymal cells (gp130MxCre-flox). Upon carbon tetrachloride (CCl4)-

induced liver fibrosis, both gp130 mutant mice developed a heightened fibrosis relative

to controls, however gp130MxCre-flox mice had a marked increase in total liver collagen

content over wt littermates and gp130AlfpCre-flox mice (Streetz et al., 2003). These results

suggest that IL-6/gp130 pathways in cells other than hepatocytes limit the developm

of liver fibrosis following injury.

More recent reports have described IL-6/gp130-mediated activities as promoting

the development and progression of liver fibrosis. By disrupting the SOCS1 or SOCS3

locus, several groups have enhanced STAT1/3 activation to assess the in vivo effects o

chronic activation of these molecules. In a study examining the progression of liver

fibrosis, SOCS1 deficiencies were found to correlate with progression of Hepatitis

virus induced liver fibrosis (HCF) (Yoshida et al., 2004). Examination of biop

liver fibrosis patients found that SOCS1 transcription was decreased rela

and this was due to methylation of the SOCS1 gene. SOCS1+/- mice were more s

to dimethylnitrosamine (DMN)-induced liver fibrosis than wt littermates. SOCS1

deficiency produced an increase in serum alanine transferase and reduced surviv

the 21 day observation period (Yoshida et al., 2004). Qualitative assessment of fibrosis

revealed that DMN treated SOCS1+/-

53

myof

(Yoshida et

ice had

s found SOCS3 levels are inversely

corre h the the

MN-

of

letion y

Cre-

sit

on d

OCS3

NA in liver tissue

(Ogata et al., 2006). These observations were not limited to the DMN-induced liver

fibrosis as examination of concanavalin-A (ConA)-induced liver fibrosis in the same

ibroblastic foci characterised by α-SMA expression and increased TGF-β1

synthesis (Yoshida et al., 2004). Mice were maintained on a choline deficient diet for 12

weeks to model chronic liver injury and assess fibrosis development. SOCS1+/- mice

developed fibrosis more rapidly and severely than wt littermates after four weeks and

liver collagen was approximately two fold greater than controls at 12 weeks

al., 2004). Molecular analysis of DMN-induced fibrosis showed SOCS1+/- m

increased pSTAT1, Irf1 and SOCS3, and decreased pSTAT3 and pERK1/2 in liver

biopsies (Yoshida et al., 2004).

Examination of patients with HCF ha

lated wit severity of fibrosis (Ogata et al., 2006). The role of SOCS3 in

development and progression of hepatic fibrosis has been assessed using the D

induced fibrosis in SOCS3+/- mice and mice with adenovirus-induced hepatic deletion

SOCS3 (AdCre-SOCS3flox/flox). Liver specific de of SOCS3 was achieved b

inserting loxP sites 5’ and 3’ of the gene (SOCS3flox/flox) and injecting an adenovirus

carrying the Cre-recombinase gene (AdCre-SOCS3flox/flox) or LacZ as a control

(AdLacZ-SOCS3flox/flox) i.v. Adenovirus has a natural tropism for the liver and the

recombinase excises DNA located between the loxP es. I.v. delivery of DMN to

SOCS3+/- and AdCre-SOCS3flox/flox reduced survival over the 30 day experimental

period compared to SOCS3+/+ and AdLacZ-SOCS3flox/flox controls (Ogata et al., 2006).

Assessment of fibrosis by liver collagen accumulati demonstrated SOCS3+/- mice ha

increased collagen compared to wt littermates and collagen was further enhanced in

AdCre-SOCS3flox/flox mice compared to AdLacZ-SOCS3flox/flox. Immunohistochemical

localisation of α-SMA showed abundant myofibroblastic foci in SOCS3+/- and S

deficient mice and this correlated with increases in TGF-β1 mR

54

umulation, increased TGF-β1 mRNA and α-

SMA

t

liver and hypoplasia of the myocardium

(Yosh

ly

ors

mice also showed enhanced collagen acc

localisation in liver tissue of ConA treated SOCS3 deficient mice compared to

controls (Ogata et al., 2006). Furthermore, adenoviral delivery of SOCS3 or a

dominant-negative STAT3 to simulate SOCS3 activity, ablated collagen accumulation

and TGF-β1 mRNA, and reduced α-SMA levels in liver tissue from DMN treated w

mice (Ogata et al., 2006).

1.13 Gp130 Mutant Mice

The role of gp130 dependent signalling pathways has been difficult to assess in

vivo as disruption of this gene leads to embryonic lethality due to significant reduction

in haematopoietic progenitors in the foetal

ida et al., 1996). Post-natal inactivation of gp130 leads to haematopoietic,

immunological, hepatic and pulmonary defects (Betz et al., 1998). In contrast,

constitutive activation of gp130 induces myocardial hypertrophy (Hirota et al., 1995).

While these studies have assessed the effects of systemic activation or inactivation

of gp130, the physiological effects of differential activation of these molecules has on

been assessed in recent years (Ernst et al., 2001, Ohtani et al., 2000). To assess the

effect of preferentially activating these pathways, Ernst et al. (2001) developed

targeting constructs that introduced a stop codon between the YxxV and membrane

proximal YxxQ motifs, truncating gp130 and ablating gp130-STAT1/3 signalling

(figure 1.4) (Jenkins et al., 2002). This colony of mice was suitably named

gp130ΔSTAT/ΔSTAT and developed a number of physiological abnormalities including

reactive synovitis, increased circulating leukocytes, reduced megakaryocyte precurs

and platelets, gastrointestinal ulceration, impaired wound healing and failure of

blastocyst implantation (Jenkins et al., 2002, Ernst et al., 2001).

55

xV

on with a phenylalanine (F),

de-activating SHP-2 phosphorylation activity of gp130 (figure 1.5) (Tebbutt et al., 2002,

Jenkins et al., 2005a). Similar to IL-6 transgenic mice, these mice (gp130757F/757F) had a

chronic inflammatory phenotype in the peritoneum, lung and liver characterised by

increased numbers of mononuclear cells and lymphocytes, and IL-6-induced serum

levels of the inflammatory markers haptoglobin and α1-acid glycoprotein. Circulating

neutrophils were more abundant in these mice than wt, as were megakaryocytes, bone

marrow and splenic progenitor cells (Jenkins et al., 2005b). Gp130 mice were

resistant to DSS-induced colonic injury and demonstrated enhanced migration and

proliferation of the colonic epithelium compared to wt and gp130 mice

(Tebbutt et al., 2002). In addition, T-lymphocyte trafficking was enhanced in

gp130 mice upon immunological challenge compared to wt and

gp130 mice (McLoughlin et al., 2005). This suggests the introduced

mutation, gp130 , enhanced the wound healing abilities and immune system of

these mice. However, they also had a clear reduction in their life expectancy to

approximately one year compared to over two years for wt littermate controls (Jenkins

et al., 2005a, Tebbutt et al., 2002).

Another group developed gp130 mutants by replacing endogenous murine gp130

with a hybrid gp130 molecule consisting of the murine extracellular and transmembrane

domains and the human intracellular domain (Ohtani et al., 2000). This group produced

knock-in mutant mice that expressed either a truncated gp130 that consisted of only the

extracellular domain (gp130 ), gp130 with hyper-morphic Shp2-ERK1/2

(gp130 ) or STAT1/3 (gp130 ) signalling. Both gp130 and

gp130 mice died as neonates within one day of birth similarly to STAT3α

knock-out mice (Ohtani et al., 2000, Maritano et al., 2004, Atsumi et al., 2002).

In a separate colony of mice, this group replaced the tyrosine residue of the Yx

motif necessary for gp130-mediated SHP-2 phosphorylati

757F/757F

ΔSTAT/ΔSTAT

757F/757F

ΔSTAT/ΔSTAT

757F/757F

D/D

FxxQ/FxxQ F759/F759 D/D

FxxQ/FxxQ

56

MEK1/2

Ras Raf Sos-Grb2

SHP2

JAK1/2-TYK2

-757-

JAK1/2-TYK2

pYpYG

p130

Gp1

30

IL-6/11Rα

IL-6/11

SOCS1/3

APRE TARGET GENESIL-6/11RE

Figure 1.4. IL-6 and IL-11-mediated intracellular signalling in gp130ΔSTAT/ΔSTAT

mice.

The intracellular domain of gp130 is truncated in gp130ΔSTAT/ΔSTAT mice, leaving only

specific α-receptor and induce homodimerisation of gp130, inducing phosphorylation

heterotrimer activates the pY in the cytoplasmic tail of gp130. This pY phosphorylat

proteins Sos-Grb2, Raf and Ras, and the cytoplasmic kinase MEK1/2. ERK1/2 binds

initiating transcription of these target genes. The pathway is inhibited ( ) through

the membrane proximal tyrosine kinase (pY) motif intact. IL-6 and IL-11 bind to their

of the membrane bound JAK1/2-TYK2 heterotrimer. Phosphorylated JAK1/2-TYK2 es

SHP2 and activates the ERK1/2 signalling cascade via the membrane bound adaptor

to IL-6/11 response elements (IL-6/11RE) encoded in the promoters of target genes,

SOCS1/3-mediated inhibition of JAK1/2-TYK2 and pY actions.

ERK1/2

57

G G

p130

p130

IL-6/11

IL-6/11Rα

STAT1/3

PTyr

-757-

-904-

-767-

-812-

-914-

JAK1/2-TYK2

pY

pY

pY

pY

pY

pY

pY

pY

PIAS1/3

Ser STAT1/3

PTyrP

Ser STAT1/3

PTyrP

SOCS1

APRE TARGET GENESIL-6/11RE

JAK1/2-TYK2

Figure 1.5. IL-6 and IL-11-mediated intracellular signalling in gp130757F/757F mice.

ithin the membrane proximal tyrosine kinase motif (pY) was lalanine (F) in gp130757F/757F mice. IL-6 and IL-11 bind to their

speciphos ylated

his

The tyrosine residue wsubstituted for a pheny

fic α-receptor and induce homodimerisation of gp130 and, susbsequently, phorylation of the membrane bound JAK1/2-TYK2 heterotrimer. Phosphor

JAK1/2-TYK2 activates the four remaining pYs in the cytoplasmic tail of gp130. These pYs phosphorylate tyrosines in STAT1/3, inducing the formation of STAT1/3 hetero or homodimers that dimerise and become phosphorylated at specific serine residues. STAT1/3 homo/heterodimers are transported to the nucleus, then bind to IL-6/11 response elements (IL-6/11RE) and acute phase resonse elements (APRE) encoded in the promoters of target genes, triggering transcription of target genes. Tpathway is inhibited ( ) by PIAS1/3 inhibition of STAT1/3 nuclear translocation and DNA binding, and SOCS1-mediated inhibition of JAK1/2-TYK2 STAT1 actions. STAT3 signalling escapes negative regulation by ERK1/2 and SOCS3 inhibition.

F F

PIAS1/3

58

these mice had

hyper

9

nd

6

ated in the

atures of the human disease including

incre ive

s

o

.,

Gp1307579/759 mice developed normally up to 11 weeks of age when they

developed splenomegaly and lymphadenopathy. By 5 months

trophic lymphoid organs in the spleen and the lymph nodes but not the thymus,

bone marrow or Peyer’s patches. As with gp130757F/757F and IL-6TG mice, gp130F757

mice displayed chronic inflammatory phenotype with approximately 3 fold more

mononuclear cells in the peritoneum, spleen and lymph nodes compared to littermate

controls. Serum levels of the IL-6/gp130-induced acute phase proteins SAA a

complement component C3 were elevated in gp130F759 mice, and stimulation with IL-

further increased the release of these proteins into the circulation compared to wt

littermates (Ohtani et al., 2000). This chronic inflammatory phenotype culmin

development of reactive arthritis in 100% of gp130F759 mice by 16 months (Atsumi et

al., 2002). This arthritis mimicked many of the fe

ased synthesis of IL-6 in the joints. However, the murine condition was distinct

due to foci of lymphocytes and plasma cells in the periarticular areas with little

infiltration of these cells in to the joints (Atsumi et al., 2002).

1.14 Transforming Growth Factor-β

Transforming growth factor (TGF)-β has a variety of physiological functions

including the modulation of wound repair, inflammation, immunity and tumourigenesi

(Border and Noble, 1994, Gauldie et al., 2006). Enhanced TGF-β levels are thought t

be the point of coalescences inflammatory and wound healing pathways to fibrosis

many tissues (Gauldie et al., 2006, Roberts et al., 1986, Connor et al., 1989, Border and

Noble, 1994, Verrecchia and Mauviel, 2007). Animal models of pulmonary fibrosis

have shown that TGF-β activity precedes the development of fibrosis, human studies

have found that TGF-β is increased in IPF and is localised to fibrotic foci (Khalil et al

59

into the

F-βRIII),

which er of

ways

1.14.1 Smad Signalling

The Smad pathway is specifically activated by TGF-β superfamily members

mily and consists of 8 proteins in mammals. Smad proteins are

approximately 500 amino acids in length and consist of one N-terminal Mad-homology

domain (MH1), a highly conserved C-terminal Mad-homology domain (MH2) and a

variable linker region located between the MH1 and MH2 domains (Massague et al.,

2005). Cytoplasmic signalling is mediated by receptor phosphorylation of C-terminal

1991, Broekelmann et al., 1991, Vanhee et al., 1994, Sime et al., 1997, Denton et al.,

2003, Whyte, 2003, Yoshida et al., 1995).

TGF-β belongs to the transforming growth factor-β family of growth factors of

which there are 5 vertebrate isoforms and 3 are expressed by mammals (TGF-β1, 2, 3).

This growth factor is synthesised as a large precursor molecule that is cleaved

active form and a latent associated peptide (LAP) that homo-dimerise (TGF-β-LAP) and

are secreted into the extracellular matrix (ECM). The TGF-β-LAP hetero-tetrameric

complex is bound by a latent TGF-β binding protein (LTBP) and stored in the ECM

(Gauldie et al., 2006). This large protein complex (TGF-β-LAP-LTBP) is cleaved to

produce active TGF-β by proteases such as matrix metalloproteinase-9 (MMP-9),

cathepsins, plasmin and integrins including αvβ6, following tissue injury or infection.

TGF-β binds to its type II receptor (TGF-βRII) which homo-dimerises and in turn binds

to type I receptor (TGF-βRI) dimers, including activin receptor-like kinase (ALK)-5.

This process can be facilitated by a 3rd receptor, the type III receptor (TG

regulates TGF-βRII binding of TGF-βRI, activation of TGFβRI, and turnov

the ligand receptor complex. Upon ligand binding, serine/threonine kinase domains

within the intracellular domain of TGF-βRI activates intracellular signalling path

that include the p38 and ERK1/2 mitogen activated kinase pathways, and the Smad

(SMA and MAD related protein) pathway (figure 1.6).

including the BMP subfa

60

CBP/p300

TG

F-βR

III

TG

RF

-βII

TG

F-βR

I

SBE TARGET GENES

TG

F-βR

IT

GR

F-β

II

pS/T

Smad7

LAP-LTBPTGF-β

TGF-β

pS/T

SARA

Smad4

Ser P

Smad2 Smad3Ser P

TGF-β

Proteases

Smad4

SerP

Smad2 Smad3Ser P

Smad2 Smad3

ERK1/2

p38 MAPK

Figure 1.6. TGF-β-Mediated Signalling.

TGF- ng peptide

odimer. The TGF-β/TGF-βRII heterotrimer induces the dimerisation eptor (TGF-βRI); this process is facilitated by the type III receptor

(TGF-βIII). Serine/threonine kinase domains (pS/T) within the cytoplasmic tail of TGF-βRI activate intracellular signalling events. Adapted from Massague et al. (2005), Genes Dev.

β is secreted into the ECM as a latent protein bound to a latent activati(LAP) and the latent TGF-β binding protein (LTBP). Proteases in the ECM cleave LAP-LTBP from TGF-β, TGF-β then binds to it’s type II receptor (TGF-βRII), which then forms a homof the type I rec

TBRE

ECM

Seri

lasmic

anslocates to the nucleus. The

C-ter

). The physical interaction of Smad2/3 with sequence specific

transcription factors and CBP/p300 activates the transcription of TGF-β target genes

(figure 1.6).

The protein-DNA and protein-protein interactions of Smad2 and Smad3 are

regulated by their MH1 and MH2 domains respectively. However, a 30 amino acid

insertion in the MH1 domain of Smad2 prevents it from binding to genomic DNA.

TGF-β promoted transcription is dependent upon Smad3 binding of the 4 base pair

minimal Smad-binding element (SBE) 5’-AGAC-3’ (Verrecchia et al., 2006, Massague

et al., 2005). TGF-β effects are terminated by the inhibitory Smad (I-Smad), Smad7.

This protein prevents Smad2/3 phosphorylation and nuclear translocation of the

Smad2/3-Smad4 complex, and recruits E3-type ubiquitin ligases to the ligand-receptor

complex leading to internalisation of the receptor and its subsequent degradation (figure

1.6) (Verrecchia et al., 2006).

1.14.2 Physiological Role of TGF-β

The physiological effects of the TGF-β family have been assessed in rodents using

permanent and transient transfection of the human gene and manipulation of

endogenous genes to delete or constitutively activate the Smad signalling pathway.

Genetic disruption of the three different TGF-β loci leads to the development of

significantly different pathologies demonstrating the different physiological roles of

61

ne-X-Serine residues on Smad2 and Smad3, known as receptor or R-Smads. The R-

Smads are recruited to activated type I receptors by the membrane bound cytop

protein SARA (Smad Anchor for Receptor Activation).

The activated Smad2/3 hetero-dimer forms a heteromeric complex with the

Common-Smad (Co-Smad), Smad4, in vertebrates, and tr

minus of Smad4 interacts with transcriptional co-activators of the AP-1 family

such as CREB-binding protein (CBP), also known as p300 (Verrecchia et al., 2006,

Massague et al., 2005

62

these growth factors (Kulkarni et al., 1993, Shull et al., 1992, Sanford et al., 1997, Taya

et al., 1999). Two independent studies found that mating of mice heterozygous for the

disrupted TGF-β1 locus (TGF cted ratios of TGF-β1-/-

pups, approximately 10%, and death due to chronic inflammation of multiple organs by

4 weeks of age (Shull et al., 1992, Kulkarni et al., 1993). The inflammation was

primarily composed of most

severely affected o

TGF-β2-/- mice display severe structural abnormalities in the lung, heart, skeleton

and kidney (Sanford et al., 1997). Approximately two-thirds of new born TGF-β2 pups

die within minutes of birth, exhibiting signs of respiratory distress and cyanosis.

Examination of the pre-natal lungs did not reveal any morphological abnormalities

compared to TGF-β2+/+ littermates, however post-natal lungs of null mice had dilated

conducting airways but collapsed terminal and respiratory bronchioles (Sanford et al.,

1997). There does appear to be some redundancy between TGF-β2 and TGF-β3 as

approximately one-quarter of TGF-β2-/- mice developed extensive cleft palate, which is

the only described feature of TGF-β3-/- mice. These features highlight the importance of

this family of growth factors in the organisation and maintenance of the ECM and the

mesenchyme (Taya et al., 1999, Sanford et al., 1997).

The role of TGF-β in modulating epithelial-mesenchymal interactions has been

assessed by the manipulation of TGF-βRII activity in stromal fibroblasts. These studies

have suggested that TGF-β signalling in stromal fibroblasts suppresses neoplastic

transformation of gastric, prostate and mammary epithelia (Bhowmick et al., 2004,

Cheng et al., 2005). These phenotypes have also been observed in mice with genetic

disruption of TGF-β signalling molecules Smad3 and Smad4 (Weinstein et al., 2000,

Zhu et al., 1998, Xu et al., 2000).

-β1+/-) lead to lower than expe

lymphocytic cells and large macrophages, with the

rgans being the stomach, liver, heart and lungs.

63

l levels of TGF-β1 are critical for normal development and lung

organogenesis. Strategies aimed at systemic over-expression of bioactive TGF-β1 have

proved to be lethal and limiting over-expression to the lung by co-ordinating transgene

expression with surfactant protein-C (SPC) synthesis leads to foetal lethality due to

disrupted branching

tivation of the TGF-βRII in fibroblasts leads to fibrosis in the

lung al constitutive activation TGF-βRI induces

sclero rterial hypertension and enhanced susceptibility to bleomycin-

induc ton et al., 2003, Sonnylal et al., 2007).

1.15

ng fibroblast proliferation and promoting differentiation of the myofibroblast

phenotype. The excessive tissue deposition observed in fibrotic processes in many

organ systems is mainly due to the increase in transcription of type I collagen genes,

COL1A1 and COL1A2 (Uitto and Kouba, 2000). The transcription of further ECM

genes have been identified as TGF-β-Smad3 targets, such as COL3A1, COL5A2,

COL6A1, COL6A3 and TIMP-1 (Verrecchia et al., 2001).

The sustained release and enhanced activation of TGF-β is a consistent finding in

animal models of pulmonary fibrosis and appears to be a critical feature of IPF.

Enhanced TGF-β reactivity and transcription has been observed in fibrotic foci in lung

tissue obtained from IPF patients and enhanced levels of this growth factor have been

measured in BALF obtained from IPF patients compared to control patients

(Broekelmann et al., 1991, Awad et al., 1998). Rodent models of pulmonary fibrosis

have uniformly found enhanced TGF-β levels in the airway and lung tissue during the

Physiologica

morphogenesis of the lung (Zhou et al., 1996, Lee et al., 2006).

However, constitutive ac

and skin. Similarly, post-nat

derma, pulmonary a

ed pulmonary fibrosis (Den

TGF-β and IPF

TGF-β is a potent promoter of ECM synthesis in vitro and in vivo. TGF-β

enhances ECM gene expression and inhibits the expression of ECM catabolic enzymes,

stimulati

64

ibition TGF-βRI or Smad3

phosp ects or limits the development of pulmonary fibrosis (Bonniaud et

al., 2 et al., 1989).

gulation of TGF-β1 activity required for mammalian

devel 06) utilised a novel triple transgenic system to control the

locati -β1 transgene expression. The location of TGF-β1

expre icted to the lung by ligating the SPC promoter to the TGF-β1

codin e

by withdrawing doxycycline. This group monitored the

morphological changes induced by the expression of the TGF-β1 transgene over three

months. Lee et al. (2006) observed many of the features found in the lungs of patients

with interstitial lung disease and IPF. Furthermore, they demonstrated that the variety of

effects attributed to TGF-β1, including apoptosis, inflammation, fibrosis, fibroblast

differentiation and alveolar remodelling, were tightly regulated over time. Within 12

hours of doxycycline administration to TGFβ1SPC-Dox mice, apoptosis of epithelial cells

was evident, peaking after 48 hours, and was followed by an inflammatory response.

This inflammatory response manifested as an increase in total cells and macrophages in

BALF and lung tissue, attaining maximum levels after ten days. During this early phase

of TGF-β1 induction, signs of fibrosis were noted in the sub-epithelial and adventitial

regions of the airways and alveolar remodelling manifested as alveolar septal

thickening, septal rupture and areas of honeycomb lung. Remodelling was assessed by

measuring the distance from the centre of the alveolar sac to the alveolar epithelium,

known as the chord length. Significant increases in this length were noted after seven

days and became more prominent over time. W s a prominent increase in α-

development and progression of fibrosis, and inh

horylation prot

004, Zhao et al., 2002, Khalil

To overcome the tight re

opment, Lee et al. (20

on and timing of TGF

ssion was restr

g region, and the timing was controlled by flanking the transgene with tetracyclin

recombinase recognition sites (TGFβ1SPC-Dox). In this way, expression of the TGF-β1

transgene was switched on in the alveoli by administering doxycycline supplemented

water and then switched off

ithin ten day

65

d. Transmission electron microscopy confirmed these cells were myofibroblasts at

all time points. The increase in myofibroblast accumulation coincided with the spread of

fibrosis from the adventitial areas of the airways into the lung parenchyma and increases

in lung collagen content over the experimental period (Lee et al., 2006).

While the aetiology of IPF remains unclear, there is significant evidence to

suggest that the multi-faceted activities of TGF-β are involved in the progression of this

disease. This is due to its direct effect of enhancing the synthesis of ECM while

simultaneously inhibiting the breakdown of existing ECM. Furthermore, the role of

TGF-β in regulating lung morphogenesis, fibroblast differentiation, inflammation and

apoptosis suggests that the hypothetical pathways towards fibrosis culminate in

enhanced TGF-β activity.

1.16

onary fibrosis is a chronic and fatal disease of unknown aetiology.

Howe

mal epithelial-mesenchymal

mal wound repair, cytokines and growth factors regulate the

magn flammation, epithelial regeneration, mesenchymal

prolif f the ECM. The IL-6 family of cytokines are important

medi ound healing responses in the lungs. Maintenance

of the

sts play a role in this process.

The bleomycin model of lung fibrosis is a powerful tool in the search for

molecular pathways involved in the development and progression of IPF. Bleomycin-

induced lung fibrosis replicates many of the features of IPF including the accumulation

SMA positive cells was observed within the lung and persisted over the experimental

perio

Thesis Overview

Idiopathic pulm

ver, clinical data suggests that IPF is the end-stage of a dysregulated wound

healing response culminating in the breakdown of nor

interactions. During nor

itude and duration of in

eration and restoration o

ators of the inflammatory and w

ECM is a dynamic process, orchestrated by adjacent fibroblasts, and it is likely

that IL-6 family cytokines secreted by fibrobla

66

of fibrillar collagens in the alveolar interstitium, myofibroblastic foci, fibroblast

accumulation and lung remodelling. In recent years, this model of lung fibrosis has been

applied to genetically modified rodents, typically mice, to gain further insights into the

molecular events involved in fibrotic lung disease.

The development of gp130 mutant mice has given new insights into the

pathogenesis of inflammatory mechanisms in the peritoneum and lymphoid tissues, in

addition to wound healing processes in the colon and liver. The objective of this thesis

is to assess the role of IL-6/gp130-mediated intracellular signalling pathways in the

ression of bleomycin-induced murine lung fibrosis in wt and

gp13 l to

is of lung fibrosis, including inflammation, myofibroblast formation and

fibrob

of this thesis are:

effect of directed gp130-mediated signalling on the

devel ycin-induced lung fibrosis, as well as fibroblast proliferation and

differ

0-mediated signalling on bleomycin-induced

t, gp130ΔSTAT/ΔSTAT and gp130757F/757F mice, and the role of

inflam

of directed gp130-mediated signalling in lung fibroblasts on

TGF- fibroblast differentiation.

ill address the hypothesis that IL-6/gp130-mediated signalling

pathw

development and prog

0 mutant mice. The ability of IL-6 and IL-11 to regulate cellular events critica

the pathogenes

last proliferation, will be examined.

The specific aims

To investigate the

opment of bleom

entiation.

To assess the effect of directed gp13

inflammation in w

matory cells in the development of bleomycin-induced lung-fibrosis.

To assess the effect

β-induced signalling and

These aims w

ays are dysregulated in the development and progression of bleomycin-induced

mouse lung fibrosis.

67

TWO

MATERIALS AND METHODS

CHAPTER

68

ons

ere supplied by Sigma-Aldrich, Australia unless specified.

Acetate-

drate

O

to 6.5 with HCl and volume the increased to 1000ml with

ddH2O.

10% Bi

ide

8

ents were mixed together with the APS added last, ensuring no air bubbles

were formed. The gel was poured into a 1.5mm spacer Bio-Rad (Protean 3) gel

assembly device. Water was layered over the top of the gel to produce a level surface on

the gel, and it was left to set at room temperature for 30-60 minutes.

MATERIALS

2.1 Buffers and Soluti

All chemicals w

Citrate Buffer

1.56M Sodium tri-hy

240mM Citric acid

12ml Glacial Acetic Acid

850mM Na H

900ml ddH2O

Solutions were mixed in the above order and the volume adjusted to 900ml with

ddH2O. The pH was adjusted

s-Acrylamide Tris-Glycine Separating Gels

4ml ddH2O

3.3ml 30% Bis-acrylam

2.5ml 1.5M Tris pH8.

100μl 10% w/v SDS

100μl 10% w/v Ammonium Persulfate (APS)

4μl N, N, N, N-Tetramethyl-Ethylenediamine

All reag

69

5% Bis-Acrylamide Tris-Glycine Stacking Gels

1.4ml ddH O

330μl 30% Bis-acrylamide

250μl 1.0M Tris pH6.8

20μl 10% w/v SDS

20μl 10% w/v APS

2μl TEMED

All reagents were mixed together with APS added last, ensuring no air bubbles

were formed. The gel was poured over 10% bis-acrylamide tris-glycine separating gels

and a 15 well comb inserted into the gel. The stacking gel was set at room temperature

for 15 minutes before use.

Breaking Buffer (BB)

This buffer was used to harvest protein from cells grown in vitro for SDS-PAGE

and western blotting.

50mM Trizma-base pH7.5

0.5mM EGTA

150mM NaCl

1% v/v Triton-X 100

25mM HEPES

2mM MgCl2

10% v/v Protease Inhibitor Cocktail (P8340)

2% v/v Phosphatase Inhibitor Cocktail I (P2850)

2% v/v Phosphatase Inhibitor Cocktail II (P5726)

50ml ddH2O

Tris, EGTA, NaCl, Triton-X 100, HEPES and MgCl2 were mixed together,

volume adjusted to 50ml ddH2O and stored at 4oC until required. Protease and

2

70

phosphatase inhibitors were added to an aliquot of breaking buffer immediately prior to

use.

Buffe

etate (HPLC grade; BDH Chemicals, U.K.)

onitrile (HPLC grade; BDH Chemicals, U.K.)

was placed on a magnetic stirring block with a flea for 5 minutes

and the pH adjusted to 6.4 with the addition of ortho-phosphoric acid drop-wise. The

total volume was adjusted to 2L with ddH O and the solution was filtered through a

0.22μm membrane (Falcon, U.K.).

Buffe

onitrile (HPLC grade; BDH Chemicals, U.K.)

repared in a fume hood, mixed thoroughly by inversion and

filtered through a 0.22μm membrane (Falcon, U.K.).

Chloramine T Reagent

56mM Chloramine T

20ml 50% n-Propanol

100ml Acetate-Citrate buffer

Chloramine T reagent was dissolved in 20ml 50% n-propanol and the volume was

adjusted to 100ml with acetate-citrate buffer.

r A - HPLC

83mM Sodium Ac

160ml Acet

2L ddH2O

Sodium acetate was dissolved in 1.5L of ddH2O and 160ml acetonitrile was

added. The solution

2

r B - HPLC

750ml Acet

250ml ddH2O

The solution was p

71

ed

sections.

trate (C6H5Na3O7)

ate was added to 900ml of ddH2O, mixed, the pH adjusted to 6

with olume brought up to 1L with ddH2O.

Comassie Blue Stain

This solution was used to stain polyacrylamide gels after electrophoresis for

zymography.

0.5% w/v Comassie Brilliant Blue R250

500ml Methanol

10ml Acetic acid

400ml ddH2O

ed in methanol-water-acetic acid

soluti

Destain Solution

This solution was used to remove Comassie blue from polyacrylamide gels.

50% v/v Methanol

10% v/v Acetic acid

40% ddH2O

Solution was typically made in 1000ml volumes by mixing all reagents and stored

at room temperature.

Citrate Buffer

Citrate buffer was used for heat-mediated antigen retrieval of paraffin embedd

tissue

10mM Tri-sodium ci

1L ddH2O

Tri-sodium citr

HCl and the v

Comassie Brilliant Blue R250 was dissolv

on and stored at room temperature.

72

Ehrlich’s Reagent

This solution was used as the chromogen in the Chloramine T assay for the

measurement of hydroxyproline in lung tissue.

1.01M p-dimethylaminobenzaldehyde

70ml n-Propanol

35ml Perchloric acid

The n-propanol and perchloric acid were mixed together in a ratio of 2:1

respectively. P-dimethylaminobenzaldehyde was dissolved in 100ml of the

This solution was prepared immediately before use.

High Molecular Weight Solution

This solution was in the isolation of DNA from whole blood.

30mM Tris-HCl pH 8

olutions were mixed, the volume was adjusted to 50ml

with was stored at room temperature.

propanol:perchloric acid solution.

50mM NaCl

10mM EDTA

50ml ddH2O

Tris, NaCl and EDTA s

ddH2O and solution

73

fer

ction

d mice.

ucose

ted to 7.4 with HCl and

the volume increased to 100ml with ddH2O. The solution was filtered through a 0.22μm

membrane and stored at 4oC.

Luria

s used for the culture of Escherichia Coli (E.coli).

tone

t extract

ml ddH2O, the pH was adjusted 7.0 with HCl and

the volume increased to 1000ml. The solution was autoclaved and stored at room

temperature.

Glucose-Potassium-Sodium (GKN) +0.002% BSA Buf

GKN buffer was used to resuspend whole bone marrow cells prior to inje

into γ-irradiate

137mM NaCl

5.37mM KCl

24.9mM Na2HPO4

6.45mM NaH2PO4

27.75mM Gl

0.002g Bovine Serum Albumin

100ml ddH2O

Chemicals were dissolved in 90ml ddH2O, the pH adjus

Bertani (LB) Broth

This solution wa

10g Bacto-tryp

5g Bacto-yeas

85mM NaCl

1000ml ddH2O

Reagents were dissolved in 900

74

ntibiotic Medium

ls.

r

roth

picillin

ded to LB broth, mixed and autoclaved. Medium was allowed to cool

to ap mixing. Medium was poured into

100m

d at 4oC overnight for approximately four weeks.

etry.

de

was dissolved in 65ml ddH2O at 60oC in a fume hood and 1M

NaOH wise until the solution became clear. The pH was adjusted to 7.2 with

HCl a

volumes (10ml or 50ml), wrapped in aluminium foil and stored at -20oC until required.

Luria Bertani (LB)-Agar-A

This solution was used for the culture of transformed competent E.Coli cel

15g Aga

1000ml LB b

100μg/ml Am

Agar was ad

proximately 55oC before adding ampicillin and

m petri-dishes to a depth of approximately 3mm. Air bubbles were removed by

flaming the surface of the medium of a Bunsen burner, allowed to harden at room

temperature and then store

4% Paraformaldehyde (PFA)

4% PFA was used for the fixation of tissues and cells in vitro. It was diluted to 1%

PFA to fix leukocytes prior to flow cytom

4g Paraformaldehy

100ml ddH2O

Paraformaldehyde

added drop

nd the volume was increased to 100ml. The solution was aliquotted into smaller

75

10x Phosphate Buffered Saline (PBS)

1.49M NaCl

160mM Na2HPO4

52mM NaH2PO4

1L ddH2O

Chemicals were dissolved in 900ml ddH2O, the pH was adjusted to 7.4 with HCl

and the volume increased to 1L with ddH2O. The solution was autoclaved and stored at

room temperature.

Scott’s Tap Water

Scott’s tap water was used to develop haematoxylin stained tissue sections.

42mM NaHCO3

164mM MgSO4

1L ddH2O

Chemical were added to 1L of ddH2O, mixed until dissolved and kept at room

temperature until used.

76

abolite Repression (SOC) Medium

This medium was used for the heat shock transformation of competent E.coli

ther reagents, pH adjusted to 7.0 and

volume was increased to 100ml.

) Solution

lation of

Super Optimal Broth-Cat

cells.

2g Bacto-tryptone

0.5g Bacto-yeast extract

100mM NaCl

25mM KCl

200mM Glucose

200mM MgSO4

100ml ddH2O

Bacto-tryptone, bacto-yeast, NaCl and KCl were dissolved in 90ml ddH2O,

autoclaved and cooled to room temperature. Glucose and MgSO4 solutions were filtered

through a 0.22μm membrane and added to o

10% Sodium Dodecyl Sulphate (SDS

10% SDS solution is a denaturing agent used in polyacrylamide gel

electrophoresis and for RNase treatment of glass and plasticware used in the iso

total RNA.

25g SDS

250ml ddH2O

SDS was added to 200ml of ddH2O, mixed with a magnetic stirring rod until

dissolved and the volume adjusted to 250ml.

77

10x SDS-PAGE Running Buffer

2.5M Tris-base

1.9M Glycine

170mM SDS

500ml ddH2O

Chemicals were mixed together in 400ml ddH2O and mixed gently for 16-18

hours. The volume was then adjusted to 500ml with ddH2O.

2

2

1x SDS-PAGE Running Buffer

100ml 10x Running Buffer

900ml ddH2O

Buffer was prepared immediately prior to use.

5x SDS-PAGE Loading Buffer

312mM Tris-base

520mM SDS

500mM Dithiothreitol

50% v/v Glycerol

0.05% w/v Bromophenol Blue

5ml ddH O

Chemicals were mixed in 3ml ddH O and 5ml of glycerol was added. The volume

was adjusted to 10ml with ddH2O and the solution mixed thoroughly. The buffer was

dispensed in 1ml aliquots and stored at -20oC until required.

78

5x SDS-PAGE Non-reducing Loading Buffer

This buffer was used for the electrophoresis of protein samples used for

zymography.

312mM Tris-base

520mM SDS

50% v/v Glycerol

0.05% w/v Bromophenol Blue

5ml ddH2O

Chemicals were mixed in 3ml ddH2O and 5ml of glycerol was added. The volume

was adjusted to 10ml with ddH2O and the solution mixed thoroughly. The buffer was

dispensed in 1ml aliquots and stored at -20 C until required.

Tissue Digest Buffer

solate DNA from tissue biopsies.

o

Tissue digest buffer was used to i

50mM Tris-HCl pH 8

100mM EDTA

100mM NaCl

1% w/v SDS

0.5mg/ml Proteinase K (Invitrogen, Australia)

The solution was prepared as a 10ml stock and aliquotted into 1ml single use

preparations and stored at -20oC.

79

Tissue Lysis Buffer (TLB)

ibitor Cocktail (P8340)

r

t

room temperature.

Tissue lysis buffer was used to isolate protein from mouse lung tissue for

zymography

20mM Tris

1mM EDTA

1mM DTT

10% v/v Protease Inh

Tris, EDTA and DTT were mixed together, volume adjusted to 50ml ddH2O and

stored at 4oC until required. Protease inhibitors were added to an aliquot of TLB

immediately prior to use.

50x Tris-Acetate-EDTA (TAE)

1x TAE buffer was used as a major constituent of agarose gels and running buffe

for the electrophoresis of nucleic acids.

5mM EDTA pH8

57.1ml Glacial Acetic Acid

242g Tris-Base

1L ddH2O

Solutions and chemicals were mixed thoroughly and stored at room temperature.

10x Tris Buffered Saline (TBS)

200mM Tris-Base

1.37M NaCl

1L ddH2O

Chemicals were dissolved in 900ml ddH2O, the pH adjusted to 7.6 with HCl and

the volume increased to 1L with ddH2O. The solution was autoclaved and stored a

80

ic

acids.

100mM Tris

1

erature.

10x Western

2

2 ml

2

10x Tris-EDTA (TE) Buffer

10x TE was diluted 1:10 with ddH2O and used to resuspend precipitated nucle

pH7.6

0mM EDTA pH8

100ml ddH2O

Tris and EDTA solutions were mixed and the volume increased to 100ml with

ddH2O, autoclaved and stored at room temp

Blot Transfer Buffer

250mM Trizma-base

1.92M Glycine

500ml ddH O

Chemicals were dissolved in 400ml of ddH O and the volume adjusted to 500

with ddH O.

1x Western Blot Transfer Buffer

100ml 10x Transfer Buffer

200ml Methanol

700ml ddH2O

81

2.2 Bleomycin Administration

rnst

ed with 5%

isoflu TM

fed

2.

r

o

2.3 Bone Marrow Reconstitution

St

m

f GKN buffer and cell counts performed.

The cells were washed with PBS and suspended to a concentration of 1x107 cells per ml

METHODS

C57BL/6 and gp130 mutant mice, generously donated by A/Prof. Matthias E

(Ludwig Institute for Cancer Research, VIC, Australia), were anaesthetis

orane supplemented air and a 50μl droplet of bleomycin (Blenoxane , Bristol-

Squibb-Meyer, Manchester, U.K.) at 15U/ml in saline was applied to the left nostril and

passively inhaled. Mice were maintained in a pathogen free environment and

acidified water and standard chow ad libitum during the course of all experiments.

Gp130 mutant mice were maintained on a mixed Sv/129 and C57BL/6 background.

Lungs were either prepared for histology as described below or snap frozen in liquid N

While immersed in liquid N2 the tissue was crushed into a fine powder with a morta

and pestle and stored at -80 C for later use.

Host and donor mice were maintained on neomycin (2mg/ml, Sigma-Aldrich,

Louis, MO, USA.) supplemented water for 2 weeks prior to reconstitution and

maintained on the supplemented water for 4 weeks after reconstitution. To ablate the

host bone marrow, mice were subjected to two doses of 5.5Gy gamma irradiation 3

hours apart. The donor mice were killed by CO2 asphyxiation and their femurs were

removed from above the knee and below the hip joints and placed in a petri dish

containing 0.002% bovine serum albumin supplemented glucose-potassium-sodiu

(GKN) buffer. Bone marrow was flushed from the femurs with GKN buffer and the

cells were collected by centrifugation at 1500rpm for 7 minutes. The supernatant was

aspirated and the cells resuspended in 1ml o

82

in PB

s before

Bone marrow reconstitution was assessed by flow cytometry quantification of

7BL/6 Ly5.2 mice, and PCR

ident

ugation

ut

r

ls were

l

tect

μl

tometer.

S. Host mice received 200μl (2x106 cells) of cell suspension i.v. Mice were

maintained under pathogen free housing conditions for a further four week

being used for experiments.

circulating leukocytes in congenic C57BL/6 Ly5.1 and C5

ification of donor DNA in whole blood and splenic tissue of gp130 mutant mice.

Whole mouse blood was collected by cardiac puncture at the time of euthanasia,

yielding between 500-1000μl of blood, which was adjusted to a total volume of 1.5ml

with PBS-1% BSA. Ficoll-paque (1ml) was added to a lidless FACS tube (Falcon,

Oxnard, CA, USA) and the blood suspension was layered gently over the top of the

Ficoll-paque. Leukocytes were separated from other circulating cells by centrif

at 1200rpm at 4oC for 20 minutes. The intermediate buffy layer was collected, witho

disturbing the upper Ficoll-paque or the lower blood layers and transferred to anothe

FACS tube. A further 2ml of PBS-1% BSA was added to the leukocyte layer, cel

resuspended in solution and collected by centrifugation at 1400rpm at 4oC for 5

minutes. The supernatant was removed. Cells were either fixed in 1% paraformaldehyde

on ice for 30 minutes, washed twice with 2ml PBS-1% BSA, resuspended in 400μ

PBS-1%BSA and stored at 4oC in the dark for 24 hours or prepared immediately for

flow cytometry. Cells were divided into 2 equal aliquots and labelled with either biotin

conjugated anti-CD45.1 or biotin conjugated anti-CD45.2 immunoglobulins in 100μl of

PBS-1% BSA on ice for 30 minutes. Leukocytes were washed twice with PBS-1% BSA

and labelled with phycoerythrin conjugated streptavidin for 30 minutes on ice to de

labelled cells. Two further washes were performed and cells were resuspended in 400

of PBS-1% BSA and analysed using the FACscanTM (Becton Dickinson, Franklin

Lakes, NJ, USA) flow cy

83

om gp130 mutant mice snap frozen in liquid N2 and

stored at -80oC. The tissue was thawed at room temperature and DNA extraction was

below. Gp130 mutant alleles and wild-type gp130 were detected

in a m

cassette in the mutant allele (5’-CTGAATGAACTGCAGGACGA-3’) and wt gp130

visualised on a 1% agarose gel with the positive mutant gp130 allele present at 700bp

The trachea was dissected free and 1cm of 0.8mm polyethylene tubing was

into the trachea and fixed in place with standard

sutur

m for 5

ed

, NSW,

k-Dip

and the slides were mounted in DePex (Merck, Middlesex, UK). At least 400

cells

Spleens were removed fr

performed as described

ulti-plex PCR reaction using a common anti-sense primer (5’-

TGAAGCCACTCGTCTTTAGC-3’) and sense primers for the neomycin resistance

(5’-CAAGTGTTCTCAAGGTCCGAGTCCAC-3’). Qiagen (Qiagen, Doncaster, VIC,

Australia) PCR reagents were used to amplify genomic DNA using the following

temperature cycles; 94oC for 10minutes, 35 cycles at 94oC for 1minute, 60oC for 1

minute, 72oC for 1 minute followed by 5 minutes at 72oC. Amplified DNA was

and a positive wt allele present at 500bp.

2.4 Broncho-alveolar Lavage (BAL)

attached to a 21 gauge needle, inserted

es. Four volumes of sterile saline (0.4ml and 3 x 0.3ml) were injected into the lung

and collected with a syringe. Cells were pelleted by centrifugation at 5000 rp

minutes at room temperature, the supernatant was transferred to a new tube and stor

at -80oC for later analysis. Cells were resuspended in 100μl of sterile PBS, quantitated

using a Nebauer chamber and collected on sialinated slides (Starfrost, Bangalow

Australia) by centrifugation at 400rpm for 3 minutes. Cells were stained with Quic

(Scot Scientific, Western Australia) staining solution to distinguish inflammatory cell

types,

per slide were counted to approximate total inflammatory cell numbers and

differentiate inflammatory cell types in BAL fluid (BALF).

84

Between 25-50mg of lung tissue was added to 2ml of 6N HCl (Sigma-Aldrich,

ours. A 20μl aliquot was added to

180μ at

plate

2.5.2 Reverse-phase High Pressure Liquid Chromatography (HPLC)

Australia) and incubated at 110oC for 16-18 hours, approximately 20mg of charcoal was

vacuum pressure in a separate 1.5ml polypropylene tube. Proteins were derivatised by

M

l.

o ted

2.5 Collagen Assays

Total lung collagen was assessed by measurement of hydroxyproline in lung

tissue. Hydroxyproline is the most abundant amino acid in mature collagen representing

approximately 8% of the total amino acid content. Hydroxyproline levels were

measured in acidified lung tissue using the chloramine T assay and by reverse phase

high pressure liquid chromatography (HPLC).

2.5.1 Chloramine T Assay

NSW, Australia) and incubated at 110oC for 16-18 h

l of chloramine T reagent and mixed thoroughly. The solution was incubated

room temperature with gentle mixing for 60 minutes, 200μl of 1M Ehrlich’s reagent

was added to the solution and incubated at 65oC with gentle mixing for 40 minutes or

until colour developed. A 100μl aliquot was then transferred to a 96 well ELISA

and scanned on a spectrophotometer at 570nm.

