the role of gp130/il-6 cytokines in the development of pulmonary fibrosis: critical determinants of...

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Pharmacology & Therapeutics 99 (2003) 327–338

The role of gp130/IL-6 cytokines in the development of pulmonary

fibrosis: critical determinants of disease susceptibility and progression?

Darryl A. Knighta,b,*, Matthias Ernstc, Gary P. Andersond,e,Yuben P. Moodleya,b, Steven E. Mutsaersa,b

aAsthma and Allergy Research Institute, Sir Charles Gairdner Hospital, Ground Floor, ‘‘E’’ Block, Verdun Street, Nedlands, Western Australia, 6009, AustraliabDepartment of Medicine, University of Western Australia, Crawley, Western Australia, 6972, AustraliacLudwig Institute for Cancer Research, Royal Melbourne Hospital, Melbourne, Victoria, 3050, Australia

dDepartment of Pharmacology, University of Melbourne, Parkville, Victoria, 3082, AustraliaeDepartment of Medicine, University of Melbourne, Parkville, Victoria, 3082, Australia

Abstract

Cryptogenic fibrosing alveolitis (CFA), also known as idiopathic pulmonary fibrosis (IPF), is the end stage of a heterogeneous group of

disorders in which the deposition of excessive amounts of collagen results in the loss of lung function and premature death. The molecular

mechanisms underlying the disease are unknown. Accordingly, there is much debate as to whether pulmonary fibrosis is the end result of (1)

a chronic inflammatory process or (2) a disturbance in normal epithelium-fibroblast cross talk, or both. In addition, it appears increasingly

likely that there is a genetic component in the development of pulmonary fibrosis. The IL-6 cytokine family is a group of pleiotropic

mediators produced by a variety of cells in response to a inflammatory stimuli. These cytokines are grouped together on the basis of weak

structural homology, overlapping functions, and shared use of the transmembrane glycoprotein b-subunit gp130 as part of their multimeric

receptor complexes. Activation of these receptor complexes results in the recruitment and phosphorylation of specific transcription factors. In

addition, membrane-proximal tyrosine residues act as docking sites for molecules involved in the activation of extracellular signal-related

kinase (ERK). However, studies in genetically engineered mice that overexpress members of this family have shown that while overlapping

biological activities exist, there are effects specific to individual cytokines. Data from both human and animal studies are now emerging to

suggest that members of this cytokine family play an important role in the pathogenesis of fibroproliferative diseases and thus represent a

novel group of cytokines implicated in pulmonary fibrosis. Importantly, manipulation of signaling pathways activated by these cytokines may

suppress fibrosis but leave innate cellular mechanisms necessary for host defense largely untouched. This may provide guides for the

development of novel pharmacological treatment for fibroproliferative diseases.

D 2003 Elsevier Inc. All rights reserved.

Keywords: Lung; Fibrosis; IL-6; gp130; Cytokine; Wound repair

Abbreviations: CFA, cryptogenic fibrosing alveolitis; CT-1, cardiotrophin-1; CNTF, ciliary neurotrophic factor; DLCO, carbon monoxide transfer potential;

ECM, extracellular matrix; ERK, extracellular signal-regulated kinase; gp130, glycoprotein 130; IPF, idiopathic pulmonary fibrosis; JAK, Janus kinase; LIF,

leukemia inhibitory factor; MAPK, mitogen-activated protein kinase; OSM, oncostatin M; PGE, prostaglandin E; PIAS, protein inhibitors of activated STAT;

SOCS, suppressors of cytokine signaling; STAT, signal transducers and activators of transcription; TGFb, transforming growth factor b

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328

2. The gp130/interleukin-6 family of cytokines. . . . . . . . . . . . . . . . . . . . . . . . . . . 328

3. gp130-dependent intracellular signal transduction pathways . . . . . . . . . . . . . . . . . . . 329

4. Negative regulators of intracellular gp130 signaling . . . . . . . . . . . . . . . . . . . . . . . 330

0163-7258/03/$ – see front matter D 2003 Elsevier Inc. All rights reserved.

doi:10.1016/S0163-7258(03)00095-0

* Corresponding author. Asthma and Allergy Research Institute, Ground Floor, ‘‘E’’ Block, Sir Charles Gairdner Hospital, Verdun Street, Nedlands,

Western Australia, 6009, Australia. Fax: +61-8-9346-2816.

E-mail address: [email protected] (D.A. Knight).

