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Cellular Players in Lung Fibrosis

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 Current Pharmaceutical Design, 2012, 18, 4093-4102 4093

Cellular Players in Lung Fibrosis

Annemarie N. Lekkerkerker, Jamil Aarbiou, Thomas van Es, Richard A.J. Janssen*

Galapagos BV, P.O. Box 127, 2300 AC Leiden, The Netherlands

Abstract: Pathogenic mechanisms involved in fibrosis of various organs share many common features. Myofibroblasts are thought to
play a major role in fibrosis through excessive deposition of extracellular matrix during wound healing processes. Myofibroblasts are ob-
served in fibrotic lesions, and whereas these derive from the hepatic stellate cells in liver, in lung they appear to originate from fibro-
blasts. The source of these fibroblasts has been the object of numerous studies over the recent years and points towards multiple sources.
First of all, resident fibroblasts are thought to differentiate into the more contractile myofibroblasts, secreting many extracellular matrix
proteins. Secondly, the epithelial to mesenchymal transition (EMT) of epithelial cells may also account for increased numbers of fibro-
blasts, though in vivo evidence in patient tissue.is still scarce. Thirdly, the enigmatic fibrocytes, stemming from the bone marrow, may
also account for increasing numbers of fibroblasts in fibrotic lesions. These pathogenic processes are further augmented by the generation
of so-called alternatively activated macrophages, which have direct and indirect effects on myofibroblast accumulation and collagen
deposition. TGF, which is produced predominantly by macrophages, plays a central role in all these processes by inducing EMT, driv-
ing differentiation of fibrocytes, and differentiation towards myofibroblasts.
This review describes the potential origins and roles of these fibrotic cells in the lung and discusses models to study these cells in vitro.
These models offer innovative approaches in target and drug discovery, aiming to uncover novel therapeutic targets that regulate the pro-
fibrotic phenotype of these cells.
Keywords: Fibrosis, IPF, EMT, epithelial cell, fibroblasts, fibrocytes, macrophages, TGF.

INTRODUCTION inflammatory and repair responses. Interestingly, hepatocytes and

There is considerable overlap between liver fibrosis and lung
fibrosis. Both diseases most likely originate from damage to the
organ. While in the liver this may be due to viral infection, toxic
compounds, or metabolic stress, in the lung the damage may be due
to viral or bacterial infection, smoking, or exposure to allergens. In
both tissues, the damage induces a cascade of cellular responses and
molecular processes, trying to control the damage, to fight the in-
fection, and to repair the damaged tissue. In most instances, these
processes are well controlled leading to proper healing without any
pathological problems. In some cases, the repair process goes awry
and the immune and repair response overreact, leading to inflam-
mation, uncontrolled apoptosis, improper tissue remodeling, and
excessive wound healing.
Fibrotic interstitial lung diseases are characterized by progres-
sive decline in lung function and premature death from respiratory
failure and include amongst others cystic fibrosis and familial pul-
monary fibrosis. In this review we focus mostly on idiopathic pul-
monary fibrosis (IPF), a rare disease with an estimated prevalence
of 14–43 per 100,000 people, depending on the case definition of
IPF. It is a progressive disorder that culminates in premature death
from respiratory failure and in which no treatment intervention has
been effective to date. The only treatment of proven benefit is lung
transplantation. IPF is characterized by progressive scarring of the
lung parenchyma, leading to areas of inflammation, excessive col-
lagen deposition and fibroblast proliferation (fibroblastic foci),
adjacent to areas of normal, uninjured, and seemingly uninvolved
lung. While normal wound healing involves signals to terminate the
accumulation of fibroblasts and the deposition of collagen, in IPF
this process fails.
In both liver and lung a similar cascade of events is induced
leading to fibrosis. The damaged hepatocytes or lung epithelial cells
lose their protective function, including detoxification of the hepa-
tocytes and barrier function of the lung epithelium. As a result,
toxic or microbial insults accumulate and exacerbate the
*Address correspondence to this author at the Galapagos BV, P.O. Box 127,
2300 AC Leiden, The Netherlands; Tel: +31-717506725;
E-mail: Richard.Janssen@glpg.com
biliary epithelial cells in the liver [1, 2] and epithelial cells in the
lung may contribute to fibrosis through the transition of epithelial
cells into mesenchymal cells (epithelial mesenchymal transition,
EMT). These mesenchymal cells can then transdifferentiate into
(myo)fibroblasts.
There is strong evidence that activated fibroblasts and myofi-
broblasts play an important role in fibrosis. Myofibroblasts are
contractile cells expressing -smooth muscle actin (SMA). These
cells contribute to excessive wound healing and to excessive depo-
sition of extracellular matrix proteins (fibronectin, collagen,
laminin). The source of these cells is the focus of intense research.
Several sources of fibroblasts have been put forward, including
proliferation of resident fibroblasts, generation of fibroblasts from
epithelial cells through epithelial mesenchymal transition, and dif-
ferentiation of circulating fibrocytes or mesenchymal progenitor
cells.
Furthermore, current studies indicate that macrophages as well
as fibrocytes are involved in disease progression by mediating fi-
broblast and myofibroblast activation. TGF, derived from macro-
phages and epithelial cells, plays an important role in all these
processes. Below we will discuss the role of the various cell types
and the contribution of TGF in the various pro-fibrotic processes.
The focus of this review is predominantly on the (de)differentiation
processes of the various cells and the profibrotic factors these cells
produce in IPF. (Fig. 1) gives an overview of the various cells dis-
cussed in this paper and how they contribute to the generation of
(myo)fibroblasts.
Epithelium of the Lung
The airway epithelium covers the inner walls of the airways and
serves as the first line of defense against potentially hazardous envi-
ronmental factors and microorganisms. We will first discuss the
physiological function of lung epithelium followed by its role in
fibrosis. The epithelium forms a pseudo stratified cell layer com-
posed of different epithelial cell types. These include basal, ciliated
and goblet cells that form the core of the epithelial layers in trachea,
as well as large and small airway. Basal cells are predominantly
located at the basolateral side and are considered as a pool of pro-