Between 25-50mg of lung tissue was immersed in 2ml 6N HCl (Sigma-Aldrich,

added, and this solution was filtered through a 0.65μm membrane. A 10μl aliquot was

diluted in 990μl of ddH2O and a 200μl aliquot of this solution was precipitated under

resuspending in 100μl dH2O, 100μl of 0.4M potassium tetraborate, and 100μl of 36m

7-chloro-4-nitrobenzo-2-oxa-1,3-diazole chloride (NBD-Cl; Merck, U.K.) in methano

Samples were incubated at 37 C for 20 minutes and colour development was termina

by the addition of 50μl of 3.33x HPLC buffer A. Three duplicate hydroxyproline

85

st the

Hydroxyproline (ρmol) = HPLC Reading x (2000μl HCl/ 200μl used) x 100

(dilution of hydrolysate) x 5 (dilution in activation)

mol) x

hydroxyproline, g/mol)/1x106 x Lung weight (mg)/Tissue sample weight (mg)

id

er 3

g

TAT3

48 hours later

red using the Dual-Luciferase Reporter Assay System

(Promega, Australia) according to the manufacturer’s instructions.

2.6 DNA Isolation from Tissue Biopsy

DNA was isolated from between 10-50mg of tissue by incubating in 50mM Tris-

a-

o

standards (50ρmol) were prepared and run as duplicates evenly dispersed among

test samples. Total lung collagen was determined using the following calculations:

Total Lung Collagen (mg) = hydroxyproline (ρmol) x 10-9 (conversion to

8.1967 (proline percentage composition of collagen) x 131 (molecular weight of

2.5.3 Collagen Luciferase Reporter Assay

Murine embryonic fibroblasts (MEFs) were seeded into 24 well cell culture plates

at a density of 5x104 cells per well in normal growth medium (NGM) for 24 hours.

Medium was aspirated and replaced with minimal serum medium (MSM) and MEFs

were transfected with 200ng pCOL1α1-Luc and 33ng pCMV-RLuc using a plasm

DNA:Fugene 6 ratio of 6:1 diluted in DMEM. Cells were incubated for a furth

hours then NGM was added to untreated controls and NGM containing growth factors

or cytokines were added to remaining cells in triplicate. To confirm gp130 signallin

mutations were preserved in MEFs, cells were transfected with 100ng of the S

specific reporter vector pAPRE-Luc and 33ng pCMV-RLuc as a transfection control.

Cells were then stimulated with cytokines or growth factors, harvested

and luciferase activity was measu

HCl, 100mM EDTA, 100mM NaCl, 1% SDS and 0.5mg/ml Proteinase K (Sigm

Aldrich, Australia) for 16-24 hours at 55 C. Samples were vortexed until the digest

appeared homogeneous, 500μl of 5M NaCl was added, samples were vortexed for 5

86

as resuspended in 1ml of ice cold PBS and washes were repeated as described

above

t

new

of 3M sodium acetate solution equivalent to 10% of the aqueous phase. The

solution was mixed and a volume of cold 100% ethanol equivalent to 200% of the

ded and mixing thoroughly. DNA was collected by

centr

ethanol

minutes and excess cellular material was collected by centrifugation for 20 minutes at

14,000 rpm at room temperature. The supernatants were transferred to separate tubes

and DNA precipitated by the addition of 0.5x volume of isopropanol and mixed

thoroughly with a pipette. DNA was collected by centrifugation at 14,000rpm for 15

minutes, the pellet washed twice with ice-cold 100% ethanol and 70% ethanol

respectively with the pellet collected by centrifugation after each wash.

DNA was isolated from 200-500μl of whole blood by increasing the volume to

5ml with cold ddH2O, mixed and incubated for 2 minutes. A further 2mls of 3.5% NaCl

(w/v) was added, mixed well and the cellular organelles were collected by

centrifugation at 4,200rpm at 4oC for 10 minutes. The supernatant was discarded, the

pellet w

. Pelleted organelles were resuspended in 5ml of HMW solution and 50μl of

10mg/ml Proteinase K and 10% SDS were added to the solution, mixed gently and

incubated for 16-24 hours at 37oC. DNA was dissolved in 5ml of Phenol and the diges

mixed vigorously for 10 minutes or until an emulsion was formed. Aqueous material

was removed from the digest by centrifugation at 4,200rpm for 10 minutes and the top

phase aspirated. A further 5ml of 1:1 phenol-chloroform solution was added to the

remaining organic phase, mixed thoroughly and the organic phase was collected by

centrifugation at 4,200rpm for 10 minutes. The aqueous phase was transferred to a

tube, while the organic phase was discarded. The DNA was precipitated by adding a

volume

aqueous phase volume was ad

ifugation at 10,000rpm at 4oC for 20 minutes. The supernatant was removed and

the DNA transferred to a 1.5ml polypropylene tube, washed with 1ml of 70%

and collected at 14,000rpm for 20 minutes.

87

.

03 cells

r 30 minutes at room

temperature. Primary antibodies were applied with no washing after the FBS block at

or one hour at room temperature. Biotin conjugated

secon

ethyl

o

S

DNA was resuspended in 50μl of either ddH2O or TE buffer, quantitatively and

qualitatively assessed by absorbance at 260/280nm and agarose-TAE gel

electrophoresis respectively.

2.7 Enzyme Linked Immuno-Sorbent Assay (ELISA)

ELISA assay was adapted from the methods described by Scaffidi et al. (2001)

Lung fibroblasts (LFs) were trypsinised (see section 2.7.1) and cell numbers were

determined using a NebauerTM chamber. Cells were seeded at a density of 5x1

per well in a tissue culture plate in MSM. Cells were incubated overnight and treated

with cytokines or growth factors for 24-72 hours, fixed in cold 100% methanol (MeOH)

and stored at -20oC until assay the was performed. Cells were thawed at room

temperature for 10 minutes, washed twice with PBS supplemented with 0.05% v/v

Tween 20TM (PBS-T) and after each subsequent step described below. Endogenous

peroxidase activity was quenched with 3% H2O2 for 5 minutes and non-specific

antibody-epitope interactions were blocked with 5% FBS fo

the optimum concentration f

dary antibodies and streptavidin conjugated horse-radish peroxidase (S-HRP;

DakoCytomation, NSW, Australia) were mixed and incubated with the cells for 1 hour.

Antibody-epitope interactions were detected by applying the HRP substrate tetram

benzidine (TMB) or 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) t

each well. The TMB reaction was terminated by applying 25μl 1M H2SO4 and the

ABTS reaction was terminated with 100μl of 1% v/v SDS. Colour development was

read on a spectrophotometer at a wavelength of 450nm and 490nm for TMB and ABT

reactions respectively.

88

ribed

37oC

USA)

as

2.8.2 Propagation of Eukaryotic Cells

Cells were seeded in 0.2μm vented cap tissue culture flasks (Falcon, USA) in

growth medium containing 10% foetal bovine serum (FBS; Invitrogen Life

itrogen Life Technologies,

USA mycin

2.8 Eukaryotic Cell Culture

2.8.1 Cell Isolation

Primary fibroblasts were obtained from the lungs of mature mice by trypsin-

collagenase serial digestion. Lungs were removed from the thoracic cavity as desc

in section 2.8, dissected free of the heart and incubated in 0.25% Trypsin-EDTA at

for 20 minutes. NGM was added to the digest to neutralise the trypsin activity. Lungs

were removed from the digest and the trachea, and major conducting airways were

dissected free. The lungs were finely minced with a sterile scalpel and the tissue was

incubated in 1mg/ml collagenase 1 (Invitrogen Life Technologies, Carlsbad, CA,

at 37oC for 2-3 hours with vigorous agitation every 30-60 minutes. Collagenase w

neutralised with NGM and the lung tissue was discarded, cells were collect from digest

by centrifugation at 1200 rpm for 5 minutes at room temperature. The supernatant was

discarded, the cells were re-suspended in 1ml of NGM and added to 2ml of NGM in a

25cm2 vented tissue culture flask.

Technologies, USA) by volume, 4mM L-glutamine (Inv

), 100U/ml penicillin (Invitrogen Life Technologies, USA), 100μg/ml strepto

(Invitrogen Life Technologies, USA), 0.25μg/ml amphotericin (Invitrogen Life

Technologies, USA) and 20mM HEPES in Dulbecco’s Modified Eagles Medium

(DMEM; Invitrogen Life Technologies, USA). Cells were maintained at 37oC in 5%

CO2.

Cells were routinely passaged by two washes of sterile phosphate buffered saline

(PBS) and incubating at 37oC with 0.05% Trypsin-EDTA-PBS solution for five

89

he

n

ere

ing to their growth rates in vitro. Cells

were stored as stocks of 1x106 cells per tube by resuspending in cryostorage serum;

50% FBS, 40% DMEM and 10% DMSO and freezing for 24 hours at -80oC prior to

long term storage in liquid N .

hich encodes the

early region of the SV40 virus under the regulation of the Rous Sarcoma virus

ted using 1μg of plasmid DNA and the Fugene 6

transf

of 50μl and the cells were incubated for 3 hours. NGM (1ml)

was added to the transfection reaction and cells were incubated at 37oC for 24 hours and

the media was replaced with NGM. Cells were grown for a further 4 weeks in 6 well

plates when signs of dysregulated growth became apparent. Foci of transfected cells

were identified by the loss of contact induced growth inhibition while foci of non-

transfected cells underwent apoptosis. Each well, including negative controls, was

passaged and distributed evenly across a 24 well plate to select for transfected cells.

Immortalised cells reached confluency rapidly and were transferred to a 6 well plate

minutes. The trypsin was neutralised by the addition of an equal volume of NGM. T

cells were mixed my repeat pipetting until a single cell suspension was attained and a

aliquot was added to 10ml growth medium in a 75cm2 culture flask. Primary cells w

routinely passaged at a ratio of 1:2 to ensure continual growth and avoid senescence,

and immortalised cell lines were passaged accord

2

2.8.3 Immortalisation of Primary Lung Fibroblasts

Primary lung fibroblasts were isolated as described above, cultured for two

passages then seeded into 6 well plates at a density of 5x104 cells per well. Cells were

grown to 60-80% confluence then transfected with pRSV-T plasmid w

promoter. Cells were transfec

ection reagent (Roche Diagnostic, IN, USA) according to the manufacturer’s

instructions. Briefly, NGM was aspirated, replaced with 1ml of MSM and the

transfection solution was added at a ratio of 6:1 (volume Fugene 6TM:mass plasmid

DNA) in a total volume

90

.

escribed

of

bloc and immersed in 4% paraformaldehyde for 30 minutes. The

immersed in 0.1% glycine-PBS for 30

minut

immersed in 70% ethanol prior to processing for embedding in paraffin wax.

o

were cut to 7μm thickness to preserve the parenchymal structures for

emistry (IHC)

xylene for 5 minutes

each, f

on slides as described below if necessary. Slides were washed twice with TBS,

where again stably transfected cells rapidly became confluent. These cells were

transferred to 25cm2 tissue culture flasks and passaged 10 times before cryostorage

2.9 Histology

Mice were euthanased by a lethal dose of pentobarbitone i.p. (140mg/kg;

Lethobarb, Fort Dodge, KS, USA) and the trachea was dissected free once pinch and

blink reflexes were absent. A 21 gauge needle was inserted into the trachea as d

in section 2.3 and the lungs were inflated with 4% paraformaldehyde under pressure

a 20cm column of fluid. The lung tissues were fixed for 5 mins and the heart and lungs

were removed en

tissue was washed thoroughly in PBS and then

es, and dehydrated in 15% sucrose-PBS for 16-24 hours. Lungs were then

Alternatively, lungs were immersed in a 1:1 PBS-OCT solution prior to embedding in

OCT compound (Scot Scientific, Western Australia) and stored at -80 C. Paraffin

embedded section were cut to a thickness of 5μm for immunohistochemistry,

haematoxylin-eosin (H&E), and Masson’s trichrome stain. OCT embedded sections

immunohistochemical labelling.

2.9.1 Immunohistoch

IHC was performed by de-waxing sections in two washes of

ollowed by rehydration through serial immersion in decreasing grades of ethanol

(100%, 70%, and 30%) and ddH2O for 5 minutes each. Antigen retrieval was performed

endogenous peroxidase was quenched with 3% H2O2 for 5 mins, and non-specific

antibody binding was blocked with 10% FCS-TBS for 30 mins at room temperature.

91

at room

oC. Slides were washed twice with TBS and biotinylated

secondary antibodies were incubated at an optimal dilution for 1 hour at room

on of 1:200 for

1 hou

strate was

%) and two

washes of xylene (Merck,

USA

the

ed

2.10 Immunocytochemistry

4 d

growth factors and/or cytokines and fixed with 500μl of 4%

Primary antibody was incubated on slides at an optimal dilution for 1-3 hours

temperature or 16-24 hours at 4

temperature, followed by S-HRP (DakoCytomation, Australia) at a diluti

r at room temperature. Slides were washed 3-4 times for 5 minutes to remove

excess S-HRP and di-aminobenzidene (DAB; Sigma-Aldrich, USA) HRP sub

added to slides to detect antibody-epitope binding. Slides were counterstained with

Mayer’s haematoxylin (Sigma-Aldrich, USA) for 30 seconds, developed in Scott’s tap

water for 2 minutes, dehydrated through graded alcohols (30%, 70%, 100

, and coverslips fixed with DePex polymer mounting medium

).

Antigen retrieval was achieved by one of two methods; either heat-mediated or

enzyme-mediated, and was performed prior to quenching of endogenous peroxidase

activity. Heat-mediated antigen retrieval was achieved by boiling either citrate buffer

(pH 6) or EDTA buffer (pH 8) in an electric pressure cooker, immersing slides in

boiling buffer and sealing the lid of the pressure cooker. Slides were maintained at

maximum temperature and pressure for an optimised period of time (5-15 minutes) then

cooled to room temperature. Enzyme-mediated antigen retrieval was performed by

incubating slides with Proteinase K solution (Dako Cytomation, Australia) for an

optimised period of time (2-10 minutes), washed in TBS and then treated as describ

above.

Cells were seeded at a density of 5x10 cells per well onto glass cover slips place

in the wells of a 12 well tissue culture plate. Cells were treated with defined

concentrations of

92

parafo

were washed twice with PBS and stored 500μl PBS until they were labelled

with protein specific antibodies. Prior to antibody labelling the cells were equilibrated to

room temperature and washed twice in TBS-0.2% Triton-X 100 (TBS-0.2Tx; Sigma-

Aldri

y-

ary antibodies were added without washes at optimised

.05T for 1 hour. Fluorophore conjugated secondary

antib

4oC for

ection

2.8.1

y

ies

atory

foci.

rmaldehyde for 5 minutes at room temperature. The PFA was removed and cells

at 4oC in

ch, Australia) to permeablise cells. Between subsequent steps two washes of TBS-

0.05T for 5 minutes were performed unless stated otherwise. Non-specific antibod

epitope reactions were blocked using 10% FBS in TBS-0.05T for 30 minutes at room

temperature and prim

concentrations diluted in TBS-0

odies directed against the host species of the primary antibody were added at an

optimised dilution for 1 hour at room temperature in the dark and were protected from

light in subsequent steps. Coverslips were washed three times with TBS-0.05T then

mounted on standard slides with Prolong GoldTM (Invitrogen, Australia), kept at

24-48 hours to allow mounting medium to polymerise and then visualised under a

fluorescent light microscope and by confocal microscopy.

2.11 In situ Inflammation

Inflammation was measured in situ using tissue sections immuno-labelled with

macrophage, T-lymphocyte and B-lymphocyte specific antigens as described in s

. Inflammatory foci were characterised as a collection of granulocytic and or

lymphocytic cells that occupied a 6.22x103μm2 field of view (equivalent to one field

with 20x objective lens). Total inflammatory cells and immuno-labelled cells were

counted within an inflammatory foci from at least three random slides chosen randoml

from sectioned tissue block. Sequential slides were immuno-labelled with antibod

recognising F4/80, CD3 or B220 antigens to assess the composition of inflamm

93

2.12.1 Preparation of Heat Shock Competent Cells

red for heat shock transformation

by tra

by

ved and

ubated

C

l of

glycerol stock into 10ml of LB broth in 15ml culture tubes (Falcon, Australia) and

o d to

an

ice for 20 minutes, transferred to a 42oC heating block for 2 minutes,

transferred to ice for 2 minutes where 900μl of SOC medium was added and transferred

2.12 Prokaryotic Cell Culture

Escherichia coli (E.coli) strain DH5α were prepa

nsferring 10μl of glycerol cell stock into 10ml of LB broth and incubating

overnight at 37oC with shaking at 225-250rpm. Approximately 2.5ml of overnight

culture was used to inoculate 250ml of LB broth and incubated at 37oC with shaking

(225-250rpm) for 2-3 hours. The cell density was assessed by optical density at a

wavelength of 600nm and the culture was stopped when the optical density of culture

was equal to 0.6. Cells were chilled on ice for 15-30 minutes to stop growth and

collected by centrifugation at 4000g at 4oC for 10 minutes. The supernatant was

removed, cells were resuspended in 25ml of ice-cold 0.1M MgCl2 and then collected

centrifugation at 2000g for 10 minutes. The supernatant was removed and cells were

resuspended in 25ml ice-cold 0.1M CaCl2 and incubated on ice for 20 minutes. Cells

were collected by centrifugation at 2000g for 10 minutes, supernatant was remo

cells were resuspended in 10 ml ice-cold 0.1M CaCl2 and 14% v/v glycerol inc

on ice for 10 minutes. Cells were dispensed as 500μl aliquots and snap frozen at -80o

for later use.

2.12.2 Heat Shock Transformation of Competent Cells

Heat transformation of DH5α E.coli was performed by transferring 10μ

incubating at 37 C for 16-24 hours. A 120μl aliquot of E.coli culture was transferre

a 1.5ml tube and approximately 500ng of plasmid encoding a luciferase protein and

antibiotic resistance cassette was added to the suspension. The transformation solution

was incubated on

94

containing 5ml LB. Cells were expanded at 37oC for 90-120

minu

2.12.3 Plasmid Extraction

A single colony of transformed cells was transferred to 5ml LB-antibiotic broth

and incubated at 37 C for 6-8 hours with shaking. A 500μl aliquot of this starter culture

was added to 500ml of LB-antibiotic broth. The cells were incubated overnight at 37oC

for 16-24 hours and the plasmid DNA was extracted using a Qiagen Maxi-prep

endotoxin free plasmid isolation kit (Qiagen, Australia) according to the manufacturer’s

instructions. Plasmid DNA was resuspended in 500μl TE or ddH2O.

2.13 Proliferation Assays

Cell proliferation was assessed by three alternative methods 1) incorporation of

the nucleotide analogue brominated deoxy-uridine 2) the total protein accumulation

(methylene blue assay) and 3) the reduction of tetrazolium salts by mitochondrial

enzymes (MTT assay).

a

lls were plated in either a T0 plate to account for cell

numbers at the start of the experiment or a T1 plate that was incubated for 24-72 hours

to determine growth rates of untreated cells, cells treated with cytokines and/or growth

factors and cells treated with NGM. Incorporation of brominated deoxy-uridine was

to 15ml culture tubes

tes, collected at 500rpm for 6 minutes at room temperature and resuspended in

100μl SOC medium that was streaked across LB-agar-antibiotic plates to select for

transformed cells. Streaked plates were incubated at 37oC for 24 hours and transformed

colonies were expanded for plasmid extraction.

o

2.13.1 Brominated Deoxy-Uridine Incorporation (BrdU) Assay

Lung fibroblasts were trypsinised and cell numbers were determined using a

NebauerTM chamber and seeded at a density of either 3x103 or 5x103 cells per well in

tissue culture plate in MSM. Ce

95

measured using the Biotrak Cell Proliferation kit (GE Healthcare, USA) according to

the manufacturer ns. Briefly, c ted w g

solutio at 37oC then fixed for 30 m at room tempe The fixative

was re were blocked for 30 oom tempe revent

non-sp anti-BrdU antibod ls. The blockin on was

aspirated and peroxidase conjugated anti-BrdU dy was added ells for 90

minute tibody wa ed from the ce the

chrom ate TMB was a r 30 minutes. T oxidase

reactio dition of 25μl 2SO4 and colo lopment was

read at

2.13.2 Methylene Blue Binding (MBB) Assay

Cells were seeded in 96 well plates at 5x103 cells per

described above for the BrdU assay. At the end e incubation per media was

aspirated and the cells fixed by the addition of 4% PFA for 10 minutes. The fixative was

d replaced with PBS and the cells were stored at 4oC until the assay was

comp

were

0.01M borate buffer and dye was eluted with 1:1 v/v,

ethan

ethyl-Tetrazolium Reduction (MTT) Assay

(MTT) was added (Mosmann, 1983,

Carmichael et al., 1987). The solution was incubated at 37oC for 3 hours for complete

reduction of methyl tetrazolium salts to formazan. Formazan crystals were eluted from

’s instructio ells were incuba ith BrdU labellin

n for 2 hours inutes rature.

moved, the cells minutes at r rature to p

ecific binding of the y to cel g soluti

antibo to the c

s at room temperature. The an s wash lls and

ogenic peroxidase substr dded fo he per

n was terminated by the ad 1M H ur deve

450nm.

a density of well as

of th iod the

aspirated an

leted. Cells were thawed to room temperature and 100μl 1% w/v methylene blue-

borate buffer solution was added to each well and incubated for 30 minutes. Cells

washed four times with

ol:0.1M HCl solution. Colour development was read at 650nm using a

spectrophotometer.

2.13.3 M

Cells were seeded at a density of 5x103 cells per well as described above. After

incubation the medium was aspirated, the cells washed three times with PBS and 100μl

of 5mg/ml methyl tetrazolium salt solution

96

cells

2.14 Reverse-Transcriptase Real-Time Polymerase Chain Reaction

Total RNA was isolated from cells that were 90-100% confluent in a 6 well plate

TM

ons. The yield of total RNA was assessed by spectrophotometry

at 260n

, and gel electrophoresis on a 2% agarose TAE gel.

RNA (μg) = Absorbance at 260nm x 40 x Dilution of RNA

A was removed from total RNA preparations by

DNase

al-time measurement of SYBR green (Sigma-

Aldrich,

se-keeping genes (HKG) glyceraldehyde pyruvate dehydrogenase

) or 18S ribosomal RNA levels. Transcript copy number was assessed by

const

n

n, Australia) and quantified by spectrophotometry at 260nm using the

formu

(μg) = Absorbance at 260nm x 50 x Dilution of DNA

The amplicon yield was converted to transcript copy number using the formula

below and serial 10 fold dilutions were made from 102 to 1010 and added to the PCR

experiment.

with 50μl DMSO and proliferation was assessed by colorimetry with a

spectrophotometer at 540nm.

using 500μl per well Trizol (Invitrogen, NSW, Australia) according to the

manufacturer’s instructi

m using the formula below. RNA quality was assessed by ratio of light absorbed

at 260nm and 280nm

Contaminating genomic DN

I digest (Promega, NSW, Australia). Complementary DNA was synthesised

using 1μg of template RNA using the Superscript IIITM first-strand synthesis system

(Invitrogen, Australia) according to the manufacturer’s instructions. Transcription of

genes of interest was assessed by re

USA) incorporation using the iCyclerTM system (Bio-rad, NSW, Australia) and

normalised to the hou

(GAPDH

ructing a standard curve for HKG and Col1α1. Amplicons for these genes were

synthesised by conventional PCR purified using the Qiaquick® DNA purificatio

system (Qiage

la below:

DNA

97

Transcript Copy Number = (Yield of DNA (μg) x 6.08 x 1023)/(Amplicon Length

try

actors and enzymes. Protein was

isolated from cell monolayers cultured in vitro or from bleomycin or control saline

2.15.

culture grade

were

quies M for 16-24 then trea r growth while

untr ls w M fo f the e

Cyto eins w shes es with i

100 ell cu ll lys ed

polyp tube. mix nch-top v bated

on tes, lubl separa lles

by ce ion at 1,40 inutes atant w a

new and s -4 we for 4 we

bed in d appro as

im f T teinase Sigma-A .

T thoroughl a bench-top v inute, inc e for

45 min nd then h for 15 h a pol

(Drem da, The le pro arated from

material by centrifug for nd superna

x 660)

2.15 Protein Biochemis

Cellular proteins were isolated to assess the activation of intracellular signalling

pathways and release and activation of growth f

mouse lung tissue.

1 Protein Isolation

Cells were seeded at approximately 1x105 cells per 100mm tissue

petri dishes (Falcon, Australia), maintained in NGM to 80-90% confluency. Cells

ced in MS hours ted with cytokines o factors,

eated contro ere maintained in MS r the duration o xperiment.

solic prot ere harvested from di after 2 wash ce cold PBS with

μl BB per c lture dish, and the ce ates were transferr to a 1.5ml

ropylene The cell lysates were ed using a be ortex and incu

ice for 5 minu and mixed again. So e re proteins we ted from organe

ntrifugat 0rpm for 10 m and the supern as transferred to

1.5ml tube tored at -20oC for 2 eks or -80oC eks or more.

Lung tissue was crushed as descri section 2.1 an ximately 50mg w

mersed in 1ml o LB containing pro inhibitors ( ldrich, Australia)

issue was mixed y on ortex for 1 m ubated on ic

utes a omogenised twice seconds wit ytron homogeniser

el, Bre Netherlands). Solub tein was sep insoluble

ation at 14,000g, 4oC 10 minutes a tant was

98

transf 1.5ml tube. yield w modified ssay

ies, Aust .

y hore

ately 25μ s used f spho by

gels a 5% acryl -Tris

stacki Samples reduc Gels at 100V

tions and then transferred to a PVDF membrane

after 2 hours at 230mA. Transfer of proteins onto PVDF membranes was assessed by

staining mem % Ponceau ma-Aldrich ution.

Membranes w ean of P tion wit -specific

antibody-pro blo emb kim milk

(Carnation, U solu m te -24 hours at

4oC. Primar incubated embranes at optimal dilution in 5% skim

for 1-3 hours at room temperature or 16-24 hours at 4oC. The

memb

secondary antibody or biotinylated conjugated secondary antibody and S-HRP were

ed at

room temperature for 1 hour or at 4oC for 16-24 hours. Membranes were then washed 3

times for 5-10 minutes before adding chemiluminescent peroxidase substrate solution

interactions were visualised with ECLTM hyperfilm (GE Biosciences, Piscataway, NJ,

USA) under safelight illumination using an automated x-ray processor. Equal lane

buffer for 30 minutes at 50oC and re-probing membranes for total protein of interest as

described above.

erred to a Protein as assessed using a Lowry A

(Bio-Rad Laborator ralia)

2.15.2 SDS-Polyacr lamide Gel Electrop sis

Approxim g protein wa or analysis of pho rylated proteins

Western blot on 10% acrylamide bis-Tris overlaid with amide bis

ng gel. were mixed 5:1 in ing sample buffer. were run

for 45-60 minutes under reducing condi

branes with 0.1 S (Sig , Australia) sol

ere washed cl

tein binding was

onceau S solu

cked by incubating m

h dH2O and non

ranes with 5% s

K) TBS-0.05T tion for 1 hour at roo

with m

mperature or 16

y antibodies were

milk TBS-0.05T

rane was washed twice with TBS-0.05T for 5 minutes, HRP conjugated

added at optimal dilutions in 5% skim milk TBS-0.05T. Incubations were conduct

(Sigma-Aldrich, USA) for 5 minutes at room temperature. Antibody-epitope

loading was assessed by stripping membranes of bound antibodies using Stripping

99

ed by

nt 5.1TM software. Changes in proteinase activity were

expre

and mixed to a

concentration of 10mM. SYBR green dye was obtained form Sigma-Aldrich (Australia)

and fluorescein calibration dye was from Bio-Rad Laboratories (Australia). All other

PCR reagents were obtained from Qiagen (Australia). Primers were resuspended in

ddH2O to a concentration of 1μg/μl. Working solutions were prepared at 100ng/μl and

both stock and working solutions were stored at -20oC. Standard and real-time PCR

conditions are outlined below.

2.15.3 Zymography

Samples (25μg) were mixed 5:1 in non-reducing sample buffer, loaded into 4%

stacking gels and resolved in 10% acrylamide bis-Tris gels containing 2mg/ml gelatine

or casein and run as described above. Gels were washed in ddH2O for 5 minutes at room

temperature followed by 3 washes in TBS-2.5Tx (Sigma-Aldrich, Australia) and then

incubated in developing buffer for 16-24 hours at 37oC. Proteins were visualis

briefly washing in ddH2O for 5 minutes at room temperature then staining in Comassie

blue solution for 30 minutes, excess stain was removed by washing in ddH2O for 30

minutes and repeated washes in destain solution until discrete bands were visible

indicative of gelatinase or caseinase activity. Gels were placed on a Canon flat-bed

scanner, images captured as tagged image format files (TIFF), and densitometry was

performed using ImageQua

ssed relative to wt levels.

2.16 Oligonucleotide Primers

All oligonucleotide primers were obtained from Invitrogen (Australia) in 50nM

quantities and desalted. dNTPs were obtained from Promega (Australia)

100

Reagents Standard PCR Real-Time PCR

10x Buffer 2.0μl 3.0μl

25mM MgCl2 0.5-2.0μl 0.5-2.0μl

10mM dNTP Mix 0.5μl 0.5μl

100ng/ml Sense Primer 0.7μl 0.7μl

100ng/ml Antisense Primer 0.7μl 0.7μl

Taq DNA Polymerase 0.2μl 0.2μl

100x SYBR Gree 0.2μl n I dye n/a

Fluorescein n/a 0.2μl

DNA 2μl 1μl

ddH2O 19.9-21.4μl 12.5-14.0μl

Total Volume 30μl 20μl

18S rRNA

18S rRNA was used to normalise the expression of genes of interest for real time

PCR the optimal annealing temperature and MgCl2 concentration was 59oC and

1.25mM respectively.

Sense 5’-GTAACCCGTTGAACCCCATT-3’; Antisense 5’-CCATCCAATCG

GTAGTAGCG-3’.

Gp130

Gp130 was used to genotype gp130 mutant mice and detect chimerism in bone

marrow transplanted mice. The optimal annealing temperature and MgCl2 concentration

was 60oC and 83nM respectively.

101

Sense (mutant) 5’-CTGAATGAACTGCAGGACGA-3’; Sense (wt) 5’- CAAG

TCCGAGTCCAC; Antisense (common) 5’-TGAAGCCACTCGT

CTTT

Glyceraldehyde 3-Phosphate Pyruvate Dehydrogenase (GAPDH)

2

Pro-C

h

C

TGTTCTCAAGG

AGC-3’.

GAPDH was used to normalise the expression of genes of interest for real time

PCR. The optimal annealing temperature and MgCl concentration was 60oC and

1.25mM respectively.

Sense 5’-TCGGTGTGAACGGATTTGGC-3’; Antisense 5’-GAATTTGCCGT

GAGTGGAGT-3’.

ollagen α1 Type 1 (Col1)

Col1 was used to assess collagen type 1 gene expression in mouse lung tissue

after bleomycin administration and lung fibroblasts grown in vitro after treatment wit

cytokines and growth factors. Optimal annealing temperature and MgCl2 concentration

was 59oC and 1.9mM respectively.

Sense 5’-GGAAGAGCGGAGAGTACTGG-3’; Antisense 5’-GTACTCGAA

GGGAATCCAT-3’.

Smad3

Smad3 was used to genotype Smad3 mutant mice after bleomycin administration.

Optimal annealing temperature and MgCl2 concentration was 60oC and 1.5mM

respectively.

Sense 5’-GACACTTTCTGTTCCTTCTTTGC-3’; Antisense 5’-GGCAGGGAC

ACACTCACAC-3’.

102

mycin

admi

CC

LacZ

LacZ primers were used to genotype Smad3 mutant mice after bleo

nistration. Optimal annealing temperature and MgCl2 concentration was 94oC and

1.5mM respectively.

Sense 5’-TCCCAACAGTTGCGCAGCCTGAATG-3’; Antisense 5’-ATAT

TGATCTTCCAGATAACTGCCG-3’.

2.17 Primary and Secondary Antibodies

All primary and secondary antibodies, and conjugate proteins were used as

described below for western blot (WB), immunohistochemistry (IHC),

immunocytochemistry (ICC) and enzyme-linked immunosorbent assay (ELISA).

Antigen Host Dilution and Use Source

P-STAT3 Rabbit Monoclonal 1:1000 WB 1:100 IHC

Cell Signaling Technology

STAT3 Rabbit Polyclonal 1:500 WB Santa Cruz Biotechnology

P-STAT1 Rabbit Polyclonal 1:500 WB Cell Signaling Technology

STAT1 Rabbit Polyclonal 1:1000 WB Cell Signaling Technology

P 1:100 IHC Technology -ERK1/2 Rabbit Monoclonal 1:1000 WB Cell Signaling

ERK2 Mouse 1:4000 WB Santa CrMonoclonal

uz Biotechnology

P-Smad3 Rabbit Polyclonal 1:500 WB Cell Signaling Technology

Smad3 Goat Polyclonal 1:1000 WB Santa Cruz Biotechnology

CD3 Rat Monoclonal 1:100 Serotec CD45R/B220 Rat Monoclonal 1:100 IHC BD Biosciences Streptavidin- n/a 1:500 ICC Alexa Fluor 546 Invitrogen

CD4 n Biosciences Rat Monoclonal 1:100 IHC Becton Dickinso

CD8 Rabbit Polyclonal 1:50 IHC Abcam Ki-67 Rabbit Polyclonal 1:100 IHC Abcam

MMP-2 Rabbit Polyclonal 1:100 IHC Chemicon Rat

Immunoglobulins- Rabbit Polyclonal 1:200 IHC 1:5000 WB Dako Cytomation

Biotin

103

Antigen Host Dilution and Use Source

Streptavidin-HRP n/a 1:4001:1000

IHC 0 WB Dako Cytomation

Mouse Immunoglobulins-

HRP Rabbit Polyclonal 1:2000 WB Dako Cytomation

α-Tubulin Mouse Monoclonal 1:2000 WB Sigma-Aldrich

α-Smooth Muscle Actin Rabbit Polyclonal 1:500 IHC,

ELISA, ICC Abcam

F4/80 Rat Monoclonal 1:50 IHC Abcam

2.18 Cytokines and Growth Factors

Protein Host Source

Recombinant Mouse IL-6

E.coli Bender Med Systems

Recombinant Mouse IL-6Rα

M.musculus myeloma cell line

R&D Systems

Recombinant Mouse IL-11

E.coli R&D Systems

Recombinant Human TGF-β1

Trichoplusia ni cell line

Sigma-Aldrich

2.19 Plasmids

Plasmid constructs were used for the luciferase reporter assays and the

immortalisation of primary lung fibroblasts. Photinus pyralis luciferase reporter

plasmids encoded the luciferase enzyme under the transcriptional control of the

COL1α1 promoter (pCOL1α1-Luc), and the STAT3 DNA binding sequence known as

the Acute Phase Response Element (pAPRE-Luc). The control Renilla reniformis

luciferase reporter plasmid was constitutively expressed by the Cytomegalovirus

promoter (pCMV-RLuc). The pAPRE-Luc and pCMV-RLuc were generously donated

by A/Prof Matthias Ernst. PLFs were immortalised using the pRSV-T plasmid,

generously donated by Prof. Bruce Howard, which encodes the natural p53 inhibitors

small and large ‘T’ antigens of Simian Virus 40.

104

2.20 TGF-β Reporter Assay

m were assessed in the laboratory of Dr Hong-Jian Zhu

versity of Melbourne by Dr Bo Wang using NIH3T3 cells stably transfected

with enerate

To investigate the TGF-β levels in the serum of the gp130 mutant mice,

s were seeded in 96-well plate for overnight. Then, the cells were

treate re

The TGF-β levels in seru

at the Uni

the Smad3 luciferase gene reporter pMLP-(CAGA)12-Luc (pCAGA). To g

pCAGA in NIH3T3 cells, pMLP-(CAGA)12-Luc and a plasmid encoding a hygromycin

resistance gene under the regulation of thymidine kinase (pTK-Hyg) were co-

transfected into NIH3T3 cells using Fugene6 TM (Roche Molecular Biochemicals, IN,

USA) as per manufacturer’s protocol, and the cells were selected in Hygromycin

(400ug/ml). Hygromycin (Roche Molecular Biochemicals) resistant clones were

isolated as single colonies. Positive clones were screened by luciferase assay using

Promega's luciferase reporter assay system for their ability to increase pCAGA activity

in the presence of TGF-β.

pCAGA/NIH3T3 cell

d with TGF-β titration or the serum (10%). After 24 h, luciferase activities we

assayed. Cell lysates (50ul/well) were used to measure the total light emission using a

microtiter plate luminometer (BMG Labtechnologies, VIC, Australia).

105

GP130-STAT3 SIGNALLING REGULATES THE

DEVELOPMENT OF BLEOMYCIN-INDUCED

CHAPTER THREE

LUNG FIBROSIS IN VIVO

106

the excessive deposition of ECM, particularly fibrillar

collagens, and the development of fibroblastic foci (Katzenstein and Myers, 1998, King

et al., 2001). The cryptogenic nature of IPF has lead to a number of theories concerning

the pathogenesis of this disease, such as an ongoing and unremitting inflammatory

reaction, and dysregulated epithelial-mesenchymal interactions, that culminate in

hypertrophy of the interstitial mesenchyme and myofibroblast differentiation (Noble,

2003, Selman et al., 2001).

The IL-6 family of cytokines are involved in these processes (Scaffidi et al., 2002)

(Yoshida et al., 1995) and are elevated in the BALF and sera of patients with IPF and

other lung diseases (Lesur et al., 1994, Takizawa et al., 1997, Pantelidis et al., 2001,

Tsantes et al., 2003, Scala et al., 2004). This family of cytokines has a significant role in

the regulation of lung morphology (Kuhn et al., 2000, O'Hara et al., 2003) and

pulmonary responses to airborne particles (Wang et al., 2000a, Xing et al., 1998).

Animal studies have demonstrated that IL-6 and IL-11 mutant animals have

morphological and physiological differences to their wt littermates (DiCosmo et al.,

1994, Kuhn et al., 2000, Tang et al., 1996, Wang et al., 2000a, Xing et al., 1998). Lung

specific over-expression of IL-6 and IL-11, in both an acute and chronic setting, induces

airspace enlargement, peri-bronchial fibrosis and thickening of the tunica adventitia of

the conducting airways (DiCosmo et al., 1994, Kuhn et al., 2000, Tang et al., 1996),

while deletion of IL-6 or IL-11Rα induces an enhanced inflammatory response to

airborne, immunogenic and injurious particles (Xing et al., 1998, Qiu et al., 2004,

Johnston et al., 2005, Betz et al., 1998). Recently, it has been shown that enhanced

3.1 Introduction

IPF is a heterogeneous disease involving severe remodelling of the lung

parenchyma culminating in

107

expression of IL-6 and gp130 is associated with the susceptibility of in-bred mouse

omycin-induced pulmonary fibrosis (Cavarra et al., 2004).

IL-6 and IL-11 induce the activation of gp130-mediated signalling pathways by

promoting gp130 homo-dimerisation and activation of Ras-ERK1/2 and STAT1/3

signalling pathways. There is limited understanding of the effects of IL-6/gp130-

mediated ERK1/2 and IL-6/gp130-mediated STAT1/3 signalling on pulmonary

physiology and morphology. However, Moodley et al. (2003) found that IL-6 induced

proliferation of lung fibroblasts derived from IPF patients and inhibited the proliferation

of lung fibroblasts derived from control patients; while IL-11 promoted the proliferation

of lung fibroblasts from IPF and control patients (Moodley et al., 2003). These different

responses were due to preferential and prolonged activation of the IL-6/gp130-ERK1/2

pathway and blunted activation of the IL-6/gp130-STAT3 pathway by lung fibroblasts

derived from IPF. Interestingly, in vivo models of fibrosis in other organ systems has

attributed the development and progression of fibrosis to preferential and prolonged

activation of gp130-STAT1/3 signalling (Streetz et al., 2003, Ezure et al., 2000, Lim et

al., 2006, Ogata et al., 2006).

gp13

y of the

r

d

strains to ble

It is the aim of this chapter to assess the role of directed gp130-ERK1/2 and

0-STAT1/3 signalling in the development of bleomycin-induced murine

pulmonary fibrosis. The bleomycin-induced lung fibrosis model replicates man

key morphological changes observed in IPF, such as the accumulation of fibrillar

collagens within the alveolar interstitium and the formation of fibroblastic foci. Directed

gp130-medidated signalling will be achieved by utilising gp130ΔSTAT/ΔSTAT and

gp130757F/757F mice developed by A/Prof. Matthias Ernst (Ludwig Inst. for Cance

Research, Victoria, Australia). These mice carry mutations that direct and enhance

gp130-mediated signalling through the ERK1/2 (gp130ΔSTAT/ΔSTAT) or the STAT1/3

(gp130757F/757F) pathways (Ernst et al., 2001, Tebbutt et al., 2002). The effect of directe

108

s gp130-mediated signalling on bleomycin-induced lung fibrosis and collagen synthesi

will be assessed using in vivo and in vitro methods.

109

ΔSTAT/ΔSTAT 757F/757F

757F/757F +/-

isofluorane and a 50μl droplet 15U/ml of

bleomycin in sterile saline or saline alone was applied to the left nostril and passively

froze hemical

er. C5

o hours. A

standard curve was c

ted

lin-eosin

3.2 Materials and Methods

Animals

Pulmonary fibrosis was induced in groups of mice (n≥3) with mutations

introduced into the intracellular region of gp130 (gp130 , gp130 and

gp130 ;STAT3 ) and wt mice by trans-nasal delivery of bleomycin as described

in section 2.2. Mice were anaesthetised with

inhaled. Lungs were either prepared for histology as described in section 2.9, or snap

n in liquid N2 and crushed into a fine powder and stored at -80oC for bioc

analysis lat 7BL/6 mice were used to optimise the isolation of viable fibroblasts

from lung tissue for in vitro experiments.

Chloramine T Assay

Powdered lung tissue from mice 30 days after bleomycin administration was

assessed for hydroxyproline (Hyp) content using the chloramine T assay as described in

section 2.5.1. Briefly, tissue was hydrolysed in 2ml 6N HCl at 110 C for 16-18

reated using serial dilutions of 1mg/ml L-hydroxyproline and tissue

hydroxyproline content was assessed as a measure of lung collagen.

Histology

Lung tissue was embedded in paraffin wax as described in section 2.9 and 5μm

serial sections were cut and numbered from tissue blocks and mounted on sialina

slides by PathWest Laboratory Medicine WA, Perth Western Australia. Tissue sections

from wt, gp130ΔSTAT/ΔSTAT, gp130757F/757F and gp130757F/757F;STAT3+/- mice 30 and 60

days after bleomycin or control saline treatment were processed for haematoxy

(H&E) and Masson’s trichrome stains by PathWest.

110

es.

ction 2.5.2.

HPLC

Real Time PCR

Total RNA was isolated from lung tissue using Trizol as stipulated by the

manufacturer. A 1μg aliquot of total RNA was used as a template for cDNA synthesis

using the Superscript III cDNA synthesis kit according to the manufacturer’s

instructions. Gene specific oligonucleotide primers for Col1α1 and the housekeeping

gene GAPDH were added to 1μl of cDNA for real-time PCR to assess collagen

synthesis in lung tissue of bleomycin treated wt, gp130 , gp130757F/757F and

gp130 7F/757F;STAT3 mice as described in section 2.14.

Matrix metalloproteinase levels were assessed by adding 2mg/ml gelatin or casein

to 10% polyacrylamide gels. Protein was isolated from mouse lung tissue 3 days after

bleomycin treatment and 50μg was added to gels and run under non-denaturing

conditions as described in section 2.15.3. Proteins were visualised by staining in

Comassie blue solution for 30 minutes, destained and images were captured using a

Canon flat-bed scanner. Densitometry was performed using ImageQuant 5.1

software.

HPLC

HPLC was performed with hydrolysate obtained from 25-50mg of crushed lung

tissue from bleomycin or saline treated mice to assess proline content of lung biopsi

Total lung collagen content was estimated from this data as described in se

analysis was performed in the laboratory of Prof. Geoffrey Laurent, University

College London, U.K.

TM

ΔSTAT/ΔSTAT

75 +/-

Zymography

TM

Luciferase Gene Reporter Assays

MEFs were generously donated by A/Prof Matthias Ernst and seeded into 24 well

tissue culture plates simultaneously transfected with pCOL1α1-Luc and pCMV-RLuc or

111

pAPRE-Luc and pCMV-RLuc. All transfections were performed using Fugene 6TM

transfection reagent as recommended by the manufacturer and luciferase reporter

activity was assessed using the Dual Luciferase Reporter System (Promega, USA) as

described in section 2.5.3.

112

3.3.1 Chloramine T Assessment of Lung Collagen Content

assessed two assays that estimate collagen content of lung tissue by

quantifying the hydrolysed proline content of mouse lungs. These were the chloramine

T chromogenic assay and HPLC. The chloramine T assay yielded linear measurements

initial experiments with approximately 20-50mg of powdered lung tissue from wt,

ΔSTAT/ΔSTAT 757F/757F

proline were below the lower threshold of the chloramine T assay. en assays

using HPLC to measure hydroxyproline levels in 20-50mg of lung tissue are frequently

hydroxyproline within these quanta of tissue. Therefore, the quantitative assessment of

leomycin-Induced Pulmonary Fibrosis

t

3.3 Results

This study

of hydroxyproline within the range of 2-20μg of the amino acid (figure 3.1). However,

gp130 and gp130 mice indicated that physiological quantities of

Collag

reported in the literature and can accurately measure the physiological levels of

collagen accumulation and fibrosis was performed by HPLC analysis of acidified lung

tissue in this study.

3.3.2 Gp130-STAT1/3 Signalling Enhances B

The first phase of this study was to evaluate the development of pulmonary

fibrosis in gp130 mutant mice and wt control mice after intra nasal delivery of

bleomycin compared to saline treated controls. Animals were culled 30 or 60 days after

treatment and lungs were removed and prepared for histology and HPLC measuremen

of collagen levels. There was no significant difference in survival between wt,

gp130ΔSTAT/ΔSTAT and gp130757F/757F mice over the 30 day period (figure 3.2).

Morphological examination of H&E and Masson’s trichrome stained lung sections from

saline treated mice showed no significant differences in lung morphology of wt and

gp130ΔSTAT/ΔSTAT mice (figure 3.3). Most regions of gp130757F/757F mouse lungs

113

o d in the g L-

ne. The chloramine T reaction measures hydroxyproline levels within the f 1-20μg (A). Hydroxyproline in 30-50mg lung tissue samples of wt,

gp130ΔSTAT/ΔSTAT 757F/757F T rea

1.0

1.5

3.0

Figure 3.1. Assessment of chloramine T assay for the measurement of collagen inmouse lung tissue.