D.A. Knight et al. / Pharmacology & Therapeutics 99 (2003) 327–338328

4.1. The suppressor of cytokine signaling family of proteins . . . . . . . . . . . . . . . . . 330

4.2. The protein inhibitors of activated signal transducers and activators of transcription

proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330

4.3. Effect of SHP2 regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330

5. Cryptogenic fibrosing alveolitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331

6. The central role of the myofibroblast in cryptogenic fibrosing alveolitis . . . . . . . . . . . . 331

7. Current therapeutic strategies used in cryptogenic fibrosing alveolitis . . . . . . . . . . . . . . 331

8. In vitro human data suggest that interleukin-6/gp130 cytokines may play an important

role in cryptogenic fibrosing alveolitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332

8.1. Interleukin-6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332

8.2. Interleukin-11. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332

8.3. Oncostatin M . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333

8.4. Leukemia inhibitory factor, ciliary neurotrophic factor, and cardiotrophin-1 . . . . . . . 333

9. Investigation with genetically engineered mice suggest that cytokines that signal through

gp130 are important in fibrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333

9.1. Interleukin-6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333

9.2. Interleukin-11. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333

9.3. Oncostatin M . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333

9.4. Leukemia inhibitory factor, ciliary neurotrophic factor, and cardiotrophin-1 . . . . . . . 334

9.5. gp130 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334

9.6. Effect of individual mutations in gp130 . . . . . . . . . . . . . . . . . . . . . . . . . 334

10. Evidence for a genetic component in cryptogenic fibrosing alveolitis . . . . . . . . . . . . . . 334

11. Genetic influences in interleukin-6/gp130 cytokine signaling in cryptogenic fibrosing alveolitis 335

12. Summary and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335

1. Introduction

Fibrosis can be defined as an excessive deposition of

extracellular matrix components that results in the destruc-

tion of normal tissue architecture and a compromise in tissue

function. If fibrosis occurs in the major organs such as lung,

it will inevitably lead to organ failure and death of the

individual.

The development of fibrosis appears to follow a similar

pathway to that of normal wound healing; however, in most

cases, there is a chronic progression of the disease without

resolution. Clearly, the normal fine control of cell function

that occurs during normal healing is disturbed. Current

therapies for fibrosis aim to inhibit inflammation, but this

is effective in only a small subset of patients. There are

currently no proven therapies targeting the fibrotic response

itself. A significant research effort is being directed towards

establishing the relative roles of growth factors and cyto-

kines that may determine the pathogenesis and outcomes of

the disease. Support for this strategy is derived from a joint

statement produced by the American Thoracic and Europe-

an Respiratory Societies in which the roles of various

growth factors and cytokines were described as ‘‘critical’’

to the process of fibrosis.

The interleukin (IL)-6/gp130 family of cytokines has the

potential to exert endocrine, autocrine, and paracrine effects

within the lung and therefore regulate local inflammatory

and subsequent healing events. The aim of this manuscript

is to review current knowledge on the role that cytokines,

which signal through gp130, play in the susceptibility to and

progression of pulmonary fibrosis.

2. The gp130/interleukin-6 family of cytokines

The IL-6 family of cytokines is a group of related

pleiotropic acting cytokines that are produced by a variety

of cells in response to inflammatory stimuli (Kishimoto et

al., 1995; Heinrich et al., 1998). Individual family members

play pivotal roles in the immune, nervous, cardiovascular,

and hemopoietic systems as well as bone metabolism,

inflammation, wound repair, acute phase response, and

development of the embryo (Hirota et al., 1995; Hirano et

al., 1997; Taga, 1997; Betz et al., 1998). The cytokine family

includes IL-6, leukemia inhibitory factor (LIF), oncostatin M

(OSM), IL-11, ciliary neurotrophic factor (CNTF), and

cardiotrophin-1 (CT-1) (Table 1). These cytokines share a

weak structural homology, because they comprise single

chain polypeptides with predicted molecular masses of 20

kb. All, with the exception of CNTF and CT-1, are secreted

proteins that are synthesized with N-terminal signal peptides

(Heinrich et al., 1998). However, all IL-6 family cytokines

engage the common transmembrane glycoprotein b-subunitgp130 as part of a multimeric (a and b) receptor complex

(Fig. 1). By itself, gp130 does not provide high-affinity

binding to any of the IL-6 family cytokines but rather

converts low-affinity binding to ‘‘specific’’ a-subunit recep-tors into a high-affinity receptor complex. In the case of IL-6,

Table 1

Properties of IL-6 cytokines and their receptors (modified from Heinrich et al., 1998 with permission)

Property IL-6 IL-6R IL-11 IL-11R LIF LIFR OSM OSMR CNTF CNTFR CT-1 gp130

Number of amino acids 184 449 178 400 180 1053 196 952 200 352 201 896

Molecular mass 21–28 80 23 43.1 45 190 28 180 21–28 72 21.5 130

mRNA (kb) 1.3 5 1.5 1.9 4 2 2 < 5 1 2 1.7 7

Number of exons 5 ? 5 12 3 20 3 ? 2 4 3 17

Chromosome

localization (human)

7p21-14 1q21 19q13 9p13 22q14 5p12 22q12 5p15 11q12 9p13 16p11 5q11

D.A. Knight et al. / Pharmacology & Therapeutics 99 (2003) 327–338 329

IL-11, and CNTF, the respective b-chains are completely

dispensable for initiation of intracellular signaling events,

which is initiated by ligand-mediated dimerization of b-chains (Heinrich et al., 1998).

The shared use of the signal transducing subunit gp130

as part of any of the corresponding multimeric receptor

complexes provides a molecular mechanism to explain

extensive functional redundancy of individual members of

the IL-6 family of ligands. Within this family, LIF and OSM

are the most closely related. This is based on both an

identical exon organization and the close proximity of their

respective genes on chromosome 22q12.2 (Jeffery et al.,

1993). These cytokines also share usage of the LIF receptor

despite the presence of a specific OSM receptor. Both LIF

and OSM are also secreted as a glycosylated protein.

3. gp130-dependent intracellular signal transduction

pathways

Following ligand-mediated gp130 homodimerization or

heterodimerization, juxtapositioning of the cytoplasmic Ja-

nus kinases (JAK), which are constitutively associated with

the receptor b-chains, results in activation and subsequent

phosphorylation of highly conserved cytoplasmic tyrosine

Fig. 1. Association of IL-6 and related cytokines with the gp130 signal transducing

or heterodimers with specific a-subunits (LIF, OSM, CNTF, and CT-1). Within th

gp130 or the LIFR/gp130 complex.

residues in gp130, LIFRb, and OSMRb (Heinrich et al.,

1998). Although gp130 associates with and leads to activa-

tion of Jak1, Jak2, and Tyk2, knockout studies suggest that in

most cell types Jak1 is the critical kinase for gp130-mediated

signal transduction (Rodig et al., 1998). The subsequent

activation of intracellular signaling is dependent on phos-

photyrosine residues in the b-chains, which act as docking

sites for SH2 domain containing intermediate signaling

molecules. The cytoplasmic protein tyrosine phosphatase

SHP2, for instance, binds to a membrane-proximal tyrosine

residue in the b-chains and is necessary and sufficient for

activation of the extracellular signal-regulated kinase (ERK)/

mitogen-activated protein kinase (MAPK) via the adaptor

proteins Grb2 and/or Gab (Stahl et al., 1995; Hibi & Hirano,

2000). In contrast, two members of the family of signal

transducers and activators of transcription (STAT), namely

STAT-1 and STAT-3, dock to several of the membrane-distal

phosphotyrosine residues (4 in gp130 and 3 in LIFRb) (Fig.2) (Lutticken et al., 1994; Stahl et al., 1994). Once tyrosine

phosphorylated, these latent transcription factors form either

homodimers or heterodimers, translocate to the nucleus, and

transactivate target genes in a DNA sequence-specific con-

text (Zhong et al., 1994; Ernst et al., 1996; Fukada et al.,

1996; Giordano et al., 1997; Hirano et al., 1997; Schiemann

et al., 1997; Ohtani et al., 2000; reviewed in Heinrich et al.,

subunit. gp130 (shown in gray) forms either homodimers (IL-6 and IL-11)

is family, OSM can interact with its own specific receptor complex OSMR/

Fig. 2. Signal transduction through gp130. Following ligand-mediated homodimerization or heterodimerization, juxtapositioning of the cytoplasmic JAK

results in activation and subsequent phosphorylation of highly conserved cytoplasmic tyrosine residues in gp130, LIFRb, and OSMRb. The subsequent

activation of intracellular signaling is dependent on particular phosphotyrosine residues. SHP2 binds to a membrane-proximal tyrosine residue and results in

activation of the ERK/MAPK. In contrast, STAT-1 and STAT-3 dock to membrane-distal phosphotyrosine residues. Once tyrosine phosphorylated, these latent

transcription factors form either homodimers or heterodimers, translocate to the nucleus, and transactivate target genes in a DNA sequence-specific context.


1998). Among the best characterized STAT-3 target genes are

hepatic acute phase response genes (Wegenka et al., 1993).

4. Negative regulators of intracellular gp130 signaling

4.1. The suppressor of cytokine signaling family of proteins

One of the key factors involved in the negative re-gulation

of gp130 signaling is the family of suppressor of cytokine

signaling (SOCS) proteins (Starr & Hilton, 1999). Generally,

individual SOCS genes are transcriptionally activated in

response to cytokine stimulation, often via STAT-mediated

mechanisms, to inhibit cytokine signaling via a classical

negative feedback loop. The SOCS proteins inhibit phos-

phorylation of receptors and their associated intermediate

signaling proteins (i.e., STAT) by at least two distinct mech-

anisms. SOCS proteins can interact with the catalytic

domains of JAK proteins and inhibit kinase activity (Yasu-

kawa et al., 1999). In addition, the SOCS proteins bind to

elongins, which form part of the E3 ubiquitin-ligase com-

plex that targets ubiquinated proteins for proteosome-me-

diated degradation (Zhang et al., 1999). Although all SOCS

proteins can inhibit STAT phosphorylation, the mechanisms

by which individual SOCS proteins inhibit STAT activity

vary. While SOCS-1 and SOCS-3 both inhibit JAK activity,

SOCS-1 binds directly to JAK with high affinity and

inhibits tyrosine kinase activity (Endo et al., 1997; Naka

et al., 1997). In contrast, SOCS-3 appears to require

interaction with receptors, such as gp130, for recruitment

to the signaling complex (Nicholson et al., 2000).