Lung tissue (30-50mg) from wt, gp130ΔSTAT/ΔSTAT and gp130757F/757F was hydrolysed in 2ml of 6N HCl at 110 C for 16 hours. A 20μl aliquot of hydrolysate was usechloramine T reaction and a standard curve was made using a range of 0.1-20μhydroxyprolilinear range o

0 5 10 15 200

0.5

and gp130 are less than the lower threshold of the chloraminection (B). Data as represented as the mean ± S.D., n=3.

A

2.0

2.5

3.5 Standards Line of Best Fit

Hydroxyproline -μg

bso

B

0

0.2

0.3

Arb

anm

0.1

ce - λ=

570n

0.4 wt gp130ΔSTAT/ΔSTAT

gp130757F/757F

Standards

Line of Best Fit

Hydroxyproline - μg

bsor

banc

e - λ

=570

n

0 0.5 1.0 1.5 2.0

Am

Fitreat

eir plot. There was no significant difference in surviv ans-nasal bleomycin

by chi-squared log-rank test, p>0.05, n≥6.

114

gu

gp130ΔSTAT/ΔSTAT

0 10 20 300

50

75

100

wt

25 gp130757F/757F

Days

et

iP

rcen

surv

val

re 3.2. Survival wt and gp130 mutant mice is not affected by bleomycin ment.

Animal survival over the 30 day experimental period was recorded in a Kaplan-Mal after tr

administration between wt, gp130ΔSTAT/ΔSTAT, and gp130757F/757F. Data was analysed

E

was

ons

there was abundant ECM in these foci but it did

not p

ed

s after

115

appeared normal while some areas of the lung in some mice showed peribronchial

mononuclear aggregates, thickened alveolar septa and exudative pneumonia in specific

lobes (figure 3.4). There were no signs of extensive fibrosis associated with these

morphological alterations (figure 3.4).

Bleomycin-induced changes in lung morphology were assessed initially by H&

stained sections from treated mice. Wt mice had significant remodelling of the lung

parenchyma with bronchiolo-centric fibrosis of the alveolar interstitium. Fibrosis

confirmed upon examination of parallel Masson’s trichrome stained lung secti

(figure 3.5). Gp130ΔSTAT/ΔSTAT mice did not have prominent remodelling of the lung

parenchyma although there appeared to be some enlargement of the airspaces and

sporadic foci of limited fibrosis emanating from the pleura and bronchioles (figure 3.6).

Masson’s trichrome sections indicated

rogress to fibrosis. There were additional foci of mononuclear cell aggregates

localised to bronchiolar-alveolar junctions (figure 3.6). Gp130757F/757F mice had

dramatic and severe remodelling of the lung parenchyma 30 days after bleomycin

treatment with hypertrophic alveolar epithelium, extensive fibrosis, mononuclear

infiltration of the parenchyma and destruction of the pulmonary acini (figures 3.7).

Quantitative assessment of collagen accumulation confirmed the histological

observations of wt, gp130ΔSTAT/ΔSTAT and gp130757F/757F mouse lungs. Bleomycin treated

wt mice had a 100% increase in total lung collagen relative to saline treated wt mice

(p<0.005), while there was no significant increase in lung collagen of bleomycin treat

gp130ΔSTAT/ΔSTAT mice compared to saline controls (figure 3.8). In contrast,

gp130757F/757F mice had 300% more lung collagen (p<0.005) after bleomycin

administration compared gp130757F/757F mice treated with saline.

The development of bleomycin-induced lung fibrosis was assessed 60 day

bleomycin administration in wt and gp130ΔSTAT/ΔSTAT mice to determine if these mice

116

C -

gpΔ

ΔE

gp1

30Δ

ΔT

AT

AF

–

130

STA

T/

STA

T, H

&

STA

T/ST

AT, M

T

H -

gp13

0ΔST

/ΔST

T, M

T

B –

wt,

H&

E

G -

E –

wt,

M

wt,

MT

(i)

(ii)

(i

(ii)

)

(i)

(ii)

D -

gpΔS

T/Δ

ST, H

&E

gp

ΔST

/ΔST

A m

ice.

ne tr

etm

ent,

ind

unde

r e

and

fixed

in 4

%

ith H

&E

(A-D

) or M

aon

’om

-HH

&E

stai

ned

lung

mor

phol

days

af

trat

men

t. M

sson

’io

n of

EC

in th

e t

o

f con

duct

ing

le b

rs =

2A

, E

, G)

m (B

130

AT

AT

Figu

ran

ir

eo

or

gp

wt o

r13

0A

TT

Trac

hd

re0

/

ter s

ali

afla

tepr

essu

rpa

rafo

Lung

wer

mf

nd 5

aine

d w

sss t

richr

e (E

). lu

ng

nST

AT

i sh

ow

nges

in

ogy

30

ter s

alin

e e

as

trich

r st

ains

of

()

)

mst

ribut

M

unic

aad

vent

itia

airw

ays

(i), p

uon

ary

bloo

d v

s (ii)

hem

ve

n=3.

Sca

a00μm

(C

, a

nd 5

0μ, D

, F, H

).

wt

&

e 3.

3. T

rs-

nasa

l sal

ne t

eatm

nt d

es n

t alte

lun

mor

holo

gy o

f

ea a

n lu

ngs w

e e

xcis

ed fr

om w

t and

gp1

3ΔS

TATΔS

TAT

mic

e 30

day

s af

rmal

dehy

de.

s e

ebe

dded

in p

araf

in a

μm se

ctio

ns w

ere

stse

ctio

s of w

t (A

,B) a

nd g

p130

Δ/Δ

STA

T (E, F

) mce

no g

ross

cha

ome

wt

C, D

and

gp1

30ΔS

TAT/ΔS

TAT

(F, G

lung

s sho

w n

oral

di

lmes

sel

, and

t a

lveo

li. I

ages

are

repr

esen

tati

of

A –

, H

E

A

C – H&E D –H&E

igns of exudative pneumonia and lymp

757F/757F ,

paraffin and 5μm sections were stained with H&E (A-D) or Masson’s trichrome (MT; E,

lobes with normal morphology (A, B). Some lobes showed altered lung morphology (C, D) onia

representative of n=3. Scale bars = 200μm (A, C, E) and 50μm (B, D, F).

117

– H&E B – H&E

E - MT F - MT

Figure 3.4. Gp130757F/757F control lungs show s

(i)

(i)

(i) (ii)

(ii)

(iii)

hocytic aggregates.

Trachea and lungs were excised from gp130 mice 30 days after saline treatmentinflated under pressure and fixed in 4% paraformaldehyde. Lungs were embedded in

F). H&E stained lung sections of gp130757F/757F mice 30 days after saline treatment show

with discrete foci of peribronchial lymphocytic aggregates (i) and exudative pneum(ii). Staining of serial sections by Masson’s trichrome (MT) show ECM accumulation inthe alveolar interstitium (iii) is associated with the exudative pneumonia. Images are

d

118

B - MTA – H&E

C - MT D - MT

(i) (i)

(ii)

Figure 3.5. Wt mice develop fibrosis 30 days after bleomycin-induced lung injury.

Trachea and lungs were excised from gp130757F/757F mice 30 days after bleomycin treatment, inflated under pressure and fixed in 4% paraformaldehyde. Lungs were embedded in paraffin and 5μm sections were stained with H&E (A, B) or Masson’s trichrome (MT; C, D). H&E stained sections of lung from wt mice 30 days after bleomycin-induced pulmonary injury (A). MT stained serial sections of lung (B) show areas of remodelled lung contain large amounts of ECM (i). High power images of MT stained lung sections show remodelled areas of lung contain dense foci of ECM (i) anhyperplastic pneumocytes (ii). Images are representative of n=3 mice. Scale bars=200μm (A, B) and 20μm (C, D).

Figu

bleo

Tractreaem

daysstainimaof mn=3 m

119

re 3.6. Gp130ΔSTAT/ΔSTAT mice show minor lung remodelling 30 days after

mycin-induced lung injury.

hea and lungs were excised from gp130ΔSTAT/ΔSTAT mice 30 days after bleomycin tment, inflated under pressure and fixed in 4% paraformaldehyde. Lungs were

bedded in paraffin and 5μm sections were stained with H&E (A, B) or Masson’s trichrome (MT; C, D). H&E stained sections of lung from gp130ΔSTAT/ΔSTAT mice 30

after bleomycin-induced pulmonary injury (A) show some lung remodelling. MT of serial lung sections shows areas of remodelling contain ECM (i). High power

ges (C, D) show these areas of remodelling contain dense ECM (i) and aggregates ononuclear cells peripheral to the small airways (ii). Images are representative of

ice. Scale bars=200μm (A, B) and 20μm (C, D).

A – H&E B - MT

(i)

D - MTC - MT

(i)

(ii)

30 days after bleomycin treatment.

cin

embedded in paraffin and 5μm sections were stained with H&E (A, B) or Masson’s d section of lung taken from gp130757F/757F mice 30 g injury (A) show there is severe remodelling of

the l in ECM

vascular spaces (iii) and within the remodelled parenchyma (iv). Images are

B - MT

120

A – H&E

D - MT

(ii

C – MT

)

(iv)

(i)

(iii)

(iv)

Figure 3.7. Gp130757F/757F mice develop severe and extensive pulmonary fibrosis

Trachea and lungs were excised from gp130757F/757F mice 30 days after bleomytreatment, inflated under pressure and fixed in 4% paraformaldehyde. Lungs were

trichrome (MT; C, D).H&E stainedays after bleomycin-induced lun

ung. MT stained serial section of lung (B) shows the remodelled lung is rich . High power images of the remodelled area (C, D) show dense ECM (i),

hyperplastic alveolar epithelium (ii) and aggregates of lymphocytic cells in the peri-

representative of n=3 mice. Scale bars = 200μm (A,B) and 20μm (C, D).

olg

121 C

lage

n –

m/lu

ng

salinebleo

5 10

0

15

30

20 25

35 wtgp130ΔSTAT/ΔSTAT

gp130757F/757F

bleo bleosalinesaline

*

*

Figure 3.8. Directed gp130-mediated signalling regulates the sensitivity to bleomycin-induced lung fibrosis.

in lung tissue (25-50mg) from bleomycin or saline treated mice. Lung collagen of wt

respectively) 30 days after bleomycin administration while there was no significant ΔSTAT/ΔSTAT 757F/757F

mice increased by approximately 300% (9.1mg vs 30.4mg, saline vs bleomycin

HPLC quantification of hydroxyproline and presented as the mean ± S.E.M., n≥8 an

Lung collagen content was assessed by HPLC measurement of hydroxyproline content

mice increased by approximately 100% (from 5.6mg to 10.6mg, saline vs bleomycin

increase in lung collagen in gp130 mice. Lung collagen of gp130

respectively) 30 days after bleomycin administration. Lung collagen was assessed by d

*p≤0.003.

es included foci of mononuclear aggregates

locali

to

3.3.3 Gp130-Mediated STAT3 Signalling Promotes Bleomycin-Induced Lung Fibrosis

The contrast in bleomycin-induced lung phenotypes between wt, gp130ΔSTAT/ΔSTAT

and gp130757F/757F mice indicated that directed gp130-mediated signalling could regulate

the development of bleomycin-induced lung fibrosis. However, the results described

above did not indicate if the absence of gp130-STAT1/3 signalling in gp130ΔSTAT/ΔSTAT

mice conferred protection from bleomycin-induced lung fibrosis or if enhanced gp130-

ERK1/2 signalling in these mice protected them from bleomycin-induced lung fibrosis.

To address this point gp130757F/757F mice were cross-bred with animals lacking a single

STAT3 allele, gp130757F/757F;STAT3+/- mice, and treated with bleomycin.

The lungs of gp130757F/757F;STAT3+/- mice treated with saline appeared normal

after 30 days, with no alterations in morphology of the airways, or the pulmonary acinus

(figure 3.10). Genetic depletion of one STAT3 allele had no effect on animal survival

after bleomycin administration over the 30 day period. Histological examination of

bleomycin injured gp130757F/757F;STAT3+/- lung tissue demonstrated significant

remodelling of lung parenchyma including the accumulation of the ECM in the alveolar

interstitium (figure 3.10). Measurement of hydroxyproline levels in the lungs by HPLC

showed there was significantly less collagen (p<0.02) in bleomycin treated

gp130757F/757F;STAT3+/- mice relative to bleomycin treated gp130757F/757F mice after 30

days (figure 3.11). Although morphological examination of the lungs suggested there

122

were resistant to bleomycin-induced fibrosis or if the onset of fibrosis was delayed. The

fibrosis observed in wt mice persisted for 60 days with no signs of progression, while in

gp130ΔSTAT/ΔSTAT mice morphological chang

sed to minor arterioles and thickened alveolar septa with abundant ECM (figure

3.9). However, this lesion was qualitatively similar to that observed 30 days after

bleomycin administration and did not represent a progression of this earlier lesion

fibrosis.

123

Trachea and lungs were excised from wt and gp 30ΔSTAT/ΔSTAT mice 60 days after aformaldehyde.

Lungs were embedded in paraffin and 5μm sections were stained with Masson’s delling (i) was evident in MT stained

ys. The development of fibrosis is inhibited in gp130ΔSTAT/ΔSTAT

Thes).

Figure 3.9. Gp130ΔSTAT/ΔSTAT mice have no signs of lung fibrosis 60 days after bleomycin-induced lung injury.

1bleomycin treatment, inflated under pressure and fixed in 4% par

trichrome (MT).Bleomycin-induced lung remotissue section of wt mice (A, C) after 60 da

mice for up to 60 days after bleomycin administration (C, D). e animals had focal accumulations of macrophages in the alveoli (ii), but no

fibrosis. Images are representative of n=2. Scale bars=200μm (A, B) and 50μm (C, D

B - gp130ΔSTAT/ΔSTAT

(ii)

(ii)

D - gp130ΔSTAT/ΔSTAT

(ii)

A - wt

(i)

(i)

C - wt

(i)

(i)

Figlung

after

). line had no effect on lung structure or morphology after 30 days. ice had foci of remodelled lung with abundant interstitial ECM

that was localised to the pleura (i) and the airways (ii). Images are representative of n=2 mice and scale = 200μm (A, B) and 50μm (C-F).

A

F

124

u

C

B

D

re 3.10. Gp130757F/757F;STAT3+/- mice show evidence of bleomycin-induced fibrosis.

Trachea and lungs were excised from gp130757F/757F;STAT3+/- mice 30 daysbleomycin or saline treatment, inflated under pressure and fixed in 4% paraformaldehyde. Lungs were embedded in paraffin and 5μm sections were stained with Masson’s trichrome (MT; C, D). MT stained lung sections of gp130757F/757F;STAT3+/- mice treated with saline (A, C, E) and bleomycin (B, D, FAdministration of saBleomycin treated m

E

(i)(i)(i)

(ii)

(ii)

(ii)

als there was no significant change in lung

collag

gp130757F/757F;STAT3+/- mice and normalised to the house-keeping gene GAPDH.

Normalised values were expressed as a ratio of wt values. COL1 transcription was

inhibited in bleomycin treated gp130ΔSTAT/ΔSTAT mice relative to wt mice; while

gp130757F/757F mice had 2.5 fold more collagen transcripts than wt. There was no

difference in the ratio of COL1:GAPDH transcripts between wt and

gp130757F/757F;STAT3+/- mice (figure 3.11). These findings demonstrated that gp130-

STAT3 signalling promoted de novo collagen synthesis in bleomycin treated lungs,

rather than the absence of gp130-ERK1/2 signalling.

3.3.4 Gp130-Mediated ERK1/2 Signalling Increases MMPs after Bleomycin-Induced

Lung Injury.

The accumulation of ECM in the lungs is a product of an imposed imbalance

between the normal mechanisms of ECM synthesis and ECM degradation. Lung tissue

was taken from wt, gp130ΔSTAT/ΔSTAT and gp130757F/757F mice three days after bleomycin

administration to assess their acute response. The rationale being that the protection

from lung fibrosis observed in gp130ΔSTAT/ΔSTAT mice was due increased proteinase

activity that prevented ECM accumulation and fibrotic lesions from becoming

established. Gelatin zymography showed there were three proteins with molecular

weights of 115kDa, 90kDa and 65kDa that corresponded to the predicted weights of

pro-MMP-9, MMP-9 and MMP-2 (figure 3.12). Densitometry of these bands

125

was evidence of some fibrosis in these anim

en after bleomycin administration to gp130757F/757F;STAT3+/- mice relative to

saline control mice (figure 3.11).

Real-time PCR analysis of collagen type 1 (COL1) mRNA transcription in 30 day

bleomycin treated lung tissue was performed to determine if gp130-mediated signalling

was promoting de novo collagen synthesis. COL1 transcription was assessed in lung

tissue from bleomycin treated wt, gp130ΔSTAT/ΔSTAT, gp130757F/757F and

126

mycin-induced lung fibrosis and collagen synthesis.

ntent

ence b

gp130 ibleomycin

transc analysis (B). COLlowe

A gp130757F/757F

Figure 3.11. Gp130-STAT3 signalling regulates the development of bleo

Lung collagen content was assessed by HPLC measurement of hydroxyproline coin lung tissue (25-50mg) from bleomycin or saline treated mice. Assessment of lung collagen shows no significant differ etween saline and bleomycin treated gp130757F/757F;STAT3+/- mice (A). However, bleomycin-induced lung collagen was significantly lower in gp130757F/757F;STAT3+/- mice than 757F/757F m ce (A). Total RNA was isolated from lung tissue (10-20mg) of mice 30 days after treatment, 1μg of total RNA was reversed ribed for real-time PCR

1α1 transcription was normalised to the housekeeping gene GAPDH. COL1α1 is r in the lung tissue of bleomycin treated gp130ΔSTAT/ΔSTAT mice and higher in

gp130757F/757F mice compared to wt mice (B). COL1α1 transcription in gp130757F/757F;STAT3+/- mice is lower compared to gp130757F/757F mice and is equivalent to wt levels 30 days post-bleomycin (B). Data is represented as the mean ± S.E.M of n≥4 mice, *p<0.02. COL1α1 transcription was normalised against GAPDH and expressed as the fold change relative to wt, n≥4.

gp130757F/757F;STAT3+/-

saline bleomycin saline bleomycin0

10

15

20

30

Col

lage

nm

g/lu

n

5

25

–

35

g

* *

0.5

2.5

0

0

1.0

1.5

2.0

3.

3.5

wt gp130ΔSTAT/ΔSTAT

gp130757F/757F

gp130757F/757F;STAT3+/-

Fold

ge C

han

B

and gp130757F/757F mice 3 days after bleomycin treatment. Casein

zymo weight of

r

brane-

cin-

3.3.5 IL-6 Gp130-STAT3 Signalling Promotes Collagen Transcription

ed from wt and gp130 mutant mice (MEFs) were

transi

ible

127

demonstrated that total MMP-9 (pro-MMP-9 and MMP-9) was significantly elevated in

the lung tissue of gp130ΔSTAT/ΔSTAT mice compared to wt and gp130757F/757F mice

(p<0.05). There was no difference in the amount of lung MMP-2 between wt,

gp130ΔSTAT/ΔSTAT

graphy identified only a protein with caseinase activity and a molecular

approximately 70kDa. This caseinase had the equivalent mass of the pro-peptides fo

MMP-15, MMP-16, MMP-24, MMP-25 pro-peptides, which belong to the mem

type MMP family (MT-MMP; figure 3.12). This band was significantly elevated in

gp130ΔSTAT/ΔSTAT lung tissue but was not detected in the lung tissue of wt or

gp130757F/757F mice 3 days after bleomycin treatment (figure 3.12). These results

suggested that the protection from bleomycin-induced lung fibrosis observed in

gp130ΔSTAT/ΔSTAT mice was associated with enhanced levels of MMP-9 and the pro-

peptides for MMP-15, MMP-16, MMP-24 and/or MMP-25 three days after bleomy

induced lung injury.

Embryonic fibroblasts deriv

ently transfected with pCOL1α1-Luc. MEFs were stimulated with the designer

cytokine hyper-IL-6, an IL-6αR and IL-6 fusion protein. Gp130757F/757F MEFs had

approximately 5-fold more pCOL1α1-Luc reporter activity compared to wt,

gp130ΔSTAT/ΔSTAT and gp130757F/757F;STAT3+/- MEFs at 100ng/ml hyper-IL-6 (Figure

3.13). To ensure this response was due to IL-6/gp130-STAT3 signalling all MEFs were

transiently transfected with the luciferase reporter encoding the acute phase response

element (APRE), a characterised DNA sequence that is recognised by STAT3

specifically. Hyper-IL-6 induced pAPRE-Luc activity was greatest in gp130757F/757F

MEFs with reduced activity in wt and gp130757F/757F;STAT3+/- MEFS and neglig

activity detected in gp130ΔSTAT/ΔSTAT MEFs (figure 3.13).

128

wt gp130ΔSTAT/ΔSTAT gp130757F/757F

A P-9

MMP-9

MMP-2

Pro-MM

Pro-MMP-15/16/24/25

C Pro-MMP-15/16/24/25

0

20

40

60

80 *

ln

Pixe

Inte

sity

x10

3

D

0

50

100

150

Pixe

l Dns

x10

Figur ΔSTAT/ΔSTAT ycin treat

gp130 mice 3 days after bleomycin treatment and protein samples (25μg)

polyacrylamide gels containing 1mg/ml gelatine (A) or casein (B) until the dye front

and MMP-2. Densitometry of these bands demonstrated that MMP-9 (empty bars)

gp130 , but there was no difference in MMP-2 (filled bars) levels. Casein P-15,

MMP-16, MMP-24 and MMP-25. Densitometry demonstrated that this caseinase (D)

gp130 mouse lungs. n≥3, *p<0.05.

*

eity

3

MMP-9 & MMP-2B

e 3.12. MMPs are elevated in gp130 mice 3 days after bleomment compared to wt and gp130757F/757F mice.

Protein was isolated from lung tissue (50mg) of wt, gp130ΔSTAT/ΔSTAT and 757F/757F

were mixed with 5X non-reducing sample buffer for electrophoresis on 10%

ran off the gel. Gelatin zymography (A) detected 3 gelatinases pro-MMP-9, MMP-9

was significantly elevated in gp130ΔSTAT/ΔSTAT mice (B) compared to wt and 757F/757F

zymography detected a caseinase (C) that corresponded to pro-peptides of MM

was significantly elevated in gp130ΔSTAT/ΔSTAT mouse lungs compared to wt and 757F/757F

Hyp

stim

lucife

129

A

0.08 0.40 2.0 10 500

1

2

wtgp130ΔSTAT/ΔSTAT

gp130757F/757F ****

*

Hyper-IL-6 - ng/ml

Fold

Cha

nge

B

wtgp130757F/757F

gp130757F/757F;STAT3+/-

Figure 3.13. Gp130-STAT3 signalling promotes collagen transcription bfibrobasts.

757F/757F

to wt and but does not induce pCOL1-Luc activity in gp130ΔSTAT/ΔSTAT MEFs after 72 757F/757F

MEFs relative to wt and gp130757F/757F;STAT3+/- MEFs after 48 hours of Hyper-IL-6

Luc; empty bars) activity (B). Firefly luciferase activity was normalised against renilla

n=3, *p<0.05.

y

er-IL-6 enhances pCOL1α1-Luc reporter activity in gp130 MEFs relative

hours (A). pCOL1α1-Luc reporter activity (filled bars) is enhanced in gp130

ulation (B). This corresponded to Hyper-IL-6-induced STAT3 reporter (pAPRE-

rase activity and expressed as fold change of untreated syngeneic cells ± S.E.M.,

Hyper-IL-6 - ng/ml10 30

0

10

20

**

*

*

*

100

30

* *

*

*

*

40

50

Fold

Cha

nge

130

t

pathwa of the

er thro cating

d

ng pa

v

oto

t nd over-

expression of IL-6 family cytokines and gp130 in the context of lung physiology

(Brandt et al., 1990, DiCosmo et al., 1994, Kuhn et al., 2000, Lentsch et al., 1999, Qiu

et al., 2004, Sheridan et al., 1999, Tang et al., 1996, Wang et al., 2000a, Waxman et al.,

1998, Xing et al., 1998, Betz et al., 1998). Targeted over-expression of IL-6 and IL-11

in the lung produces; lymphocytic aggregates focussed around the airways, lymphocytic

pneumonia, airspace enlargement and fibrosis of the airways (Xing et al., 1994, Yoshida

et al., 1995). Genetic disruption of IL-6 and gp130 elicits an enhanced inflammatory

response after injury and age-related airspace enlargement respectively (Xing et al.,

3.4 Discussion

The findings in this chapter have demonstrated that gp130-mediated signalling

can regula e disparate bleomycin-induced lung phenotypes depending upon the

activation of the ERK1/2 or STAT3 ys. Directed activation gp130-STAT3

pathway enhanced lung fibrosis by increasing collagen transcription and accumulation

following bleomycin treatment. It was also shown that IL-6 activated the collagen

promot ugh gp130-STAT3 signalling impli IL-6 in de novo collagen

synthesis. Ablated STAT3 activation (directed gp130-ERK1/2 signalling) conferred

protection from lung fibrosis following bleomycin treatment. Protection from lung

fibrosis was associated with increased MMP9 and MT-MMP activity and decrease

collagen transcription in vivo. This report is the first study to describe both enhanced

fibrosis and protection from fibrosis through the directed activation of different

signalli thway by a cell bound receptor.

There is a growing body of evidence associating the IL-6 family of cytokines with

lung physiology including the de elopment of pulmonary fibrosis. IL-6 is elevated in

human lung disease and following bleomycin-induced lung injury in rodents (Sakam

et al., 2002, Olman et al., 2004, Tabata et al., 2007, Lesur et al., 1994). There are a

varie y of reports describing the physiological effects of genetic disruption a

131

). Mice transiently over-expressing the IL-6 family cytokine

Oncostatin M (OSM) in the lung also develop severe interstitial fibrosis (Langdon et al.,

y

0-

f

r wt

r

reduc

e

ns

eak and

1998, Betz et al., 1998

2003).

To further investigate the role of the IL-6 family in the development of pulmonar

fibrosis this study utilized mice with mutant gp130 molecules that direct gp13

mediated signalling through ERK (gp130ΔSTAT/ΔSTAT) or STAT1/3 pathways

(gp130757F/757F). Recent reports characterising these gp130 mutant mice found there

were no phenotypic differences in the morphology of the lungs of adult wt and

gp130ΔSTAT/ΔSTAT mice but the lifespan of gp130757F/757F mice was approximately 50%

of wt controls (Ernst et al., 2001, Jenkins et al., 2005a). While a definitive cause o

death was not described by Jenkins et al. (2005a), gp130757F/757F mice had developed

gastric adenomas by 6 weeks of age and lymphocytic accumulations in the lung, liver

and peritoneum by eight months of age (Jenkins et al., 2005b). Neither IL-6 nor IL-11

transgenic mice are reported to have a shortened life expectancy compared to thei

littermates (Waxman et al., 1998, Tang et al., 1996, DiCosmo et al., 1994), howeve

mice that co-expressed IL-6 and IL-6Rα transgenes (IL-6TG;IL-6RαTG) had a similar

tion in life expectancy (Maione et al., 1998). These reports suggest that enhanced

activation of gp130-STAT1/3 signalling cascades contributes to shortened lif

expectancy.

Lymphocytic accumulations were noted in the lungs of some gp130757F/757F mice

less than eight months of age in my study, 30 days after saline treatment. Targeted over-

expression of IL-6 or IL-11 in the lungs produced similar lymphocytic accumulatio

by two months of age (Ray et al., 1997, Kuhn et al., 2000) and adenoviral vectors

encoding IL-6 or IL-11 induced similar accumulations within two weeks (Xing et al.,

1994). It may be that the process of trans-nasal saline administration induces a w

transient inflammatory stimulus that elevates levels of IL-6 family cytokines and

132

757F/757F mice with

reduc

ults

g et

Qiu et al., 2004, Johnston et al., 2005). However, hyperplastic

epithelium and monocytic infiltration was noted in the gastric mucosa of gp130757F/757F

contributes to the development of these lymphocytic accumulations. However, as

Jenkins and colleagues (2005a) did not examine uninjured gp130757F/757F lungs earlier

than 8 months of age it is not clear if the lymphocytic accumulations I observed were a

result of saline delivery or were innately present in the lung. This still needs to be

confirmed.

Lymphocytic aggregates were not detected in the lungs of gp130

ed STAT3 activation (gp130757F/757F;STAT3+/- mice). Similarly Jenkins et al.

(2005) found that the distinctive features of gp130757F/757F mice, such as shortened life

expectancy and lymphocytic accumulations in the lung, liver and peritoneum, were

absent or reduced in gp130757F/757F;STAT3+/- mice (Jenkins et al., 2005a). These res

suggest that the accumulation of lymphocytes in the lung is due to a chronic and mild

increase in the activation of the gp130-STAT3 pathway by IL-6 family cytokines.

Histological assessment of lung morphology has shown that gp130-STAT3

signalling promoted remodelling of the lung parenchyma following bleomycin-induced

lung injury. Previous studies have shown that the gp130 ligands, IL-6 and IL-11, can

induce morphological changes to the lung parenchyma (Yoshida et al., 1995, Xin

al., 1994, Tang et al., 1996, Kuhn et al., 2000, DiCosmo et al., 1994, Betz et al., 1998).

Directed gp130-STAT1/3 signalling promoted significant changes in lung morphology,

including hyperplasia of alveolar epithelial cells, accumulation of ECM in the alveolar

interstitium and monocytic infiltration of the alveoli in gp130757F/757F mice 30 days after

bleomycin treatment. Hyperplasia of the alveolar epithelium and ECM accumulation in

the pulmonary interstitium has not been described in rodents over-expressing IL-6

family cytokines or in rodents with perturbations in genes encoding IL-6 family

cytokines (DiCosmo et al., 1994, Kopf et al., 1994, Xing et al., 1998, Maione et al.,

1998, Kuhn et al., 2000,

133

mice

a

in

f

sue, but

ility in

ld

ould

tological data that gp130-mediated signalling regulated the development

of fib

30 to activate

during the development of gastric adenomas and the livers of mice co-expressing

IL-6 and IL-6αR transgenes (IL-6TG;IL-6αRTG) (Maione et al., 1998, Jenkins et al.,

2005a). This suggests these features are due to enhanced activation of gp130-STAT3

signalling by IL-6 family cytokines. Additionally, reduced STAT3 activation in

gp130757F/757F mice (gp130757F/757F;STAT3+/-) correlated with less severe lung

remodelling including reduced hyperplastic epithelium, accumulation of interstitial

ECM and monocytic infiltration. This data suggests that gp130-STAT3 signalling has

vital role in regulating downstream cytokine and growth factor responses that promote

remodelling of the lung parenchyma.

Measuring Hyp levels is an accurate method for estimating collagen content

tissue. The most accurate methods for measuring levels of Hyp in lung tissue are the

chloramine T reaction and HPLC analysis. Reports describing the use of the chloramine

T reaction for the assessment of lung Hyp use an entire lobe, approximately 100mg o

tissue, to make reliable measurements (Geraci et al., 1992, Keane et al., 1999, Sisson et

al., 1999). Measuring tissue Hyp by HPLC uses smaller biopsies, 20-50mg of tis

requires specialised equipment (Tabata et al., 2007, Chua et al., 2007, Chaudhary et al.,

2006). Both assays were assessed to determine their reliability and reproducib

measuring Hyp in 25-50mg of mouse lung tissue. Where as the HPLC method cou

routinely measure Hyp in small quantities of lung tissue the chloramine T reaction c

not. Due to the limited number of gp130 mutant mice and lung tissue available for this

study, the HPLC assay was chosen for the measurement of all further lung Hyp levels.

Measurement of Hyp levels in the lung tissue of bleomycin treated mice

confirmed his

rosis after bleomycin treatment. This effect was due to the ability of gp130-

activated pathways to regulate both the production and breakdown of the ECM.

Collagen synthesis and accumulation correlated with the ability of gp1

134

nalling

rly,

broblasts to activate STAT3. Recent

studie gen

.

have a

ne et

L-

by

of

., 2007, Tang et al., 2007, Liu et al., 2006, Mulsow et al., 2005).

This effect of the ERK1/2 pathway has typically been a response to TGF-β, however IL-

STAT3 signalling in mice. Animals with normal or enhanced gp130-STAT3 sig

(wt and gp130757F/757F mice respectively) developed lung fibrosis and animals with

ablated or reduced gp130-STAT3 signalling (gp130ΔSTAT/ΔSTAT and

gp130757F/757F;STAT3+/- mice respectively) were protected from lung fibrosis. Simila

collagen reporter assays demonstrated that IL-6 stimulated COL1α1 promoter activity

in MEFs correlated with the ability of these fi

s have demonstrated that IL-6 and increased STAT3 activation increase colla

synthesis by keloid fibroblasts in vitro and in vivo (Ghazizadeh et al., 2007, Lim et al.,

2006).

IL-6 does not directly induce fibrosis of the pulmonary interstitium in vivo but

over-expression of IL-6 and IL-11 does induced fibrosis in the sub-epithelium of the

conducting airways (Tang et al., 1996, Ray et al., 1997). Transient over-expression of

OSM in the lung though does induce severe interstitial fibrosis (Langdon et al., 2003)

This effect is not restricted to the lung as IL-6 and gp130-mediated signalling

role in fibrotic diseases of the skin, liver, and pancreas (Ogata et al., 2006, Kuratsu

al., 2007, Bamber et al., 1998, Ghazizadeh et al., 2007). Targeted over-expression of I

6 to the skin induces thickening of the ECM rich layer of the epidermis know as the

stratum corneum (Turksen et al., 1992), and fibroblasts isolated from keloid scars

express higher levels of IL-6 than control fibroblasts (Ghazizadeh et al., 2007). Mice

transiently over-expressing OSM in the lung also develop severe interstitial fibrosis

(Langdon et al., 2003). Therefore, bleomycin-induced lung injury promotes the

development of lung fibrosis through a gp130-STAT3 pathway that may be driven

increased IL-6 family cytokines in the lung.

A variety of reports have implicated ERK1/2 signalling in the development

fibrosis (Pannu et al

135

6-ind

/2

ng cells. In

addit ll

red.

ollagen

s

e

a

ll

uced ERK1/2 signalling has also been implicated in the pathogenesis of fibrosis

and IPF (Knight et al., 2003, Moodley et al., 2003a, Moodley et al., 2003b). Recent

studies transferring lung fibroblasts isolated from patients with IPF to mice lacking an

immune response have found that chemokine-induced ERK1/2 signalling was involved

in the progression of lung fibrosis in their model (Pierce et al., 2007b, Pierce et al.,

2007a). Moodley et al. (2003) examined the effects of IL-6 on the proliferation-

apoptosis axis in human lung fibroblasts in vitro and concluded that gp130-ERK1

signalling was responsible for increased proliferation and reduced apoptosis in IPF

fibroblasts. This was not consistent with the observation that bleomycin-induced

fibrosis was gp130-STAT3 dependent. However, the assumption that in vitro data

reflects what is happening in vivo should be interpreted with caution. It is difficult to

interpret the physiological implications of in vitro data in relation to the pathogenesis of

a complex condition such as pulmonary fibrosis, as this is likely to involve cross-talk

between a variety of cell types including epithelia, mesenchyme and circulati

ion, there appears to be heterogeneity in the lung fibroblast population and ce

responses to stimuli may differ depending which specific cell population is cultu

This concept is discussed further in chapter six.

My findings suggested that gp130-ERK1/2 signalling is involved in the protection

of bleomycin-induced fibrosis and airspace enlargement through inhibition of c

synthesis and increased ECM degradation by increasing total MMP-9 and pro-peptide

for MT-MMPs MMP-15, MMP-16, MMP24 and/or MMP-25. The MT-MMPs are

membrane bound proteinases that are known to activate pro-MMPs sequestered in th

ECM and directly degrade adjacent ECM (Garcia-Alvarez et al., 2006, Pardo and

Selman, 2006, Hernandez-Barrantes et al., 2002). These MT-MMPs are produced by

variety of lung epithelial cells, fibroblasts and neutrophils and have vital roles in ce

migration, invasion and tissue remodelling (Garcia-Alvarez et al., 2006, Hernandez-

136

atic

ent of

ts

mycin-induced pulmonary fibrosis by decreasing collagen

transc

arch

ensitivity

et

on

Barrantes et al., 2002). While this study did not identify which specific MT-MMPs

where increased in gp130ΔSTAT/ΔSTAT mice, the increase in this group of proteins

suggests that gp130-ERK1/2 signalling has a significant role in regulating the

breakdown of the pulmonary ECM.

The results described here show that gp130-mediated signalling has a dram

effect on the development and progression of bleomycin-induced mouse lung fibrosis

through two mechanisms. Firstly, gp130-STAT3 signalling drives the developm

bleomycin-induced pulmonary fibrosis by promoting collagen accumulation and

transcription of collagen αI type I mRNA. Secondly, gp130-ERK1/2 signalling preven

the development of bleo

ription and increasing MMP9 and MT-MMP levels preventing the accumulation

of collagen and ECM in vivo. This study has contributed to a growing body of rese

suggesting that gp130-mediated signalling can confer resistance and enhance s

to fibrogenic stimuli depending on the pathways involved (Ezure et al., 2000, Streetz

al., 2003, Lim et al., 2006). Furthermore, the results presented in this chapter build up

the work of Moodley et al. (2003) suggesting that gp130-mediated signalling plays a

significant role in the development and progression of IPF (Knight et al., 2003,

Moodley et al., 2003).

137

TAT3 SIGNALLING LINKS

PROGRESSION OF BLEOMYCIN-INDUCED LUNG

CHAPTER FOUR

GP130-S

INFLAMMATION TO THE DEVELOPMENT AND

FIBROSIS IN VIVO

138

f

t regulate wound healing. Under normal conditions the

inflammatory response is tightly regulated, however if inflammation persists and

becomes chronic it can cause severe pathologies (Rose-John et al., 2006). While there is

no single aetiological agent associated with IPF, the literature suggests that an isolated

or ongoing injurious event is pivotal to the pathogenesis of this disease (Thannickal et

al., 2004, Selman et al., 2001, Gauldie et al., 2007). Animal models have demonstrated

that a severe acute or moderate persistent inflammation can induce lung remodelling

and fibrosis (Kolb et al., 2001, Sueoka et al., 1998). Genetic studies have shown that

people who contain polymorphisms in the TNF-α gene are up to 14 times more likely to

develop IPF than the rest of the population (Riha et al., 2004). Furthermore, pro-

inflammatory cytokines and growth factors such as IL-6, IL-1β and TNF-α are elevated

in the serum and BALF of IPF patients (Tsantes et al., 2003, Lesur et al., 1994). These

studies have lead many authors to hypothesize that a dysregulated or persistent

inflammatory response cultivates the development and progression of IPF (Thannickal

et al., 2004, Gauldie et al., 2006).

Animal models of lung fibrosis have demonstrated a link between the acute

inflammatory response and subsequent fibrosis (Gauldie et al., 2006, Bonniaud et al.,

2005). For example over-expression of inflammatory cytokines, such as IL-6, IL-1β and

TNF-α, by gene delivery to rodent lungs induced an interstitial pneumoniae with

fibrosis (Yoshida et al., 1995, Kolb et al., 2001, Sueoka et al., 1998). In addition,

fibrogenic compounds like bleomycin induce an inflammatory response in the lung

characterised by increased levels of pro-inflammatory cytokines and increased numbers

of inflammatory cells within the airways and lung parenchyma (Cavarra et al., 2004,

4.1 Introduction

Inflammation is an essential part of the tissue repair process as it initiates many o

the mechanisms tha

139

Tabata et al., 2007, Chaudhary et al., 2006, Chua et al., 2005). In several studies,

treatment of animals with corticosteroids and tyrosine kinase inhibitors inhibits the early

inflammatory response and am ibrosis (Chaudhary et al.,

2006, Bonniaud et al., 2005, Dik et al., 2003).

IL-6 family cytokines are intimately associated with the acute inflammatory

respon d

that I Th1

an

with mutations tricate

mechanism of proteins

(DiCosmo et al., 1994, Xing et al., 1998, Wang et al., 2000a, Knight, 2001, Qiu et al.,

2004, Olman et al., 2004, McLoughlin et al., 2005). IL-6 over-expression within the

haematopoietic compartment of mice caused granulocytosis and myeloproliferative

disease (Brandt et al., 1990, Hawley et al., 1992) and similarly, targeted over-expression

of IL-6 and IL-11 in the lung induced the accumulation of lymphocytic and monocytic

inflammatory cells in mice (Wang et al., 2000b, Wang et al., 2000a, Qiu et al., 2004),

while loss of IL-6 reduced the number of circulating T-lymphocytes (Kopf et al., 1994).

IL-6 and IL-11 can also inhibit the inflammatory processes associated with

immunological challenge in the lung (Qiu et al., 2004, Wang et al., 2000a, Xing et al.,

1998, DiCosmo et al., 1994, Lentsch et al., 1999, Sheridan et al., 1999). IL-6-/- mice

showed approximately two fold more airways neutrophilia, TNF-α and MIP-2 than wt

mice following LPS challenge (Xing et al., 1998). Similarly, treating rats with IL-11

following LPS administration reduced TNF-α and neutrophil infiltration of the lung by

half compared to controls (Sheridan et al., 1999). IL-6 also inhibited the type 2 immune

response initiated by sensitisation to ovalbumin (OVA). IL-6-/- mice demonstrate a

marked increase in Th2 cytokines, such as IL-4, IL-5 and IL-13 in the airways compared

eliorated the development of f

se that follows infection and injury (Rose-John et al., 2006). Initial studies foun

L-6 knock-out mice had a defective acute inflammatory response and defective

d Th2 immunological responses (Kopf et al., 1994). Subsequent studies using rodents

in the genes of IL-6 family cytokines have elucidated an in

pro and anti-inflammatory effects mediated by this family of

140

to OVA sensitised wt mice (Wang et al., 2000a). In contrast, over-expression of IL-6 in

C10-TG) of OVA sensitised mice caused a marked decrease in airways

IL-4,

the airways (IL-6C

IL-5 and IL-13 (Wang et al., 2000a).

This chapter will examine the effect of directed gp130-mediated signalling on

inflammation following bleomycin treatment. The level of inflammation following

bleomycin administration will be assessed in situ and the role of directed gp130-

mediated signalling in bone marrow-derived cells in bleomycin-induced lung fibrosis

will be assessed in vivo.

141

Animals

Mice with mutations introduced into the intracellular region of gp130

(gp130 , wt, gp130 and gp130 ;STAT3 ) were used to assess

bleomycin-induced pulmonary inflammation and fibrosis as described previously.

Congenic Ly5.1 and Ly5.2 C57BL/6 mice were used as bone marrow recipients and

donors respectively, to evaluate bone marrow reconstitution protocols and assess the

impact of this procedure on the development of bleomycin-induced lung fibrosis in

these mice. The mice were maintained on neomycin supplemented water for 4 weeks

after lethal irradiation. All animals were maintained in specific pathogen free

environments, fed acidified water and standard chow ad libitum during the course of all

experiments. Lungs were either prepared for histology as described in section 2.8 or

snap frozen in liquid N2. While immersed in liquid N2 tissue was crushed into a fine

powder using a mortar a pestle and stored at -80 C for later use.

sue

stry (IHC)

nd

4.2 Materials and Methods

ΔSTAT/ΔSTAT 757F/757F 757F/757F +/-

o

Measurement of Lung Collagen by HPLC

HPLC was performed with hydrolysate obtained from 25-50mg of lung tis

from bleomycin or saline treated mice to assess hydroxyproline as a measure of lung

collagen as described in section 2.9.2.

Histology and Immunohistochemi

Lung tissue was embedded in paraffin wax as described in section 2.8 and 5μm

serial sections were cut and mounted on sialinated slides. Tissue sections from 3, 21 a

30 days bleomycin or control saline treated wt, gp130ΔSTAT/ΔSTAT, gp130757F/757F and

gp130757F/757F;STAT3+/- mice were stained with H&E or Masson’s trichrome stain.

142

ase K mediated antigen retrieval respectively.

Apop

he

of

inflammatory foci and the frequency of immuno-labelled inflammatory cells as

described in section 2.10.

Bone Marrow Reconstitution

Bone marrow reconstitution experiments were performed as described in section

2.2. Briefly, mice were subjected to two doses of 5.5Gy gamma irradiation 3 hours apart

to ablate the bone marrow of host mice. Donor bone marrow was obtained from the

femurs of congenic mice and host mice received 2x106 cells in 200μl PBS i.v. Mice

were maintained on neomycin supplemented water 2 weeks before total body irradiation

which was continued up to 4 weeks after reconstitution.

Bone marrow reconstitution was assessed by flow cytometry quantification of

circulating CD45.1 positive and CD45.2 positive leukocytes. Bone marrow

reconstitution was assessed in gp130 mutant mice by PCR identification of donor DNA

in whole blood of recipients and lung tissue of gp130 mutant mice as described in

sections 2.2 and 2.5.

Immunohistochemistry was performed on lung sections from mice as described in

section 2.8.1. Macrophages were detected using the cell specific antigen F4/80 at a

dilution of 1:100 following 2 minutes of Proteinase K-mediated antigen retrieval. T-

lymphocytes and B-lymphocytes were differentiated using CD3 (1:50) and B220

(1:100) cell specific antigens respectively. The detection of T-lymphocytes and B-

lymphocytes was dependent on 10 minutes of heat-mediated antigen retrieval with

citrate buffer and 5 minutes of Protein

totic cells were detected using the TUNEL in situ detection system (Roche,

Australia) and proliferating cells were detected using antibodies directed against t

Ki67 antigen (1:50) following 10 minutes of heat-mediated antigen retrieval with citrate

buffer. Inflammation was measured in situ by counting the frequency and distribution

143

Broncho-alveolar Lavage (BAL)

saline (1 x 0.4ml and 3 x 0.3ml) were injected into the

lung and collected as described in section 2.3. Cells were collected by centrifugation

and the supernatant was transferred to a new tube and stored at -80oC for later analysis.

Cells were counted as described in section 2.3 using a Neubauer chamber and collected

on sialylated slides by centrifugation at 400rpm for 3 minutes. Differential cell counts

were performed with Quick-Dip stained BAL cells on at least 400 cells per slide.

Four volumes of sterile

144

4.3 R

4.3.1 The Bleomycin-Induced Inflammatory Response

The inflammatory response was assessed in the BALF and lung tissue three days

after administration of bleomycin or saline in wt and gp130 mutant mice. In saline

treated animals there was a significant increase in the total cellularity of the

gp130 BALF compared to wt mice (p<0.05) which was due to a significant

acrophages (p<0.05) and neutrophils (p<0.02) (figure 4.1).