4.2. The protein inhibitors of activated signal

transducers and activators of transcription proteins

Another group of intracellular inhibitors of gp130 signal-

ing are proteins of the protein inhibitors of activated STAT

(PIAS) family (Chung et al., 1997; Liu et al., 1998). Each of

these proteins is specific for a STAT protein (e.g., PIAS-3

inhibits STAT-3-mediated gene activation) but has no effect

on STAT-1-mediated transcription (Chung et al., 1997).

These proteins associate with activated tyrosine-phosphory-

lated STAT and induce loss of STAT DNA binding activity.

4.3. Effect of SHP2 regulation

Another regulator of gp130 signaling is the protein

tyrosine phosphatase SHP2. Recruitment of this pathway

occurs following activation of the IL-6 receptor complex

with association of SHP2 to tyrosine 759 in the gp130

transmembrane receptor subunit (Stahl et al., 1994). It

appears that SHP2 and SOCS-3 are functionally linked,

with activation of one inhibitor modulating the activity of

the other; for example, inhibition of SHP2 activation results

in increased SOCS-3 mRNAwhile the converse also occurs

(Schmitz et al., 2000). Prevention of SHP2 binding results

in enhanced and prolonged phosphorylation of gp130, Jak,

and STAT, with increased DNA binding by the STAT

proteins and up-regulation of the acute phase response

(Kim et al., 1998).

While most studies confirm a negative regulatory role of

SHP2 on gp130 (Symes et al., 1997; Burdon et al., 1999),

others have found that SHP-2 activation may modulate


gp130 signaling in a positive fashion, possibly through the

MAPK pathway (Fukada et al., 1996). It is possible that

SHP2 will function either as a positive or as a negative

regulator of gp130 signaling, depending on the tissue or cell

type studied.

5. Cryptogenic fibrosing alveolitis

Cryptogenic fibrosing alveolitis (CFA), also known as

idiopathic pulmonary fibrosis (IPF), is a specific form of

chronic diffuse interstitial lung disease of unknown etiol-

ogy (American Thoracic Society, 2000). The disease is

confined to the lung and is characterized pathologically by

areas of inflammation and excessive deposition of extra-

cellular matrix (ECM) in the lung parenchyma (King et al.,

2001; Selman et al., 2001). The disease occurs predomi-

nantly from middle age onwards, with two-thirds of

patients older than 60 years of age at the time of presen-

tation. Its incidence varies with ethnicity and is � 7 per

100,000 for women and 10 per 100,000 for Caucasian

men. Although the molecular mechanisms underlying the

disease are unknown, it has been suggested that CFA is

either the end result of (1) a chronic inflammatory pro-

cesses or (2) a disturbance in normal epithelium-fibroblast

cross talk or both. However, by its nature, knowledge of

the pathogenesis of CFA is unknown; thus, the end stage

of the disease may arise through multiple etiologies (Mason

et al., 1999).

A definitive diagnosis of CFA requires open lung biopsy,

and because of typically late presentation and nonspecificity

of clinical features, it remains a diagnosis of exclusion

(American Thoracic Society, 2000; King et al., 2001;

Crystal et al., 2002). Biopsy specimens from individuals

with CFA typically have the appearance of usual interstitial

pneumonia (UIP). The key histological features of IUP are

distinct areas of excessive interstitial collagen deposition

and areas of active fibroblast proliferation called fibroblastic

foci (Mason et al., 1999; American Thoracic Society, 2000;

Crystal et al., 2002). In other forms of interstitial pneumo-

nias, these foci are absent and the disease progression is far

more homogenous.

6. The central role of the myofibroblast in cryptogenic

fibrosing alveolitis

Fibroblastic foci are generally located with the interstitial

space directly beneath the alveolar epithelium often at the

boundary of areas of collagen deposition and normal appear-

ing lung (Bjoraker et al., 1998; Mason et al., 1999). These

foci are characterized by fibroblast/myofibroblast accumu-

lation through proliferation and decreased apoptosis as well

as enhanced release of and response to profibrotic growth

factors. At these sites, there is impaired reepithelialization

and inappropriate deposition of ECM.