Insuff

ing

ant in

ity

0.02) and neutrophil (p≤0.001) content of bleomycin treated

gp130ΔSTAT/ΔSTAT

BALF recovered from wt and gp130757F/757F mice. The trend of the inflammatory cell

757F/757F +/-

757F/757F

rding

to the frequency and location of inflammatory foci and the composition of these foci. In

nce in the inflammatory response within the

lung gp130 . Ther

he

esults

ΔSTAT/ΔSTAT

increase in the number of m

icient numbers of mice were available to accurately measure the inflammatory

cell content in the BALF of gp130757F/757F and gp130757F/757F;STAT3+/- mice follow

saline treatment, however there was trend of increased eosinophils in gp130757F/757F

mice (figure 4.1).

Following bleomycin treatment there was an increase in the cellularity of wt and

gp130ΔSTAT/ΔSTAT BALF relative to saline treatment although this was only signific

wt animals (p≤0.02). There was also a significant increase in the total cellular

(p<0.01), macrophage (p≤

mice compared to bleomycin treated wt mice (figure 4.1).

Interestingly, there was no apparent difference in the inflammatory cell content of

profile of BALF recovered from bleomycin treated gp130 ;STAT3 mice was

similar to bleomycin treated wt and gp130 mice (figure 4.1).

The inflammatory response within the lung parenchyma was measured acco

contrast to the BALF, there was no differe

parenchyma of wt and ΔSTAT/ΔSTAT mice e was a trend of increased

parenchymal inflammation in gp130757F/757F mice with a notable increase in t

145

ΔSTAT/ΔSTATgp130salinebleomyci

gp130757F/757F

nsalinebleomycin gp130757F;STAT3+/- saline

bleomycin

salinebleomycin

wt

Total

50

150 200

lls/m

0

100

350 400

Ce

x103

250 300

l –

450

*

* A

Neutrophil 0

*

10 20 30 40 50 60 70 80 90

Cel

ls/m

l – x

103

* C

Eosinophil0

5.0

7.5

12.5

x103

*

ERK1/2 signalling 3 days after saline or bleomycin treatment.

BAL was performed on all mice 3 days after bleomycin or saline treatment and cecounts were performed to measure total inflammatory cell content. Inflammatory

2.5

10.0

Cel

ls/m

l –

Figure 4.1. Inflammatory cell content of BALF is increased by directed gp130-

ll cell

types were characterised based on cell morphology on Diff-quikTM stained cytospins. ad a significant increase in total cellularity of BALF 3 days after

bleomycin treatment compared to wt mice (A). This increase in BALF cellularity was due t relative to bleomycin treated wt mice. Data are represented as the mean ± S.E.M., n>3 for

E

Gp130ΔSTAT/ΔSTAT mice h

o significant increases in macrophages (B), neutrophils (C) and eosinophils (D)

all mice except saline treated gp130757F/757F (n=1) and saline and bleomycin treatedgp130757F/757F;STAT3+/- mice (n=2).

Macrophages

*50

100150200

lls/m

*250300350400

Ce

l – x

103

450

0

B

Lymphocytes0

s/

102030405060708090

Cel

lm

l – x

103

D

Basophil0

5.0

7.5

12.5

x103

2.5

10.0

Cel

ls/m

l –

F

or these experiments did not allow statistical analysis to be performed (figure

4.2).

The composition of inflammatory foci was assessed by morphological

characterisation of neutrophils and immuno-labelling of macrophages and T and B

lymphocytes (figure 4.2). There were significantly more T-lymphocytes in the

inflammatory foci of gp130ΔSTAT/ΔSTAT mice compared to wt mice (p≤0.05, figure 4.3).

There were notably more B-lymphocytes within inflammatory foci of gp130757F/757F

mice than all other mice (figure 4.3). The trend was for the number of inflammatory foci

to be lower in gp130757F/757F;STAT3+/- mice relative to gp130757F/757F mice, however the

cellularity of inflammatory foci was similar between these two mouse genotypes (figure

4.3). There were less macrophages within inflammatory foci of gp130757F/757F and

gp130757F/757F;STAT3+/- mice three days after bleomycin treatment compared to other

mice (figure 4.3), however there were insufficient mice to examine this statistically.

4.3.2 Inflammation and Fibrosis

Results in chapter three (section 3.3.2) suggested that the enhanced bleomycin-

induced fibrosis observed in gp130757F/757F mice at 30 days following bleomycin

treatment was associated with high numbers of lymphocytic cells. To determine if the

role of inflammatory cells in the lungs of bleomycin treated mice may play a role in

regulating the fibrotic response wt, gp130ΔSTAT/ΔSTAT and gp130757F/757F mice lungs were

assessed immunohistochemically for the presence of macrophages, T-lymphocytes and

B-lymphocytes. Macrophages were not detected in bleomycin-induced pulmonary

lesions of wt, gp130ΔSTAT/ΔSTAT or gp130757F/757F mice at 30 days. However, T and B-

lymphocytes were present within pulmonary lesions of all mouse genotypes. T-

lymphocytes appeared more abundant in bleomycin-induced lesions of gp130ΔSTAT/ΔSTAT

146

frequency of peri-vascular and peri-airway inflammatory foci (figure 4.2). This trend

was reduced in gp130757F/757F;STAT3+/- mice, however the small number of mice

available f

147

0

50

100

Num

ber

150 of F

oci 200

250

wt ΔSTAT 757F 757F;Stat3+/-

Airway Lumen AirwayVascular Alveolar

Figure 4.2. Gp130-STAT3 signalling regulates the frequency and distribution of

lung sections of bleomycin treated wt and gp130 mutant mice 3 days after bleomycin foci in gp130757F/757F

mice compared to all other mice. The increase in inflammatory foci was most pronounced around the vasculature and airways of gp130757F/757F mice compared to

gp130 ;STAT3 mice compared to all other mice. Data are represented as the f n≥3 mice with the exception of gp130757F/757F mice (n=2) and

gp130757F/757F;STAT3+/- mice (n=1).

bleomycin-induced inflammatory foci in situ.

The location and frequency of inflammatory foci was measured in paraffin embedded

treatment. There was an increase in the number of inflammatory

other mice. There was a trend of more inflammatory foci in the alveolar region of 757F/757F +/-

mean o

30

Num

ll

148

A

C D

B

10

20

40

ber

of C

es x

102 Neuts

F4/80 CD3

0 wt ΔSTAT 757F 757F;Stat3+/-

B220

E

Figuinfla

ice 3

ΔSTAT/ΔSTAT

757F/757F +/- 757F/757F

p130757F/757F;STAT3+/- mice (E). There was a trend for macrophage composition lammatory foci to decrease in gp130757F/757F and gp130757F/757F;STAT3+/- mice

respegp13 t

re 4.3. Directed gp130-STAT3 signalling increases the cellularity of mmatory foci 3 days after bleomycin treatment.

The composition of inflammatory foci was assessed in lung sections taken from mdays after bleomycin treatment. Neutrophils (A) were characterised based on their morphology and macrophages, T-lymphocytes and B-lymphocytes were detected by immuno-labelling of cell specific antigens F4/80 (B), CD3 (C) and B220 (D) respectively. There was a significant increase in the number of CD3 positive T-lymphocytes (p=0.05) in inflammatory foci of gp130 mice compared to wt mice and a trend of increased T-lymphocytes in gp130757F/757F and gp130 ;STAT3 mice (E). Gp130 mice had a notable increase in B220 positive B-lymphocytes in inflammatory foci compared to wt, gp130ΔSTAT/ΔSTAT mice and gof inf

ctively (E). Data are represented as the mean of n=4 mice with the exception of 0757F/757F mice (n=2) and gp130757F/757F;STAT3+/- mice (n=1). Scale bars represen

10μm (A, B) and 20μm (C, D).

mice

tes in

4.3.3 The Effect of Bone Marrow Transfer on Bleomycin-Induced Lung Fibrosis

The previous results show an association between the presence of inflammation

and the development of interstitial fibrosis in the mouse bleomycin model. To further

assess the role of inflammatory cells in this model, bone marrow transfer experiments

were performed. It must be noted that recent studies have implicated other bone-marrow

derived cells, such as fibrocytes, in the development of lung fibrosis. Therefore, these

experiments actually assessed the role of bone marrow derived cells rather than just

inflammatory cells in the development of bleomycin-induced fibrosis.

Preliminary experiments used C57BL/6 mice as bone marrow donors and

rrow transfer. Furthermore,

irradi ts

se

d (figure

following bleomycin or saline treatment. Flow cytometry was performed on leukocytes

149

and gp130757F/757F mice than wt mice (figure 4.4) and B-lymphocytes appeared more

abundant in fibrotic foci of gp130757F/757F mice than wt and gp130ΔSTAT/ΔSTAT

(figure 4.5). These results confirm an association between lymphocytic aggrega

the interstitium of bleomycin treated mice and fibrosis although they were associated

with both the protection from fibrosis, as seen in the gp130ΔSTAT/ΔSTAT mice, and the

enhanced fibrosis seen in gp130757F/757F mice.

congenic host mice to validate the method of bone ma

ation of the thorax causes interstitial pneumonitis in mice, so these experimen

assessed if the animals would tolerate the combination of γ-irradiation and bleomycin-

induced lung injury and survive the 30 day experimental period. In addition, these

experiments determined if there was a significant difference in lung collagen

accumulation between bleomycin and saline treated mice following bone marrow

transfer. C57BL/6 Ly5.1 and Ly5.2 congenic mice expressing the CD45.1 and CD45.2

cell surface antigens, respectively, on circulating leukocytes were used for the

experiments. All animals survived the 30 day bone marrow reconstitution perio

4.6). Whole blood was collected from mice 60 days after bone marrow transfer, 30 days

150

after bl treatm

nt were y

ΔSTAT/ΔSTAT 757F/757F of

A, C,

Figure 4.4. T-lymphocytes are more prevalent in gp130 mutant mice than wt mice 30 days eomycin ent.

Lung sections from wt and gp130 mutant mice 30 days after bleomycin treatmeimmuno-labelled with the T-lymphocyte marker CD3. CD3 positive cells were diffuseldistributed in wt mice (A, B) and appeared more prevalent in bleomycin-induced lesionsof gp130 (C, D) and gp130 mice (E, F). Images are representative n=2 mice, scale bars=50μm ( E) and 20μm (B, D, F).

B

D

F

C

E

A

Figu

757F/757F

,

151

re 4.5. B-Lymphocytes are more prevalent in gp130 mutant mice than wt

A B

C D

E F

mice 30 days after bleomycin treatment.

Lung sections from wt and gp130 mutant mice 30 days after bleomycin treatment were immuno-labelled with the B-lymphocyte marker B220. B220 positive cells appeared more prevalent in bleomycin-induced lesions of gp130 mice (E, F) compared to wt (A, B) and gp130ΔSTAT/ΔSTAT (C, D) mice. B220 cells were diffusely distributed in pulmonary lesions of wt and gp130ΔSTAT/ΔSTAT mice and appeared to form aggregatesin gp130757F/757F mice. Images are representative of n=2 mice, scale bars=50μm (A, CE) and 20μm (B, D, F).

figure

ure 4.6).

eomycin treated

bone

ling within Regul om

e, to

eras. All gp130ΔSTAT/ΔSTAT host mice died during the 30 day reconstitution

perio

confirm that

utant gp130

allele fied

152

isolated from whole blood to examine the extent of bone marrow reconstitution (

4.6). Approximately 84% of circulating cells were derived from donor bone-marrow

and only approximately 10% of circulating cells were derived from the host (fig

After bone marrow transfer there was a significant reduction in the survival of

bleomycin treated mice compared to saline treated mice (p<0.05, figure 4.6). Analysis

of collagen content of lung tissue by measuring hydroxyproline levels using HPLC

demonstrated that there was a significant increase in lung collagen in bl

marrow reconstituted mice compared to saline treated bone marrow reconstituted

controls (p≤0.01, figure 4.6).

4.3.4 Gp130-Mediated Signal the Lung ates Ble ycin-Induced Lung

Fibrosis

Bone marrow was transferred from wt, gp130ΔSTAT/ΔSTAT and gp130757F/757F donor

mice into whole body irradiated wt, gp130ΔSTAT/ΔSTAT or gp130757F/757F host mic

create chim

d making it impossible to assess the effect of reinstating gp130-STAT1/3

signalling within bone marrow derived cells on bleomycin-induced fibrosis. At least

70% of wt and gp130757F/757F mice survived bone marrow reconstitution (figure 4.7).

However, there was a significant decrease in the proportion of wt mice with

gp130ΔSTAT/ΔSTAT bone marrow (wtΔ/Δ) that survived bone marrow reconstitution

compared to wt mice with wt bone marrow (p<0.05, wtwt, figure 4.7). To

bone marrow reconstitution of chimeric mice was successful, wt and m

s were examined in DNA isolated from whole blood. Both alleles were identi

in whole blood DNA of chimeric mice and donor alleles appeared to be more abundant

(figure 4.7).

Fibrosis was assessed 21 days after bleomycin treatment rather than 30 days as

animals were showing signs of distress earlier than mice used in the initial bone marrow

153

1 10 10 10 10

FSC

PE 2 3 4

84%16%250

Ly5.1 A

250

0

50

PE

0

50

100FS100

200 200

C 150 150

91%

1 10 10 10 104

o –

m

Sa eo

10

9%

2 3

D

g/lu

ng 20

Ly5.2B

Clla

gen

line Bl0

*

C

50

75

10 20 30 40 50 600

25

100

SalineBleomycin *

BMT

Day

Figure 4.6. C57BL/6 mice survive bone marrow reconstitution and develop lung fibrosis 30 days after bleomycin treatment.

Ly5.2 C57BL/6 mice were depleted of their bone marrow by a lethal dose of γ-

C57BL/6 cong

Perc

enrv

l

irradiation and their bone marrow was reconstituted with bone marrow from Ly5.1 enic mice. Circulating leukocytes were isolated from whole blood of

C57BL/6 mice after bone marrow reconstitution and labelled with phycoerythrin (PE) conjuderived Ly5.2 antigen (B). Flow cytometry analysis measured forward scatter (FSC)

Flow cytometry showed that approximately 84% of circulating leukocytes expressed

leukocytes expressed the host derived Ly5.2 antigen (B). There was 100% survival of ere was a significant

decrease in survival of bleomycin treated mice compared to saline treated mice 30 days after BMT (p=0.032, C). Bleomycin treatment increased total lung collagen

e representative histograms of n=6 mice. C-D n≥8, D – data is presented as the mean ±

.

t su

iva

gated antibodies recognising either donor derived Ly5.1 antigen (A) or the host

or cell granularity on the y-axis and PE flourescence intensity on the x-axis (A, B).

the bone marrow donor derived Ly5.1 antigen (A) while approximately 9% of

C57BL/6 mice 30 days after bone marrow transfer (BMT) but th

levels in BMT mice compared to saline treated BMT mice (p=0.01, D). A-B ar

S.E.M., *p<0.05

50

75

154

A

10 20 300

25

100wtwt

wtF/F

wtΔ/Δ *

F/Fwt

F/FF/F

F/FΔ/Δ

Days

Pece

nt su

rvi

ar

vl

Figure 4.7. Wt and gp130757F/757F mice survive bone marrow reconstitution.

Animal survival was recorded during the bone marrow reconstitution phase 30 days after to -irradiat rvival of wt a gp130757F/757F mice is represented in a Kaplangp130Δ

tal body γ ion. Su nd -Meir plot (A). There was a significant reduction in survival of wt mice with STAT/ΔSTAT bone marrow (wtΔ/Δ) over the reconstitution period (p=0.047).

Successful reconstitution of host mice was assessed by genotyping leukocytes in whole blood upon termination of bleomycin experiments, 51 days after bone marrow transfer. Multiplex PCR was performed to identify wt and/or donor mutant gp130 alleles (500 and 700bp respectively) in DNA from whole blood (B). Mutant gp130 alleles were detected in whole blood DNA from wtΔ/Δ mice (B). A - wtwt = wt with wt donor bone marrow, wtΔ/Δ = wt with gp130ΔSTAT/ΔSTAT bone marrow, wtF/F = wt with gp130757F/757F donor bone marrow, F/FF/F = gp130757F/757F with gp130757F/757F donor bone marrow, F/Fwt = gp130757F/757F with wt donor bone marrow, F/FΔ/Δ= gp130757F/757F with gp130ΔSTAT/ΔSTAT bone marrow. n≥4, *p<0.05 by chi-squared analysis. B - Lane 1 = DNA ladder, lane 2 = wt DNA, lanes 3-7 = whole blood DNA from wtΔ/Δ mice.

Whole blood DNA 1 2 3 4 5 6 7

donor gp130 allele

B

wt gp130 allele 500bp 700bp

This was evident in the bleomycin treated wtΔ/Δ mice, which had

mycin treated wtwt mice during the 21 day

experimental period (p<0.05, figure 4.8). The survival of bleomycin treated

gp130 host mice with gp130757F/757F bone marrow (F/FF/F) was decreased

comp

e

f

cted

as

g wt

donor mice (figure 4.8). While only two F/FF/F mice

survi

omycin

F/F

155

transfer experiments.

significantly more deaths than bleo

757F/757F

ared to wtwt mice (p<0.05, figure 4.8) indicating that the combination of γ-

irradiation and bleomycin treatment had a significant effect on gp130757F/757F mice.

There was a trend for gp130757F/757F host mice to have more deaths than the wt host mic

following bleomycin treatment (figure 4.8). This suggests the presence of the Y757F

mutation in the lung exacerbates any pulmonary damage caused by the combination o

γ-irradiation and bleomycin treatment. No changes in lung collagen levels were dete

between groups of bleomycin treated wt chimeric mice (figure 4.8). However, there w

reduced lung collagen in bleomycin treated gp130757F/757F chimeric mice containin

and gp130ΔSTAT/ΔSTAT bone marrow that correlated with reduced gp130-STAT1/3

signalling in the bone marrow of

ved the 21 day experimental period there was a trend for decreased total lung

collagen in F/Fwt mice compared to F/FF/F mice (figure 4.8). There was a significant

decrease in total lung collagen of bleomycin treated F/FΔ/Δ mice compared to ble

treated wt mice (p<0.01, figure 4.8).

3 6 9 12 15 18 21

40

156

60

80

0

20

100

F/FF/F – bleomycin *F/Fwt - bleomycinF/F - bleomycinΔ/Δ

Dayser

ceP

nrv

t su

ival

wtwt - bleomycin wtΔ/Δ - saline

wtF/F - bleomycin

3 6 9 12 15 18 210

20

40

60

80

100

wt

Perc

enrv

wt - salinet su

ival

A B

wtΔ/Δ – bleomycin *

Days

Saline Bleo Saline Bleo Bleo Bleo Bleo Bleo

wtwt wtΔ/Δ

0

20

30

40

Col

lage

n –

mg/

lung

*

wtF/F F/FF/F F/Fwt F/FΔ/Δ

10

C

Figuresident and bone marrow-derived cells enhances bleomycin-induced fibrosis.

an-TAT

mycin tively).

g

nd F/FF/F mice (n=2). *p<0.05.

re 4.8. The combination of directed gp130-STAT1/3 signalling in lung

The survival of wt (A) and gp130757F/757F (F/F, B) bone marrow chimeric mice was recorded for 21 days after bleomycin or saline treatment and represented as KaplMeir plots. There was a trend of reduced survival of wt mice with gp130ΔSTAT/ΔS

bone marrow (wtΔ/Δ) and gp130757F/757F host mice after bleomycin and saline treatment (A and B respectively). The survival of bleomycin treated wtΔ/Δ and gp130757F/757F mice with gp130757F/757F bone marrow (F/FF/F) was significantly lower than bleotreated wt mice with wt bone marrow (wtwt, p=0.033, A and p=0.033, B respecHPLC measurement of lung hydroxyproline content was performed to assess collagenaccumulation in lung tissue 21 days after bleomycin or saline treatment (C). There was a trend for lung collagen to increase in bleomycin treated wtwt mice compared to saline controls but this was not significant (p=0.26). There was no significant difference in bleomycin-induced lung collagen levels between wtΔ/Δ, wtF/F and wtwt control mice (p=0.47, p=0.86 respectively, C). There was a trend of reduced total luncollagen levels in bleomycin treated gp130757F/757F mice with bone marrow from wt (F/Fwt) and gp130ΔSTAT/ΔSTAT (F/FΔ//Δ) mice (C). Bleomycin treated F/FΔ/Δ mice demonstrated a significant reduction in lung collagen compared to F/Fwt mice (p=0.008). For A and B n≥4, for C n≥5 with the exception of F/Fwt and F/FΔ/Δ mice (n=3) a

157

4.4 D

ly an

lmona

7, Moore et al., 2001,

Dik e

gnalling

e was

leomy

se in t

p130 mpar

The two fold

increase in BALF cellularity between wt and gp130ΔSTAT/ΔSTAT mice is approximately

iscussion

The role of the inflammatory response in the pathogenesis of IPF is current

issue of conjecture. It has long been thought that IPF is the result of chronic

inflammation due to an unknown injurious agent provoking an unremitting

inflammatory response driving the progression of pu ry fibrosis (Gauldie et al.,

2006, Thannickal et al., 2004, Selman et al., 2001). However, there is a growing body of

literature that suggests that inflammation is not a significant feature of IPF and other

aspects of wound healing are more important to the pathogenesis of this disease

(Scotton and Chambers, 2007, Pardo and Selman, 2006, White et al., 2003). Numerous

studies have demonstrated that inflammatory cells are intimately associated with the

development of bleomycin-induced lung fibrosis (Chua et al., 200

t al., 2003). The experiments described in this chapter examined the role of

directed gp130-mediated signalling on the acute inflammatory response following

bleomycin treatment and correlated this with the development of pulmonary fibrosis.

The results described in this chapter demonstrate that gp130-mediated si

regulates the magnitude of the inflammatory response and the trafficking of

inflammatory cells into and through the lung after bleomycin treatment. Examination of

the bleomycin-induced inflammatory response in the airways and lung parenchyma

produced contrasting results. Assessment of airways inflammation by measuring BALF

cellularity indicated that the inflammatory response in gp130ΔSTAT/ΔSTAT mic

approximately two fold greater than wt and gp130757F/757F mice following b cin

treatment. However, examination of the inflammatory respon he lung parenchyma

suggested an increase in inflammatory cells in g 757F/757F mice co ed to

gp130ΔSTAT/ΔSTAT and wt mice. The number of inflammatory cells in gp130ΔSTAT/ΔSTAT

and wt mice following bleomycin treatment was approximately equal.

158

equal

supported by Sano and Li and colleagues who

show

.,

l.,

+/-),

h

ory

e

ped

t

um

into t

e

to the reported increase in the number of circulating leukocytes between wt and

gp130ΔSTAT/ΔSTAT mice (Jenkins et al., 2002). It is known that bleomycin causes damage

to the pulmonary vasculature (Orr et al., 1986, Chen and Stubbe, 2005, Adamson and

Bowden, 1974), therefore it is likely that the increased cellularity in gp130ΔSTAT/ΔSTAT

BALF is due to enhanced permeability of the pulmonary vasculature.

The increased inflammation seen in gp130757F/757F mice has previously been

reported and attributed to hyper-activation of the gp130-STAT3 pathway (Judd et al.,

2006a, McLoughlin et al., 2005). This is

ed that mice carrying a transgene that encodes a constitutively active STAT3

(STAT3C) had enhanced inflammation in tissues with and without injury (Sano et al

2005, Li et al., 2007). Furthermore, injured epithelium adjacent to the inflammation in

these mice resulted in severe tissue remodelling and fibrosis (Sano et al., 2005, Li et a

2007). In gp130757F/757F mice with monoallelic loss of STAT3 (gp130757F/757F;STAT3

there were fewer inflammatory foci three days after bleomycin administration, whic

correlated with protection from fibrosis. This data supports the previous studies

showing that enhanced STAT3 signalling correlated with increased lung parenchymal

inflammation and subsequent fibrosis. The observation that the number of inflammat

cells was higher in the lung parenchyma but lower in the BALF of gp130757F/757F mic

compared with gp130ΔSTAT/ΔSTAT mice suggests that the inflammatory cells are trap

in the lung parenchyma of gp130757F/757F mice. In contrast, the inflammatory cells pass

through the lung parenchyma into the airways of gp130ΔSTAT/ΔSTAT mice. Therefore, the

protection from fibrosis seen in gp130ΔSTAT/ΔSTAT mice following bleomycin treatmen

may be related to the ability of inflammatory cells to pass out of the lung interstiti

he alveolar space.

The recruitment of inflammatory cells to an injury or infection is regulated by th

expression of chemokines. Chemokines secreted by epithelial, endothelial and stromal

159

using

or

elium

elevated

olar

ncreased inflammatory cells in

the B

on

,

t

and

ric

ely that chemokine expression

regulated through a gp130-STAT3 mechanism induced by IL-6 promotes the retention

cells bind to chemokine receptors expressed on specific inflammatory cells to recruit

them from the vasculature and keep them at a site of injury or infection. Studies

animal models demonstrated that IL-6 enhances the expression of chemokines f

lymphocytes and granulocytes as well as chemokine receptors on inflammatory cells

(Judd et al., 2006a, McLoughlin et al., 2004, McLoughlin et al., 2005, Nowell et al.,

2003). If the expression of chemokines was downregulated in the pulmonary epith

of gp130ΔSTAT/ΔSTAT` mice then this may explain why inflammatory cells were

in the BALF and not in the lung parenchyma following bleomycin treatment. The

corollary would be that increased production of chemokines released into the alve

space in gp130ΔSTAT/ΔSTAT mice would also have lead to i

ALF. It was reported that genetic disruption of IL-6 reduces the expression of

chemokine receptors and ligands for T-lymphocytes, neutrophils and macrophages

inflammatory cells and epithelial surfaces in experimental models of peritoneal

inflammation and arthritis (McLoughlin et al., 2005, Nowell et al., 2003, McLoughlin et

al., 2004). The probability of increased levels of chemokines in the BALF of

gp130ΔSTAT/ΔSTAT animals is supported by several studies using animal models of

pulmonary inflammation and asthma, which have shown that chemokine levels are

elevated in the BALF of IL-6-/- mice compared to wt controls (DiCosmo et al., 1994

Qiu et al., 2004, Wang et al., 2000a, Xing et al., 1998). This creates an environment tha

draws inflammatory cells into the airway lumen and away from the damaged

epithelium.

Judd and colleagues (2006) have shown that gene expression of the neutrophil

macrophage chemokines CCL-1, CCL-12 and CXCL-2 are upregulated in the gast

mucosa of gp130757F/757F mice compared to wt, gp130ΔSTAT/ΔSTAT and

gp130757F/757F;STAT3+/- mice. Therefore, it is lik

160

of inf lary

ugh these

),

s are

).

dhesion to

Chen et al.,

hat the enhanced gp130-STAT3 signalling in gp130757F/757F mice

leads s

5

ry cells

and their effects on lung fibrosis, whole bone marrow from each mouse genotype was

transplanted into whole body γ-irradiated wt, gp130ΔSTAT/ΔSTAT and gp130757F/757F mice.

lammatory cells in the lungs of gp130757F/757F mice after bleomycin. The corol

being, the loss of IL-6-induced gp130-STAT3 signalling in gp130ΔSTAT/ΔSTAT mice

causes dysregulated recruitment of inflammatory cells leading to their accumulation in

the airway lumen after bleomycin treatment.

Inflammatory cells in the lung parenchyma of gp130757F/757F mice aggregate

around the vasculature, between the endothelium and tunica adventitia and below the

epithelium of the airways. This suggests the transit of inflammatory cells tho

structures are impeded. Movement from the vasculature into the tissues requires

interactions with adhesion molecules such as the selectins (L-selectin and P-selectin

cellular adhesion molecules (VCAM and ICAM) and integrins. These molecule

involved in cell-cell and cell-ECM adhesion respectively. There is evidence to show

that IL-6 family cytokines enhance the expression of these molecules under

physiological conditions. Targeted over-expression of IL-6 in the central nervous

system increases the expression of α5 and β4 integrins (Milner and Campbell, 2006

These integrins regulate cell adhesion to the ECM and cell-cell adhesion, crucial to

epithelial integrity. Blocking IL-6 family actions also inhibits lymphocyte a

venules by impairing L-selectin expression on these cells (Chen et al., 2006,

2004). It is likely t

to upregulation of these adhesion molecules and recruitment of inflammatory cell

to pulmonary venules. Upon reaching the pulmonary venules it is likely that their

transport through the vascular serosa is hampered by increased expression of α

integrins and then they are trapped below the airway epithelium due to enhanced

epithelial integrity.

To further examine the role of gp130-mediated signalling in inflammato

161

If fibrosis was regulated by bone marrow-derived cells then it would be expected that wt

or gp130ΔSTAT/ΔSTAT mice transplanted with gp130757F/757F bone marrow will have

increased fibrosis in their lungs ent whereas gp130757F/757F

host mice with wt or gp130ΔSTAT/ΔSTAT bone marrow will have reduced fibrosis.

Gp130757F/757F mice reconstituted with wt and gp130ΔSTAT/ΔSTAT bone marrow did show

reduced fibrosis compa F/F ent with expectation.

However, wt mice reco 7F/757F bone marrow

did not show any differences in fibrosis following bleomycin treatment compared with

wtwt treated mice. These results suggest that bone marrow-derived cells are important in

the development of fibrosis but the capacity of lung resident cells to activate gp130-

STAT1/3 signalling pathways limits the fibrogenic properties of bleomycin.

Although bone marrow contains precursors for inflammatory cells, it is not

conclusive that the inflammatory cells within the bone marrow are important in

regulating the lung fibrosis. Early studies examining the pathogenesis of lung fibrosis

found that the absence of inflammatory cells was not an impediment to the development

and progression of fibrosis (Adamson et al., 1988). More recent studies have

demonstrated that circulating cells not of the hematopoietic lineage are critical to the

development and progression of lung fibrosis (Phillips et al., 2004, Hashimoto et al.,

2004, Epperly et al., 2003). These circulating cells are known as fibrocytes and are

believed to be derived from the bone marrow and contribute to the development of

fibrosis (Phillips et al., 2004, Lama and Phan, 2006, Bucala et al., 1994). It has been

shown that approximately 80% of collagen producing cells in bleomycin-induced

mouse lung fibrosis are derived from the bone marrow and express fibrocyte markers

(Hashimoto et al., 2004, Phillips et al., 2004). Chemokines, in particular the chemokine

CXCL12, also known as stromal derived factor-1 (SDF-1), released after injury signal

these cells to migrate out of the circulation to the wound site to resolve the injury

following bleomycin treatm

red with control F/F treated mice, consist

nstituted with gp130ΔSTAT/ΔSTAT and gp13075

162

dd et al. (2006) showed that

transcription of the CXCL12 gene was upregulated in the gastric epithelium of

and that transcription of this gene was regulated through a gp130-

STAT

hing the stem cell niche required for effective

haem

cytes

(Strieter et al., 2007, Lama and Phan, 2006). Ju

gp130757F/757F mice

3 pathway. This suggests that the enhanced fibrosis in bleomycin-treated

gp130757F/757F mice may be due to increased recruitment and retainment of circulating

fibrocytes at the injury site. While this area requires further investigation it is outside

the scope of this thesis.

Attempts to generate gp130ΔSTAT/ΔSTAT bone marrow chimeras were unsuccessful

suggesting that gp130-mediated signalling pathways are crucial to recovery from

systemic γ-irradiation. Haematopoietic pathologies have not been reported in

gp130ΔSTAT/ΔSTAT mice. However, a number of studies have shown that IL-6 family

cytokines and gp130 are vital for the growth of bone marrow stromal cells and their

induction of haematopoiesis in vitro and in vivo (Nemunaitis et al., 1989, Rodriguez

Mdel et al., 2004, Tanaka et al., 2003, Yao et al., 2005). Colony formation of bone

marrow stromal cells is critical to establis

atopoiesis. Early work by Nemunaitis et al. (1989) demonstrated that colony

formation of human bone marrow stromal cell lines could be ablated with function

blocking IL-6 antibodies in vitro. Later studies by Carmen Rodriguez et al. (2004)

investigated the role of IL-6 in bone marrow cultures isolated from IL-6-/- mice. These

studies demonstrated that the loss of IL-6 significantly reduced the growth of bone

marrow stromal cells and delayed the onset of haematopoiesis (Rodriguez Mdel et al.,

2004, Yao et al., 2005). Furthermore, γ-irradiation of IL-6-/- stromal cell cultures

impaired their ability to support the proliferation of donated stromal cells and the

proliferation and differentiation of haematopoietic progenitor cells. Targeted deletion of

gp130 in endothelial cells of mice leads to increased numbers of circulating leuko

and abnormalities in nearly all haematopoietic organs (Yao et al., 2005). These mice

163

one

targeted disruption of

gp13

f

bone

ed

nalling increased retention of inflammatory

cells in the lung parenchyma but not the airways, suggesting the presence of

inflammatory cells in the lung parenchyma is central to the progression of lung fibrosis

in this model. However, directed gp130-STAT1/3 signalling within inflammatory cells

alone was not enough to enhance the development of lung fibrosis. The severity of

fibrosis was regulated by the extent to whi ls could activ

gp130-STAT3 pathway. It was the combination of directed gp130-STAT1/3 signalling

in the lung parenchyma and bone marrow derived cells that exacerbated bleomycin-

induced lung fibrosis in this model. Therefore, gp130-STAT3 signalling regulates the

developm eomycin- induced fibrosis through the interaction of bone marrow-

died within 12 months of birth, which was associated with hypocellularity in the b

marrow. Furthermore, bone marrow cultures from mice with

0 failed to produce haematopoietic progenitors in vitro (Yao et al., 2005).

Gp130ΔSTAT/ΔSTAT mice have a normal life expectancy, similar to mice lacking gp130 in

endothelial cells, and increased circulating leukocytes. The evidence indicates that loss

of IL-6 or targeted disruption of gp130 adversely effects homeostasis within the bone

marrow niche culminating in impaired haematopoiesis. This impairment does not

produce any pathology under normal conditions, however an injury to the bone marrow

niche, such as γ-irradiation, causes irreparable damage. It is likely that the loss o

gp130-STAT1/3 signalling in gp130ΔSTAT/ΔSTAT mice has reduced the ability of the

marrow niche to recover from γ-irradiation. The damage to the bone marrow niche in

gp130ΔSTAT/ΔSTAT mice resulted in 100% mortality in the bone marrow reconstitution

experiments.

The results described in this chapter have demonstrated that gp130-mediated

STAT1/3 links the inflammatory response to the development of bleomycin-induc

lung fibrosis. Directed gp130-STAT1/3 sig

ch lung resident cel ate the

ent of bl

164

derived cells, including inflammatory cells and/or mesenchymal precursors, and lung

parenchymal cells.

165

DIRECTED GP130-MEDIATED SIGNALLING

OF MOUSE LUNG FIBROBLASTS

CHAPTER FIVE

REGULATES THE GROWTH AND PHENOTYPE

166

s alternating

White et

of carbon monoxide, and patient mortality

(Nich ic lung,

is

s

rom the

d

nt

MA),

5.1 Introduction

One of the histopathological hallmarks of IPF at low magnification i

areas of normal lung, fibroblastic foci and dense fibrosis (Selman et al., 2001,

al., 2003). Fibroblastic foci are accurate predictors of clinical symptoms of IPF, such as

forced vital capacity and lung diffusion

olson et al., 2002, King et al., 2001). The border between normal and fibrot

where foci of fibroblasts and myofibroblasts are found, is thought to represent the active

lesion in IPF (White et al., 2003). Fibroblasts within these foci appear to be

heterogenous and demonstrate the characteristics typical of proliferating cells and

myofibroblasts (Kuhn and McDonald, 1991, Zhang et al., 1996). The significance of

these foci to the progression of IPF was highlighted recently by Cool et al. (2006). Th

investigation demonstrated that these foci were not discrete accumulations of fibroblast

and myofibroblasts, but were part of an interconnected reticulum that extended f

junction between normal and fibrotic lung to the pleura (Cool et al., 2006).

A number of studies have demonstrated that the fibroblast population in the lungs

of IPF patients is altered compared to normal lungs (Moodley et al., 2003a, Moodley et

al., 2003b, Moodley et al., 2004, Ramos et al., 2001, Raghu et al., 1988, Jordana et al.,

1988). The IPF fibroblast population is highly synthetic, producing a number of growth

factors, cytokines and ECM molecules and is enriched with myofibroblasts (Kuhn an

McDonald, 1991, Ramos et al., 2001). Myofibroblasts are typically a transie

population of cells, characterised by the expression of α-smooth muscle actin (α-S

that develop during the wound repair process and then undergo apoptosis upon

resolution of the injury (Desmouliere et al., 2003, Hinz et al., 2007). Myofibroblasts

secrete more collagen and other ECM proteins than normal lung fibroblasts (Ramos et

al., 2001, Hinz et al., 2007) and it is thought these cells have a significant role in the

167

devel

l

.,

i

myof

lasts

fferent

to

e to it’s

effec

sts

.

opment and progression of this disease (Thannickal et al., 2004). The persistence

of these cells is a distinctive feature of IPF. Proliferating fibroblasts, that are distinct

from myofibroblasts, have been identified in these fibroblastic foci in IPF and anima

models of lung fibrosis (Hashimoto et al., 2004, Moodley et al., 2003a, Hagood et al

2005). It is likely that the milieu of cytokines and growth factors within these foc

stimulates proliferation of fibroblasts and fibroblast differentiation into myofibroblasts,

contributing to the progression of the disease.

IL-6 levels are elevated in the lungs of patients with IPF and in animal models of

lung fibrosis (Lesur et al., 1994, Olman et al., 2004, Kolb et al., 2001, Cavarra et al.,

2004). IL-6 can regulate fibroblast proliferation and their differentiation into

ibroblasts (Fries et al., 1994, Moodley et al., 2003a, Gallucci et al., 2006). The

studies of Moodley et al. (2003) are most pertinent to this chapter. These studies

demonstrated that IL-6 inhibited the proliferation of control human lung fibroblasts

while IPF lung fibroblasts proliferated in response to IL-6 stimulation (Moodley et al.,

2003a). The corollary was IL-6 promoted apoptosis of control human lung fibrob

and inhibited apoptosis of IPF lung fibroblasts (Moodley et al., 2003b). The di

effects of IL-6 on normal and IPF lung fibroblasts was specific to IL-6 and not due

homo-dimerisation of gp130, as IL-11 promoted the proliferation and inhibited

apoptosis of both control and IPF lung fibroblasts. These studies demonstrated that IL-

6-induced ERK1/2 signalling was pivotal to both fibroblast proliferation and

myofibroblast differentiation.

TGF-β is a potent promoter of lung fibrosis and it is thought that this is du

ts on lung fibroblasts (Thannickal et al., 2004, Phan, 2003, Gauldie et al., 2007).

Lung fibroblasts proliferate, produce collagen and differentiate into myofibrobla

upon stimulation with TGF-β (Khalil et al., 2005, Hetzel et al., 2005, Roy et al., 2001)

The pro-fibrotic effects of TGF-β are thought to be mediated by the Smad2/3

168

ed

5a,

005)

ach

ributes to

on TGF-

cytoplasmic signalling pathway (Gauldie et al., 2007, Roberts et al., 2006, Bonniaud et

al., 2004). Recent publications have shown there is cross-talk between IL-6-induc

signalling pathways and TGF-β activated signalling pathways (Jenkins et al., 200

Becker et al., 2004, Zhang et al., 2005, Ogata et al., 2006). While Becker et al. (2

and Jenkins et al. (2005) demonstrated that TGF-β and IL-6 inhibit the actions of e

other. Ogata et al. (2006) suggested that IL-6 enhances TGF-β levels and cont

the progression of fibrosing diseases.

The aim of this chapter is to assess the role of IL-6 and IL-11-induced gp130-

mediated signalling pathways on fibroblast proliferation and differentiation into

myofibroblasts. Furthermore, the effects of directed gp130-mediated signalling

β-induced proliferation and differentiation of lung fibroblasts will be investigated.

169

Histology and Immunohistochemistry (IHC)

Lung tissue was embedded in paraffin wax as described previously and 5μm serial

sections were cut from tissue blocks and mounted on sialinated slides by PathWest

Laboratory Medicine WA. Tissue sections from wt, gp130 , gp130 and

gp130 ;STAT3 mice 30 days after bleomycin or control saline treatment were

processed for H&E and Masson’s trichrome stains by PathWest. Immunohistochemistry

was performed on the lung sections from these mice as described in section 2.13.1.

Antibodies for the detection of p-STAT3, Ki67 and α-SMA were used at a dilution of

1:100, 1:100 and 1:500 respectively. Heat-mediated antigen retrieval with citrate buffer

was performed for 10 minutes, 15 minutes and 10 minutes for the detection of p-

STAT3, Ki67 and α-SMA respectively as described previously.

th a

5.2 Materials and Methods

ΔSTAT/ΔSTAT 757F/757F

757F/757F +/-

Isolation and Immortalisation of Lung Fibroblasts

Cultures of primary lung fibroblasts (PLFs) were established from the lungs of

untreated C57BL/6, gp130 mutant and gp130 wt mice by trypsin-collagenase serial

digestion as described previously. Cells were routinely passaged in normal growth

medium once they had attained 80-95% confluence as described in chapter two.

Primary mouse lung fibroblasts from wt, gp130ΔSTAT/ΔSTAT, gp130757F/757F and

gp130757F/757F;STAT3+/- mice were immortalised (MLFs) by transient transfection wi

plasmid vector encoding the early region of the SV-40 virus genome under the

transcriptional regulation of the Rous Sarcoma virus putative promoter as described

previously.

170

Grow

MBB Proliferation Assay

Primary lung fibroblasts (PLFs) were isolated from wt, gp130 , and

gp130 mice as described in section 2.12.1 and expanded in vitro to obtain

sufficient cells to perform this assay. Cells were seeded at a density of 5x10 cells/well

in 96 well tissue culture plates and the experiment was performed as described in

section 2.13.. All treatments were performed in triplicate. Cells were fixed with 50μl of

4% paraformaldehyde (PFA) and plates were stored at 4 C until proliferation was

assessed by methylene blue binding (MBB) as described in section 2.13.2.

T and gp130757F/757F mice were seeded into 96 well

tissue culture plates as described above for the MBB proliferation assay. Mitochondrial

reduction of methyl tertrazolium salts to formazan was assessed as described in section

2.16.3 and untreated cells were grown in MSM. Positive controls and untreated controls

were maintained in NGM and MSN respectively. Experimental wells were

supplemented with 1-100ng/ml recombinant mouse IL-6 or IL-11 for 48 hours.

to 96 well

th Rate of Mouse Lung Fibroblasts

Briefly, 1x106 MLFs derived from wt, gp130ΔSTAT/ΔSTAT and

gp130757F/757F;STAT3+/- mice and 0.5x106 gp130757F/757F MLFs were seeded into 75cm2

tissue culture flasks. When MLFs reached 90-95% confluency, cell counts were

performed and the cells were re-seeded into 75cm2 tissue culture flasks. Cell growth

was recorded as a cumulative index of the number of times the seeding cell population

doubled to attain 90-95% confluency.

ΔSTAT/ΔSTAT

757F/757F

3

o

MTT Proliferation Assay

PLFs from wt, gp130ΔSTAT/ΔSTA

BrdU Incorporation Proliferation Assay

PLFs from wt, gp130ΔSTAT/ΔSTAT and gp130757F/757F mice were seeded in

plates at a density of 5x103 cells/well and stimulated with 0.01-200ng/ml of

171

ay was performed as

recom

α-SMA ELISA

Myofibroblast differentiation was detected as described previously (Scaffidi et al.,

2001). Briefly, PLFs were obtained from wt, gp130 and gp130 mice

and routinely used between passage 2 and 6. Cells were seeded into 96 well tissue

culture plates at a density of 5x10 cells/well and maintained in MSM for 18-24 hours.

Cells were treated with 1-100ng/ml recombinant mouse IL-6 or IL-11 or 0.01-10ng/ml

recombinant human TGF-β1. All treatments and controls were performed in triplicate

and the α-SMA ELISA was performed as described in section 2.7.

K1/2,

recombinant IL-6 or IL-11 or 0.01-10ng/ml recombinant TGF-β1 in triplicate for 48

hours. Upon termination of the experiment a BrdU ass

mended by the manufacturer (GE Biosciences, USA). BrdU incorporation was

assessed by measuring absorbance at a wavelength of 450nm.

ΔSTAT/ΔSTAT 757F/757F

3

Western Blot

Western blots were performed as described in sections 2.15.1 and 2.15.2. Briefly,

cytosolic proteins were isolated from PLFs or MLFs after stimulation with cytokines as

described previously. Proteins were separated on 10% polyacrylamide gels by

electrophoresis and detected using antibodies directed against phosphorylated ER

STAT1, STAT3 and Smad3 at the dilutions stipulated in section 2.15. Protein loading

was confirmed by re-probing stripped membranes with antibodies directed against

unphosphorylated forms of ERK1/2, STAT1, STAT3 and Smad2/3 or α-Tubulin.

TGF-β Reporter Assay

This assay was performed as described in section 2.18 by Dr Bo Wang in the

laboratory of Dr Hong Jian Zhu of the Department of Surgery at the University of

Melbourne, Victoria, Australia. Briefly, NIH3T3 cells were stably transfected with the

Smad3 luciferase gene reporter p(CAGA)12-Luc. Serum obtained from wt or gp130

172

ed mutant mice was diluted in PBS and added to the cells. A titration of TGF-β1 was add

to the cells to determine a standard curve. Luciferase activity was measured after 24

hours.

173

5.3.1 Optimisation of Lung Fibroblast Isolations

To determine the optimal method for isolating lung fibroblasts from mature mice,

lungs were excised from C57BL/6 mice and subjected to serial enzymatic digestion

with 0.25% trypsin-EDTA and 0.4- 3mg/ml type I collagenase. There was no significant

difference (p>0.05) in the number of viable cells recovered or the rate of growth of the

cells in vitro between the different concentrations of collagenase type I (figure 5.1).

Therefore, lung fibroblasts were obtained by serial digestion with 0.25% trypsin-EDTA

and 1mg/ml collagenase type 1 for all subsequent experiments.

d from wt, gp130ΔSTAT/ΔSTAT and

gp13

d

7F

n of ERK1/2 compared to wt PLFs. Phosphorylated STAT1 was not

detect

5.3 Results

5.3.2 Cytoplasmic Signalling in Primary Lung Fibroblasts from Wt and Gp130 Mutant

Mice

Primary lung fibroblasts (PLFs) were harveste

0757F/757F mice and IL-6-induced activation of gp130-mediated ERK1/2 and

STAT1/3 signalling pathways were assessed by western blot. IL-6 stimulated wt cells

showed phosphorylation of the ERK1/2 and STAT3 signalling pathways 15 minutes

after stimulation but was not detectable at 60 minutes. IL-6 stimulation of

gp130ΔSTAT/ΔSTAT and gp130757F/757F PLFs confirmed that the signalling mutations

introduced into these animals were preserved in these cells (figure 5.2).