Myofibroblasts have a phenotype resembling smooth

muscle cells (Powell et al., 1999). They are contractile,

express markers of the myogenic regulatory family such as

MyoD as well as muscle structural proteins including a-smooth muscle actin (Schmitt-Graff et al., 1994; Zhang et

al., 1994; Serini & Gabbiani, 1999). Myofibroblasts are

central to several normal processes through mesenchymal-

epithelial interactions, are key effectors cells in organogen-

esis and tissue morphogenesis (Leslie et al., 1992; Fries et

al., 1994), and play a central role in wound healing most

probably as a natural extension of their role in normal

growth and differentiation (Bostrom et al., 1996; Holgate

et al., 2000). These cells are pivotal in the formation and

turnover of the ECM as well as proliferation and differen-

tiation of epithelial, vascular (Villaschi & Nicosia, 1994),

and even neural cells. Normal healing is facilitated by the

contractile properties of myofibroblasts, which assists in

reducing the amount of denuded/damaged surface area of

wound tissue.

Myofibroblasts also play a fundamental role in many

disease states, through either activation, proliferation, or

deletion. For example, myofibroblasts play a fundamental

role in inflammation by producing chemokines and cyto-

kines (Berschneider & Powell, 1992; Pang et al., 1994;

Hogaboam et al., 1998a, 1998b) as well as prostanoids,

most notably prostaglandin E (PGE2) (McAnulty et al.,

1995; Gilroy et al., 2001), which may function as endoge-

nous braking mechanism. On activation, myofibroblasts

express cell adhesion molecules, facilitating recruitment

and attachment of inflammatory cells (Pang et al., 1994).

Myofibroblasts also express a large repertoire of antigens,

which serve to bind these cells to and transduce signals from

the ECM. Myofibroblasts are also the main source of ECM

proteins, such as collagen, fibronectin, and tenascin. The

reader is referred to Mutsaers et al. (1997) and Powell et al.

(1999) for excellent reviews of this area.

7. Current therapeutic strategies used in cryptogenic


Initial hypotheses on the progression of CFA involved

the accumulation of inflammatory and immune cells

around the damaged lung parenchymal tissue, and as such,

conventional treatment of CFA has been based on inhibit-

ing inflammation and inducing immunosuppression. How-

ever, aggressive treatment regimens using corticosteroids

and immunosuppressive or cytotoxic agents are only useful

in a small percentage of patients with CFA. In the majority

of cases, the disease remains progressive and irreversible.

The reader is referred to several excellent reviews on the

current therapeutic approaches to the treatment of CFA

(Mason et al., 1999; Selman et al., 2001; Selman, 2003).

A consensus has developed that future therapies should

target the fibroproliferative response or restitution of the

alveolar epithelium. In addition, given the critical role of


specific growth factors (e.g., transforming growth factor b,TGFb) in the initiation and progression of fibrosis, more

targeted approaches need to be developed. In this context,

the relevance of more recently identified cytokines needs to

be assessed.

8. In vitro human data suggest that

interleukin-6/gp130 cytokines may play an

important role in cryptogenic fibrosing alveolitis

8.1. Interleukin-6

IL-6 has a variety of well-documented direct effects

that are potentially relevant to inflammation and remodel-

ing, although its potential role in fibrosis is largely

unknown. IL-6 is released following stimulation by profi-

brogenic growth factors such as TGFb (Eickelberg et al.,

1999a, 1999b) and may even mediate some cellular effects

attributable to these molecules (Roth et al., 1995). IL-6

has been shown to stimulate dermal fibroblasts to produce

collagen, and enhanced production of IL-6 by mononu-

clear cells is observed in systemic sclerosis (Hasegawa et

al., 1999; Kawaguchi et al., 1999). In a recent study, we

have shown that primary cultures of human lung fibroblasts

derived from patients with CFA have abnormal responses to

IL-6 compared with cells derived from normal donors. For

example, IL-6 inhibited the proliferation of normal fibro-

blasts due to the sustained activation of STAT-3 and produc-

tion of the cyclin-dependent kinase inhibitor p19INK4D. In

contrast, IL-6 was mitogenic for fibroblasts derived from

patients with CFA due to the sustained activation of ERK,

Fig. 3. Summary of current research examining the effects of IL-6 on normal-Fb

activation of STAT-3 and production of p19INK4D. In contrast, IL-6 is mitogenic fo

allowing activation of cyclin D1. The ability of IL-6 to lock normal fibroblasts in

enhanced expression of Bax. In contrast, IL-6 significantly inhibited apoptosis of

which in turn inhibited the production of p27Kip1, allowing

activation of cyclin D1 (Moodley et al., 2003a, 2003b). The

ability of IL-6 to lock normal fibroblasts in cell cycle arrest

correlated with its capacity to promote apoptosis via the

enhanced expression of Bax (Moodley et al., 2003a,

2003b). In keeping with its mitogenic potential on CFA

fibroblasts, IL-6 significantly inhibited apoptosis of these

cells by inducing the expression of the antiapoptotic protein

Bcl-2 (Fig. 3). Both the effects on proliferation and

apoptosis were related to the kinetics of ERK and STAT-

3 activation. In normal cells, ERK was only transiently

expressed following IL-6 exposure, whereas STAT-3 was

detected for at least 6 hr. In contrast, in CFA fibroblasts,

ERK remained activated for 12 hr, whereas expression of

STAT-3 was transient.