Gp130ΔSTAT/ΔSTAT showed prolonged IL-6-induced phosphorylation of ERK1/2 an

reduced phosphorylation of STAT3 compared to wt PLFs. Conversely, gp130757F/75

PLFs showed prolonged phosphorylation of STAT3, although there was also prolonged

phosphorylatio

ed after IL-6 stimulation.

Figu

f

endent of

B) and t ate of lun obto

174

re 5.1. Optimisation of lung fibroblast isolation.

The concentration of collagenase required to isolate optimal numbers of viable lungfibroblasts was determined by cell counts and cell growth rate in vitro. The number oviable cells recovered from the lung in collagenase digest fluid was indepcollagenase concentration over the range tested (A). The number of days to passage of primary lung fibroblasts was recorded ( he growth r g fibr lasts in culture was not affected by the collagenase concentration (0.4mg/ml to 3mg/ml) up passage 2 (B). Data represents the mean ± S.E.M, n=3.

1 20

B

10

20

0.4 mg/ml 1.0 mg/ml 1.5 mg/ml

2.0 mg/ml3.0 mg/ml

Passage

Cu

to R

eah

oA

2.5

Day

s in

lture

c

Cnf

luen

ce

Collagenase - mg/ml0.4 1 1.5 2 3

0

5.0

7.5

- x1

06C

ells

/ml

175

Figure 5.2. Gp130-mediated signalling mutations are preserved in vitro.

Wt, gp130ΔSTAT/ΔSTAT and gp130757F/757F PLFs were stimulated with 50ng/ml IL-6 for 15-60 minutes (15’-60’) to assess IL-6-induced phosphorylation of ERK1/2 (A) and STAT3 (B). Western blots probing for phosphorylated ERK1/2 (pERK1/2) and phosphorylated STAT3 (pSTAT3) demonstrated that gp130 signalling mutations were preserved in these cells. Even protein loading was assessed after stripping PVDF membranes and re-probing for total ERK1/2 and STAT3. Blots are representative of

UT 15’ 30’ 60’ UT 15’ 30’ 60’ UT 15’ 30’ 60’ pSTAT3

STAT3

B

two experiments. UT – untreated.

wt gp130ΔSTAT/ΔSTAT gp130757F/757F

UT 15’ 30’ 60’ UT 15’ 30’ 60’ UT 15’ 30’ 60’

wt gp130ΔSTAT/ΔSTAT gp130757F/757F

pERK1/2

/2

A

ERK1

176

nt

m

ments.

LF

genot LF

T

ice

lates

5.3.4 Derivation and Characterisation of Immortalised Lung Fibroblast Cell Lines

Immortalised lung fibroblast cell lines were derived from PLFs to serve as a

model of PLFs when these cells were not available due to shortage of animals for

preparing cell isolations. PLFs were transiently transfected with pRSV-T and cultured

for up to a four weeks in a single vessel. Non-transfected cells apoptosed leaving

immortalised mouse lung fibroblasts (MLFs). These cells were passaged routinely and

mainta

and

MLFs derived from gp130 mutant mice (figure 5.4). MLFs derived from gp130757F/757F

mice were much larger in size reaching confluence with approximately 0.9 x106 cells

5.3.3 Growth and Phenotype of Primary Lung Fibroblasts from Wt and Gp130 Muta

Mice

The growth and phenotype of PLFs from all mice were assessed under normal

growth conditions in vitro. PLFs from wt mice had typical spindle shape morphology,

characteristic of fibroblasts in culture. Gp130757F/757F PLFs were larger than PLFs fro

all other mice and adopted a stellate shape, with pronounced intermediate fila

Cell counts performed on confluent PLF cultures demonstrated that gp130757F/757F PLF

cultures contained fewer cells confirming that they were larger than other P

ypes (figure 5.3). In contrast, gp130ΔSTAT/ΔSTAT and gp130757F/757F;STAT3+/- P

cultures at confluence contained more cells (figure 5.3) and appeared smaller under

phase contrast microscopy. In addition to cell size, there was a difference in growth rate

of the PLFs from the different mouse genotypes. PLFs obtained from gp130ΔSTAT/ΔSTA

and gp130757F/757F;STAT3+/- mice grew faster than cells from wt and gp130757F/757F m

(figure 5.3). These findings suggest that directed gp130-mediated signalling modu

lung fibroblast phenotype and growth rate.

ined the cell phenotype characteristic of syngeneic PLFs at all passages (figure

5.4).

There were obvious differences in the cell size and shape between wt MLFs

177

Passage

wtgp130ΔSTAT/ΔSTAT

gp130757F/757F

gp130757F/757F;STAT3+/-

1 2 3 4 50

25

50

75

100

125

ays

n C

ultr

e t

Rea

h C

onflu

ence

B

Figure 5.3. Gp130-STAT3 signalling modulatesfibroblasts.

D i

oc

the size and growth of lung

The c ell counts and days to reach in a confluence in 75cm culture flask respectively. There

than PLFs from all other mice with these cells attaining confluence with fewer cells

with higher cell numbers in vitro. Under normal cell culture conditions there was a

more rapidly (B). This suggested that gp130-STAT3 signalling inhibited the growth

Differences between means were not statistically significant. A – n=6 with the re

error bars are present, n=2 in other instances.

2

7

llm

6

ell number and growth rate of primary lung fibroblasts were measured by c2

was a trend for primary lung fibroblasts (PLFs) from gp130757F/757F mice to be larger

(A). Impairment of gp130-STAT3 signalling resulted in PLFs attaining confluency

trend for PLFs from gp130ΔSTAT/ΔSTAT and gp130757F/757F;STAT3+/- mice to grow

of PLFs under normal cell culture conditions. Data is presented as mean ± S.E.M.

exception of gp130757F/757F (n=3) and gp130757F/757F;STAT3+/- (n=4). B – n≥3 whe

0 1

3 4 5 6

8

Ce

Nu

ber

- x10

A

per 7

f these cells

Fs

757F

T3+/-

een

or

growt

5.3.5 Assessment of Fibroblast Proliferation Assays

Three different methods were evaluated on control fibroblasts to assess cell

proliferation; MTT, MBB, and BrdU incorporation assays. Proliferation of PLFs from

C57BL/6 mice maintained in control medium and NGM was assessed after 48 hours.

The MBB and BrdU assays demonstrated a dose dependent increase in absorbance for

PLFs grown in NGM compared to cells grown in MSM (figure 5.6). However,

experiments with the MTT reaction did not demonstrate a dose dependent increase in

absorbance and suggested cells maintained in control medium had enhanced

proliferation relative to cells grown in NGM (figure 5.6). The MBB and BrdU

incorporation assays showed normal growth kinetics of cells grown in NGM. The BrdU

incorporation assay was chosen for future experiments as it was more sensitive to

relative changes in cell growth.

178

5cm2 culture flask (figure 5.4). In contrast, wt, gp130ΔSTAT/ΔSTAT and

gp130757F/757F;STAT3+/- MLFs reached confluency with approximately 2.5 x106, 10

x106 and 12 x106 cells respectively (figure 5.4). High power examination o

using phase contrast microscopy showed that gp130757F/757F fibroblasts had more

prominent intermediate filaments than wt fibroblasts and they were not visible in

gp130ΔSTAT/ΔSTAT and gp130757F/757F;STAT3+/- fibroblasts.

There was a significant difference in the growth of wt and gp130 mutant ML

under normal growth conditions over a period of six weeks (figure 5.5). Gp130757F/

MLFs grew significantly slower than wt MLFs (p<0.01), while gp130757F/757F;STA

MLFs grew faster than wt MLFs (p<0.05). There was no significant difference betw

the growth rates of wt and gp130ΔSTAT/ΔSTAT MLFs (p>0.15).

MLFs shared the phenotypic and growth characteristics of syngeneic PLFs

making MLFs a useful system to assess lung fibroblast responses to cytokines and

h factors when PLFs were not available.

179

wt gp130ΔSTAT/ΔSTAT

gp130757F/757F gp1 ;STAT3+/-30757F/757F

E

15

0

5

Cel

l Nu

ber

– x1

0

*10

m6

*

*

Figure 5.4. Immortalised lung fibroblasts from wt and gp130 mutant mice are different sizes in vitro.

Phase contrast microscopy and cell counts were performed on immortalised lung fibroblasts (MLFs) to assess cell morphology and measure cell size in confluent 75cmculture flasks. Lung fibroblasts from wt (A), gp130ΔSTAT/ΔSTAT (B) and gp130 ;STAT3 (D) mice had the typical spindle shape characteristic of fibroblasts in culture. Gp130757F/757F MLFs (C) had more stellate shape in culture. MLFs derived from gp130 and gp130 ;STAT3 mice were smaas they achieved 90-95% confluency in 75cm2 culture flasks with a higher populadensity that wt MLFs (p<0.001, E). Conversely, MLFs dervived from gp130PLFs were larger than other MLFs as they a nfluency in 2 at a signifcantly lower population density than other cells (p<0.001, E). Images are representative of n=3 and scale bars=100μm. *p<0.001, n=6.

2

757F/757F +/-

ΔSTAT/ΔSTAT 757F/757F +/- ller tion

757F/757F chieved co 75cm

C

A B

D

roblasts from wt and gp130 mutant mice.

Thermal growth conditions. Gp130ΔSTAT/ΔSTAT and

asts demonstrated rapid growth compared to wt cells (p=0 7F/757F lung fibro is

flask

wt *

180

ΔSTAT/ΔSTATgp130

gp130757F/757F * gp130757F/757F;STAT3+/-

0 10 20 30

25

50

100

400

75

125

150

ings

Poa

Dl

*p<0.01

pul

tion

oub

Days

Figure 5.5 Growth of immortalised lung fib

growth rate of immortalised lung fibroblasts from wt and gp130 mutant mice was monitored in vitro under nogp130757F/757F;STAT3+/- fibrobl

.17 and p=0.04 respectively). In contrast the growth rate of gp13075

blasts was inhibited compared to wt lung fibroblasts (p<0.01). Datarepresented as the mean doubling of the starting cell number per 75cm2 culture ± S.E.M, n≥3.

Figu

Lun

wer

hadprescell

181

re 5.6. Comparison of fibroblast proliferation assays.

ibroblasts were isolated from C57BL/6 mice and maintained in MSM or NGMg f

e a M or NGM

a l re ens g

for 48 hours to measure cell proliferation using the MTT and MBB, and BrdU incorporation assays. Cells were seeded in MSM and basal levels of cell proliferation

ssessed after 24 hours (T0). Remaining cells were maintained in MSfor a further 48 hours. Cells grown in NGM had a higher absorbance than cells maintained in MSM using the MBB and BrdU assays. However, cells grown in NGM

ower absorbance than cells grown in MSM using the MTT reaction. Data ated as the mean ± S.E.M., n≥4 and analysed by Student’s t-test compared to rown in MSM, *p<0.05.

T0 MSM-48hrs NGM- 48hrs0

0.25

0.50

0.75

1.00

1.25 MTTMBBBrdU

Abs

n

*

*

ce

orba

*

182

T

esults

are repr nse to IL-6 and IL-

11 vari

inhibit

I p130 and wt PLFs with maximum

effect a

signific p130

PLFs,

demon

prolife ference between IL-6 and IL-11-

induce

P ue of wt, gp130 ,

gp130 mice 30 days after bleomycin treatment using

the nuc s were detected in the

respira e

in the

fibrotic

Im T3) in bleomycin treated lung

tissue of all mice showed that pSTAT3 was present within fibrotic foci of wt and

5.3.6 IL-6 and IL-11 Differentially Regulate Fibroblast Proliferation

he effects of IL-6 and IL-11 on cell proliferation were assessed in PLFs from wt

and gp130 mutant mice using the BrdU incorporation assay (figure 5.7). These r

esentative of three experiments where the magnitude of respo

ed between isolations of PLFs, but the trend of proliferation versus growth

ion was maintained across experiments.

L-6 stimulated the proliferation of g 757F/757F

t 50 (p<0.05) and 100ng/ml (p<0.005) respectively. IL-6 inhibited the

proliferation of gp130ΔSTAT/ΔSTAT PLFs although the inhibition was not statistically

ant. Interestingly, IL-11 inhibited proliferation of both wt and g ΔSTAT/ΔSTAT

while it stimulated a biphasic response in gp130757F/757F PLFs with maximal

proliferation at 0.01 and 100ng/ml (p<0.05 respectively). One-way ANOVA

strated there was a significant difference between IL-6 and IL-11-induced

ration of wt PLFs (p<0.01), but there was no dif

d proliferation of gp130ΔSTAT/ΔSTAT and gp130757F/757F PLFs (p>0.05).

5.3.7 Proliferating Cells are Associated with Fibrotic Lung Tissue

roliferation was assessed in the lung tiss ΔSTAT/ΔSTAT

757F/757F and gp130757F/757F;STAT3+/-

lear proliferation marker Ki67. Ki67 positive cell

tory epithelium and inflammatory cells within the lungs of all mice, and in th

fibrotic foci of wt and gp130757F/757F mice (figure 5.8). A variety of cells with

foci were positive for the Ki67 antigen and morphological examination

suggested these cells included macrophages, lymphocytes and fibroblasts.

5.3.8 Phosphorylated STAT3 and α-SMA Co-localise in Fibrotic Foci

munostaining for phosphorylated STAT3 (pSTA

183

ry lung f

Prolif a BrdU incorporation assay

cells. ated with IL-6 (A) and IL-11

prolifprolif ed by IL-6 and IL-11, however,

repres *p<0.

Figure 5.7. IL-6 and IL-11 have different effects on the proliferation of primaibroblasts.

eration of PLFs was assessed after 48 hours usingand is expressed as a percentage change in growth relative to untreated syngeneic

PLFs from wt and gp130 mutant mice were incub(B). IL-6 promoted the proliferation of wt PLFs while IL-11 inhibited their

eration. Gp130 mutant PLFs had similar responses to IL-6 as IL-11. The eration of gp130ΔSTAT/ΔSTAT PLFs was inhibit

both IL-6 and IL-11 promoted the proliferation of gp130757F/757F PLFs. Results are ented as a percent change relative to syngeneic untreated (UT) PLFs ± S.E.M,05 and are representative of n≥3 experiments.

wt gp130ΔSTAT/ΔSTAT gp130757F/757F

A

Con

trol

60

80

Perc

0.01 0.1 1.0 10 50 100 200-20

0

ent o

f UT

20

40

100

* * *

* *

B

*

IL-6 - ng/ml

Perc

ent o

f U

T

-20 0

20 40

Con

trol

l

60 80

100

0.01 0.1 1.0 10 50 100 200-40 *

IL-11 - ng/ml

*

* *

*

gp130 d with the pr otic foci o ages are

184

A B

DC

Figure 5.8. Proliferating cells are common in fibrotic foci.

Proliferation within fibrotic tissue was assessed in lung sections from wt and 757F/757F mice 30 days after bleomycin treatment. Cells immuno-labelleoliferation marker Ki67 (arrows) were identified within and adjacent to fibrf wt (A, C) and gp130757F/757F (B, D) mice after bleomycin treatment. Im

representative of n=3 mice and scale bars represent 10μm (A, B) and 5μm (C, D).

gp130

ion

of α-SM ic

myofib dant within these fibrotic foci and that pSTAT3 and α-SMA

appear

G y

could r

gp1307 LFs after 48 hours but had no effect on gp130 and

gp130

SMA e

microf ;STAT3 MLFs was diffuse and

nuclea

I Fs was also quantified using an

ELISA

presen magnitude of response

to stim expression

after st these isolations of PLFs. After 48 hours IL-6

stimula in wt, gp130 and

gp130

wt or g he previous results obtained from

micros

185

757F/757F mice and areas of remodelled lung tissue in gp130757F/757F;STAT3+/- mice

but it was not detected in remodelled lung tissue in gp130ΔSTAT/ΔSTAT mice. Examinat

A expression in parallel lung sections showed filamentous staining in fibrot

foci adjacent to areas of unaffected lung tissue. This staining pattern suggested that

roblasts were abun

to co-localise in fibroblast-like cells within these foci (figure 5.9).

5.3.9 Gp130-STAT3 Signalling Regulates Fibroblast-Myofibroblast Differentiation

p130-mediated myofibroblastic differentiation of lung fibroblasts was initiall

assessed by immunocytochemistry in MLFs to determine if gp130-mediated signalling

egulate this process. IL-6 promoted the expression of α-SMA by wt and

57F/757F M ΔSTAT/ΔSTAT

757F/757F;STAT3+/- MLFs (figure 5.10). This data suggested that IL-6 induced α-

xpression is dependent upon gp130-STAT3 signalling. Labelling of α-SMA

ilaments in gp130ΔSTAT/ΔSTAT and gp130757F/757F +/-

r-centric, which was in contrast to the organised, reticular fibres crossing the

cytoplasm of wt and gp130757F/757F MLFs (figure 5.10).

L-6 and IL-11-induced α-SMA expression in PL

assay to validate the preliminary results observed in MLFs. The results

ted here are representative of three experiments where the

ulation varied with isolations of PLFs. However, the trend of α-SMA

imulation was the same for all

ted a significant increase in α-SMA expression ΔSTAT/ΔSTAT

757F/757F PLFs with a maximal effect at 100ng/ml IL-6 (p<0.05). Although

significant, stimulation of α-SMA by IL-6 in gp130ΔSTAT/ΔSTAT PLFs was far less than

p130757F/757F fibroblasts. Considering t

copic analysis of IL-6-stimulated gp130ΔSTAT/ΔSTAT MLFs, this small induction of

186

Fibrotimmudemo s present within this fibrotic focus (B) and fibroblast-like

ntative of n=

A

Figur within fibrotic foci.

ic lung tissue from gp130757F/757F mice 30 days after bleomycin treatment no-labelled for α-SMA (A) and pSTAT3 (B). A serial section of lung tissue nstrates that pSTAT3 i

e 5.9. α-SMA expression and pSTAT3 co-localise in fibroblasts

cells show immuno-reactivity for α-SMA and pSTAT3 (i-iv). Images are represe3 mice and scale bars represent 10μm.

(i) (ii)

(iii)

(iv)

B

(i)(ii)

(iii)

(iv)

187 A

– w

t, U

T

E –

wt

Figu

re-6

p

Imm

uno-

r 48

rs.

Unt

reat

edw

t (

-L

prom

oted

esct

oes

MLF

are

repr

es

n

- gp

ΔST

AT

/Δ, U

T

gp13

057

FD

7F/7

5A

, UT

+ IL

-6

5.1

0. IL

labe

lli (U

T)

α-S

MA

enta

tive

rom

ng o

f α-S

(A),

g e

xpr

of tw

o otes

α

MA

exp

rp1

30ΔS

T

sion

in w

t aex

perim

e

-SM

A e

xpr

essi

on

AT/ΔS

TAT

nd g

p130

nts a

nd

B F - g es

si

by M

LF (B

), 75

scal

e ba

130

p130 on

thro

ugh

gp

s fro

m w

t gp

13075

7F/7

57F

7F/7

57F M

LFs

rs re

pres

e

STA

T

ΔST

AT

/ΔST

AT

130-

ST

and

gp13

0 C

) and

gp1

30 (E

and

G re

t 20μ

m

+ IL

-6 A

T3

sign

mut

ant m

757F

/7

spec

tive

C –

G –

allin

g.

ice.

Fib

robl

a57

F ;STA

T3+/

ly),

but h

ad

757F

/7

gp13

0757F

/

sts w

ere

(D) M

LFno

eff

e

, UT

757F

+ IL

stim

ulat

eds d

id n

ot

n α-

SMA

-6

with

50n

g/co

nstit

uti

expr –

gp1

3

H –

gp

ml

vely

sion

in o

ther

075

1307

IL-6

fo e

xpre

ss α

7F;S

T

57F/

757F

;S

hou

-SM

A. I

s. Im

a

T3+/

-

TA

T3+/

-

-6

ges

+ IL

-6

to

functio

gp1307

s

after 48 no effect on

α-SMA

d

gp1307

5.3.10

I β are implicated in the development of lung fibrosis, so

basal le

TGF-β ad3 gene luciferase reporter pCAGA -

Luc (fi

howev f gp130 mice relative to controls

(p<0.0

T ced activation of the Smad2/3 pathway was assessed in MLFs

derived

TGF-β

minute lated Smad3 was not detected in gp130 MLFs (figure 5.12).

These ical levels

of TGF

188

α-SMA is likely to reflect it’s nuclear-centric distribution rather than organisation in

nal micr-filaments. IL-6 had no effect on α-SMA expression in

57F/757F;STAT3+/- PLFs (figure 5.11).

IL-11 was a potent stimulus of α-SMA expression in wt and gp130757F/757F PLF

hours with maximal effect at 100ng/ml (figure 5.11). IL-11 but had

expression in gp130ΔSTAT/ΔSTAT or gp130757F/757F;STAT3+/- PLFs (figure 5.11).

Interestingly, the magnitude of stimulation of α-SMA expression by IL-11 in wt an

57F/757F PLFs was significantly greater than following IL-6 stimulation (p<0.001).

Directed Gp130-Mediated Signalling Regulates TGF-β-Induced Smad3

Responses

ncreased levels of TGF-

vels of this growth factor were measured in the sera of all mouse genotypes.

levels were assessed in sera using the Sm 12

gure 5.12). Serum levels of TGF-β were similar in wt and gp130ΔSTAT/ΔSTAT mice,

er TGF-β was elevated in the sera o 757F/757F

1, figure 5.12). There was a trend for TGF-β levels to decrease in

gp130757F/757F;STAT3+/- mice but this was not statistically significant.

GF-β1-indu

from wt and gp130 mutant mice after stimulation with 5ng/ml TGF-β1 (figure

5.12). Phosphorylation of Smad3 (pSmad3) was detected in wt MLFs by 60 minutes of

1 stimulation and in gp130ΔSTAT/ΔSTAT and gp130757F/757F;STAT3+/- MLFs by 30

s. Phosphory 757F/757F

results indicate that gp130-mediated signalling modulates the physiolog

-β and the subsequent activation of Smad3.

189

5.3.11 I

eration

of PLF GF-β1

promo -

10ng/m ay ANOVA

demon

contras

(p<0.0

concen ce. The

LFs

(figure trend

e

(figure

ntly

increas

/ml

TGF-β

5.3.12 -Myofibroblast

Differe

increas ns of 1-

uniform increase in α-SMA expression, at least 75% greater than unstimulated controls,

L-6 and IL-11 Modulate TGF-β1-Induced Fibroblast Proliferation

The effect of directed gp130 mediated signalling on TGF-β1 induced prolif

s after 48 hours was assessed using the BrdU proliferation assay. T

ted the proliferation of wt and gp130757F/757F PLFs at a concentration of 1

l and 10ng/ml respectively (p<0.05, figure 5.13). Two-w

strated there was a significant increase in the proliferation of wt PLFs treated

with a combination of 0.01ng/ml TGF-β1 and 50ng/ml IL-6 (p<0.001, figure 5.13). In

t, 50ng/ml IL-11 inhibited the proliferative effects of TGF-β1 on wt PLFs

01, figure 5.13).

There was a trend for gp130ΔSTAT/ΔSTAT PLFs to proliferate in response to low

trations of TGF-β1, but these increases did not reach statistical significan

combination of IL-6 and TGF-β1 had no significant effect on gp130ΔSTAT/ΔSTAT P

5.13) and although the combination of IL-11 and TGF-β1 demonstrated a

for an increase in proliferation in these cells it did not reach statistical significanc

5.13).

Co-stimulation of gp130757F/757F PLFs with IL-6 and TGF-β did not significa

e cell proliferation above TGF-β alone (p>0.05). There was a trend of increased

gp130757F/757F PLF proliferation with the combination of 50ng/ml IL-11 and 10ng

1 but this was not statistically significant (figure 5.13).

Gp130 Signalling Mediates TGF-β1-Induced Fibroblast

ntiation

The effect of directed gp130-mediated signalling on TGF-β1-induced α-SMA

expression after 48 hours was assessed using an α-SMA ELISA. TGF-β1 significantly

ed α-SMA expression in wt and all gp130 mutant PLFs at concentratio

10ng/ml 48 hours after stimulation. Interestingly, gp130757F/757F;STAT3+/- PLFs had a

190

fibrob

Prima

gp130n

repres*p<0.0

Figure 5.11. IL-6 and IL-11 promote myofibroblast differentiation of lung lasts.

ry lung fibroblasts were incubated with IL-6 (A) or IL-11 (B) for 48 hours and α-SMA expression was assessed by ELISA. IL-6 and IL-11 promoted α-SMA expression in wt fibroblasts after 48 hours (A). IL-6 induced α-SMA expression in

ΔSTAT/ΔSTAT and gp130757F/757F lung fibroblasts (A) and IL-11 promoted α-SMA 757F/757Fexpression in wt and gp130 fibroblasts (B) by 48 hours. α-SMA expressio

was not stimulated in gp130757F/757F;STAT3+/- PLFs by IL-6 or IL-11. Results are ented as a percent change relative to syngeneic untreated PLFs ± S.E.M, 5 relative to untreated syngeneic PLFs and are representative of 3 experiments

performed in triplicate.

wt gp130ΔSTAT/ΔSTAT

gp130757F/757F gp130757F/757F;STAT3+/-

IL-6 - ng/ml

**40

60

80

f UT

Con

t

**** 0

20 *

Perc

ent

o

A

100

rol

1 10 50 100-20

IL-11 - ng/ml

* 0

50

Perc

ent o

1 10 50 100-50

*100 150

f UT

**

B

250 300

ol * *

* 200

Con

tr

191

0.6

F/F F/F;ST3+/-wt Δ/Δ0

0.1 0.2

TG

F-

0.3 0.4 0.5

β –

ng/m

*A

l

wt Δ/Δ F/F F/F;ST3+/-

UT 30’ 60’ UT 30’ 60’ UT 30’ 60’ UT 30’ 60’

pSmad3

B

α-Tubulin

Figuractiva

TGF-βdemon

gp130 mice w

± S.E.

e 5.12. Directed gp130-induced signalling regulates TGF-β levels and tion of the Smad3 pathway.

A Smad3 gene luciferase reporter (pCAGA12-Luc) was used to assess basal levels of in the serum of untreated wt and gp130 mutant mice (A). This assay strated there was significantly more TGF-β in the serum of untreated

757F/757F ΔSTAT/ΔSTATgp130 mice compared to wt and gp130 mice (p=0.009). There was a trend for TGF-β levels to be reduced in gp130757F/757F;STAT3+/- mice compared to

757F/757F mice, however this was not significant. MLFs from wt and gp130 mutantere stimulated with 5ng/ml TGF-β1 and phosphorylation of Smad3 (pSmad3)

was assessed by western blot (B). Smad3 phosphorylation was enhanced in gp130ΔSTAT/ΔSTAT and gp130757F/757F;STAT3+/- MLFs compared to wt cells, while

757F/757FpSmad3 was not detected in TGF-β1 stimulated gp130 MLFs. Even loading of protein was confirmed upon detection of α-Tubulin. A – data is represented as the mean

M. n=4, *p<0.05. B – blots are representative of two experiments.

TGF-β1

produc Fs after 48

hours, ificant

differe

gp1307 PLFs, however the TGF-β1-induced expression of α-SMA was

lower i

T n of 50ng/ml IL-6 and 0.01-0.1ng/ml TGF-β1 significantly

enhanc

TGF-β id not enhance expression of

α-SMA

of IL-6 -β1 had no additional effects on TGF-β1-induced α-SMA expression by

gp130 ure

5.14).

192

at all concentrations of TGF-β1 tested relative to controls (p<0.05, figure 5.14).

ed only a modest increase in α-SMA expression in gp130757F/757F PL

approximately 30% greater than unstimulated controls. There was no sign

nce in TGF-β1-induced α-SMA expression in wt, gp130ΔSTAT/ΔSTAT and

57F/757F;STAT3+/-

n gp130757F/757F PLFs relative to wt cells (p<0.05, figure 5.14).

he combinatio

ed α-SMA expression in wt PLFs relative to PLFs stimulated with 0.01-0.1ng/ml

1 alone (p<0.05, figure 5.14). The addition of IL-6 d

by wt PLFs at higher concentrations of TGF-β1 (figure 5.14). The combination

and TGF

ΔSTAT/ΔSTAT, gp130757F/757F or gp130757F/757F;STAT3+/- PLFs after 48 hours (fig

193

TGF-β TGF-β + IL-11TGF-β + IL-6

* 100

150

ntro

l # A - wt

**

0

50

of U

T C

o

0.01 0.1 1.0 10-50 Pe

rcen

t

# #

B - gp130

100

150

UT

Con

trol

ΔSTAT/ΔSTAT

-50

0

0.01 0.1 1.0 10

Perc

ent o

f 50

Figure 5.13. Effect of IL-6 and IL-11 on TGF-β1-induced proliferation of PLFs.

1-induced proliferation was assessed in wt and gp130 mutant PLFs after 48 y BrdU incorporation assay. Synergy between TGF-β-induced effects and activate

TGF-βhours b

PLFs w

nt

of gp1combi

StudenANOV

gp130 d signalling was assessed by co-stimulating cells with 50ng/ml IL-6 or IL-11. TGF-β1 stimulated the proliferation of wt PLFs at 1-10ng/ml (A, p<0.05). Wt

ere stimulated to proliferate in response to a combination of low doses of TGF-β1, 0.01-0.1ng.ml and 50ng/ml IL-6 (p<0.001, A). Conversely, co-stimulation

with 50ng/ml IL-11 inhibited the proliferation observed at 1-10ng/ml TGF-β1 01, A). TGF-β1 alone or com(p<0.0 bination with IL-6 or IL-11, had no significa

effect on the proliferation of gp130ΔSTAT/ΔSTAT PLFs. TGF-β1-induced proliferation 30757F/757F PLFs, relative to untreated controls, at 10ng/ml (p<0.05, C). The nation of 10ng/ml TGF-β1 and 50 ng/ml IL-6 or IL-11 was not significantly nt to the response to TGF-β1 alone. Data are pdiffere resented as the mean ± S.E.M,

and are representative of n=3. *p<0.05 relative to untreated syngeneic PLFs by t’s t-test. #p<0.001 relative to TGF-β1 stimulated syngeneic PLFs by two-way A Bonferroni post-tests.

**100

UT

Con

150 C – gp130

trol

757F/757F

0.01 0.1 1.0 10-50

TGF-β1 - ng/ml

Pe

0

50

rcen

t of

194

wtwt + IL-6

gp130ΔSTAT/ΔSTAT

gp130ΔSTAT/ΔSTAT + IL-6

gp130757F/757F;STAT3+/- + IL-6gp130757F/757F;STAT3+/-

gp130 7F/757F + IL-675gp130757F/757F

Figur y lung fibroblasts 48 hours after stimulation.

e 5.14. TGF-β-induced α-SMA expression in primar

0.01 0.1 1.0 10

α-SMA expression was measured in primary lung fibroblasts from wt and gp130 t mice following stimulation with TGF-β1 and the combination of TGF-β1y ELISA. TGF-β1 promoted α-SMA expression in wt, gp130ΔSTAT/ΔSTAT, 757F/757F and gp130757F/757F;STAT3+/- PLFs after 48

mutan and IL-6 bgp130 hours (p<0.05). The addition

0.1nggp130 or gp130 ;STAT3 PLFs after 48 hours. Data

d to untrea -β1 alone.

of 50 ng/ml IL-6 enhanced TGF-β-induced α-SMA expression in wt PLFs at 0.01-/ml TGF-β1, however it had no additive effect on α-SMA expression in ΔSTAT/ΔSTAT, gp130757F/757F 757F/757F +/-

is representative of n=3 experiments performed in triplicate, *p<0.05 compareted syngeneic cells, #p<0.05 compared to syngeneic cells treated with TGF

-25

0

25

50

75

100

125 **

** ** **

ent C

hang

e * **

**

*

#

#

** *

**

Perc **

*

TGF-β1 - ng/ml

195

5.4 Dis

interfa

f

lung fi

fibrobl 130-

d

myofib

betwee

grow at o

e

literatu

fibrobl

IPF

lung fi

Ramos and gp130757F/757F

cussion

Fibroblastic foci are a distinctive feature of IPF and are typically found at the

ce between unaffected lung and remodelled lung in IPF patients. The location of

these foci and the presence of myofibroblasts have led many authors to speculate that

ibrobthese f lastic foci represent active lesions in IPF. This chapter investigated two

mechanisms that could lead to the formation of fibroblastic foci; the proliferation o

broblasts and the differentiation of lung fibroblasts into myofibroblasts. The

described demonstrate that gp130-mediated signallinresults g pathways can regulate

ast proliferation and the formation of myofibroblasts. Furthermore, gp

mediated signalling can modulate the effects of TGF-β1 on fibroblast proliferation an

roblast differentiation.

Characterisation of primary lung fibroblasts and immortalised fibroblast cell lines

from all mice showed there were differences in cell phenotype and growthderived

n the genotypes under standard culture conditions. There was a trend for

gp130757F/757F primary lung fibroblasts and immortalised lung fibroblast cell lines to

a slower rate than other cell genotypes. Gp130757F/757F lung fibroblasts were als

larger with a stellate phenotype and prominent intermediate filaments compared to the

shape of wt, gp130ΔSTAT/ΔSTAT and gp130757F/757F;STAT3+/- lung fibrospindle blasts. Th

re on the differences between normal lung fibroblasts and lung fibroblasts from

IPF patients, in some instances, appears to be contradictory (Jordana et al., 1988;

ey et al., 2003a; Ramos et al., 2001). However, it has been shown Moodl that lung

asts from IPF patients grow more slowly and are more prone to apoptosis than

human lung fibroblasts (Ramos et al., 2001, Hetzel et al., 2005). Incontrol addition,

broblasts constitutively express more intermediate filaments and the

microfilament α-SMA than control human lung fibroblasts (Moodley et al., 2003a,

et al., 2001). These similarities between IPF lung fibroblasts

196

lung fi sis.

serum,

follow vated as part of the wound repair

proces ammation

).

Further y

(Cavar on of wt and gp130 mice 30

days af

myofib

fibrobl

s of

IL-6 an the proliferation of wt PLFs, as IL-6 promoted proliferation and IL-

11 inhi

disrupt sting that there are differences in the magnitude and the

duratio

molecu ubsequent

activat to be identical (Kurth et al.,

1999, D hese

cytokin ferences between IL-6 and IL-11 (Moodley et

al., 200 andurkar et al.,

1997).

P

throug

fibroblasts suggest that gp130-STAT3 signalling confers a pro-fibrotic phenotype upon

broblasts which has a significant effect on the development of lung fibro

Standard in vitro growth conditions consist of medium supplemented with bovine

containing a mixture of bioactive molecules at homeostatic levels. However,

ing an injury many bioactive molecules are ele

s. IL-6 is increased following injury and participates in the ensuing infl

and wound repair process (Kopf et al., 1994, Xing et al., 1998, Rose-John et al., 2006

more, it is known that IL-6 levels are elevated in bleomycin-induced lung injur

ra et al., 2004, Tabata et al., 2007). Examinati 757F/757F

ter bleomycin treatment showed that cell proliferation and the expression of

roblast marker α-SMA were intimately associated with fibroblastic foci. The

experiments described in this chapter assessed what effect IL-6 and IL-11 may have on

ast behaviour following an injury.

The results of these experiments showed that there is a dichotomy in the effect

d IL-11 on

bited the proliferation of these cells. Directed gp130-mediated signalling

ed this dichotomy, sugge

n of ERK1/2 and STAT1/3 signalling activated by IL-6 and IL-11. The

lar interactions between IL-6 and IL-11 binding of gp130 and the s

ion of intracellular signalling pathways are reported

ahmen et al., 1998), yet studies examining the physiological effects of t

es show there are significant dif

3, Kopf et al., 1994, Howlett et al., 2005, Kuhn et al., 2000, N

revious studies showed that IL-6 inhibited the proliferation of lung fibroblasts

h preferential activation of the STAT3 pathway compared to the ERK1/2

197

ver,

directe

by STA

t

al., 200 of

the ER ed proliferation of lung

fibrobl

T the lung is heterogeneous, and it has been

demon

fibrobl ed in IPF (Moodley et al., 2003a, Moodley et al., 2003b,

Ramos

is

(Mona

fibrobl

phosph treated wt and gp130 mice.

IL-6 an hours,

but did ce α-SMA expression in gp130 or gp130 ;STAT3

PLFs,

is depe

in the

skin an

pathway (Moodley et al., 2003a). In contrast, my findings suggest that fibroblast

proliferation is stimulated by IL-6-induced activation of the STAT3 pathway. Howe

d gp130-STAT1/3 signalling in PLFs (gp130757F/757F) also showed activation of

the ERK1/2 pathway suggesting that cross-activation of these pathways can be mediated

T3. It has been demonstrated that cross-activation of these pathways by gp130 is

crucial to maintaining homeostasis in a variety of organs (Jenkins et al., 2004, Jenkins e

5a, Tebbutt et al., 2002). Therefore, dysregulated gp130-mediated activation

K1/2 and STAT3 pathways may culminate in increas

asts and contribute to the development of fibrosis in vivo.

he fibroblast population within

strated by a number of independent laboratories that the normal distribution of

ast sub-types is alter

et al., 2001, Phan, 2003). A hallmark of UIP/IPF is the formation of a

myofibroblastic focus and the frequency of these foci is indicative of a poor prognos

ghan et al., 2004). Furthermore, myofibroblasts are more abundant in the lungs of

patients with IPF than control patients and are known to produce more collagen than

asts (Moodley et al., 2003a, Ramos et al., 2001). α-SMA expression is the

defining characteristic of myofibroblasts and this protein co-localised with

orylated STAT3 in fibrotic foci of bleomycin 757F/757F

d IL-11 induced α-SMA expression in wt and gp130757F/757F PLFs after 48

not indu ΔSTAT/ΔSTAT 757F/757F +/-

suggesting that IL-6/11-induced myofibroblast differentiation of lung fibroblasts

ndent on a complete gp130-STAT3 signalling capacity.

IL-6 and IL-11 have been reported to promote myofibroblast differentiation

d lung, but these studies have not attributed the effects of these cytokines to the

activation of the STAT3 pathway (Chen et al., 2005, Gallucci et al., 2006, Tang et al.,

198

1996).

signall ilieu of

bleomy d lung injury, the lung fibroblasts of gp130 mice are shunted

toward

active n wound resolution.

Emerg

on of

some d

2005a, hapter show that IL-6 and

TGF-β

low co

interac

GF-β1-

induce

As gp1

possibl

showed

Smad-

mediat

stimula

Myofibroblast differentiation is typically a transient event at a wound site that is

critical for the contraction of the opposing wound edges. Directed gp130-STAT3

ing induces myofibroblast differentiation and, in the inflammatory m

cin-induce 757F/757F

s this fibrogenic cell phenotype.

Cross-talk between cytokines and growth factors is vital for the transition from an

process of wound healing to normal tissue function upo

ing data is describing a complex interaction between IL-6 and TGF-β in various

pathological situations that suggests these molecules cooperate in the progressi

isease states while antagonism between these molecules contributes to other

diseases (Becker et al., 2004, Ogata et al., 2006, Jenkins et al., 2007, Jenkins et al.,

Zhang et al., 2005). The results described in this c

1 have a positive effect on the proliferation and α-SMA expression in wt PLFs at

ncentrations of TGF-β1. IL-11 inhibited TGF-β1-induced proliferation of wt

PLFs at all concentrations tested. Directed gp130-mediated signalling disrupted the

tion between IL-6, IL-11 and TGF-β1-induced proliferation observed in wt

fibroblasts. However, there was a trend for gp130-ERK1/2 signalling to inhibit T

d proliferation while gp130-STAT1/3 permitted TGF-β1-induced proliferation.

30757F/757F;STAT3+/- mice were not available for these experiments it was not

e to assess the role of the STAT3 pathway specifically in these responses.

Examination of the effects of combining IL-6 and TGF-β1 on α-SMA expression

that IL-6-induced gp130-STAT3 signalling reduced the immediate effects of

TGF-β1. Other studies have shown that IL-6 synergises with TGF-β1 to enhance

ed transcription in vitro (Zhang et al., 2005). Ogata et al. (2006) showed in their

model of hepatic fibrosis that enhanced levels of STAT3 increased fibrosis by

ting TGF-β synthesis (Ogata et al., 2006). However, gastric models of

199

(Jenkin

A

expres

s. This

sugges nduced α-SMA expression through other

cytokin

T a

molecu l models of fibrosis (Bonniaud et al., 2004,

Zhao et

asal

serum

er, the

results

molecu gp130 lung fibroblast cell lines while

phosph brosis resistant

gp130

effects

inhibit Smad3 phosphorylation. These results suggest that TGF-β-induced

respon

transcr

2004, R forms hetero-dimers

with th

tumorigenesis have demonstrated that TGF-β and IL-6 are mutual antagonists in vivo

s et al., 2005a, Becker et al., 2004). Interestingly, monoallelic loss of STAT3 in

gp130757F/757F PLFs (gp130757F/757F;STAT3+/-) enhanced TGF-β1-induced α-SM

sion independent of concentration. Loss of a single STAT3 allele also

significantly ablated IL-6 and IL-11-induced α-SMA expression in mouse PLF

ts that STAT3 inhibits TGF-β1-i

es and growth factors that activate this pathway.

GF-β is a potent stimulus for the synthesis of ECM and fibrogenesis (Verrecchi

and Mauviel, 2007, Bonniaud et al., 2005). TGF-β and it’s intracellular signalling

les Smad2/3 are increased in anima

al., 2002, Sime et al., 1997) and in fibrotic foci and BALF of patients with IPF

(Khalil et al., 1991, Broekelmann et al., 1991, Awad et al., 1998). Interestingly, b

levels of TGF-β were increased in gp130757F/757F mice compared to other mice

suggesting gp130757F/757F mice may be pre-disposed to enhanced fibrosis. Howev

described here show that directed gp130-STAT3 signalling disrupts TGF-β-

mediated signalling and functions. TGF-β1-induced phosphorylation of the signalling

le Smad3 was undetected in 757F/757F

orylation of Smad3 was enhanced in MLFs derived from fi

ΔSTAT/ΔSTAT and gp130757F/757F;STAT3+/- mice. Furthermore, TGF-β1-induced

were observed in fibroblasts from gp130757F/757F mice despite constitutive

ion of

ses may occur independently of Smad3.

Previous studies have shown that the effects of TGF-β1 are dependent upon

iptional responses elicited by Smad3 (Bonniaud et al., 2005, Bonniaud et al.,

oberts et al., 2006, Sato et al., 2003). However, Smad3

e structurally related molecule Smad2 to activate cytoplasmic signalling

200

pathwa

p38 mi

7),

describ l.,

et

al. (20 focal

adhesio pro-

studies

charac

phosph

T contributes to the

develo viour

and ph cted gp130-STAT3 signalling confers a proliferative and

myofib

contrib e, IL-6 enhances the

prolife

ys (Gauldie et al., 2007, Roberts et al., 2006, Massague et al., 2005). It is known

that there is cross-talk between Smad2 and other cytoplasmic signalling molecules, such

togen activated protein kinase, focal adhesion kinase, Ras and c-Abl

(Kretzschmar et al., 1999, Daniels et al., 2004, Liu et al., 2006, Horowitz et al., 200

and it is therefore possible that these cytoplasmic interactions with Smad2 regulate

TGF-β1 induced responses. Furthermore, there is a growing body of evidence

ing TGF-β-induced responses independent of Smad2/3 signalling (Pannu et a

2007, Mulsow et al., 2005, Khalil et al., 2005, Thannickal et al., 2003). Thannickal

03) showed TGF-β-induced myofibroblast differentiation was regulated by

n kinase signalling. More recently Pannu et al. (2007) showed that the

fibrotic effects of TGF-β were regulated by Smad1 and ERK1/2 pathways. These

support the results described in this chapter describing pro-fibrotic

teristics of lung fibroblasts in the absence of TGF-β1-induced Smad3

orylation.

his chapter has demonstrated that gp130-mediated signalling

pment and progression of pulmonary fibrosis by regulating fibroblast beha

enotype. Dire

roblastic phenotype upon lung fibroblasts which are consistent with them

uting to the formation of fibroblastic foci. Furthermor

ration and myofibroblastic differentiation of PLFs induced by TGF-β1.

201

CHAPTER SIX

GENERAL DISCUSSION

202

6.1.1 In

F e of a group of heterogeneous lung diseases known as

idiopat

UIP is 0,

s of IPF

remain wever the persistent accumulation of collagen and the formation of

fibrobl ity

(Mona

of the i sforming growth factor-β

(TGF-β , Phan,

y new

treatme

fibrotic r

polymo kine levels, and the nature of fibrotic disease, they

shed lit

n of

IL-6 fa 7, Qiu

et al., 2

that is

have b anism of IL-6/gp130-induced progression of lung

fibrosi

6.1 GENERAL DISCUSSION

troduction

ibrosis is the end stag

hic pulmonary fibrosis (IPF) that includes usual interstitial pneumonia (UIP).

always fatal and current treatments do not improve patient survival (ATS, 200

Knight et al., 2003, Fellrath and du Bois, 2003). The aetiology and pathogenesi

s uncertain, ho

astic foci are features of this disease that are causally linked to patient mortal

ghan et al., 2004, Cool et al., 2006). Previous studies have focussed on the roles

nflammatory response, fibroblast behaviour and tran

) activity in the development and progression of IPF (Gauldie et al., 2007

2003, Thannickal et al., 2004). Further understanding of these mechanisms and their

relationship with the development and progression of IPF is required to identif

nts and improve patient survival.