8.2. Interleukin-11

Apart from fibroblasts, IL-11 is released in large amounts

from epithelial cells, airway smooth muscle cells, and

infiltrating macrophages. Notably, one of the most potent

inducers of IL-11 release is TGFb (Elias et al., 1994), which

is thought to be a central mediator in the development of

fibrosis (Zhang & Phan, 1996; Eickelberg et al., 1999a,

1999b; Chambers et al., 2003). However, human data

examining the effect of IL-11 in CFA are lacking. In our

studies, we have found that exposure to IL-11 induced

proliferation and inhibited apoptosis of both normal and

CFA-derived fibroblasts (Moodley et al., 2003a, 2003b).

These data suggest that although this cytokine family has

many overlapping functions, in specific disease states, their

effects are quite divergent.

and CFA-Fb. IL-6 inhibits proliferation of normal-Fb due to the sustained

r CFA-Fb due to the sustained activation of ERK and inhibition of p27Kip1,

cell cycle arrest correlated with its capacity to promote apoptosis via the

CFA-Fb by inducing the expression of Bcl-2.


8.3. Oncostatin M

OSM was first described as a product of inflammatory

cells such as T-lymphocytes, monocyte/macrophages (Rose

& Bruce, 1991), and more recently neutrophils (Grenier et

al., 1999). A variety of studies have provided indirect

evidence to suggest that OSM may play an important role

in pulmonary fibrosis. In cultured human lung epithelial

cells, OSM is a potent inducer of the protease inhibitors a1antichymotrypsin and a1 protease inhibitor (Cichy et al.,

1998). We have shown that OSM is a cofactor for the

augmented release of two other epithelium-derived mole-

cules, PGE2 and nitric oxide (Knight et al., 2000). This

effect on antiprotease release also extends to fibroblasts,

where OSM has been shown to be a powerful agonist for

the release of tissue inhibitor of metalloprotease (TIMP)-1

(Richards et al., 1993). In dermal fibroblasts, OSM is

mitogenic and this effect is intimately dependent on ERK

activation (Ihn & Tamaki, 2000). In these cells, OSM

induces transcriptional activation of the collagen a2(1)promoter (Ihn et al., 1997) as well as promotes collagen

and glycosaminoglycan production (Duncan et al., 1995).

We have recently demonstrated that in primary cultures of

normal human lung fibroblasts, OSM is mitogenic, inhibits

spontaneous and FasL-induced apoptosis, and induces col-

lagen production (Scaffidi et al., 2002). These effects did

not appear to involve downstream PGE2 or IL-6 production

but were completely suppressed by the ERK inhibitor

PD98059 and the tyrosine kinase inhibitors genestein and

herbimycin.

More recently, Somasundaram et al. (2002) demonstrat-

ed that collagens I, III, and VI could specifically sequester

and bind OSM. This interaction was not shared by other

members of the IL-6/gp130 family. Furthermore, the col-

lagen-bound OSM retained functional activity. These find-

ings suggest that in fibrosis the increased deposition of

collagens may serve to store and prolong the activity of

OSM.

8.4. Leukemia inhibitory factor, ciliary

neurotrophic factor, and cardiotrophin-1

Although human lung fibroblasts synthesize and release

LIF (Knight et al., 1999) and CT-1 (Langdon et al., 2003), a

role for these cytokines in pulmonary fibrosis has not been

described to date.

9. Investigation with genetically engineered mice suggest

that cytokines that signal through gp130 are important

in fibrosis

The use of genetically engineered mice that overexpress

IL-6 cytokines show that while they share overlapping

biological activities there are also distinct effects specific

to each cytokine.

9.1. Interleukin-6

Mice that have been engineered to overexpress IL-6 in

the lung reveal a marked peribronchiolar mononuclear cell

inflammatory response but of a diffuse nature (DiCosmo et

al., 1994). However, these mice appear resistant to airway

hyperresponsiveness and showed only weak signs of fibro-

sis. This is supported by data from studies where human IL-

6 was overexpressed in rat airways (Yoshida et al., 1995). In

these animals, IL-6 induced lymphocytic alveolitis without

marked fibroblast proliferation. Thus, specific contributions

of IL-6 to fibrotic lesions in animal models have not been

forthcoming. However, because IL-6 is an important cofac-

tor for inflammatory cell recruitment and activation, the

contribution of IL-6 via this indirect mechanism may have

been overlooked. In support of this, IL-6, together with

TNFa, modulates expression of the profibrotic chemokine

MIP1a (Smith et al., 1998). However, the effects of IL-6

appeared to be temporal in as much as neutralizing anti-

bodies to IL-6 only influenced MIP1a expression if admin-

istered early during the inflammatory phase.