It is well established that the IL-6/gp130 cytokine family is associated with

lung diseases including IPF (Takizawa et al., 1997, Pantelidis et al., 2001, Lesu

et al., 1994). However, while several studies have demonstrated associations between

rphisms in the IL-6 gene, cyto

tle light on the mechanisms involved. With the advent of genetically modified

animals, a number of groups have shown causal relationships between the expressio

mily cytokines and lung remodelling (Yoshida et al., 1995, Ray et al., 199

004). However, these models did not replicate the extensive interstitial fibrosis

indicative of UIP and IPF. In recent years there have been reports emerging that

egun to describe a mech

s. These reports have demonstrated that IL-6 promotes the proliferation and

203

inhibit

T

fibrosi s

respon nt of lung fibrosis in vivo. Activation of STAT3

signall en in the

myofib -SMA. I have also demonstrated inhibitory cross-talk between

gp130-

inhibit A in lung fibroblasts independent of

Smad3

bleomy

6.1.2 D

signall

ce gp130-

mediat

pathwa Reports characterising these mice have demonstrated

that the

eding

of gp13 imals lacking a single STAT3 allele yielded offspring

(gp130

s apoptosis of lung fibroblasts from patients with IPF but not normal lung

fibroblasts (Moodley et al., 2003a, Moodley et al., 2003b).

he experiments outlined in this thesis have examined the role of gp130-mediated

Shp2-ERK1/2 and STAT1/3 signalling on the development of bleomycin-induced lung

s in vivo and mechanisms of fibrogenesis using fibroblasts isolated from the lung

of these mice. I have shown that gp130-STAT3 signalling regulates the inflammatory

se and the subsequent developme

ing by gp130 also regulated the transcription and accumulation of collag

lung following bleomycin treatment. IL-6-induced activation of gp130-STAT3

signalling facilitated the proliferation of lung fibroblasts and the expression of the

roblast marker α

mediated signalling pathways and Smad3 signalling. This cross-talk led to

ion of TGF-β-induced expression of α-SM

signalling. This is the first study to examine the effect of preferential activation

of receptor-mediated cytoplasmic signalling pathways in the development of

cin-induced murine lung fibrosis.

irected Gp130-STAT3 Signalling Regulates Lung Remodelling

In the first of the results chapters (chapter three) the role of gp130-mediated

ing in the development of lung fibrosis was investigated in vivo. Genetically

engineered mice carrying knock-in mutations in gp130 that direct and enhan

ed activation of the ERK1/2 (gp130ΔSTAT/ΔSTAT) or STAT1/3 (gp130757F/757F)

y were used in this study.

se signalling mutations are functional in vivo and are expressed in all cultured

cells isolated from these mice (Ernst et al., 2001, Tebbutt et al., 2002). Cross-bre

0757F/757F mice with an

757F/757F;STAT3+/-) with reduced gp130-STAT3 activation compared to

204

gp1307 udy as

they w .

the lun c

n

o

the lym

have re

s

clear th lung remodelling than other

mouse

I that directed gp130-STAT3 signalling promoted the

develo

less co

ms

have su

fibroge

mice la gene (Ezure et al., 2000) and decreased levels of the negative

feedba

with liv d

(Sano e s

IL-6 family cytokines activate STAT1/3 signalling with ERK1/2 signalling functioning

57F/757F mice (Jenkins et al., 2005a). These animals were also used in this st

ere useful tool for assessing the specific role of gp130-STAT3 signalling in vivo

Histological examination of the lungs of 30 day control wt and gp130 mutant

mice showed normal lung morphology in wt and gp130ΔSTAT/ΔSTAT mice. Interestingly,

g phenotype of gp130757F/757F mice demonstrated accumulations of lymphocyti

cells adjacent to airways and blood vessels, similar to that induced by over-expressio

of IL-6 and IL-11 (Xing et al., 1994, Ray et al., 1997, Kuhn et al., 2000). In addition t

phocytic accumulations, control gp130757F/757F mice had thickened alveolar septa

with apparent thickening of the interstitial ECM in some areas of the lung. This may

sulted from increased levels of IL-6 family cytokines induced by saline treatment

or more likely, as the result of increased basal gp130-STAT3 activity. However, it i

at the gp130757F/757F mice are more susceptible to

genotypes examined.

t is clear from my studies

pment of interstitial lung fibrosis, collagen transcription and accumulation

following bleomycin treatment, while, directed gp130-ERK1/2 signalling resulted in

llagen transcription but increased levels of MMPs and signs of airspace

enlargement. Investigations into the development of fibrosis in other organ syste

ggested STAT1/3 pathways activated by gp130 ligands are central to

nesis. For example, the development of hepatic biliary fibrosis was reduced in

cking the IL-6

ck inhibitor of gp130-STAT3 signalling, SOCS3, have been observed in patients

er fibrosis (Ogata et al., 2006). Enhanced levels of IL-6 and phosphorylate

STAT3 have been reported in fibrotic skin diseases such as psoriasis and keloid scars

t al., 2005, Lim et al., 2006, Ghazizadeh et al., 2007). Under normal condition

205

pta et

al., 199

in wt a

fibrosi

U

absent osis; or

enhanc

in gp13

(gp130

STAT3 g bleomycin treatment. These

mice m

ese

nd

progre

.1.3 Directed Gp130-STAT3 Links Inflammation to Fibrosis

Chapter four examined the inflammatory response that preceded fibrosis in wt and

gp130 mutant mice. Retention of inflammat ls within the lung parenchyma was

associated with and possibly necessary for the development of bleomycin-induced lung

fibrosis. The frequency and cellularity of inflammatory foci in gp130757F/757F mice was

significantly greater than all other mice three days after bleomycin treatment, suggesting

a role for STAT1/3 in interstitial inflammation. This finding is supported by Sano et al.

(2005) who showed that enhanced STAT3 activation increased inflammatory cell

infiltration of the epidermis and this was associated with thickening of the epidermis

and fibrosis. Furthermore, previous studies with gp130 mutant mice showed that

as a negative regulator of the STAT1/3 pathway (Ernst and Jenkins, 2004, Sengu

8). Therefore, it follows that normal and enhanced activation of gp130-STAT1/3

nd gp130757F/757F mice respectively, leads to fibrosis, and the inability of

gp130ΔSTAT/ΔSTAT mice to signal through gp130-STAT1/3 confers protection from

s.

sing these mice I could not identify whether enhanced STAT1/3 signalling or

ERK1/2 signalling in gp130757F/757F mice drove the development of fibr

ed ERK1/2 and absent STAT1/3 signalling in gp130ΔSTAT/ΔSTAT mice conferred

protection from fibrosis. To address these possibilities, STAT3 activation was decreased

0757F/757F mice by cross-breeding with animals lacking a STAT3 allele

757F/757F;STAT3+/-). This enabled me to perform a physiological titration of

and examine its effect on lung fibrosis followin

orphologically demonstrated less fibrosis and biochemically had significantly

less collagen in their lungs than gp130757F/757F mice, with levels approaching wt. Th

experiments demonstrated that gp130-STAT3 signalling regulated the development a

ssion of lung fibrosis in this model in vivo.

6

ory cel

206

inflammation is enhanced in the colon and peritoneum of gp130757F/757F mice, due to

enhanced gp130-STAT3 signalling (Judd et al., 2006b, McLoughlin et al., 2005).

Importantly, I showed that the number of inflammatory foci decreased in

gp130757F/757F;STAT3+/- mice suggesting that the recruitment and retention of

inflammatory cells is an important feature of bleomycin-induced fibrosis and occurs

through a gp130-STAT3 mechanism.

The inflammatory response is a tightly regulated process that involves the

recruitment of inflammatory cells from the circulation, migration of these cells through

the vasculature and tissue, and their retention and activation at the wound site.

Migration is achieved along a chemokine gradient, binding to adhesion molecules on

endothelial surfaces and migrating through the cell-cell junctions and ECM (Strieter et

al., 2007). My findings showed that directed gp130-mediated ERK1/2 and STAT1/3

signalling disrupted the normal recruitment and trafficking of inflammatory cells after

bleomycin treatment. In the lung, chemokines are secreted into the airways and

airspaces by epithelial and stromal cells to assist in the resolution of an injury and

prevent opportunistic infections (Strieter et al., 2007). Disruption of the specific

chemokine-chemokine receptor interaction in granulocytes and lymphocytes has been

shown to reduce or ablate bleomycin-induced fibrosis (Jiang et al., 2004, Moore et al.,

2001). Judd and colleagues (2006) demonstrated that directed gp130-STAT3 signalling

promoted gene expression of neutrophil and macrophage chemokines in the gastric

mucosa of gp130757F/757F mice. However, this study did not show that these

transcriptional changes translated into increased chemokine levels. Interestingly,

inflammatory foci were typically located in the serosa of the vasculature and airways of

gp130757F/757F mice. This suggests that either chemokine gradients from the vasculature

to the air spaces and airways are dysregulated or the migration of inflammatory cells

through the serosa of the vasculature and the airways was being impeded in these mice.

207

Recent studies have also demonstrated that IL-6 increases the levels of adhesion

molecules and integrins expressed by endothelial and epithelial cells, that facilitate the

transport of lymphocytes through venules (Chen et al., 2006, Chen et al., 2004) and

regulate epithelial integrity and cell-ECM adhesion under physiological conditions

(Milner and Campbell, 2006). Increased expression of these molecules creates a

pulmonary micro-environment that facilitates movement of inflammatory cells from the

vasculature and impedes the trafficking of these cells through the vascular serosa and

pulmonary epithelium. Increased expression of cell adhesion molecules and integrins on

interstitial cells in gp130757F/757F mice may also explain why more inflammatory cells

were retained in the lung interstitium three days after bleomycin treatment.

The frequency and cellularity of inflammatory foci in gp130ΔSTAT/ΔSTAT mice was

equivalent to wt mice three days after bleomycin treatment, however inflammatory cell

content in the BALF of gp130ΔSTAT/ΔSTAT mice was significantly increased. The findings

in chapter four suggest that recruitment of granulocytes into the air spaces and airways

of gp130ΔSTAT/ΔSTAT mice and their retention at the wound site was dysregulated

compared to wt mice. The findings of previous studies using IL-6-/- mice have

demonstrated that loss of IL-6 increased the levels of macrophage, neutrophil and

eosinophil chemokines in the airways of mice in experimental models of pulmonary

inflammation (Xing et al., 1998, Wang et al., 2000a, Sheridan et al., 1999, Wang et al.,

2000b). My findings showed some parallels with these studies and suggest that the

secretion of granulocyte chemokines into the air spaces and airways may be enhanced in

gp130ΔSTAT/ΔSTAT mice compared to wt mice following bleomycin treatment.

Notable differences in macrophage numbers were observed between wt and gp130

mutant mice three days after bleomycin treatment in both the BALF and tissue.

Macrophages have been reported to promote the development of bleomycin-induced

fibrosis (Khalil et al., 1989) which can be prevented if macrophage recruitment to the

208

lung is blocked (Okuma et al., 2004, Gharaee-Kermani et al., 2003, Moore et al., 2001).

The increased numbers of macrophages in the airways and inflammatory foci in

fibrosis-resistant gp130ΔSTAT/ΔSTAT mice and the absence of macrophages in the

inflammatory foci of fibrosis-sensitive gp130757F/757F mice supports the idea that

macrophage function may be altered by gp130-mediated signalling. Jenkins et al.

(2004) showed that macrophage proliferation and differentiation, induced by

macrophage colony stimulating factor (M-CSF), was partially inhibited by IL-6 in cells

from wt mice, was completely inhibited in gp130757F/757F cells but had no effect on

gp130ΔSTAT/ΔSTAT cells. These responses were associated with gp130-ERK1/2 dependent

regulation of the gene encoding the receptor for M-CSF. Recent reports have

demonstrated that STAT3 activation regulated the differentiation of macrophages from

a M1 to an M2 phenotype (Mantovani, 2006, Sica and Bronte, 2007) although these

effects appeared to be mediated by IL-10-induced activation of STAT3. The role of IL-

6-mediated activation of STAT3 in macrophage differentiation needs not be examined

but is beyond the scope of this thesis. Nevertheless, my data suggests that bleomycin-

induced lung fibrosis is not causally associated with increased macrophage proliferation

and differentiation.

Several studies have demonstrated that if inflammation is impeded or ablated,

protection from bleomycin-induced fibrosis is observed (Chaudhary et al., 2006, Dik et

al., 2003, Moore et al., 2001, Okuma et al., 2004). Interestingly, inflammatory foci were

a prominent feature of fibrotic lesions in gp130757F/757F mice but were also present in the

lung parenchyma of gp130ΔSTAT/ΔSTAT mice. Lymphocytes appeared to be the most

abundant cell type in inflammatory foci and fibrotic lesions of gp130ΔSTAT/ΔSTAT and

gp130757F/757F mice 30 days after bleomycin treatment. There were no obvious

differences in the number of T-lymphocytes and B-lymphocytes between the two mouse

genotypes although further studies need to be performed to confirm this. These results

209

suggest that there may be functional differences in the inflammatory cells of

gp130ΔSTAT/ΔSTAT and gp130757F/757F mice.

To address the relationship between inflammatory cell function and the

development of fibrosis in these mice, I created bone marrow chimeric mice using wt,

gp130ΔSTAT/ΔSTAT and gp130757F/757F host mice transplanted with bone marrow from one

of these three genotypes. I hypothesized that if inflammatory cells drove the progression

of lung fibrosis then adoptive transfer of bone marrow from gp130757F/757F mice into

gp130ΔSTAT/ΔSTAT (Δ/ΔF/F) or wt (wtF/F) mice would promote or enhance the

development of fibrosis in host mice respectively. The corollary being that transfer of

gp130ΔSTAT/ΔSTAT or wt bone marrow into gp130757F/757F mice (F/FΔ/Δ and F/Fwt

respectively) or gp130ΔSTAT/ΔSTAT bone marrow into wt mice (wtΔ/Δ ) would confer

resistance to fibrosis. The transfer of gp130757F/757F bone marrow into wt mice (wtF/F)

did not enhance fibrosis, nor was there any amelioration of fibrosis in wtΔ/Δ mice

compared to controls, suggesting that lung resident cells rather than bone marrow-

derived cells were driving the development of fibrosis. However, there was a trend for

fibrosis to progressively decrease in F/Fwt and F/FΔ/Δ chimeric mice compared with

F/FF/F mice following bleomycin treatment to near wt levels. These experiments showed

that although resident cells in the lung drove the development of fibrosis, bone marrow-

derived cells potentiated but were not essential to the development of lung fibrosis.

Taken together these results suggest that it is necessary for both resident lung cells and

bone marrow-derived cells to carry the gp130757F/757F mutation, i.e. enhanced gp130-

STAT3 signalling, for the enhancement of fibrosis. This is consistent with Sano et al.

(2005) who showed the development of spontaneous fibrotic lesions in the epidermis in

mice was dependent upon enhanced STAT3 activity in keratinocytes and lymphocytes

(Sano et al., 2005).

210

An alternative explanation is based on recent studies suggesting that

fibroblast/myofibroblast precursor cells derived from the bone marrow are responsible

for de novo collagen synthesis in experimental models of lung fibrosis (Phillips et al.,

2004, Hashimoto et al., 2004, Epperly et al., 2003). These precursors, or fibrocytes, are

identified as CD34+/col-1+ and have been implicated in the progression of fibrosis in

humans and animal models. For example, the number of fibrocytes are elevated in the

circulation of IPF patients (Mehrad et al., 2007) and expression of fibrocyte chemokines

(eg CXCL12) have been described in fibrotic foci of IPF patients (Gomperts and

Strieter, 2007, Strieter et al., 2007). Furthermore, transfer of human fibrocytes to

immuno-deficient mice produced IPF like lesions in these animals (Pierce et al., 2007b,

Phillips et al., 2004). Therefore my observation in chimeric mice showing that enhanced

gp130-STAT3 signalling within bone-marrow-derived cells promotes bleomycin-

induced lung fibrosis may not necessarily be inflammatory cell mediated but due to

increased recruitment and activity of fibrocytes in the lung. In order to elucidate the role

these cells play in fibrogenesis in this model it would be necessary to determine if there

was increased recruitment of these cells into the lung of bleomycin treated gp130757F/757F

mice and if IL-6 family cytokines could regulate their differentiation into fibroblasts or

myofibroblasts (see future studies).

118B6.1.4 Gp130-Mediated Signalling Regulates Fibroblast Growth

It is widely accepted that the dysregulated proliferation and differentiation of

resident fibroblasts and the recruitment of fibrocytes are crucial to the development and

progression of lung fibrosis (Scotton and Chambers, 2007, Hinz et al., 2007, Phan,

2003). In this study, fibroblastic cells in fibrotic foci of bleomycin treated lung tissue

proliferated as demonstrated by positive staining for the proliferation marker Ki67. This

finding was in agreement with previous studies that have reported enhanced

proliferative capacity of lung fibroblasts isolated from patients with IPF (Hagood et al.,

211

2005, Moodley et al., 2003a, Jordana et al., 1988) and lead me to investigate the effects

of IL-6 and IL-11 on fibroblast proliferation in vitro.

Moodley et al. (2003a, 2003b) showed that the effects of IL-6 and IL-11 on the

proliferation-apoptosis axis in fibroblasts isolated from IPF patients were altered

compared to cells from healthy lungs. In IPF fibroblasts IL-6-induced activation of

ERK1/2 signalling was prolonged and STAT3 signalling was truncated. My findings

were consistent with previous studies that demonstrated that IL-6 stimulated the

proliferation of normal primary lung fibroblasts (Jordana et al., 1988, Olman et al.,

2004) and suggested this was due to prolonged gp130-STAT3 signalling. This appeared

to conflict with results of Moodley et al. (2003), however both studies found that

enhanced IL-6-mediated fibroblast proliferation was associated with a different fibrotic

phenotype. The different findings regarding fibroblast proliferation identified by

Moodley et al. (2003) and myself suggest that these studies were measuring

proliferation in different fibroblast phenotypes.

Desmouliere and colleagues (2003) outlined three distinct fibroblast phenotypes

that have specific roles under homeostatic conditions and during normal wound healing;

the fibroblast, which is a quiescent cell that maintains the ECM and serves as a

progenitor for other fibroblast phenotypes, the proto-myofibroblast, that readily

proliferates and expresses intracellular stress fibres in response to mechanical stress and

injury, and the myofibroblast, which is an activated phenotype that expresses α-SMA,

synthesises ECM, has a prominent cytoskeleton and possesses contractile properties but

proliferates very slowly. Desmouliere et al. (2003) suggested that only fibroblasts and

proto-myofibrobasts are present under normal conditions. Following injury, these cells

differentiate into myofibroblasts and play important roles in wound closure and scar

formation. In certain pathologies, including IPF, these cells persist forming excessive

scar tissue and fibrosis, which results in impaired organ function. It has been understood

212

for many years that the fibroblast population in IPF patients is heterogeneous and the

differentiation of lung fibroblast phenotypes is dysregulated (Cool et al., 2006, Hinz et

al., 2007, Jordana et al., 1988, Monaghan et al., 2004). This characteristic of IPF

fibroblasts provides an explanation for the different responses to IL-6 family cytokines

described in this thesis and the study by Moodley and colleagues (2003). Thus, the cells

used by Moodley et al. (2003) have characteristics of proto-myofibroblasts and

demonstrate IL-6-induced ERK1/2 mediated proliferation. Whereas in my study the

cells resembled either basal fibroblast-like cells or myofibroblast-like cells and gp130-

mediated STAT1/3 signalling mediated relative increases in the proliferation of these

cells. The different responses of these different fibroblast phenotypes to a common

ligand suggest that dysregulated responses to IL-6 or other IL-6 family cytokines may

be involved in the progression of lung fibrosis.

119B6.1.5 Gp130-Mediated Signalling Regulates Fibroblast Phenotype

I assessed the effects of gp130-mediated signalling on fibroblast phenotype by

establishing cell lines from mice with normal gp130-mediated signalling (wt) and mice

with directed gp130-mediated signalling (gp130ΔSTAT/ΔSTAT, gp130757F/757F and

gp130757F/757F;STAT3+/-). Fibroblasts with enhanced gp130-STAT3 signalling

(gp130757F/757F) were large, stellate shaped, slow-growing cells. Fibroblasts with ablated

or reduced gp130-STAT3 signalling (gp130ΔSTAT/ΔSTAT and gp130757F/757F;STAT3+/-

respectively) were small, spindle shaped and fast-growing cells that were comparable to

wt cells. These primary fibroblasts were early passage and hence were more likely to

display phenotypes consistent with those found in vivo. Applying Desmouliere’s model

of fibroblast differentiation (Desmouliere et al., 2003) gp130757F/757F fibroblasts

(enhanced gp130-STAT3 signalling) represent a myofibroblast-like phenotype and

gp130ΔSTAT/ΔSTAT and gp130757F/757F;STAT3+/- fibroblasts (ablated or reduced gp130-

STAT3 signalling) a proto-myofibroblast-like phenotype. However, gp130757F/757F

Comment [SM1]: Check with Rob

Comment [SM2]:

213

fibroblasts did not show significant increases in α-SMA expression (Lau, unpublished

data) suggesting that these cells were not terminally differentiated myofibroblasts.

Recent publications have demonstrated that enhanced STAT3 activity increases α-

SMA expression in interstitial fibroblasts in the kidney and liver (Ogata et al., 2006,

Kuratsune et al., 2007). It is possible that the myofibroblast-like phenotype of lung

fibroblasts in gp130757F/757F mice predisposed them to the development of fibrosis. IL-6

and IL-11-induced expression of α-SMA and it’s organisation into reticular fibres

through gp130 activation of STAT3 signalling. Previous studies have shown IL-6

stimulates α-SMA expression in dermal fibroblasts and pancreatic stellate cells but this

response was associated with ERK1/2 activation and not STAT3 activation (Gallucci et

al., 2006, Mews et al., 2002). The reasons behind this discrepancy are not clear.

However phosphorylated STAT3 and α-SMA were co-localised in fibroblastic foci after

bleomycin treatment indicating that in my system gp130-STAT3 signalling may be

important for myofibroblast differentiation in vivo.

Cross-talk between gp130-mediated ERK1/2 and the STAT1/3 signalling is

important for regulating cellular response to IL-6 family cytokines (Symes et al., 1997,

Sengupta et al., 1998, Kim et al., 1998). The mutations introduced into gp130 disrupt

the necessary interaction of these proteins in co-ordinating a response to IL-6/11

activation of gp130. Furthermore, the Y757F mutation in gp130757F/757F mice alters the

binding motif for the negative feedback regulator SOCS3 (Schmitz et al., 2000,

Heinrich et al., 2003). The presence of specific mutations in gp130 has not been

identified in patients with IPF. However, it is conceivable that a mutation causing

dysregulated gp130 activation of the STAT3 pathway could promote the progression of

lung fibrosis.

Considering fibroblasts and myofibroblasts are thought to be cells that are actively

involved in the progression of IPF (Scotton and Chambers, 2007, Hinz et al., 2007,

214

Thannickal et al., 2004) these result suggest gp130-STAT3 signalling should be

investigated as a therapeutic target for the treatment of IPF. Nevertheless, this finding is

the first to describe fundamental differences in cell phenotype and growth stimulated by

manipulating cytoplasmic signalling pathways activated by a cell bound receptor.

120B6.1.6 Directed Gp130-STAT3 Inhibits TGF-β-Induced Smad3 Signalling

A number of cytokines and growth factors have been shown to promote the

development of pulmonary fibrosis (Scotton and Chambers, 2007, Strieter et al., 2007),

although these stimuli all appear to culminate in increased levels of active TGF-β and

enhanced activation of the Smad3 pathway (Gauldie et al., 2007, Selman et al., 2001,

Verrecchia and Mauviel, 2007, Hinz et al., 2007). We demonstrated that basal levels of

active TGF-β were elevated in the serum of gp130757F/757F mice compared to wt or

gp130ΔSTAT/ΔSTAT mice, providing a potential mechanism for why these mice developed

more severe fibrosis following bleomycin treatment than the other mice. This finding is

consistent with the report that increased levels of IL-6 and STAT3 promote increases in

TGF-β1 (Ogata et al., 2006, Becker et al., 2004). My observation that there was a trend

for reduced basal TGF-β levels in the sera of gp130757F/757F;STAT3+/- mice further

supports a role for STAT3 in TGF-β expression.

TGF-β1-induced effects are regulated by the immediate activation of several

pathways including the Smad2/3 pathway, whose transcriptional effects are regulated by

Smad3 binding of target gene promoters. However, in this study, TGF-β1-induced

phosphorylation of Smad3 was inhibited in lung fibroblasts from gp130757F/757F mice

compared to fibroblasts from wt, gp130ΔSTAT/ΔSTAT and gp130757F/757F;STAT3+/- mice.

Further evidence that gp130 activation of STAT3 inhibits TGF-β-induced Smad2/3

signalling was demonstrated by showing that deletion of one STAT3 allele in

gp130757F/757F mice restored phosphorylation of Smad2 and Smad3 to wt levels (Jenkins

et al., 2005a). Inhibition of Smad2/3 signalling was associated with gp130-STAT3

215

dependent induction of the negative feedback regulator Smad7 (Jenkins et al., 2005a).

These findings support previous studies that have elegantly described the pro-fibrotic

effects of TGF-β, but suggest that the effects of gp130/STAT3 are independent of

Smad3 signalling.

The reduced expression of α-SMA stimulated by TGF-β1 in primary lung

fibroblasts from gp130757F/757F mice correlated with reduced Smad3 phosphorylation in

lung fibroblasts from these mice. This finding correlated Smad3 activation with altered

fibroblast differentiation and is consistent with previous studies that have shown

myofibroblast differentiation of fibroblasts is regulated by the Smad2/3 pathway (Hinz

et al., 2007, Desmouliere et al., 2003, Tomasek et al., 2002). Previous studies have

shown that increased STAT3 activation increased active TGF-β levels and α-SMA

expression in fibrotic tissue (Ogata et al., 2006, Kuratsune et al., 2007). However, these

studies did not show how the activation of STAT3 signalling increased TGF-β levels or

α-SMA expression in cells. My findings have demonstrated mechanistic interaction for

the inhibition of TGF-β1-induced α-SMA expression and gp130-STAT3 activation.

In this study I showed that TGF-β1 promoted the proliferation of lung fibroblasts,

as has been reported previously (Khalil et al., 2005), but this response was partly

moderated by IL-6 and IL-11. The combination of IL-6 and TGF-β1 enhanced TGF-β1-

induced proliferation and the combination of IL-11 and TGF-β1 inhibited TGF-β1-

induced proliferation. These results suggest that cross-talk between gp130-mediated

ERK1/2 and STAT1/3 signalling was necessary to co-ordinate TGF-β-induced

proliferation. These findings did not correlate with the altered Smad3 activation

reported in these fibroblasts, suggesting that TGF-β1 stimulated proliferation was

regulated through another pathway.

A number of studies have demonstrated that binding of TGF-β to its cognate

receptors activates a variety of cytoplasmic signalling pathways in addition to the

216

Smad2/3 pathway, including the p38 and ERK1/2 mitogen activated protein kinase

(MAPK) pathways, Gli1/2, focal adhesion kinase and Akt pathways (Dennler et al.,

2007, Horowitz et al., 2007, Khalil et al., 2005, Chen and Khalil, 2006). These reports

indicate that the MAPK pathways, including p38 and ERK1/2 pathways, are crucial for

TGF-β1 stimulated cell proliferation (Chen and Khalil, 2006, Horowitz et al., 2007,

Khalil et al., 2005). Therefore, it is possible that enhanced TGF-β1-induced

proliferation stimulated by the combination of IL-6 and TGF-β in lung fibroblasts is due

to the combined activation of MAPK pathways by both of these proteins.

In conclusion the work presented in this thesis has demonstrated that gp130-

mediated signalling can regulate the development and progression of lung fibrosis in

vivo and links the bone marrow and resident fibroblast population to this disease. Gp130

is able to regulate disparate bleomycin-induced lung phenotypes through differential

activation of the ERK1/2 and STAT1/3 pathways. Activation of the gp130-STAT3

pathway by gp130 ligands regulates the accumulation of ECM in vivo, fibroblast

proliferation and differentiation, and the inflammatory response following bleomycin

treatment of mice. Gp130-mediated signalling pathways also negatively regulates the

effects of TGF-β/Smad3 activation and fibroblast behaviour. This thesis suggests that

differential activation of cytoplasmic signalling pathways by gp130 is involved in the

development and progression of lung fibrosis independent of classical TGF-β signalling.

70B6.2 Future Studies

In this thesis I have clearly demonstrated a role for gp130-STAT3 signalling in

bleomycin-induced pulmonary fibrosis. However, many questions still need to be

answered regarding the role of IL-6 family cytokines and the activation of gp130-

mediated ERK1/2 and STAT1/3 signalling in pulmonary fibrosis.

217

The bleomycin-induced model of lung fibrosis is an efficient model of pulmonary

fibrosis as it induces lesions within a relatively short period of time (14-30 days) and

replicates many of the features seen in the human disease. However, bleomycin-induced

fibrosis is an acute lung injury and does not accurately reproduce the myofibroblastic

foci or honeycomb changes observed in IPF. Therefore, it will be necessary to

investigate other models of interstitial pulmonary fibrosis such as radiation, asbestos

and silica-induced models as these stimuli also cause interstitial fibrosis in humans.

These studies would assess the role of gp130-mediated signalling in other fibrotic

respiratory diseases that are clinically relevant.

While bleomycin treatment stimulates increased levels of IL-6 family cytokines in

the lung, it also stimulates an increase in a milieu of other cytokines and growth factors.

This confounds attempts to identify the specific role of IL-6 family cytokines and their

signalling pathways in lung fibrosis. It is crucial to determine which IL-6 family

cytokines can drive the development of pulmonary fibrosis. To determine which

cytokines are in involved in pulmonary fibrogenesis it will be necessary to investigate if

genetic depletion of IL-6 family cytokines, such as IL-6, IL-11 and OSM, from wt and

gp130757F/757F mice (e.g. IL-6-/- and gp130757F/757F;IL-6-/- respectively) can inhibit the

development of bleomycin-induced lung fibrosis.

Previous studies have shown that transient gene delivery of IL-6 promotes

morphological changes in the lung including fibrosis of the conducting airways (Xing et

al., 1998, Xing et al., 1994). However, these studies did not investigate the intracellular

signalling pathways activated by IL-6 gene transfer or the contribution of cytoplasmic

signalling pathways to the lung phenotype. In addition, using adenoviral vectors to

transiently over-express IL-6 family cytokines alone to wt, gp130ΔSTAT/ΔSTAT and

gp130757F/757F mice would determine if gp130-STAT3 activation by these cytokines

alone could induce lung fibrosis.

218

Lim et al. (2006) showed that pharmacological inhibition of STAT3 activity

inhibited collagen production, proliferation and migration of keloid fibroblasts in a

mouse model of epidermal fibrosis. To investigate the possibility of using

pharmacological inhibition of STAT3 signalling in the treatment of lung fibrosis,

cucurbitacin would be administered to wt and gp130757F/757F mice after bleomycin

treatment. Previous work by Chaudhary et al. (2006) has found that bleomycin

treatment produces an inflammatory reaction that persists for ten days which then

recedes and is replaced by a fibrotic reaction (Chaudhary et al., 2006). This thesis

implicated gp130-STAT3 signalling in the enhanced inflammation that preceded

fibrosis in addition to the progression of fibrosis. Therefore cucurbitacin would be

administered under three regimes; the first intended to inhibit the pro-inflammatory

effects of STAT3, the second intended to inhibit the pro-fibrotic effects of STAT3 and

the third intended to inhibit both the inflammatory and fibrotic effects of STAT3.

The effects of gp130-STAT1 signalling could not be assessed in this thesis. A

recent paper has suggested that STAT1 signalling limits the development of bleomycin-

induced fibrosis (Walters et al., 2005). The gp130757F/757F mice have been cross-bred

with animals carrying a null mutation in the gene encoding STAT1

(gp130757F/757F;STAT1-/-). To assess the role of gp130-STAT1 signalling in this model

of lung fibrosis bleomycin would be administered to wt, gp130757F/757F and

gp130757F/757F;STAT1-/- mice and inflammation and the development of fibrosis would

be assessed three and 30 days following bleomycin treatment respectively.

Increased activity of TGF-β is believed to be central to the pathogenesis of IPF

and other fibrotic diseases (Verrecchia and Mauviel, 2007, Gauldie et al., 2007, Roberts

et al., 2006). Increased TGF-β activity is also believed to be crucial for the development

of bleomycin-induced lung fibrosis (Zhao et al., 2002, Khalil et al., 2005). The results

outlined in chapter five suggest that the enhanced fibrosis observed in gp130757F/757F

219

mice is not dependent upon TGF-β induced Smad3 signalling. To critically assess this,

gp130757F/757F mice have been cross-bred with mice carrying null mutations in the

Smad3 gene (gp130757F/757F;Smad3-/-). Bleomycin would be administered to wt, Smad3-/-

, gp130757F/757F and gp130757F/757F;Smad3-/- mice and fibrosis assessed by morphological

and biochemical methods 30 days later. TGF-β may also promote fibrosis through an

alternative pathway to Smad3. Therefore, pharmacological inhibitors of the TGF-β type

1 receptor (ALK-5) could be administered to bleomycin treated wt and gp130757F/757F

mice to determine what, if any, role TGF-β plays in fibrogenesis in this model.

Pharmacological inhibitors could also be used to assess the interaction of STAT3 and

TGF-β on bleomycin-induced lung fibrosis by incorporating gp130757F/757F;STAT3+/-

mice into the above experiment. The converse of these studies would be to determine if

gp130ΔSTAT/ΔSTAT mice were resistant to the fibrotic effects of TGF-β as they are

resistant to the fibrotic effects of bleomycin. Using transient gene transfer methods

TGF-β1 would be delivered to the lungs of wt, gp130ΔSTAT/ΔSTAT and gp130757F/757F mice

and morphological and biochemical changes would be assessed up to 60 days later.

Recent publications have shown that fibrocytes are elevated in the circulation of

patients with IPF (Mehrad et al., 2007) and fibrocyte chemokines are expressed in

fibrotic foci in IPF (Xu et al., 2007, Choi et al., 2006). It has already been shown that

neutralising antibodies and pharmacological agents that inhibit fibrocyte chemokine

function can ameliorate the development and progression of experimental fibrosis in

vivo (Pierce et al., 2007b, Phillips et al., 2004, Watanabe et al., 2007). Application of

these tools to wt and gp130 mutant mice in the bleomycin-induced lung fibrosis model

would provide exciting insights into the role gp130-mediated signalling plays in the

recruitment and activation of fibrocytes in vivo. Furthermore, fibrocytes could be

isolated from the bone marrow of wt, gp130ΔSTAT/ΔSTAT and gp130757F/757F mice and

adoptively transferred to littermates of a different genotype to assess if increased

Comment [SM3]:

220

circulating fibrocytes with directed gp130-mediated signalling could induce lung

fibrosis.

These experiments described above will:

1) Evaluate whether gp130-ERK1/2 and gp130-STAT1/3 pathways regulate the

development of fibrosis in other models of pulmonary injury.

2) Identify which IL-6 cytokine family members promote the development and

progression of lung fibrosis and evaluate whether activation of gp130-ERK1/2 or

gp130-STAT1/3 signalling by specific IL-6 family cytokines (e.g. IL-6, IL-11 and

OSM) in the lung will promote morphological changes in vivo including fibrosis,

lymphocytic accumulations and, or airspace enlargement.

3) Determine if pharmacological inhibition of STAT3 signalling has clinical

applications for the treatment of lung fibrosis.

4) Evaluate the effect of gp130-STAT1 signalling on the bleomycin-induced

inflammation and lung fibrosis.

5) Assess the dependence of bleomycin-induced fibrosis in wt and

gp130757F/757F mice upon Smad3 signalling and TGF-β-induced intracellular

responses.

6) Determine if gp130-ERK1/2 signalling inhibits the development of TGF-β1-

induced pulmonary fibrosis in vivo.

7) Determine if fibrocyte recruitment and activation is regulated by gp130-

mediated signalling in bleomycin-induced lung fibrosis and the role gp130-mediated

signalling in the activation of fibrocytes in vivo.

221

21BREFERENCES

ADAMSON, I. & BOWDEN, D. (1974) The pathogenesis of bleomycin-induced pulmonary fibrosis in mice. Am J Pathol., 77, 185-197.

ADAMSON, I. Y., YOUNG, L. & BOWDEN, D. H. (1988) Relationship of alveolar epithelial injury and repair to the induction of pulmonary fibrosis. Am J Pathol, 130, 377-83.

ALEXANDER, W. S., STARR, R., FENNER, J. E., SCOTT, C. L., HANDMAN, E., SPRIGG, N. S., CORBIN, J. E., CORNISH, A. L., DARWICHE, R., OWCZAREK, C. M., KAY, T. W., NICOLA, N. A., HERTZOG, P. J., METCALF, D. & HILTON, D. J. (1999) SOCS1 is a critical inhibitor of interferon gamma signaling and prevents the potentially fatal neonatal actions of this cytokine. Cell, 98, 597-608.

ALONZI, T., MARITANO, D., GORGONI, B., RIZZUTO, G., LIBERT, C. & POLI, V. (2001) Essential role of STAT3 in the control of the acute-phase response as revealed by inducible gene inactivation [correction of activation] in the liver. Mol Cell Biol, 21, 1621-32.

ANDO, M., MIYAZAKI, E., FUKAMI, T., KUMAMOTO, T. & TSUDA, T. (1999) Interleukin-4-producing cells in idiopathic pulmonary fibrosis: an immunohistochemical study. Respirology, 4, 383-91.

ASOKANANTHAN, N., GRAHAM, P. T., FINK, J., KNIGHT, D. A., BAKKER, A. J., MCWILLIAM, A. S., THOMPSON, P. J. & STEWART, G. A. (2002) Activation of protease-activated receptor (PAR)-1, PAR-2, and PAR-4 stimulates IL-6, IL-8, and prostaglandin E2 release from human respiratory epithelial cells. J Immunol, 168, 3577-85.

ATS (2000) American Thoracic Society. Idiopathic pulmonary fibrosis: diagnosis and treatment. International consensus statement. American Thoracic Society (ATS), and the European Respiratory Society (ERS). Am. J. Respir. Crit. Care Med., 161, 646-64.

ATSUMI, T., ISHIHARA, K., KAMIMURA, D., IKUSHIMA, H., OHTANI, T., HIROTA, S., KOBAYASHI, H., PARK, S. J., SAEKI, Y., KITAMURA, Y. & HIRANO, T. (2002) A point mutation of Tyr-759 in interleukin 6 family cytokine receptor subunit gp130 causes autoimmune arthritis. J Exp Med, 196, 979-90.

AWAD, M. R., EL-GAMEL, A., HASLETON, P., TURNER, D. M., SINNOTT, P. J. & HUTCHINSON, I. V. (1998) Genotypic variation in the transforming growth factor-beta1 gene: association with transforming growth factor-beta1 production, fibrotic lung disease, and graft fibrosis after lung transplantation. Transplantation, 66, 1014-20.

BAMBER, B., REIFE, R., HAUGEN, H. & CLEGG, C. (1998) Oncostatin M stimulates excessive extracellular matrix accumulation in a transgenic mouse model of connective tissue disease. J Mol Med, 76, 61-69.

BARBARIN, V., XING, Z., DELOS, M., LISON, D. & HUAUX, F. (2005) Pulmonary overexpression of IL-10 augments lung fibrosis and Th2 responses induced by silica particles. Am J Physiol Lung Cell Mol Physiol, 288, L841-8.

BAUMANN, H., SYMES, A., COMEAU, M., MORELLA, K., WANG, Y., FRIEND, D., ZIEGLER, S., FINK, J. & GEARING, D. (1994) Multiple regions within the cytoplasmic domains of the leukemia inhibitory factor receptor and gp130 cooperate in signal transduction in hepatic and neuronal cells. Mol Cell Biol, 14, 138-146.

222

BAUMGARTNER, K. B., SAMET, J. M., COULTAS, D. B., STIDLEY, C. A., HUNT, W. C., COLBY, T. V. & WALDRON, J. A. (2000) Occupational and environmental risk factors for idiopathic pulmonary fibrosis: a multicenter case-control study. Collaborating Centers. Am J Epidemiol, 152, 307-15.

BAUMGARTNER, K. B., SAMET, J. M., STIDLEY, C. A., COLBY, T. V. & WALDRON, J. A. (1997) Cigarette smoking: a risk factor for idiopathic pulmonary fibrosis. Am J Respir Crit Care Med, 155, 242-8.

BECKER, C., FANTINI, M. C., SCHRAMM, C., LEHR, H. A., WIRTZ, S., NIKOLAEV, A., BURG, J., STRAND, S., KIESSLICH, R., HUBER, S., ITO, H., NISHIMOTO, N., YOSHIZAKI, K., KISHIMOTO, T., GALLE, P. R., BLESSING, M., ROSE-JOHN, S. & NEURATH, M. F. (2004) TGF-beta suppresses tumor progression in colon cancer by inhibition of IL-6 trans-signaling. Immunity, 21, 491-501.

BENNETT, A. M., HAUSDORFF, S. F., O'REILLY, A. M., FREEMAN, R. M. & NEEL, B. G. (1996) Multiple requirements for SHPTP2 in epidermal growth factor-mediated cell cycle progression. Mol Cell Biol, 16, 1189-202.

BERG, R. A. & PROCKOP, D. J. (1973) The thermal transition of a non-hydroxylated form of collagen. Evidence for a role for hydroxyproline in stabilizing the triple-helix of collagen. Biochem Biophys Res Commun, 52, 115-20.

BERG, R. A., SCHWARTZ, M. L. & CRYSTAL, R. G. (1980) Regulation of the production of secretory proteins: intracellular degradation of newly synthesized "defective" collagen. Proc Natl Acad Sci U S A, 77, 4746-50.

BETZ, U., BLOCH, W., VAN DEN BROEK, M., YOSHIDA, K., TAGA, T., KISHIMOTO, T., ADDICKS, K., RAJEWSKY, K. & MULLER, W. (1998) Postnatally induced inactivation of gp130 in mice results in neurological, cardiac, hematopoietic, immunological, hepatic, and pulmonary defects. Journal of Experimental Medicine, 188, 1955-1965.

BHOWMICK, N. A., CHYTIL, A., PLIETH, D., GORSKA, A. E., DUMONT, N., SHAPPELL, S., WASHINGTON, M. K., NEILSON, E. G. & MOSES, H. L. (2004) TGF-beta signaling in fibroblasts modulates the oncogenic potential of adjacent epithelia. Science, 303, 848-51.

BIENKOWSKI, R. S. (1984) Collagen degradation in human lung fibroblasts: extent of degradation, role of lysosomal proteases, and evaluation of an alternate hypothesis. J Cell Physiol, 121, 152-8.

BIENKOWSKI, R. S. (1989) Intracellular degradation of newly synthesized collagen. Revis Biol Celular, 21, 423-43.

BILLINGS, C. G. & HOWARD, P. (1994) Hypothesis: exposure to solvents may cause fibrosing alveolitis. Eur Respir J, 7, 1172-6.

BJORBAEK, C., ELMQUIST, J. K., FRANTZ, J. D., SHOELSON, S. E. & FLIER, J. S. (1998) Identification of SOCS-3 as a potential mediator of central leptin resistance. Mol Cell, 1, 619-25.

BONNIAUD, P., KOLB, M., GALT, T., ROBERTSON, J., ROBBINS, C., STAMPFLI, M., LAVERY, C., MARGETTS, P. J., ROBERTS, A. B. & GAULDIE, J. (2004) Smad3 null mice develop airspace enlargement and are resistant to TGF-beta-mediated pulmonary fibrosis. J Immunol, 173, 2099-108.

BONNIAUD, P., MARGETTS, P. J., ASK, K., FLANDERS, K., GAULDIE, J. & KOLB, M. (2005) TGF-beta and Smad3 signaling link inflammation to chronic fibrogenesis. J Immunol, 175, 5390-5.

BORDER, W. A. & NOBLE, N. A. (1994) Transforming Growth Factor {beta} in Tissue Fibrosis. N Engl J Med, 331, 1286-1292.

223

BRANDT, S. J., BODINE, D. M., DUNBAR, C. E. & NIENHUIS, A. W. (1990) Dysregulated interleukin 6 expression produces a syndrome resembling Castleman's disease in mice. J Clin Invest, 86, 592-9.

BROCKER, V., LANGER, F., FELLOUS, T. G., MENGEL, M., BRITTAN, M., BREDT, M., MILDE, S., WELTE, T., EDER, M., HAVERICH, A., ALISON, M. R., KREIPE, H. & LEHMANN, U. (2006) Fibroblasts of Recipient Origin Contribute to Bronchiolitis Obliterans in Human Lung Transplants. Am. J. Respir. Crit. Care Med., 173, 1276-1282.

BROEKELMANN, T. J., LIMPER, A. H., COLBY, T. V. & MCDONALD, J. A. (1991) Transforming growth factor beta 1 is present at sites of extracellular matrix gene expression in human pulmonary fibrosis. Proc Natl Acad Sci U S A, 88, 6642-6.

BROEKEMA, M., HARMSEN, M. C., VAN LUYN, M. J. A., KOERTS, J. A., PETERSEN, A. H., VAN KOOTEN, T. G., VAN GOOR, H., NAVIS, G. & POPA, E. R. (2006) Bone Marrow-Derived Myofibroblasts Contribute to the Renal Interstitial Myofibroblast Population and Produce Procollagen I after Ischemia/Reperfusion in Rats. J Am Soc Nephrol, ASN.2005070730.

BUCALA, R., SPIEGEL, L. A., CHESNEY, J., HOGAN, M. & CERAMI, A. (1994) Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair. Mol Med, 1, 71-81.

BULLEN, D. V., DARWICHE, R., METCALF, D., HANDMAN, E. & ALEXANDER, W. S. (2001) Neutralization of interferon-gamma in neonatal SOCS1-/- mice prevents fatty degeneration of the liver but not subsequent fatal inflammatory disease. Immunology, 104, 92-8.

CALDENHOVEN, E., VAN DIJK, T. B., SOLARI, R., ARMSTRONG, J., RAAIJMAKERS, J. A., LAMMERS, J. W., KOENDERMAN, L. & DE GROOT, R. P. (1996) STAT3beta, a splice variant of transcription factor STAT3, is a dominant negative regulator of transcription. J Biol Chem, 271, 13221-7.

CARMICHAEL, J., DEGRAFF, W. G., GAZDAR, A. F., MINNA, J. D. & MITCHELL, J. B. (1987) Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of radiosensitivity. Cancer Res, 47, 943-6.

CAVARRA, E., CARRARO, F., FINESCHI, S., NALDINI, A., BARTALESI, B., PUCCI, A. & LUNGARELLA, G. (2004) Early response to bleomycin is characterized by different cytokine and cytokine receptor profiles in lungs. Am J Physiol Lung Cell Mol Physiol, 287, L1186-92.

CAWSTON, T. E. (1996) Metalloproteinase inhibitors and the prevention of connective tissue breakdown. Pharmacol Ther, 70, 163-82.

CHAUDHARY, N. I., SCHNAPP, A. & PARK, J. E. (2006) Pharmacologic Differentiation of Inflammation and Fibrosis in the Rat Bleomycin Model. Am. J. Respir. Crit. Care Med., 173, 769-776.

CHEN, E. S., GREENLEE, B. M., WILLS-KARP, M. & MOLLER, D. R. (2001) Attenuation of lung inflammation and fibrosis in interferon-gamma-deficient mice after intratracheal bleomycin. Am J Respir Cell Mol Biol, 24, 545-55.

CHEN, G. & KHALIL, N. (2006) TGF-beta1 increases proliferation of airway smooth muscle cells by phosphorylation of map kinases. Respir Res, 7, 2.

CHEN, J. & STUBBE, J. (2005) Bleomycins: towards better therapeutics. Nat Rev Cancer, 5, 102-12.

CHEN, Q., FISHER, D. T., CLANCY, K. A., GAUGUET, J. M., WANG, W. C., UNGER, E., ROSE-JOHN, S., VON ANDRIAN, U. H., BAUMANN, H. & EVANS, S. S. (2006) Fever-range thermal stress promotes lymphocyte

224

trafficking across high endothelial venules via an interleukin 6 trans-signaling mechanism. Nat Immunol, 7, 1299-308.

CHEN, Q., RABACH, L., NOBLE, P., ZHENG, T., LEE, C. G., HOMER, R. J. & ELIAS, J. A. (2005) IL-11 receptor alpha in the pathogenesis of IL-13-induced inflammation and remodeling. J Immunol, 174, 2305-13.

CHEN, Q., WANG, W. C., BRUCE, R., LI, H., SCHLEIDER, D. M., MULBURY, M. J., BAIN, M. D., WALLACE, P. K., BAUMANN, H. & EVANS, S. S. (2004) Central role of IL-6 receptor signal-transducing chain gp130 in activation of L-selectin adhesion by fever-range thermal stress. Immunity, 20, 59-70.

CHENG, N., BHOWMICK, N. A., CHYTIL, A., GORKSA, A. E., BROWN, K. A., MURAOKA, R., ARTEAGA, C. L., NEILSON, E. G., HAYWARD, S. W. & MOSES, H. L. (2005) Loss of TGF-beta type II receptor in fibroblasts promotes mammary carcinoma growth and invasion through upregulation of TGF-alpha-, MSP- and HGF-mediated signaling networks. Oncogene, 24, 5053-68.

CHOI, E. S., PIERCE, E. M., JAKUBZICK, C., CARPENTER, K. J., KUNKEL, S. L., EVANOFF, H., MARTINEZ, F. J., FLAHERTY, K. R., MOORE, B. B., TOEWS, G. B., COLBY, T. V., KAZEROONI, E. A., GROSS, B. H., TRAVIS, W. D. & HOGABOAM, C. M. (2006) Focal interstitial CC chemokine receptor 7 (CCR7) expression in idiopathic interstitial pneumonia. J Clin Pathol, 59, 28-39.

CHUA, F., DUNSMORE, S. E., CLINGEN, P. H., MUTSAERS, S. E., SHAPIRO, S. D., SEGAL, A. W., ROES, J. & LAURENT, G. J. (2007) Mice Lacking Neutrophil Elastase Are Resistant to Bleomycin-Induced Pulmonary Fibrosis. Am J Pathol, 170, 65-74.

CHUA, F., GAULDIE, J. & LAURENT, G. J. (2005) Pulmonary fibrosis: searching for model answers. Am J Respir Cell Mol Biol, 33, 9-13.

CHUNG, C., LIAO, J., LIU, B., RAO, X., JAY, P., BERTA, P. & SHUAI, K. (1997) Specific inhibition of Stat3 signal transduction by PIAS3. Science, 278, 1803-1805.

COLGAN, J. & ROTHMAN, P. (2006) All in the family: IL-27 suppression of T(H)-17 cells. Nat Immunol, 7, 899-901.

CONNOR, T. B., JR., ROBERTS, A. B., SPORN, M. B., DANIELPOUR, D., DART, L. L., MICHELS, R. G., DE BUSTROS, S., ENGER, C., KATO, H., LANSING, M. & ET AL. (1989) Correlation of fibrosis and transforming growth factor-beta type 2 levels in the eye. J Clin Invest, 83, 1661-6.

COOL, C. D., GROSHONG, S. D., RAI, P. R., HENSON, P. M., STEWART, J. S. & BROWN, K. K. (2006) Fibroblast Foci Are Not Discrete Sites of Lung Injury or Repair: The Fibroblast Reticulum. Am. J. Respir. Crit. Care Med., 174, 654-658.

COULTAS, D. B., ZUMWALT, R. E., BLACK, W. C. & SOBONYA, R. E. (1994) The epidemiology of interstitial lung diseases. Am J Respir Crit Care Med, 150, 967-72.

CROKER, B. A., KREBS, D. L., ZHANG, J. G., WORMALD, S., WILLSON, T. A., STANLEY, E. G., ROBB, L., GREENHALGH, C. J., FORSTER, I., CLAUSEN, B. E., NICOLA, N. A., METCALF, D., HILTON, D. J., ROBERTS, A. W. & ALEXANDER, W. S. (2003) SOCS3 negatively regulates IL-6 signaling in vivo. Nat Immunol, 4, 540-5.

CUTRONEO, K. R. (2003) How is Type I procollagen synthesis regulated at the gene level during tissue fibrosis. J Cell Biochem, 90, 1-5.

DAHMEN, H., HORSTEN, U., KUSTER, A., JACQUES, Y., MINVIELLE, S., KERR, I. M., CILIBERTO, G., PAONESSA, G., HEINRICH, P. C. & MULLER-NEWEN, G. (1998) Activation of the signal transducer gp130 by interleukin-11

225

and interleukin-6 is mediated by similar molecular interactions. Biochem J, 331 (Pt 3), 695-702.

DANIELS, C. E., WILKES, M. C., EDENS, M., KOTTOM, T. J., MURPHY, S. J., LIMPER, A. H. & LEOF, E. B. (2004) Imatinib mesylate inhibits the profibrogenic activity of TGF-beta and prevents bleomycin-mediated lung fibrosis. J Clin Invest, 114, 1308-16.

DAVIES, B. H. & TUDDENHAM, E. G. (1976) Familial pulmonary fibrosis associated with oculocutaneous albinism and platelet function defect. A new syndrome. Q J Med, 45, 219-32.

DE SOUZA, D., FABRI, L. J., NASH, A., HILTON, D. J., NICOLA, N. A. & BACA, M. (2002) SH2 domains from suppressor of cytokine signaling-3 and protein tyrosine phosphatase SHP-2 have similar binding specificities. Biochemistry, 41, 9229-36.

DEMOPOULOS, K., ARVANITIS, D. A., VASSILAKIS, D. A., SIAFAKAS, N. M. & SPANDIDOS, D. A. (2002) MYCL1, FHIT, SPARC, p16(INK4) and TP53 genes associated to lung cancer in idiopathic pulmonary fibrosis. J Cell Mol Med, 6, 215-22.

DENNLER, S., ANDRE, J., ALEXAKI, I., LI, A., MAGNALDO, T., TEN DIJKE, P., WANG, X. J., VERRECCHIA, F. & MAUVIEL, A. (2007) Induction of sonic hedgehog mediators by transforming growth factor-beta: Smad3-dependent activation of Gli2 and Gli1 expression in vitro and in vivo. Cancer Res, 67, 6981-6.

DENTON, C. P., ZHENG, B., EVANS, L. A., SHI-WEN, X., ONG, V. H., FISHER, I., LAZARIDIS, K., ABRAHAM, D. J., BLACK, C. M. & DE CROMBRUGGHE, B. (2003) Fibroblast-specific Expression of a Kinase-deficient Type II Transforming Growth Factor {beta} (TGF{beta}) Receptor Leads to Paradoxical Activation of TGF{beta} Signaling Pathways with Fibrosis in Transgenic Mice. J. Biol. Chem., 278, 25109-25119.

DERDAK, S., PENNEY, D. P., KENG, P., FELCH, M. E., BROWN, D. & PHIPPS, R. P. (1992) Differential collagen and fibronectin production by Thy 1+ and Thy 1- lung fibroblast subpopulations. Am J Physiol, 263, L283-90.

DESMOULIERE, A., DARBY, I. A. & GABBIANI, G. (2003) Normal and pathologic soft tissue remodeling: role of the myofibroblast, with special emphasis on liver and kidney fibrosis. Lab Invest, 83, 1689-707.

DICOSMO, B., GEBA, G., PICARELLA, D., ELIAS, J., RANKIN, J., STRIPP, B., WHITSETT, J. & FLAVELL, R. (1994) Airway epithelial cell expression of interleukin-6 in transgenic mice. Uncoupling of airway inflammation and bronchial hyperreactivity. J Clin Invest, 94, 2028-2035.

DIK, W. A., MCANULTY, R. J., VERSNEL, M. A., NABER, B. A., ZIMMERMANN, L. J., LAURENT, G. J. & MUTSAERS, S. E. (2003) Short course dexamethasone treatment following injury inhibits bleomycin induced fibrosis in rats. Thorax, 58, 765-71.

EGAN, J. J., WOODCOCK, A. A. & STEWART, J. P. (1997) Viruses and idiopathic pulmonary fibrosis. Eur Respir J, 10, 1433-7.

ENGEL, J. & PROCKOP, D. J. (1991) The zipper-like folding of collagen triple helices and the effects of mutations that disrupt the zipper. Annu Rev Biophys Biophys Chem, 20, 137-52.

EPPERLY, M. W., GUO, H., GRETTON, J. E. & GREENBERGER, J. S. (2003) Bone marrow origin of myofibroblasts in irradiation pulmonary fibrosis. Am. J. Respir. Cell Mol. Biol., 29, 213-224.

ERNST, M., INGLESE, M., WARING, P., CAMPBELL, I., BAO, S., CLAY, F., ALEXANDER, W., WICKS, I., TARLINTON, D., NOVAK, U., HEATH, J. &

226

DUNN, A. (2001) Defective gp130-mediated signal transducer and activator of transcription (STAT) signaling results in degenerative joint disease, gastrointestinal ulceration, and failure of uterine implantation. J Exp Med, 194, 189-203.

ERNST, M. & JENKINS, B. J. (2004) Acquiring signalling specificity from the cytokine receptor gp130. Trends in Genetics, 20, 23.

EZURE, T., SAKAMOTO, T., TSUJI, H., LUNZ, J. R., MURASE, N., FUNG, J. & DEMETRIS, A. (2000) The development and compensation of biliary cirrhosis in interleukin-6-deficient mice. Am J Pathol., 156, 1627-1639.

FALFAN-VALENCIA, R., CAMARENA, A., JUAREZ, A., BECERRIL, C., MONTANO, M., CISNEROS, J., MENDOZA, F., GRANADOS, J., PARDO, A. & SELMAN, M. (2005) Major histocompatibility complex and alveolar epithelial apoptosis in idiopathic pulmonary fibrosis. Hum Genet, 118, 235-44.

FELLRATH, J. M. & DU BOIS, R. M. (2003) Idiopathic pulmonary fibrosis/cryptogenic fibrosing alveolitis. Clin Exp Med, 3, 65-83.

FLAHERTY, K. R., MUMFORD, J. A., MURRAY, S., KAZEROONI, E. A., GROSS, B. H., COLBY, T. V., TRAVIS, W. D., FLINT, A., TOEWS, G. B., LYNCH, J. P., 3RD & MARTINEZ, F. J. (2003) Prognostic implications of physiologic and radiographic changes in idiopathic interstitial pneumonia. Am J Respir Crit Care Med, 168, 543-8.

FLEISCHMAN, R. W., BAKER, J. R., THOMPSON, G. R., SCHAEPPI, U. H., ILLIEVSKI, V. R., COONEY, D. A. & DAVIS, R. D. (1971) Bleomycin-induced interstitial pneumonia in dogs. Thorax, 26, 675-82.

FRIES, K. M., FELCH, M. E. & PHIPPS, R. P. (1994) Interleukin-6 is an autocrine growth factor for murine lung fibroblast subsets. Am J Respir Cell Mol Biol, 11, 552-60.

FUJITA, M., SHANNON, J. M., MORIKAWA, O., GAULDIE, J., HARA, N. & MASON, R. J. (2003) Overexpression of tumor necrosis factor-alpha diminishes pulmonary fibrosis induced by bleomycin or transforming growth factor-beta. Am J Respir Cell Mol Biol, 29, 669-76.

GAHL, W. A., BRANTLY, M., KAISER-KUPFER, M. I., IWATA, F., HAZELWOOD, S., SHOTELERSUK, V., DUFFY, L. F., KUEHL, E. M., TROENDLE, J. & BERNARDINI, I. (1998) Genetic defects and clinical characteristics of patients with a form of oculocutaneous albinism (Hermansky-Pudlak syndrome). N Engl J Med, 338, 1258-64.

GALLUCCI, R. M., LEE, E. G. & TOMASEK, J. J. (2006) IL-6 modulates alpha-smooth muscle actin expression in dermal fibroblasts from IL-6-deficient mice. J Invest Dermatol, 126, 561-8.

GARCIA-ALVAREZ, J., RAMIREZ, R., SAMPIERI, C. L., NUTTALL, R. K., EDWARDS, D. R., SELMAN, M. & PARDO, A. (2006) Membrane type-matrix metalloproteinases in idiopathic pulmonary fibrosis. Sarcoidosis Vasc Diffuse Lung Dis, 23, 13-21.

GAULDIE, J., BONNIAUD, P., SIME, P., ASK, K. & KOLB, M. (2007) TGF-beta, Smad3 and the process of progressive fibrosis. Biochem Soc Trans, 35, 661-4.

GAULDIE, J., KOLB, M., ASK, K., MARTIN, G., BONNIAUD, P. & WARBURTON, D. (2006) Smad3 signaling involved in pulmonary fibrosis and emphysema. Proc Am Thorac Soc, 3, 696-702.

GERACI, J. P., MARIANO, M. S. & JACKSON, K. L. (1992) Radiation hepatology of the rat: microvascular fibrosis and enhancement of liver dysfunction by diet and drugs. Radiat Res, 129, 322-32.

227

GHARAEE-KERMANI, M., MCCULLUMSMITH, R. E., CHARO, I. F., KUNKEL, S. L. & PHAN, S. H. (2003) CC-chemokine receptor 2 required for bleomycin-induced pulmonary fibrosis. Cytokine, 24, 266-76.

GHAZIZADEH, M., TOSA, M., SHIMIZU, H., HYAKUSOKU, H. & KAWANAMI, O. (2007) Functional implications of the IL-6 signaling pathway in keloid pathogenesis. J Invest Dermatol, 127, 98-105.

GIRI, S. N., HYDE, D. M. & MARAFINO, B. J., JR. (1986) Ameliorating effect of murine interferon gamma on bleomycin-induced lung collagen fibrosis in mice. Biochem Med Metab Biol, 36, 194-7.

GOMPERTS, B. N. & STRIETER, R. M. (2007) Fibrocytes in lung disease. J Leukoc Biol, 82, 449-56.

GOTKIN, M. G., RIPLEY, C. R., LAMANDE, S. R., BATEMAN, J. F. & BIENKOWSKI, R. S. (2004) Intracellular trafficking and degradation of unassociated proalpha2 chains of collagen type I. Exp Cell Res, 296, 307-16.

GUERRY-FORCE, M. L., MULLER, N. L., WRIGHT, J. L., WIGGS, B., COPPIN, C., PARE, P. D. & HOGG, J. C. (1987) A comparison of bronchiolitis obliterans with organizing pneumonia, usual interstitial pneumonia, and small airways disease. Am Rev Respir Dis, 135, 705-12.

GUNAJE, J. J. & BHAT, G. J. (2001) Involvement of tyrosine phosphatase PTP1D in the inhibition of interleukin-6-induced Stat3 signaling by alpha-thrombin. Biochem Biophys Res Commun, 288, 252-7.

GUSCHIN, D., ROGERS, N., BRISCOE, J., WITTHUHN, B., WATLING, D., HORN, F., PELLEGRINI, S., YASUKAWA, K., HEINRICH, P., STARK, G. R. & ET AL. (1995) A major role for the protein tyrosine kinase JAK1 in the JAK/STAT signal transduction pathway in response to interleukin-6. Embo J, 14, 1421-9.

HAGIMOTO, N., KUWANO, K., INOSHIMA, I., YOSHIMI, M., NAKAMURA, N., FUJITA, M., MAEYAMA, T. & HARA, N. (2002) TGF-beta 1 as an enhancer of Fas-mediated apoptosis of lung epithelial cells. J Immunol, 168, 6470-8.

HAGOOD, J. S., MANGALWADI, A., GUO, B., MACEWEN, M. W., SALAZAR, L. & FULLER, G. M. (2002) Concordant and discordant interleukin-1-mediated signaling in lung fibroblast thy-1 subpopulations. Am J Respir Cell Mol Biol, 26, 702-8.

HAGOOD, J. S., PRABHAKARAN, P., KUMBLA, P., SALAZAR, L., MACEWEN, M. W., BARKER, T. H., ORTIZ, L. A., SCHOEB, T., SIEGAL, G. P., ALEXANDER, C. B., PARDO, A. & SELMAN, M. (2005) Loss of fibroblast Thy-1 expression correlates with lung fibrogenesis. Am J Pathol, 167, 365-79.

HASHIMOTO, N., JIN, H., LIU, T., CHENSUE, S. & PHAN, S. (2004) Bone marrow-derived progenitor cells in pulmonary fibrosis. J Clin Invest, 113, 243-252.

HASLAM, P. L., POULTER, L. W., ROSSI, G. A., BAUER, W., DE ROSE, V., ECKERT, H., OLIVIERI, D. & TESCHLER, H. (1990) The clinical role of BAL in idiopathic pulmonary fibrosis. Eur Respir J, 3, 940-2, 961-9.

HASTON, C. K. & TRAVIS, E. L. (1997) Murine susceptibility to radiation-induced pulmonary fibrosis is influenced by a genetic factor implicated in susceptibility to bleomycin-induced pulmonary fibrosis. Cancer Res, 57, 5286-91.

HAWLEY, R. G., FONG, A. Z., BURNS, B. F. & HAWLEY, T. S. (1992) Transplantable myeloproliferative disease induced in mice by an interleukin 6 retrovirus. J. Exp. Med., 176, 1149-1163.

HEINRICH, P., BEHRMANN, I., MULLER-NEWEN, G., SCHAPER, F. & GRAEVE, L. (1998) Interleukin-6-type cytokine signalling through the gp130/Jak/STAT pathway. Biochem J, 334, 297-314.

228

HEINRICH, P. C., BEHRMANN, I., HAAN, S., HERMANNS, H. M., MULLER-NEWEN, G. & SCHAPER, F. (2003) Principles of interleukin (IL)-6-type cytokine signalling and its regulation. Biochem J, 374, 1-20.

HERNANDEZ-BARRANTES, S., BERNARDO, M., TOTH, M. & FRIDMAN, R. (2002) Regulation of membrane type-matrix metalloproteinases. Semin Cancer Biol, 12, 131-8.

HETZEL, M., BACHEM, M., ANDERS, D., TRISCHLER, G. & FAEHLING, M. (2005) Different effects of growth factors on proliferation and matrix production of normal and fibrotic human lung fibroblasts. Lung, 183, 225-37.

HINZ, B., PHAN, S. H., THANNICKAL, V. J., GALLI, A., BOCHATON-PIALLAT, M. L. & GABBIANI, G. (2007) The Myofibroblast. One Function, Multiple Origins. Am J Pathol.

HIROTA, H., YOSHIDA, K., KISHIMOTO, T. & TAGA, T. (1995) Continuous activation of gp130, a signal-transducing receptor component for interleukin 6-related cytokines, causes myocardial hypertrophy in mice. Proc Natl Acad Sci U S A, 92, 4862-4866.

HOKUTO, I., IKEGAMI, M., YOSHIDA, M., TAKEDA, K., AKIRA, S., PERL, A. K., HULL, W. M., WERT, S. E. & WHITSETT, J. A. (2004) Stat-3 is required for pulmonary homeostasis during hyperoxia. J Clin Invest, 113, 28-37.

HOROWITZ, A. L., FRIEDMAN, M., SMITH, J., YAGODA, A., LAMONTE, C. & WATSON, R. C. (1973) The pulmonary changes of bleomycin toxicity. Radiology, 106, 65-8.

HOROWITZ, J. C., ROGERS, D. S., SHARMA, V., VITTAL, R., WHITE, E. S., CUI, Z. & THANNICKAL, V. J. (2007) Combinatorial activation of FAK and AKT by transforming growth factor-beta1 confers an anoikis-resistant phenotype to myofibroblasts. Cell Signal, 19, 761-71.

HOWLETT, M., JUDD, L. M., JENKINS, B., LA GRUTA, N. L., GRAIL, D., ERNST, M. & GIRAUD, A. S. (2005) Differential regulation of gastric tumor growth by cytokines that signal exclusively through the coreceptor gp130. Gastroenterology, 129, 1005-18.

HUAUX, F., LOUAHED, J., HUDSPITH, B., MEREDITH, C., DELOS, M., RENAULD, J. C. & LISON, D. (1998) Role of interleukin-10 in the lung response to silica in mice. Am J Respir Cell Mol Biol, 18, 51-9.

HUBBARD, R. & VENN, A. (2000) Adult height and cryptogenic fibrosing alveolitis: a case-control study using the UK general practice research database. Thorax, 55, 864-6.

HUBBARD, R., VENN, A., LEWIS, S. & BRITTON, J. (2000) Lung cancer and cryptogenic fibrosing alveolitis. A population-based cohort study. Am J Respir Crit Care Med, 161, 5-8.

INAGAKI, K., NOGUCHI, T., MATOZAKI, T., HORIKAWA, T., FUKUNAGA, K., TSUDA, M., ICHIHASHI, M. & KASUGA, M. (2000) Roles for the protein tyrosine phosphatase SHP-2 in cytoskeletal organization, cell adhesion and cell migration revealed by overexpression of a dominant negative mutant. Oncogene, 19, 75-84.

JENKINS, B. J., GRAIL, D., INGLESE, M., QUILICI, C., BOZINOVSKI, S., WONG, P. & ERNST, M. (2004) Imbalanced gp130-Dependent Signaling in Macrophages Alters Macrophage Colony-Stimulating Factor Responsiveness via Regulation of c-fms Expression. Mol. Cell. Biol., 24, 1453-1463.

JENKINS, B. J., GRAIL, D., NHEU, T., NAJDOVSKA, M., WANG, B., WARING, P., INGLESE, M., MCLOUGHLIN, R. M., JONES, S. A., TOPLEY, N., BAUMANN, H., JUDD, L. M., GIRAUD, A. S., BOUSSIOUTAS, A., ZHU, H. J. & ERNST, M. (2005a) Hyperactivation of Stat3 in gp130 mutant mice

229

promotes gastric hyperproliferation and desensitizes TGF-beta signaling. Nat Med, 11, 845-52.

JENKINS, B. J., QUILICI, C., ROBERTS, A. W., GRAIL, D., DUNN, A. R. & ERNST, M. (2002) Hematopoietic abnormalities in mice deficient in gp130-mediated STAT signaling. Experimental Hematology, 30, 1248.

JENKINS, B. J., ROBERTS, A. W., GREENHILL, C. J., NAJDOVSKA, M., LUNDGREN-MAY, T., ROBB, L., GRAIL, D. & ERNST, M. (2007) Pathologic consequences of STAT3 hyperactivation by IL-6 and IL-11 during hematopoiesis and lymphopoiesis. Blood, 109, 2380-8.

JENKINS, B. J., ROBERTS, A. W., NAJDOVSKA, M., GRAIL, D. & ERNST, M. (2005b) The threshold of gp130-dependent STAT3 signaling is critical for normal regulation of hematopoiesis. Blood, 105, 3512-3520.

JIANG, D., LIANG, J., HODGE, J., LU, B., ZHU, Z., YU, S., FAN, J., GAO, Y., YIN, Z., HOMER, R., GERARD, C. & NOBLE, P. W. (2004) Regulation of pulmonary fibrosis by chemokine receptor CXCR3. J Clin Invest, 114, 291-9.

JOHNSTON, R. A., SCHWARTZMAN, I. N., FLYNT, L. & SHORE, S. A. (2005) Role of interleukin-6 in murine airway responses to ozone. Am J Physiol Lung Cell Mol Physiol, 288, L390-7.

JORDANA, M., SCHULMAN, J., MCSHARRY, C., IRVING, L. B., NEWHOUSE, M. T., JORDANA, G. & GAULDIE, J. (1988) Heterogeneous proliferative characteristics of human adult lung fibroblast lines and clonally derived fibroblasts from control and fibrotic tissue. Am Rev Respir Dis, 137, 579-84.

JUDD, L. M., BREDIN, K., KALANTZIS, A., JENKINS, B. J., ERNST, M. & GIRAUD, A. S. (2006a) STAT3 Activation Regulates Growth, Inflammation, and Vascularization in a Mouse Model of Gastric Tumorigenesis. Gastroenterology, 131, 1073.

JUDD, L. M., BREDIN, K., KALANTZIS, A., JENKINS, B. J., ERNST, M. & GIRAUD, A. S. (2006b) STAT3 activation regulates growth, inflammation, and vascularization in a mouse model of gastric tumorigenesis. Gastroenterology, 131, 1073-85.

JUNICHO, A., MATSUDA, T., YAMAMOTO, T., KISHI, H., KORKMAZ, K., SAATCIOGLU, F., FUSE, H. & MURAGUCHI, A. (2000) Protein inhibitor of activated STAT3 regulates androgen receptor signaling in prostate carcinoma cells. Biochem Biophys Res Commun, 278, 9-13.

KASPER, M., KOSLOWSKI, R., LUTHER, T., SCHUH, D., MULLER, M. & WENZEL, K. W. (1995) Immunohistochemical evidence for loss of ICAM-1 by alveolar epithelial cells in pulmonary fibrosis. Histochem Cell Biol, 104, 397-405.

KATZENSTEIN, A. L. (1985) Pathogenesis of "fibrosis" in interstitial pneumonia: an electron microscopic study. Hum Pathol, 16, 1015-24.

KATZENSTEIN, A. L. & MYERS, J. L. (1998) Idiopathic pulmonary fibrosis: clinical relevance of pathologic classification. Am J Respir Crit Care Med, 157, 1301-15.

KAWANAMI, O., FERRANS, V. J. & CRYSTAL, R. G. (1982) Structure of alveolar epithelial cells in patients with fibrotic lung disorders. Lab Invest, 46, 39-53.

KEANE, M. P., BELPERIO, J. A., ARENBERG, D. A., BURDICK, M. D., XU, Z. J., XUE, Y. Y. & STRIETER, R. M. (1999) IFN-{gamma}-Inducible Protein-10 Attenuates Bleomycin-Induced Pulmonary Fibrosis Via Inhibition of Angiogenesis. J Immunol, 163, 5686-5692.

KEOGH, B. A. & CRYSTAL, R. G. (1982) Alveolitis: the key to the interstitial lung disorders. Thorax, 37, 1-10.

230

KHALIL, N., BEREZNAY, O., SPORN, M. & GREENBERG, A. H. (1989) Macrophage production of transforming growth factor beta and fibroblast collagen synthesis in chronic pulmonary inflammation. J Exp Med, 170, 727-37.

KHALIL, N., O'CONNOR, R. N., UNRUH, H. W., WARREN, P. W., FLANDERS, K. C., KEMP, A., BEREZNAY, O. H. & GREENBERG, A. H. (1991) Increased production and immunohistochemical localization of transforming growth factor-beta in idiopathic pulmonary fibrosis. Am J Respir Cell Mol Biol, 5, 155-62.

KHALIL, N., XU, Y. D., O'CONNOR, R. & DURONIO, V. (2005) Proliferation of Pulmonary Interstitial Fibroblasts Is Mediated by Transforming Growth Factor-beta1-induced Release of Extracellular Fibroblast Growth Factor-2 and Phosphorylation of p38 MAPK and JNK. J. Biol. Chem., 280, 43000-43009.

KIDA, H., YOSHIDA, M., HOSHINO, S., INOUE, K., YANO, Y., YANAGITA, M., KUMAGAI, T., OSAKI, T., TACHIBANA, I., SAEKI, Y. & KAWASE, I. (2005) Protective effect of IL-6 on alveolar epithelial cell death induced by hydrogen peroxide. Am J Physiol Lung Cell Mol Physiol, 288, L342-349.

KIM, H., HAWLEY, T., HAWLEY, R. & BAUMANN, H. (1998) Protein tyrosine phosphatase 2 (SHP-2) moderates signaling by gp130 but is not required for the induction of acute-phase plasma protein genes in hepatic cells. Mol Cell Biol, 18, 1525-1533.

KING, T. J., SCHWARZ, M., BROWN, K., TOOZE, J., COLBY, T., WALDRON, J. J., FLINT, A., THURLBECK, W. & CHERNIACK, R. (2001) Idiopathic pulmonary fibrosis: relationship between histopathologic features and mortality. Am J Respir Crit Care Med, 164, 1025-1032.

KISHIMOTO, T., AKIRA, S., NARAZAKI, M. & TAGA, T. (1995) Interleukin-6 family of cytokines and gp130. Blood, 86, 1243-1254.

KNIGHT, D. (2001) Leukaemia inhibitory factor (LIF): a cytokine of emerging importance in chronic airway inflammation. Pulm Pharmacol Ther, 14, 169-76.

KNIGHT, D., ERNST, M., ANDERSON, G., MOODLEY, Y. & MUTSAERS, S. (2003) The role of gp130/IL-6 cytokines in the development of pulmonary fibrosis: critical determinants of disease susceptibility and progression? Pharmacol Ther, 99, 327-338.

KOHASE, M., MAY, L. T., TAMM, I., VILCEK, J. & SEHGAL, P. B. (1987) A cytokine network in human diploid fibroblasts: interactions of beta-interferons, tumor necrosis factor, platelet-derived growth factor, and interleukin-1. Mol Cell Biol, 7, 273-80.

KOLB, M., BONNIAUD, P., GALT, T., SIME, P. J., KELLY, M. M., MARGETTS, P. J. & GAULDIE, J. (2002) Differences in the fibrogenic response after transfer of active transforming growth factor-beta1 gene to lungs of "fibrosis-prone" and "fibrosis-resistant" mouse strains. Am J Respir Cell Mol Biol, 27, 141-50.

KOLB, M., MARGETTS, P. J., ANTHONY, D. C., PITOSSI, F. & GAULDIE, J. (2001) Transient expression of IL-1beta induces acute lung injury and chronic repair leading to pulmonary fibrosis. J Clin Invest, 107, 1529-36.

KOPF, M., BAUMANN, H., FREER, G., FREUDENBERG, M., LAMERS, M., KISHIMOTO, T., ZINKERNAGEL, R., BLUETHMANN, H. & KOHLER, G. (1994) Impaired immune and acute-phase responses in interleukin-6-deficient mice. Nature, 368, 339-42.

KRADIN, R. L., SAKAMOTO, H., JAIN, F., ZHAO, L. H., HYMOWITZ, G. & PREFFER, F. (2004) IL-10 inhibits inflammation but does not affect fibrosis in the pulmonary response to bleomycin. Exp Mol Pathol, 76, 205-11.

231

KRETZSCHMAR, M., DOODY, J., TIMOKHINA, I. & MASSAGUE, J. (1999) A mechanism of repression of TGFbeta / Smad signaling by oncogenic Ras. Genes Dev., 13, 804-816.

KUHN, C., 3RD, BOLDT, J., KING, T. E., JR., CROUCH, E., VARTIO, T. & MCDONALD, J. A. (1989) An immunohistochemical study of architectural remodeling and connective tissue synthesis in pulmonary fibrosis. Am Rev Respir Dis, 140, 1693-703.

KUHN, C., 3RD, HOMER, R. J., ZHU, Z., WARD, N., FLAVELL, R. A., GEBA, G. P. & ELIAS, J. A. (2000) Airway hyperresponsiveness and airway obstruction in transgenic mice. Morphologic correlates in mice overexpressing interleukin (IL)-11 and IL-6 in the lung. Am J Respir Cell Mol Biol, 22, 289-95.

KUHN, C. & MCDONALD, J. A. (1991) The roles of the myofibroblast in idiopathic pulmonary fibrosis. Ultrastructural and immunohistochemical features of sites of active extracellular matrix synthesis. Am J Pathol, 138, 1257-65.

KULKARNI, A. B., HUH, C. G., BECKER, D., GEISER, A., LYGHT, M., FLANDERS, K. C., ROBERTS, A. B., SPORN, M. B., WARD, J. M. & KARLSSON, S. (1993) Transforming growth factor beta 1 null mutation in mice causes excessive inflammatory response and early death. Proc Natl Acad Sci U S A, 90, 770-4.

KURATSUNE, M., MASAKI, T., HIRAI, T., KIRIBAYASHI, K., YOKOYAMA, Y., ARAKAWA, T., YORIOKA, N. & KOHNO, N. (2007) Signal transducer and activator of transcription 3 involvement in the development of renal interstitial fibrosis after unilateral ureteral obstruction. Nephrology (Carlton), 12, 565-71.

KURTH, I., HORSTEN, U., PFLANZ, S., DAHMEN, H., KUSTER, A., GROTZINGER, J., HEINRICH, P. C. & MULLER-NEWEN, G. (1999) Activation of the signal transducer glycoprotein 130 by both IL-6 and IL-11 requires two distinct binding epitopes. J Immunol, 162, 1480-7.

KUWANO, K., HAGIMOTO, N., TANAKA, T., KAWASAKI, M., KUNITAKE, R., MIYAZAKI, H., KANEKO, Y., MATSUBA, T., MAEYAMA, T. & HARA, N. (2000a) Expression of apoptosis-regulatory genes in epithelial cells in pulmonary fibrosis in mice. J Pathol, 190, 221-9.

KUWANO, K., KAWASAKI, M., MAEYAMA, T., HAGIMOTO, N., NAKAMURA, N., SHIRAKAWA, K. & HARA, N. (2000b) Soluble form of fas and fas ligand in BAL fluid from patients with pulmonary fibrosis and bronchiolitis obliterans organizing pneumonia. Chest, 118, 451-8.

LAMA, V. N. & PHAN, S. H. (2006) The extrapulmonary origin of fibroblasts: stem/progenitor cells and beyond. Proc Am Thorac Soc, 3, 373-6.

LANG, R., PAULEAU, A. L., PARGANAS, E., TAKAHASHI, Y., MAGES, J., IHLE, J. N., RUTSCHMAN, R. & MURRAY, P. J. (2003) SOCS3 regulates the plasticity of gp130 signaling. Nat Immunol, 4, 546-50.

LANGDON, C., KERR, C., TONG, L. & RICHARDS, C. D. (2003) Oncostatin M Regulates Eotaxin Expression in Fibroblasts and Eosinophilic Inflammation in C57BL/6 Mice. J Immunol, 170, 548-555.

LAURENT, G. J. (1982) Rates of collagen synthesis in lung, skin and muscle obtained in vivo by a simplified method using [3H]proline. Biochem J, 206, 535-44.

LAWSON, W. E., GRANT, S. W., AMBROSINI, V., WOMBLE, K. E., DAWSON, E. P., LANE, K. B., MARKIN, C., RENZONI, E., LYMPANY, P., THOMAS, A. Q., ROLDAN, J., SCOTT, T. A., BLACKWELL, T. S., PHILLIPS, J. A., 3RD, LOYD, J. E. & DU BOIS, R. M. (2004) Genetic mutations in surfactant protein C are a rare cause of sporadic cases of IPF. Thorax, 59, 977-80.

232

LEE, C. G., KANG, H. R., HOMER, R. J., CHUPP, G. & ELIAS, J. A. (2006) Transgenic modeling of transforming growth factor-beta(1): role of apoptosis in fibrosis and alveolar remodeling. Proc Am Thorac Soc, 3, 418-23.

LEHMANN, U., SCHMITZ, J., WEISSENBACH, M., SOBOTA, R. M., HORTNER, M., FRIEDERICHS, K., BEHRMANN, I., TSIARIS, W., SASAKI, A., SCHNEIDER-MERGENER, J., YOSHIMURA, A., NEEL, B. G., HEINRICH, P. C. & SCHAPER, F. (2003) SHP2 and SOCS3 contribute to Tyr-759-dependent attenuation of interleukin-6 signaling through gp130. J Biol Chem, 278, 661-71.

LENTSCH, A. B., CROUCH, L. D., JORDAN, J. A., CZERMAK, B. J., YUN, E. C., GUO, R., SARMA, V., DIEHL, K. M. & WARD, P. A. (1999) Regulatory effects of interleukin-11 during acute lung inflammatory injury. J Leukoc Biol, 66, 151-157.

LESUR, O. J., MANCINI, N. M., HUMBERT, J. C., CHABOT, F. & POLU, J. M. (1994) Interleukin-6, interferon-gamma, and phospholipid levels in the alveolar lining fluid of human lungs. Profiles in coal worker's pneumoconiosis and idiopathic pulmonary fibrosis. Chest, 106, 407-13.

LI, W., NISHIMURA, R., KASHISHIAN, A., BATZER, A. G., KIM, W. J., COOPER, J. A. & SCHLESSINGER, J. (1994) A new function for a phosphotyrosine phosphatase: linking GRB2-Sos to a receptor tyrosine kinase. Mol Cell Biol, 14, 509-17.

LI, Y., DU, H., QIN, Y., ROBERTS, J., CUMMINGS, O. W. & YAN, C. (2007) Activation of the Signal Transducers and Activators of the Transcription 3 Pathway in Alveolar Epithelial Cells Induces Inflammation and Adenocarcinomas in Mouse Lung. Cancer Res, 67, 8494-8503.

LIAN, X., QIN, Y., HOSSAIN, S. A., YANG, L., WHITE, A., XU, H., SHIPLEY, J. M., LI, T., SENIOR, R. M., DU, H. & YAN, C. (2005) Overexpression of Stat3C in pulmonary epithelium protects against hyperoxic lung injury. J Immunol, 174, 7250-6.

LIM, C. P., PHAN, T. T., LIM, I. J. & CAO, X. (2006) Stat3 contributes to keloid pathogenesis via promoting collagen production, cell proliferation and migration. Oncogene, 25, 5416-25.

LIU, B., LIAO, J., RAO, X., KUSHNER, S., CHUNG, C., CHANG, D. & SHUAI, K. (1998) Inhibition of Stat1-mediated gene activation by PIAS1. Proc Natl Acad Sci U S A, 95, 10626-10631.

LIU, B., MINK, S., WONG, K. A., STEIN, N., GETMAN, C., DEMPSEY, P. W., WU, H. & SHUAI, K. (2004) PIAS1 selectively inhibits interferon-inducible genes and is important in innate immunity. Nat Immunol, 5, 891-8.

LIU, B. & SHUAI, K. (2001) Induction of apoptosis by protein inhibitor of activated Stat1 through c-Jun NH2-terminal kinase activation. J Biol Chem, 276, 36624-31.

LIU, J. Y., SIME, P. J., WU, T., WARSHAMANA, G. S., POCIASK, D., TSAI, S. Y. & BRODY, A. R. (2001) Transforming growth factor-beta(1) overexpression in tumor necrosis factor-alpha receptor knockout mice induces fibroproliferative lung disease. Am J Respir Cell Mol Biol, 25, 3-7.

LIU, T., CHUNG, M. J., ULLENBRUCH, M., YU, H., JIN, H., HU, B., CHOI, Y. Y., ISHIKAWA, F. & PHAN, S. H. (2007) Telomerase activity is required for bleomycin-induced pulmonary fibrosis in mice. J. Clin. Invest., 117, 3800-3809.

LIU, T., NOZAKI, Y. & PHAN, S. H. (2002) Regulation of telomerase activity in rat lung fibroblasts. Am J Respir Cell Mol Biol, 26, 534-40.

LIU, X., SUN, S. Q., HASSID, A. & OSTROM, R. S. (2006) cAMP inhibits transforming growth factor-beta-stimulated collagen synthesis via inhibition of

233

extracellular signal-regulated kinase 1/2 and Smad signaling in cardiac fibroblasts. Mol Pharmacol, 70, 1992-2003.

LONG, J., WANG, G., MATSUURA, I., HE, D. & LIU, F. (2004) Activation of Smad transcriptional activity by protein inhibitor of activated STAT3 (PIAS3). Proc Natl Acad Sci U S A, 101, 99-104.

LUTTICKEN, C., WEGENKA, U., YUAN, J., BUSCHMANN, J., SCHINDLER, C., ZIEMIECKI, A., HARPUR, A., WILKS, A., YASUKAWA, K., TAGA, T. & AL., E. (1994) Association of transcription factor APRF and protein kinase Jak1 with the interleukin-6 signal transducer gp130. Science, 263, 88-92.

MA, B., BLACKBURN, M. R., LEE, C. G., HOMER, R. J., LIU, W., FLAVELL, R. A., BOYDEN, L., LIFTON, R. P., SUN, C. X., YOUNG, H. W. & ELIAS, J. A. (2006) Adenosine metabolism and murine strain-specific IL-4-induced inflammation, emphysema, and fibrosis. J Clin Invest, 116, 1274-83.

MAEYAMA, T., KUWANO, K., KAWASAKI, M., KUNITAKE, R., HAGIMOTO, N., MATSUBA, T., YOSHIMI, M., INOSHIMA, I., YOSHIDA, K. & HARA, N. (2001) Upregulation of Fas-signalling molecules in lung epithelial cells from patients with idiopathic pulmonary fibrosis. Eur Respir J, 17, 180-9.

MAHER, T. M., WELLS, A. U. & LAURENT, G. J. (2007) Idiopathic pulmonary fibrosis: multiple causes and multiple mechanisms? Eur Respir J, 30, 835-9.

MAIONE, D., DI CARLO, E., LI, W., MUSIANI, P., MODESTI, A., PETERS, M., ROSE-JOHN, S., DELLA ROCCA, C., TRIPODI, M., LAZZARO, D., TAUB, R., SAVINO, R. & CILIBERTO, G. (1998) Coexpression of IL-6 and soluble IL-6R causes nodular regenerative hyperplasia and adenomas of the liver. Embo J, 17, 5588-97.

MANNINO, D. M., ETZEL, R. A. & PARRISH, R. G. (1996) Pulmonary fibrosis deaths in the United States, 1979-1991. An analysis of multiple-cause mortality data. Am J Respir Crit Care Med, 153, 1548-52.

MANTOVANI, A. (2006) Macrophage diversity and polarization: in vivo veritas. Blood, 108, 408-409.

MARINE, J. C., MCKAY, C., WANG, D., TOPHAM, D. J., PARGANAS, E., NAKAJIMA, H., PENDEVILLE, H., YASUKAWA, H., SASAKI, A., YOSHIMURA, A. & IHLE, J. N. (1999a) SOCS3 is essential in the regulation of fetal liver erythropoiesis. Cell, 98, 617-27.

MARINE, J. C., TOPHAM, D. J., MCKAY, C., WANG, D., PARGANAS, E., STRAVOPODIS, D., YOSHIMURA, A. & IHLE, J. N. (1999b) SOCS1 deficiency causes a lymphocyte-dependent perinatal lethality. Cell, 98, 609-16.

MARITANO, D., SUGRUE, M. L., TINININI, S., DEWILDE, S., STROBL, B., FU, X., MURRAY-TAIT, V., CHIARLE, R. & POLI, V. (2004) The STAT3 isoforms alpha and beta have unique and specific functions. Nat Immunol, 5, 401-9.

MASON, R., SCHWARZ, M., HUNNINGHAKE, G. & MUSSON, R. (1999) NHLBI Workshop Summary. Pharmacological therapy for idiopathic pulmonary fibrosis. Past, present, and future. Am J Respir Crit Care Med, 160, 1771-1777.

MASSAGUE, J., SEOANE, J. & WOTTON, D. (2005) Smad transcription factors. Genes Dev, 19, 2783-810.

MCANULTY, R. J., HERNANDEZ-RODRIGUEZ, N. A., MUTSAERS, S. E., COKER, R. K. & LAURENT, G. J. (1997) Indomethacin suppresses the anti-proliferative effects of transforming growth factor-beta isoforms on fibroblast cell cultures. Biochem J, 321 (Pt 3), 639-43.

MCANULTY, R. J. & LAURENT, G. J. (1987) Collagen synthesis and degradation in vivo. Evidence for rapid rates of collagen turnover with extensive degradation of newly synthesized collagen in tissues of the adult rat. Coll Relat Res, 7, 93-104.

234

MCLOUGHLIN, R. M., HURST, S. M., NOWELL, M. A., HARRIS, D. A., HORIUCHI, S., MORGAN, L. W., WILKINSON, T. S., YAMAMOTO, N., TOPLEY, N. & JONES, S. A. (2004) Differential regulation of neutrophil-activating chemokines by IL-6 and its soluble receptor isoforms. J Immunol, 172, 5676-83.

MCLOUGHLIN, R. M., JENKINS, B. J., GRAIL, D., WILLIAMS, A. S., FIELDING, C. A., PARKER, C. R., ERNST, M., TOPLEY, N. & JONES, S. A. (2005) IL-6 trans-signaling via STAT3 directs T cell infiltration in acute inflammation. Proc Natl Acad Sci U S A, 102, 9589-94.

MEHRAD, B., BURDICK, M. D., ZISMAN, D. A., KEANE, M. P., BELPERIO, J. A. & STRIETER, R. M. (2007) Circulating peripheral blood fibrocytes in human fibrotic interstitial lung disease. Biochemical and Biophysical Research Communications, 353, 104.

MERRILL, W. W. & REYNOLDS, H. Y. (1983) Bronchial lavage in inflammatory lung disease. Clin Chest Med, 4, 71-84.

METCALF, D., MIFSUD, S., DI RAGO, L. & ALEXANDER, W. S. (2003) The lethal effects of transplantation of Socs1-/- bone marrow cells into irradiated adult syngeneic recipients. Proc Natl Acad Sci U S A, 100, 8436-41.

MEWS, P., PHILLIPS, P., FAHMY, R., KORSTEN, M., PIROLA, R., WILSON, J. & APTE, M. (2002) Pancreatic stellate cells respond to inflammatory cytokines: potential role in chronic pancreatitis. Gut, 50, 535-41.

MILLER, E. J. (1985) The structure of fibril-forming collagens. Ann N Y Acad Sci, 460, 1-13.

MILNER, R. & CAMPBELL, I. L. (2006) Increased expression of the beta4 and alpha5 integrin subunits in cerebral blood vessels of transgenic mice chronically producing the pro-inflammatory cytokines IL-6 or IFN-alpha in the central nervous system. Mol Cell Neurosci, 33, 429-40.

MIYAHARA, M., NJIEHA, F. K. & PROCKOP, D. J. (1982) Formation of collagen fibrils in vitro by cleavage of procollagen with procollagen proteinases. J Biol Chem, 257, 8442-8.

MONAGHAN, H., WELLS, A., COLBY, T., DU BOIS, R., HANSELL, D. & NICHOLSON, A. (2004) Prognostic implications of histologic patterns in multiple surgical lung biopsies from patients with idiopathic interstitial pneumonias. Chest, 125, 522-526.