9.2. Interleukin-11

Mice with targeted overexpression of IL-11 to the lung

also show areas of peribronchiolar mononuclear cell

inflammation. However, in contrast to IL-6, these mice

also reveal marked deposition of types I and III collagen

and a concomitant increase in the number of myofibro-

blasts, suggesting that IL-11 may play a central role in

the developing a fibrotic response (Tang et al., 1996; Ray

et al., 1997).

9.3. Oncostatin M

Recently, evidence has accumulated to suggest that OSM

possesses intrinsic fibrogenic activity and may play an

important role in wound healing.

Overexpression of OSM into pancreatic islets of trans-

genic mice produced a phenotype with many similarities to

connective tissue disease, including profound fibrosis and

lymphocytic accumulation (Bamber et al., 1998). The profi-

brotic potential of OSM was not related to downstream

production of mediators such as TGFb, PDGF, or bFGF.

This finding has been supported by Richards et al. (2002)

using intranasal administration of adenoviral constructs to

induce local overexpression of OSM in the lung. In this

model, overexpression of OSM led to a profound elevation

in neutrophil numbers, peribronchial mononuclear cell in-

filtration, and increased deposition of collagen adjacent to

areas of inflammation. Within the fibrotic lesions, cells

staining positive for a-smooth muscle actin (presumably

myofibroblasts) were observed and persisted for at least 70

days. The fibrotic changes observed in these animals may

involve effects of OSM on collagen synthesis or direct

regulation of TIMP-1 expression or a combination of both.


9.4. Leukemia inhibitory factor, ciliary neurotrophic factor,

and cardiotrophin-1

In knockout mice for these cytokines, all three cyto-

kines have been shown to play important roles for the

development of motor and sensory neurons during em-

bryogenesis (Li et al., 1995; Cheng & Patterson, 1997;

Cafferty et al., 2001). However, studies examining their

potential role in fibrosis have been lacking. Bamber et al.

(1998) examined the effect of local overproduction of LIF

in the pancreas and unlike the results observed for OSM

could find no evidence of fibrosis response in response to

LIF.

9.5. gp130

Mice generated with targeted deletion of gp130 are

embryonically lethal. Embryos die due to hypoplasia of

the ventricular myocardium and greatly reduced numbers

of hemopoietic progenitors (Yoshida et al., 1996). How-

ever, mice generated with inducibly inactivated gp130 via

Cre-loxP-mediated recombination exhibited pulmonary

defects and lung derangement, although the early observa-

tions of these mice suggest that alveolar enlargement,

resembling human emphysema, is a prominent feature

(Betz et al., 1998). In contrast, continuous activation of

gp130 in vivo is associated with myocardial hypertrophy

(Hirota et al., 1995). Evidence of fibrosis has not been

reported.

9.6. Effect of individual mutations in gp130

A number of in vitro studies indicate that gp130-medi-

ated activation of the SHP2/ERK pathway results in the

transduction of proliferative signals. In contrast, STAT-1 and

STAT-3 activation appears to be critical for regulation of

cellular differentiation, apoptosis, and gene transcription

associated with terminally differentiated cells. Recently,

these studies have been refined using murine gene targeting

strategies to introduce subtle mutations into the individual

signaling ‘‘modules’’ of gp130, permitting the dissection of

the individual contribution of STAT-1/3 and the SHP2/ERK

cascades, respectively.

The SHP2 signal-deficient mice (gp130F757) displayed

splenomegaly, lymphadenopathy, enhanced acute phase

response, and spontaneous development of gastric adeno-

mas (Tebbutt et al., 2002). By contrast, gp130DSTAT mice,

which carry an engineered truncation mutation in gp130

that selectively abolishes all STAT-mediated signaling,

developed gastrointestinal ulceration and impaired colonic

mucosal wound healing and showed compromised immune

and acute phase responses and failure of blastocyst im-

plantation (Ernst et al., 2001). Surprisingly, these studies

revealed a tight reciprocal negative regulation between

JAK-STAT and SHP2/ERK pathways in response to

gp130 activation, suggesting that balanced signaling from

each pathway is critical for the generation of physiological

responses to IL-6 family cytokines (Ohtani et al., 2000).

Hence, some of the pathology noted in gp130DSTAT or

gp130757F mice is not observed in mice lacking all gp130-

dependent signaling to a particular IL-6 family ligand. We

are currently undertaking experiments aimed at determin-

ing whether these subtle and targeted alterations in gp130

signaling have effects in the development of bleomycin-

induced fibrosis.