MOODLEY, Y., CATERINA, P., SCAFFIDI, A., MISSO, N., PAPADIMITRIOU, J., MCANULTY, R., LAURENT, G., THOMPSON, P. & KNIGHT, D. (2004) Comparison of the morphological and biochemical changes in normal human lung fibroblasts and fibroblasts derived from lungs of patients with idiopathic pulmonary fibrosis during FasL-induced apoptosis. J Pathol, 202, 486-495.

MOODLEY, Y., MISSO, N., SCAFFIDI, A., FOGEL-PETROVIC, M., MCANULTY, R., LAURENT, G., THOMPSON, P. & KNIGHT, D. (2003b) Inverse effects of interleukin-6 on apoptosis of fibroblasts from pulmonary fibrosis and normal lungs. Am J Respir Cell Mol Biol, 29, 490-498.

MOODLEY, Y., SCAFFIDI, A., MISSO, N., KEERTHISINGAM, C., MCANULTY, R., LAURENT, G., MUTSAERS, S., THOMPSON, P. & KNIGHT, D. (2003a) Fibroblasts isolated from normal lungs and those with idiopathic pulmonary fibrosis differ in interleukin-6/gp130-mediated cell signaling and proliferation. Am J Pathol., 163, 345-354.

MOODLEY, Y. P., SCAFFIDI, A. K., MISSO, N. L., KEERTHISINGAM, C., MCANULTY, R. J., LAURENT, G. J., MUTSAERS, S. E., THOMPSON, P. J. & KNIGHT, D. A. (2003) Fibroblasts isolated from normal lungs and those with

235

idiopathic pulmonary fibrosis differ in interleukin-6/gp130-mediated cell signaling and proliferation. Am J Pathol, 163, 345-54.

MOORE, B. B., PAINE, R., 3RD, CHRISTENSEN, P. J., MOORE, T. A., SITTERDING, S., NGAN, R., WILKE, C. A., KUZIEL, W. A. & TOEWS, G. B. (2001) Protection from pulmonary fibrosis in the absence of CCR2 signaling. J Immunol, 167, 4368-77.

MOSMANN, T. (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods, 65, 55-63.

MOSSMAN, B. T. & CHURG, A. (1998) Mechanisms in the pathogenesis of asbestosis and silicosis. Am J Respir Crit Care Med, 157, 1666-80.

MULSOW, J. J., WATSON, R. W., FITZPATRICK, J. M. & O'CONNELL, P. R. (2005) Transforming growth factor-beta promotes pro-fibrotic behavior by serosal fibroblasts via PKC and ERK1/2 mitogen activated protein kinase cell signaling. Ann Surg, 242, 880-7, discussion 887-9.

MUSK, A. W., ZILKO, P. J., MANNERS, P., KAY, P. H. & KAMBOH, M. I. (1986) Genetic studies in familial fibrosing alveolitis. Possible linkage with immunoglobulin allotypes (Gm). Chest, 89, 206-10.

NAKA, T., NARAZAKI, M., HIRATA, M., MATSUMOTO, T., MINAMOTO, S., AONO, A., NISHIMOTO, N., KAJITA, T., TAGA, T., YOSHIZAKI, K., AKIRA, S. & KISHIMOTO, T. (1997) Structure and function of a new STAT-induced STAT inhibitor. Nature, 387, 924-929.

NANDURKAR, H. H., ROBB, L., TARLINTON, D., BARNETT, L., KONTGEN, F. & BEGLEY, C. G. (1997) Adult mice with targeted mutation of the interleukin-11 receptor (IL11Ra) display normal hematopoiesis. Blood, 90, 2148-59.

NEMUNAITIS, J., ANDREWS, D. F., MOCHIZUKI, D. Y., LILLY, M. B. & SINGER, J. W. (1989) Human marrow stromal cells: response to interleukin-6 (IL-6) and control of IL-6 expression. Blood, 74, 1929-35.

NICHOLSON, A. G., FULFORD, L. G., COLBY, T. V., DU BOIS, R. M., HANSELL, D. M. & WELLS, A. U. (2002) The relationship between individual histologic features and disease progression in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med, 166, 173-7.

NJIEHA, F. K., MORIKAWA, T., TUDERMAN, L. & PROCKOP, D. J. (1982) Partial purification of a procollagen C-proteinase. Inhibition by synthetic peptides and sequential cleavage of type I procollagen. Biochemistry, 21, 757-64.

NOBLE, P. W. (2003) Idiopathic pulmonary fibrosis. New insights into classification and pathogenesis usher in a new era therapeutic approaches. Am J Respir Cell Mol Biol, 29, S27-31.

NOGUCHI, T., MATOZAKI, T., HORITA, K., FUJIOKA, Y. & KASUGA, M. (1994) Role of SH-PTP2, a protein-tyrosine phosphatase with Src homology 2 domains, in insulin-stimulated Ras activation. Mol Cell Biol, 14, 6674-82.

NOWELL, M. A., RICHARDS, P. J., HORIUCHI, S., YAMAMOTO, N., ROSE-JOHN, S., TOPLEY, N., WILLIAMS, A. S. & JONES, S. A. (2003) Soluble IL-6 receptor governs IL-6 activity in experimental arthritis: blockade of arthritis severity by soluble glycoprotein 130. J Immunol, 171, 3202-9.

NOZAKI, Y., LIU, T., HATANO, K., GHARAEE-KERMANI, M. & PHAN, S. H. (2000) Induction of telomerase activity in fibroblasts from bleomycin-injured lungs. Am J Respir Cell Mol Biol, 23, 460-5.

O'HARA, K. A., KEDDA, M. A., THOMPSON, P. J. & KNIGHT, D. A. (2003) Oncostatin M: an interleukin-6-like cytokine relevant to airway remodelling and the pathogenesis of asthma. Clin Exp Allergy, 33, 1026-32.

236

OGATA, H., CHINEN, T., YOSHIDA, T., KINJYO, I., TAKAESU, G., SHIRAISHI, H., IIDA, M., KOBAYASHI, T. & YOSHIMURA, A. (2006) Loss of SOCS3 in the liver promotes fibrosis by enhancing STAT3-mediated TGF-beta1 production. Oncogene, 25, 2520-30.

OHTANI, T., ISHIHARA, K., ATSUMI, T., NISHIDA, K., KANEKO, Y., MIYATA, T., ITOH, S., NARIMATSU, M., MAEDA, H., FUKADA, T., ITOH, M., OKANO, H., HIBI, M. & HIRANO, T. (2000) Dissection of signaling cascades through gp130 in vivo: reciprocal roles for STAT3- and SHP2-mediated signals in immune responses. Immunity, 12, 95-105.

OKAMOTO, T., AMANO, T., WADA, T., HARADA, M. & TANAKA, T. (1970) [Experimental pulmonary fibrosis induced by bleomycin]. Igaku To Seibutsugaku, 80, 299-301.

OKUMA, T., TERASAKI, Y., KAIKITA, K., KOBAYASHI, H., KUZIEL, W. A., KAWASUJI, M. & TAKEYA, M. (2004) C-C chemokine receptor 2 (CCR2) deficiency improves bleomycin-induced pulmonary fibrosis by attenuation of both macrophage infiltration and production of macrophage-derived matrix metalloproteinases. J Pathol, 204, 594-604.

OLMAN, M. A., WHITE, K. E., WARE, L. B., CROSS, M. T., ZHU, S. & MATTHAY, M. A. (2002) Microarray analysis indicates that pulmonary edema fluid from patients with acute lung injury mediates inflammation, mitogen gene expression, and fibroblast proliferation through bioactive interleukin-1. Chest, 121, 69S-70S.

OLMAN, M. A., WHITE, K. E., WARE, L. B., SIMMONS, W. L., BENVENISTE, E. N., ZHU, S., PUGIN, J. & MATTHAY, M. A. (2004) Pulmonary edema fluid from patients with early lung injury stimulates fibroblast proliferation through IL-1 beta-induced IL-6 expression. J Immunol, 172, 2668-77.

ORR, F. W., ADAMSON, I. Y. R. & YOUNG, L. (1986) Promotion of Pulmonary Metastasis in Mice by Bleomycin-induced Endothelial Injury. Cancer Res, 46, 891-897.

PANNU, J., NAKERAKANTI, S., SMITH, E., TEN DIJKE, P. & TROJANOWSKA, M. (2007) Transforming growth factor-beta receptor type I-dependent fibrogenic gene program is mediated via activation of Smad1 and ERK1/2 pathways. J Biol Chem, 282, 10405-13.

PANTELIDIS, P., FANNING, G., WELLS, A., WELSH, K. & DU BOIS, R. (2001) Analysis of tumor necrosis factor-alpha, lymphotoxin-alpha, tumor necrosis factor receptor II, and interleukin-6 polymorphisms in patients with idiopathic pulmonary fibrosis. Am J Respir Crit Care Med, 163, 1432-1436.

PARDO, A. & SELMAN, M. (2006) Matrix metalloproteases in aberrant fibrotic tissue remodeling. Proc Am Thorac Soc, 3, 383-8.

PAUL, C., SEILIEZ, I., THISSEN, J. P. & LE CAM, A. (2000) Regulation of expression of the rat SOCS-3 gene in hepatocytes by growth hormone, interleukin-6 and glucocorticoids mRNA analysis and promoter characterization. Eur J Biochem, 267, 5849-57.

PEREZ, A., ROGERS, R. M. & DAUBER, J. H. (2003) The prognosis of idiopathic pulmonary fibrosis. Am J Respir Cell Mol Biol, 29, S19-26.

PFLANZ, S., TIMANS, J. C., CHEUNG, J., ROSALES, R., KANZLER, H., GILBERT, J., HIBBERT, L., CHURAKOVA, T., TRAVIS, M., VAISBERG, E., BLUMENSCHEIN, W. M., MATTSON, J. D., WAGNER, J. L., TO, W., ZURAWSKI, S., MCCLANAHAN, T. K., GORMAN, D. M., BAZAN, J. F., DE WAAL MALEFYT, R., RENNICK, D. & KASTELEIN, R. A. (2002) IL-27, a Heterodimeric Cytokine Composed of EBI3 and p28 Protein, Induces Proliferation of Naive CD4+ T Cells. Immunity, 16, 779.

237

PHAN, S. H. (2003) Fibroblast phenotypes in pulmonary fibrosis. Am J Respir Cell Mol Biol, 29, S87-92.

PHILLIPS, R. J., BURDICK, M. D., HONG, K., LUTZ, M. A., MURRAY, L. A., XUE, Y. Y., BELPERIO, J. A., KEANE, M. P. & STRIETER, R. M. (2004) Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis. J Clin Invest, 114, 438-46.

PIERCE, E. M., CARPENTER, K., JAKUBZICK, C., KUNKEL, S. L., EVANOFF, H., FLAHERTY, K. R., MARTINEZ, F. J., TOEWS, G. B. & HOGABOAM, C. M. (2007a) Idiopathic pulmonary fibrosis fibroblasts migrate and proliferate to CC chemokine ligand 21. Eur Respir J, 29, 1082-93.

PIERCE, E. M., CARPENTER, K., JAKUBZICK, C., KUNKEL, S. L., FLAHERTY, K. R., MARTINEZ, F. J. & HOGABOAM, C. M. (2007b) Therapeutic targeting of CC ligand 21 or CC chemokine receptor 7 abrogates pulmonary fibrosis induced by the adoptive transfer of human pulmonary fibroblasts to immunodeficient mice. Am J Pathol, 170, 1152-64.

QIU, Z., FUJIMURA, M., KURASHIMA, K., NAKAO, S. & MUKAIDA, N. (2004) Enhanced airway inflammation and decreased subepithelial fibrosis in interleukin 6-deficient mice following chronic exposure to aerosolized antigen. Clin Exp Allergy, 34, 1321-8.

RAGHU, G., BROWN, K. K., BRADFORD, W. Z., STARKO, K., NOBLE, P. W., SCHWARTZ, D. A. & KING, T. E., JR. (2004) A placebo-controlled trial of interferon gamma-1b in patients with idiopathic pulmonary fibrosis. N Engl J Med, 350, 125-33.

RAGHU, G., CHEN, Y. Y., RUSCH, V. & RABINOVITCH, P. S. (1988) Differential proliferation of fibroblasts cultured from normal and fibrotic human lungs. Am Rev Respir Dis, 138, 703-8.

RAMOS, C., MONTANO, M., GARCIA-ALVAREZ, J., RUIZ, V., UHAL, B. D., SELMAN, M. & PARDO, A. (2001) Fibroblasts from idiopathic pulmonary fibrosis and normal lungs differ in growth rate, apoptosis, and tissue inhibitor of metalloproteinases expression. Am J Respir Cell Mol Biol, 24, 591-8.

RAY, P., TANG, W., WANG, P., HOMER, R., KUHN, C., 3RD, FLAVELL, R. A. & ELIAS, J. A. (1997) Regulated overexpression of interleukin 11 in the lung. Use to dissociate development-dependent and -independent phenotypes. J Clin Invest, 100, 2501-11.

RIHA, R. L., YANG, I. A., RABNOTT, G. C., TUNNICLIFFE, A. M., FONG, K. M. & ZIMMERMAN, P. V. (2004) Cytokine gene polymorphisms in idiopathic pulmonary fibrosis. Intern Med J, 34, 126-9.

ROBB, L., LI, R., HARTLEY, L., NANDURKAR, H. H., KOENTGEN, F. & BEGLEY, C. G. (1998) Infertility in female mice lacking the receptor for interleukin 11 is due to a defective uterine response to implantation. Nat Med, 4, 303-8.

ROBERTS, A. B., SPORN, M. B., ASSOIAN, R. K., SMITH, J. M., ROCHE, N. S., WAKEFIELD, L. M., HEINE, U. I., LIOTTA, L. A., FALANGA, V., KEHRL, J. H. & ET AL. (1986) Transforming growth factor type beta: rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc Natl Acad Sci U S A, 83, 4167-71.

ROBERTS, A. B., TIAN, F., BYFIELD, S. D., STUELTEN, C., OOSHIMA, A., SAIKA, S. & FLANDERS, K. C. (2006) Smad3 is key to TGF-beta-mediated epithelial-to-mesenchymal transition, fibrosis, tumor suppression and metastasis. Cytokine Growth Factor Rev, 17, 19-27.

ROBERTS, A. W., ROBB, L., RAKAR, S., HARTLEY, L., CLUSE, L., NICOLA, N. A., METCALF, D., HILTON, D. J. & ALEXANDER, W. S. (2001) Placental

238

defects and embryonic lethality in mice lacking suppressor of cytokine signaling 3. Proc Natl Acad Sci U S A, 98, 9324-9.

RODIG, S., MERAZ, M., WHITE, J., LAMPE, P., RILEY, J., ARTHUR, C., KING, K., SHEEHAN, K., YIN, L., PENNICA, D., JOHNSON, E. J. & SCHREIBER, R. (1998) Disruption of the Jak1 gene demonstrates obligatory and nonredundant roles of the Jaks in cytokine-induced biologic responses. Cell, 93, 373-383.

RODRIGUEZ MDEL, C., BERNAD, A. & ARACIL, M. (2004) Interleukin-6 deficiency affects bone marrow stromal precursors, resulting in defective hematopoietic support. Blood, 103, 3349-54.

ROSE-JOHN, S., SCHELLER, J., ELSON, G. & JONES, S. A. (2006) Interleukin-6 biology is coordinated by membrane-bound and soluble receptors: role in inflammation and cancer. J Leukoc Biol, 80, 227-36.

ROY, S. G., NOZAKI, Y. & PHAN, S. H. (2001) Regulation of alpha-smooth muscle actin gene expression in myofibroblast differentiation from rat lung fibroblasts. Int J Biochem Cell Biol, 33, 723-34.

SACHDEV, S., BRUHN, L., SIEBER, H., PICHLER, A., MELCHIOR, F. & GROSSCHEDL, R. (2001) PIASy, a nuclear matrix-associated SUMO E3 ligase, represses LEF1 activity by sequestration into nuclear bodies. Genes Dev, 15, 3088-103.

SAKAMOTO, H., ZHAO, L. H., JAIN, F. & KRADIN, R. (2002) IL-12p40(-/-) mice treated with intratracheal bleomycin exhibit decreased pulmonary inflammation and increased fibrosis. Exp Mol Pathol, 72, 1-9.

SANFORD, L. P., ORMSBY, I., GITTENBERGER-DE GROOT, A. C., SARIOLA, H., FRIEDMAN, R., BOIVIN, G. P., CARDELL, E. L. & DOETSCHMAN, T. (1997) TGFbeta2 knockout mice have multiple developmental defects that are non-overlapping with other TGFbeta knockout phenotypes. Development, 124, 2659-70.

SANO, S., CHAN, K. S., CARBAJAL, S., CLIFFORD, J., PEAVEY, M., KIGUCHI, K., ITAMI, S., NICKOLOFF, B. J. & DIGIOVANNI, J. (2005) Stat3 links activated keratinocytes and immunocytes required for development of psoriasis in a novel transgenic mouse model. Nat Med, 11, 43-9.

SASAKI, A., YASUKAWA, H., SHOUDA, T., KITAMURA, T., DIKIC, I. & YOSHIMURA, A. (2000) CIS3/SOCS-3 suppresses erythropoietin (EPO) signaling by binding the EPO receptor and JAK2. J Biol Chem, 275, 29338-47.

SASAKI, A., YASUKAWA, H., SUZUKI, A., KAMIZONO, S., SYODA, T., KINJYO, I., SASAKI, M., JOHNSTON, J. A. & YOSHIMURA, A. (1999) Cytokine-inducible SH2 protein-3 (CIS3/SOCS3) inhibits Janus tyrosine kinase by binding through the N-terminal kinase inhibitory region as well as SH2 domain. Genes Cells, 4, 339-51.

SATO, M., MURAGAKI, Y., SAIKA, S., ROBERTS, A. B. & OOSHIMA, A. (2003) Targeted disruption of TGF-beta1/Smad3 signaling protects against renal tubulointerstitial fibrosis induced by unilateral ureteral obstruction. J Clin Invest, 112, 1486-94.

SAXTON, T. M., HENKEMEYER, M., GASCA, S., SHEN, R., ROSSI, D. J., SHALABY, F., FENG, G. S. & PAWSON, T. (1997) Abnormal mesoderm patterning in mouse embryos mutant for the SH2 tyrosine phosphatase Shp-2. Embo J, 16, 2352-64.

SCAFFIDI, A., MOODLEY, YP., WEISCHELBAUM, M., THOMPSON, PJ, KNIGHT, DA. (2001) Regulation of human lung fibroblast phenotype and function by vitronectin and vitronectin integrins. J Cell Sci, 114; 3507-3516.

239

SCAFFIDI, A., MUTSAERS, S., MOODLEY, Y., MCANULTY, R., LAURENT, G., THOMPSON, P. & KNIGHT, D. (2002) Oncostatin M stimulates proliferation, induces collagen production and inhibits apoptosis of human lung fibroblasts. B J Pharmacol, 136, 793-801.

SCALA, E., PALLOTTA, S., FREZZOLINI, A., ABENI, D., BARBIERI, C., SAMPOGNA, F., DE PITA, O., PUDDU, P., PAGANELLI, R. & RUSSO, G. (2004) Cytokine and chemokine levels in systemic sclerosis: relationship with cutaneous and internal organ involvement. Clin Exp Immunol, 138, 540-6.

SCHAEFER, T. S., SANDERS, L. K. & NATHANS, D. (1995) Cooperative transcriptional activity of Jun and Stat3 beta, a short form of Stat3. Proc Natl Acad Sci U S A, 92, 9097-101.

SCHAPER, F., GENDO, C., ECK, M., SCHMITZ, J., GRIMM, C., ANHUF, D., KERR, I. M. & HEINRICH, P. C. (1998) Activation of the protein tyrosine phosphatase SHP2 via the interleukin-6 signal transducing receptor protein gp130 requires tyrosine kinase Jak1 and limits acute-phase protein expression. Biochem J, 335 (Pt 3), 557-65.

SCHMIDT, M., SUN, G., STACEY, M. A., MORI, L. & MATTOLI, S. (2003) Identification of circulating fibrocytes as precursors of bronchial myofibroblasts in asthma. J Immunol, 171, 380-9.

SCHMITZ, J., WEISSENBACH, M., HAAN, S., HEINRICH, P. & SCHAPER, F. (2000) SOCS3 exerts its inhibitory function on interleukin-6 signal transduction through the SHP2 recruitment site of gp130. J Biol Chem, 275, 12848-12856.

SCHWARTZ, D. A., HELMERS, R. A., GALVIN, J. R., VAN FOSSEN, D. S., FREES, K. L., DAYTON, C. S., BURMEISTER, L. F. & HUNNINGHAKE, G. W. (1994a) Determinants of survival in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med, 149, 450-4.

SCHWARTZ, D. A., VAN FOSSEN, D. S., DAVIS, C. S., HELMERS, R. A., DAYTON, C. S., BURMEISTER, L. F. & HUNNINGHAKE, G. W. (1994b) Determinants of progression in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med, 149, 444-9.

SCOTT, J., JOHNSTON, I. & BRITTON, J. (1990) What causes cryptogenic fibrosing alveolitis? A case-control study of environmental exposure to dust. Bmj, 301, 1015-7.

SCOTTON, C. J. & CHAMBERS, R. C. (2007) Molecular targets in pulmonary fibrosis: the myofibroblast in focus. Chest, 132, 1311-21.

SEKI, Y., INOUE, H., NAGATA, N., HAYASHI, K., FUKUYAMA, S., MATSUMOTO, K., KOMINE, O., HAMANO, S., HIMENO, K., INAGAKI-OHARA, K., CACALANO, N., O'GARRA, A., OSHIDA, T., SAITO, H., JOHNSTON, J. A., YOSHIMURA, A. & KUBO, M. (2003) SOCS-3 regulates onset and maintenance of T(H)2-mediated allergic responses. Nat Med, 9, 1047-54.

SELMAN, M., KING, T. & PARDO, A. (2001) Idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy. Ann Intern Med, 134, 136-151.

SENGUPTA, T. K., TALBOT, E. S., SCHERLE, P. A. & IVASHKIV, L. B. (1998) Rapid inhibition of interleukin-6 signaling and Stat3 activation mediated by mitogen-activated protein kinases. Proc Natl Acad Sci U S A, 95, 11107-12.

SHAW, L. M. & OLSEN, B. R. (1991) FACIT collagens: diverse molecular bridges in extracellular matrices. Trends Biochem Sci, 16, 191-4.

SHERIDAN, B. C., DINARELLO, C. A., MELDRUM, D. R., FULLERTON, D. A., SELZMAN, C. H. & MCINTYRE, R. C., JR. (1999) Interleukin-11 attenuates

240

pulmonary inflammation and vasomotor dysfunction in endotoxin-induced lung injury. Am J Physiol Lung Cell Mol Physiol, 277, L861-867.

SHULL, M. M., ORMSBY, I., KIER, A. B., PAWLOWSKI, S., DIEBOLD, R. J., YIN, M., ALLEN, R., SIDMAN, C., PROETZEL, G., CALVIN, D. & ET AL. (1992) Targeted disruption of the mouse transforming growth factor-beta 1 gene results in multifocal inflammatory disease. Nature, 359, 693-9.

SICA, A. & BRONTE, V. (2007) Altered macrophage differentiation and immune dysfunction in tumor development. J. Clin. Invest., 117, 1155-1166.

SIEGEL, R. C., PINNELL, S. R. & MARTIN, G. R. (1970) Cross-linking of collagen and elastin. Properties of lysyl oxidase. Biochemistry, 9, 4486-92.

SILVERA, M. R., SEMPOWSKI, G. D. & PHIPPS, R. P. (1994) Expression of TGF-beta isoforms by Thy-1+ and Thy-1- pulmonary fibroblast subsets: evidence for TGF-beta as a regulator of IL-1-dependent stimulation of IL-6. Lymphokine Cytokine Res, 13, 277-85.

SIME, P. J., XING, Z., GRAHAM, F. L., CSAKY, K. G. & GAULDIE, J. (1997) Adenovector-mediated gene transfer of active transforming growth factor-beta1 induces prolonged severe fibrosis in rat lung. J Clin Invest, 100, 768-76.

SISSON, T. H., HATTORI, N., XU, Y. & SIMON, R. H. (1999) Treatment of bleomycin-induced pulmonary fibrosis by transfer of urokinase-type plasminogen activator genes. Hum Gene Ther, 10, 2315-23.

SONNYLAL, S., DENTON, C. P., ZHENG, B., KEENE, D. R., HE, R., ADAMS, H. P., VANPELT, C. S., GENG, Y. J., DENG, J. M., BEHRINGER, R. R. & DE CROMBRUGGHE, B. (2007) Postnatal induction of transforming growth factor beta signaling in fibroblasts of mice recapitulates clinical, histologic, and biochemical features of scleroderma. Arthritis Rheum, 56, 334-44.

STAHL, N., BOULTON, T., FARRUGGELLA, T., IP, N., DAVIS, S., WITTHUHN, B., QUELLE, F., SILVENNOINEN, O., BARBIERI, G., PELLEGRINI, S. & AL., E. (1994) Association and activation of Jak-Tyk kinases by CNTF-LIF-OSM-IL-6 beta receptor components. Science, 263, 92-95.

STAHL, N., FARRUGELLA, T. J., BOULTON, T. G., ZHONG, Z., JR, J. E. D. & YANCOPOULOS, G. D. (1995) Choice of STATs and Other Substrates Specified by Modular Tyrosine-Based Motifs in Cytokine Receptors. Science, 267, 1349.

STARR, R., METCALF, D., ELEFANTY, A. G., BRYSHA, M., WILLSON, T. A., NICOLA, N. A., HILTON, D. J. & ALEXANDER, W. S. (1998) Liver degeneration and lymphoid deficiencies in mice lacking suppressor of cytokine signaling-1. Proc Natl Acad Sci U S A, 95, 14395-9.

STARR, R., WILLSON, T. A., VINEY, E. M., MURRAY, L. J., RAYNER, J. R., JENKINS, B. J., GONDA, T. J., ALEXANDER, W. S., METCALF, D., NICOLA, N. A. & HILTON, D. J. (1997) A family of cytokine-inducible inhibitors of signalling. Nature, 387, 917-21.

STEINMANN, B., RAO, V. H. & GITZELMANN, R. (1981) Intracellular degradation of newly synthesized collagen is conformation-dependent. FEBS Lett, 133, 142-4.

STEWART, C. L., KASPAR, P., BRUNET, L. J., BHATT, H., GADI, I., KONTGEN, F. & ABBONDANZO, S. J. (1992) Blastocyst implantation depends on maternal expression of leukaemia inhibitory factor. Nature, 359, 76-9.

STREETZ, K., TACKE, F., LEIFELD, L., WUSTEFELD, T., GRAW, A., KLEIN, C., KAMINO, K., SPENGLER, U., KREIPE, H., KUBICKA, S., MULLER, W., MANNS, M. & TRAUTWEIN, C. (2003) Interleukin 6/gp130-dependent pathways are protective during chronic liver diseases. Hepatology, 38, 218-219.

241

STRIETER, R. M., GOMPERTS, B. N. & KEANE, M. P. (2007) The role of CXC chemokines in pulmonary fibrosis. J Clin Invest, 117, 549-56.

SUEOKA, N., SUEOKA, E., MIYAZAKI, Y., OKABE, S., KUROSUMI, M., TAKAYAMA, S. & FUJIKI, H. (1998) Molecular pathogenesis of interstitial pneumonitis with TNF-alpha transgenic mice. Cytokine, 10, 124-31.

SURATT, B. T., COOL, C. D., SERLS, A. E., CHEN, L., VARELLA-GARCIA, M., SHPALL, E. J., BROWN, K. K. & WORTHEN, G. S. (2003) Human Pulmonary Chimerism after Hematopoietic Stem Cell Transplantation. Am. J. Respir. Crit. Care Med., 168, 318-322.

SYMES, A., STAHL, N., REEVES, S., FARRUGGELLA, T., SERVIDEI, T., GEARAN, T., YANCOPOULOS, G. & FINK, J. (1997) The protein tyrosine phosphatase SHP-2 negatively regulates ciliary neurotrophic factor induction of gene expression. Curr Biol, 7, 697-700.

TABATA, C., TABATA, R., KADOKAWA, Y., HISAMORI, S., TAKAHASHI, M., MISHIMA, M., NAKANO, T. & KUBO, H. (2007) Thalidomide prevents bleomycin-induced pulmonary fibrosis in mice. J Immunol, 179, 708-14.

TAKAHASHI, Y., CARPINO, N., CROSS, J. C., TORRES, M., PARGANAS, E. & IHLE, J. N. (2003) SOCS3: an essential regulator of LIF receptor signaling in trophoblast giant cell differentiation. Embo J, 22, 372-84.

TAKEDA, K., CLAUSEN, B. E., KAISHO, T., TSUJIMURA, T., TERADA, N., FORSTER, I. & AKIRA, S. (1999) Enhanced Th1 activity and development of chronic enterocolitis in mice devoid of Stat3 in macrophages and neutrophils. Immunity, 10, 39-49.

TAKEDA, K., KAISHO, T., YOSHIDA, N., TAKEDA, J., KISHIMOTO, T. & AKIRA, S. (1998) Stat3 activation is responsible for IL-6-dependent T cell proliferation through preventing apoptosis: generation and characterization of T cell-specific Stat3-deficient mice. J Immunol, 161, 4652-60.

TAKEDA, K., NOGUCHI, K., SHI, W., TANAKA, T., MATSUMOTO, M., YOSHIDA, N., KISHIMOTO, T. & AKIRA, S. (1997) Targeted disruption of the mouse Stat3 gene leads to early embryonic lethality. Proc Natl Acad Sci U S A, 94, 3801-4.

TAKIZAWA, H., SATOH, M., OKAZAKI, H., MATSUZAKI, G., SUZUKI, N., ISHII, A., SUKO, M., OKUDAIRA, H., MORITA, Y. & ITO, K. (1997) Increased IL-6 and IL-8 in bronchoalveolar lavage fluids (BALF) from patients with sarcoidosis: correlation with the clinical parameters. Clin Exp Immunol, 107, 175-81.

TAN, J. A., HALL, S. H., HAMIL, K. G., GROSSMAN, G., PETRUSZ, P. & FRENCH, F. S. (2002) Protein inhibitors of activated STAT resemble scaffold attachment factors and function as interacting nuclear receptor coregulators. J Biol Chem, 277, 16993-7001.

TANAKA, M., HIRABAYASHI, Y., SEKIGUCHI, T., INOUE, T., KATSUKI, M. & MIYAJIMA, A. (2003) Targeted disruption of oncostatin M receptor results in altered hematopoiesis. Blood, 102, 3154-62.

TANG, M., ZHANG, W., LIN, H., JIANG, H., DAI, H. & ZHANG, Y. (2007) High glucose promotes the production of collagen types I and III by cardiac fibroblasts through a pathway dependent on extracellular-signal-regulated kinase 1/2. Mol Cell Biochem, 301, 109-14.

TANG, W., GEBA, G., ZHENG, T., RAY, P., HOMER, R., KUHN, C. R., FLAVELL, R. & ELIAS, J. (1996) Targeted expression of IL-11 in the murine airway causes lymphocytic inflammation, bronchial remodeling, and airways obstruction. J Clin Invest, 98, 2845-2853.

242

TAYA, Y., O'KANE, S. & FERGUSON, M. W. (1999) Pathogenesis of cleft palate in TGF-beta3 knockout mice. Development, 126, 3869-79.

TEBBUTT, N., GIRAUD, A., INGLESE, M., JENKINS, B., WARING, P., CLAY, F., MALKI, S., ALDERMAN, B., GRAIL, D., HOLLANDE, F., HEATH, J. & ERNST, M. (2002) Reciprocal regulation of gastrointestinal homeostasis by SHP2 and STAT-mediated trefoil gene activation in gp130 mutant mice. Nat Med, 8, 1089-1097.

THANNICKAL, V. J., LEE, D. Y., WHITE, E. S., CUI, Z., LARIOS, J. M., CHACON, R., HOROWITZ, J. C., DAY, R. M. & THOMAS, P. E. (2003) Myofibroblast differentiation by transforming growth factor-beta1 is dependent on cell adhesion and integrin signaling via focal adhesion kinase. J Biol Chem, 278, 12384-9.

THANNICKAL, V. J., TOEWS, G. B., WHITE, E. S., LYNCH, J. P., 3RD & MARTINEZ, F. J. (2004) Mechanisms of pulmonary fibrosis. Annu Rev Med, 55, 395-417.

THOMAS, A. Q., LANE, K., PHILLIPS, J., 3RD, PRINCE, M., MARKIN, C., SPEER, M., SCHWARTZ, D. A., GADDIPATI, R., MARNEY, A., JOHNSON, J., ROBERTS, R., HAINES, J., STAHLMAN, M. & LOYD, J. E. (2002) Heterozygosity for a surfactant protein C gene mutation associated with usual interstitial pneumonitis and cellular nonspecific interstitial pneumonitis in one kindred. Am J Respir Crit Care Med, 165, 1322-8.

TOMASEK, J. J., GABBIANI, G., HINZ, B., CHAPONNIER, C. & BROWN, R. A. (2002) Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat Rev Mol Cell Biol, 3, 349-63.

TROJANOWSKA, M., LEROY, E. C., ECKES, B. & KRIEG, T. (1998) Pathogenesis of fibrosis: type 1 collagen and the skin. J Mol Med, 76, 266-74.

TSAKIRI, K. D., CRONKHITE, J. T., KUAN, P. J., XING, C., RAGHU, G., WEISSLER, J. C., ROSENBLATT, R. L., SHAY, J. W. & GARCIA, C. K. (2007) Adult-onset pulmonary fibrosis caused by mutations in telomerase. Proc Natl Acad Sci U S A, 104, 7552-7.

TSANTES, A., TASSIOPOULOS, S., PAPADHIMITRIOU, S. I., BONOVAS, S., KAVALIEROU, L., VAIOPOULOS, G. & MELETIS, I. (2003) Suboptimal erythropoietic response to hypoxemia in idiopathic pulmonary fibrosis. Chest, 124, 548-53.

TURKSEN, K., KUPPER, T., DEGENSTEIN, L., WILLIAMS, I. & FUCHS, E. (1992) Interleukin 6: Insights to Its Function in Skin by Overexpression in Transgenic Mice. PNAS, 89, 5068-5072.

UITTO, J. & KOUBA, D. (2000) Cytokine modulation of extracellular matrix gene expression: relevance to fibrotic skin diseases. J Dermatol Sci, 24 Suppl 1, S60-9.

UYTTENHOVE, C., COULIE, P. G. & VAN SNICK, J. (1988) T cell growth and differentiation induced by interleukin-HP1/IL-6, the murine hybridoma/plasmacytoma growth factor. J. Exp. Med., 167, 1417-1427.

VANCHERI, C., SORTINO, M. A., TOMASELLI, V., MASTRUZZO, C., CONDORELLI, F., BELLISTRI, G., PISTORIO, M. P., CANONICO, P. L. & CRIMI, N. (2000) Different expression of TNF-alpha receptors and prostaglandin E(2)Production in normal and fibrotic lung fibroblasts: potential implications for the evolution of the inflammatory process. Am J Respir Cell Mol Biol, 22, 628-34.

VANHEE, D., GOSSET, P., WALLAERT, B., VOISIN, C. & TONNEL, A. B. (1994) Mechanisms of fibrosis in coal workers' pneumoconiosis. Increased production of platelet-derived growth factor, insulin-like growth factor type I, and

243

transforming growth factor beta and relationship to disease severity. Am J Respir Crit Care Med, 150, 1049-55.

VASAKOVA, M., STRIZ, I., SLAVCEV, A., JANDOVA, S., DUTKA, J., TERL, M., KOLESAR, L. & SULC, J. (2007) Correlation of IL-1alpha and IL-4 gene polymorphisms and clinical parameters in idiopathic pulmonary fibrosis. Scand J Immunol, 65, 265-70.

VASSILAKIS, D. A., SOURVINOS, G., SPANDIDOS, D. A., SIAFAKAS, N. M. & BOUROS, D. (2000) Frequent genetic alterations at the microsatellite level in cytologic sputum samples of patients with idiopathic pulmonary fibrosis. Am J Respir Crit Care Med, 162, 1115-9.

VERGNON, J. M., VINCENT, M., DE THE, G., MORNEX, J. F., WEYNANTS, P. & BRUNE, J. (1984) Cryptogenic fibrosing alveolitis and Epstein-Barr virus: an association? Lancet, 2, 768-71.

VERRECCHIA, F., CHU, M. L. & MAUVIEL, A. (2001) Identification of novel TGF-beta /Smad gene targets in dermal fibroblasts using a combined cDNA microarray/promoter transactivation approach. J Biol Chem, 276, 17058-62.

VERRECCHIA, F. & MAUVIEL, A. (2007) Transforming growth factor-beta and fibrosis. World J Gastroenterol, 13, 3056-62.

VERRECCHIA, F., MAUVIEL, A. & FARGE, D. (2006) Transforming growth factor-beta signaling through the Smad proteins: role in systemic sclerosis. Autoimmun Rev, 5, 563-9.

WALTERS, D. M., ANTAO-MENEZES, A., INGRAM, J. L., RICE, A. B., NYSKA, A., TANI, Y., KLEEBERGER, S. R. & BONNER, J. C. (2005) Susceptibility of signal transducer and activator of transcription-1-deficient mice to pulmonary fibrogenesis. Am J Pathol, 167, 1221-9.

WANG, J., HOMER, R. J., CHEN, Q. & ELIAS, J. A. (2000a) Endogenous and exogenous IL-6 inhibit aeroallergen-induced Th2 inflammation. J Immunol, 165, 4051-61.

WANG, J., HOMER, R. J., HONG, L., COHN, L., LEE, C. G., JUNG, S. & ELIAS, J. A. (2000b) IL-11 selectively inhibits aeroallergen-induced pulmonary eosinophilia and Th2 cytokine production. J Immunol, 165, 2222-31.

WANG, R., RAMOS, C., JOSHI, I., ZAGARIYA, A., PARDO, A., SELMAN, M. & UHAL, B. D. (1999) Human lung myofibroblast-derived inducers of alveolar epithelial apoptosis identified as angiotensin peptides. Am J Physiol, 277, L1158-64.

WATANABE, M., MATSUYAMA, W., SHIRAHAMA, Y., MITSUYAMA, H., OONAKAHARA, K., NOMA, S., HIGASHIMOTO, I., OSAME, M. & ARIMURA, K. (2007) Dual effect of AMD3100, a CXCR4 antagonist, on bleomycin-induced lung inflammation. J Immunol, 178, 5888-98.

WAXMAN, A. B., EINARSSON, O., SERES, T., KNICKELBEIN, R. G., WARSHAW, J. B., JOHNSTON, R., HOMER, R. J. & ELIAS, J. A. (1998) Targeted Lung Expression of Interleukin-11 Enhances Murine Tolerance of 100% Oxygen and Diminishes Hyperoxia-induced DNA Fragmentation. J. Clin. Invest., 101, 1970-1982.

WEINSTEIN, M., YANG, X. & DENG, C. (2000) Functions of mammalian Smad genes as revealed by targeted gene disruption in mice. Cytokine Growth Factor Rev, 11, 49-58.

WHITE, E. S., LAZAR, M. H. & THANNICKAL, V. J. (2003) Pathogenetic mechanisms in usual interstitial pneumonia/idiopathic pulmonary fibrosis. J Pathol, 201, 343-54.

WHYTE, M., HUBBARD, R., MELICONI, R., WHIDBORNE, M., EATON, V., BINGLE, C., TIMMS, J., DUFF, G., FACCHINI, A., PACILLI, A., FABBRI,

244

M., HALL, I., BRITTON, J., JOHNSTON, I. & DI GIOVINE, F. (2000) Increased risk of fibrosing alveolitis associated with interleukin-1 receptor antagonist and tumor necrosis factor-alpha gene polymorphisms. Am J Respir Crit Care Med, 162, 755-8.

WHYTE, M. K. (2003) Genetic factors in idiopathic pulmonary fibrosis: transforming growth factor-beta implicated at last. Am J Respir Crit Care Med, 168, 410-1.

WU, T. R., HONG, Y. K., WANG, X. D., LING, M. Y., DRAGOI, A. M., CHUNG, A. S., CAMPBELL, A. G., HAN, Z. Y., FENG, G. S. & CHIN, Y. E. (2002) SHP-2 is a dual-specificity phosphatase involved in Stat1 dephosphorylation at both tyrosine and serine residues in nuclei. J Biol Chem, 277, 47572-80.

WYNN, T. A. (2007) Common and unique mechanisms regulate fibrosis in various fibroproliferative diseases. J. Clin. Invest., 117, 524-529.

XING, Z., BRACIAK, T., JORDANA, M., CROITORU, K., GRAHAM, F. L. & GAULDIE, J. (1994) Adenovirus-mediated cytokine gene transfer at tissue sites. Overexpression of IL-6 induces lymphocytic hyperplasia in the lung. J Immunol, 153, 4059-4069.

XING, Z., GAULDIE, J., COX, G., BAUMANN, H., JORDANA, M., LEI, X. F. & ACHONG, M. K. (1998) IL-6 is an antiinflammatory cytokine required for controlling local or systemic acute inflammatory responses. J Clin Invest, 101, 311-20.

XU, J., MORA, A., SHIM, H., STECENKO, A., BRIGHAM, K. L. & ROJAS, M. (2007) Role of the SDF-1/CXCR4 axis in the pathogenesis of lung injury and fibrosis. Am J Respir Cell Mol Biol, 37, 291-9.

XU, X., BRODIE, S. G., YANG, X., IM, Y. H., PARKS, W. T., CHEN, L., ZHOU, Y. X., WEINSTEIN, M., KIM, S. J. & DENG, C. X. (2000) Haploid loss of the tumor suppressor Smad4/Dpc4 initiates gastric polyposis and cancer in mice. Oncogene, 19, 1868-74.

YAO, L., YOKOTA, T., XIA, L., KINCADE, P. W. & MCEVER, R. P. (2005) Bone marrow dysfunction in mice lacking the cytokine receptor gp130 in endothelial cells. Blood, 106, 4093-101.

YASUKAWA, H., MISAWA, H., SAKAMOTO, H., MASUHARA, M., SASAKI, A., WAKIOKA, T., OHTSUKA, S., IMAIZUMI, T., MATSUDA, T., IHLE, J. & YOSHIMURA, A. (1999) The JAK-binding protein JAB inhibits Janus tyrosine kinase activity through binding in the activation loop. EMBO J, 18, 1309-1320.

YOSHIDA, K., TAGA, T., SAITO, M., SUEMATSU, S., KUMANOGOH, A., TANAKA, T., FUJIWARA, H., HIRATA, M., YAMAGAMI, T., NAKAHATA, T., HIRABAYASHI, T., YONEDA, Y., TANAKA, K., WANG, W.-Z., MORI, C., SHIOTA, K., YOSHIDA, N. & KISHIMOTO, T. (1996) Targeted disruption of gp130, a common signal transducer for the interleukin 6 family of cytokines, leads to myocardial and hematological disorders. PNAS, 93, 407-411.

YOSHIDA, M., SAKUMA, J., HAYASHI, S., ABE, K., SAITO, I., HARADA, S., SAKATANI, M., YAMAMOTO, S., MATSUMOTO, N., KANEDA, Y. & AL., E. (1995) A histologically distinctive interstitial pneumonia induced by overexpression of the interleukin 6, transforming growth factor beta 1, or platelet-derived growth factor B gene. Proc Natl Acad Sci U S A, 92, 9570-9574.

YOSHIDA, T., OGATA, H., KAMIO, M., JOO, A., SHIRASHI, H., TOKUNAGA, Y., SATA, M., NAGAI, H. & YOSHIMURA, A. (2004) SOCS1 is a suppressor of liver fibrosis and hepatitis-induced carcinogenensis. Journal of Experimental Medicine.

245

ZAMO, A., POLETTI, V., REGHELLIN, D., MONTAGNA, L., PEDRON, S., PICCOLI, P. & CHILOSI, M. (2005) HHV-8 and EBV are not commonly found in idiopathic pulmonary fibrosis. Sarcoidosis Vasc Diffuse Lung Dis, 22, 123-8.

ZHANG, H. Y., GHARAEE-KERMANI, M. & PHAN, S. H. (1997) Regulation of lung fibroblast alpha-smooth muscle actin expression, contractile phenotype, and apoptosis by IL-1beta. J Immunol, 158, 1392-9.

ZHANG, H. Y., GHARAEE-KERMANI, M., ZHANG, K., KARMIOL, S. & PHAN, S. H. (1996) Lung fibroblast alpha-smooth muscle actin expression and contractile phenotype in bleomycin-induced pulmonary fibrosis. Am J Pathol, 148, 527-37.

ZHANG, J., FARLEY, A., NICHOLSON, S., WILLSON, T., ZUGARO, L., SIMPSON, R., MORITZ, R., CARY, D., RICHARDSON, R., HAUSMANN, G., KILE, B., KENT, S., ALEXANDER, W., METCALF, D., HILTON, D., NICOLA, N. & BACA, M. (1999) The conserved SOCS box motif in suppressors of cytokine signaling binds to elongins B and C and may couple bound proteins to proteasomal degradation. Proc Natl Acad Sci U S A, 96, 2071-2076.

ZHANG, X. L., TOPLEY, N., ITO, T. & PHILLIPS, A. (2005) Interleukin-6 regulation of transforming growth factor (TGF)-beta receptor compartmentalization and turnover enhances TGF-beta1 signaling. J Biol Chem, 280, 12239-45.

ZHAO, J., SHI, W., WANG, Y. L., CHEN, H., BRINGAS, P., JR., DATTO, M. B., FREDERICK, J. P., WANG, X. F. & WARBURTON, D. (2002) Smad3 deficiency attenuates bleomycin-induced pulmonary fibrosis in mice. Am J Physiol Lung Cell Mol Physiol, 282, L585-93.

ZHOU, L., DEY, C. R., WERT, S. E. & WHITSETT, J. A. (1996) Arrested lung morphogenesis in transgenic mice bearing an SP-C-TGF-beta 1 chimeric gene. Dev Biol, 175, 227-38.

ZHU, Y., RICHARDSON, J. A., PARADA, L. F. & GRAFF, J. M. (1998) Smad3 mutant mice develop metastatic colorectal cancer. Cell, 94, 703-14.

ZIESCHE, R., HOFBAUER, E., WITTMANN, K., PETKOV, V. & BLOCK, L. H. (1999) A preliminary study of long-term treatment with interferon gamma-1b and low-dose prednisolone in patients with idiopathic pulmonary fibrosis. N Engl J Med, 341, 1264-9.