10. Evidence for a genetic component in cryptogenic


It appears increasingly likely that there is a genetic

predisposition to CFA. This hypothesis is supported by the

existence of familial forms of the disease (Marshall et al.,

1997, 2000). In addition, there is often marked variation

in the fibrotic response observed between individuals

exposed to particulates and chemotherapeutic agents, such

as asbestos and bleomycin, despite similar levels of

exposure.

The pattern of inheritance is not certain but appears to

be autosomal dominant with variable penetrance. Further-

more, there does not appear to be sex linkage, because the

male/female ratio is close to 1:1. 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 (Hermansky et al., 1975) and familial hypercal-

cemic hypocaluria (Auwerx et al., 1985) have been

reported.

Genetic studies examining the etiology of pulmonary

fibrosis have focused on known genetic loci with a high

degree of polymorphism and those involved in the inflam-

matory response. Of these, the HLA system, located on

chromosome 6, has been the most widely studied. However,

none of these studies have been conclusive and the majority

has provided conflicting results.

More recently, microsatellite instability and loss of het-

erozygosity has been demonstrated in DNA from patients

with familial CFA, suggesting that allelic imbalance may

occur in the disease (Thomas et al., 2002). The loss of

heterozygosity was shown to occur in microsatellite markers

immediately adjacent to putative tumor suppressor genes.

Given that the relative risk of developing lung cancer is 7

times higher if CFA is also diagnosed, these data suggest

that DNA damage or dysfunctional repair may be involved

in the pathogenesis of the disease.

Perhaps the most compelling argument for a genetic

component to CFA is found in studies using fibrosis-prone

and fibrosis-resistant strains of mice. C57BL/6 and CBA

mice respond to fibrotic agents such as bleomycin or

radiation with a profound lung fibrosis. In contrast,

BALB/c and C3H/He mice develop very little fibrosis

(Ward et al., 1989; Brass et al., 1999; Kolb et al., 2002).


11. Genetic influences in interleukin-6/gp130

cytokine signaling in cryptogenic fibrosing alveolitis

Although IL-6 may promote fibrogenesis and levels of

IL-6 are markedly increased in BAL fluid from patients with

CFA compared with sarcoidosis (Shahar et al., 1996), a

genetic component to the role of IL-6/gp130 cytokines in

the pathogenesis of CFA remains largely unexplored. How-

ever, genetic studies in mice have shown strong association

between IL-6 and the development of fibrosis (Baecher-

Allan & Barth, 1993). Perhaps more convincingly, in a study

of 74 patients, analysis of TNFa, TNFa receptor, lympho-

toxin-1, and IL-6 revealed that the only genotype associated

with impaired carbon monoxide transfer (DLCO) and CFA

was an intronic polymorphism in the IL-6 gene (Pantelidis et

al., 2001). The functional significance of this is unknown.

However, this allele is in tight linkage disequilibrium with

another polymorphism in the promoter region of IL-6

associated with lower levels of cytokine expression. These

data would suggest that the reduction in DLCO observed in

the pathogenesis of CFA is associated with lower levels of

IL-6. To our knowledge, similar studies on other members of

this cytokine family have not been conducted.

12. Summary and future directions

Current thinking suggests that the pathogenesis of CFA

involves an influx of inflammatory and immune cells,

which, together with activated resident cells, release medi-

ators to induce fibroblasts to proliferate and produce excess

collagen. However, it remains unclear why a particular

insult results in an ongoing fibrotic response rather than

self-limiting wound healing. Current therapies to treat CFA

aim to inhibit inflammation, but this is effective in only a

small subset of patients. Under normal conditions, the

progressive repair of damaged tissue is orchestrated, at least

in part, by a cascade of cytokines that stimulate, maintain, or

down-regulate inflammatory events. Given that many of the

cytokines and growth factors involved in normal wound

healing and fibrosis are the same, it is clear that the normal

control of cell function is dysregulated. Therefore, the key

lies in dissecting the molecular mechanisms that occur

downstream of receptor binding by these mediators. The

IL-6/gp130 family represents a novel group of cytokines

involved in the pathogenesis of CFA. There is emerging

evidence to suggest that cytokines, which signal through

gp130, may be critical determinants of disease susceptibility

and progression and that aberrant signaling from gp130 is a

likely determinant of CFA operating independently from

airway inflammation. There are currently no approved

therapies targeting the fibrotic response itself. However,

manipulation of gp130 signaling pathways that would

suppress fibrosis but leave intact cellular mechanisms nec-

essary for host defense may provide guides for novel

pharmacological intervention.

Acknowledgments

This work was supported by the National Health and

Medical Research Council of Australia. YM is a recipient of

the SAPS/3M respiratory fellowship.

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