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4094 Current Pharmaceutical Design, 2012, Vol. 18, No. 27
EMT
A
Epithelial cells Fibroblasts
Fibrocyte to fibroblast differentiation
B
Fibrocytes
Fig. (1). Overview of the various cell types in the lung that contribute to fibrosis through the generation of myofibroblasts. 1A: epithelial mesenchymal transi-
tion. 1B: fibrocyte differentiation. 1C: fibroblast to myofibroblast conversion.
genitor cells that proliferate and subsequently can differentiate into
various epithelial cell types and thereby restore the composition and
function of the epithelial layer. Goblet cells contain large vacuoles
filled with mucus. This mucus primarily consists of large glycopro-
teins, called mucins that form a sticky substance. The mucus is
secreted from the vacuoles into the lumen where it traps inhaled
noxious agents.
Ciliated cells are characterized by the presence of a brush of
cilia that move in a coordinated fashion (ciliary beat). In this way,
exogenous agents that are trapped in the mucus, are actively trans-
ported out of the lungs. The more distally located small airways
(terminal bronchioli) also harbor so called Clara cells. These cells
are characterized by the expression of Clara cell secretory protein
(CCSP, also designated CC10) and are thought to have diverse
functions in lung homeostasis including roles in xenobiotic metabo-
lism, immune system regulation, and progenitor cell activity [3].
Like basal cells, Clara cells are believed to act as progenitors for
regeneration of the airway epithelium upon injury [4, 5].
The airways form an immense structure of branching ducts,
ending up in the alveolar spaces separated by alveolar septa. The
alveolar septa consist of a thin single cell wall primarily formed by
two cell types, alveolar type I and type II cells. The branching and
compartmentalization of the alveolar spaces provides an enormous
area enlargement where gas exchange takes place between the lu-
men and capillary vessels in the alveolar septa.
Another way the epithelium provides protection is by its barrier
function. The different cell types that comprise the epithelial layer
form so called tight junctions that restrict free entrance of exoge-
nous agents through the epithelial layer. These tight junctions con-
sist of various intercellular proteins formed by adjacent cells,
thereby creating an epithelial layer that only allows active transport
of molecules through the cells. Invasion of the subepithelial tissue
by inhaled microorganisms may thereby only take place when the
epithelial integrity is disturbed, e.g. by injury, or when microorgan-
isms such as rhinoviruses and respiratory syncytial viruses have
adapted in such a way that they can enter epithelial cells.
The fact that our lungs are not continuously invaded, while we
are constantly exposed to these microorganisms, already indicates
Lekkerkerker et al.
Myofibroblast transition
Myofibroblasts
Myofibroblast transition
Myofibroblast transition
that protective measures of the airway epithelium are quite effec-
tive. Indeed the airway epithelium has a big arsenal of antimicrobial
defense mechanisms. Upon encountering potential invaders, the
airway epithelium reacts by producing and releasing various factors
that help neutralize the invader. The epithelial cells express various
pattern recognition receptors, such as Toll-like receptors (TLRs),
that recognize and bind a variety of viruses and bacteria [6]. The
interaction of pathogens exerts a cascade of signaling pathways,
leading to the production of numerous antimicrobial peptides and
proteins, such as defensins, SLPI, elafin, lysozyme and lactoferrin
[7]. These predominantly cationic molecules show a broad spec-
trum antibiotic capacity and are considered to act as the first line of
defense, while innate, and at a later stage adaptive immune cells,
are recruited. The airway epithelium plays a prominent role in the
recruitment of innate immune cells such as neutrophils, mono-
cytes/macrophages, and eosinophils. They produce CXCL8 (IL-8),
CXCL1 (Gro-), and CXCL5 (ENA-78) to attract neutrophils [8-
10] to the site of inflammation, while monocyte and macrophage
recruitment is induced by the release of IL-1, CCL3 (MIP-1),
CCL2 (MCP-1), and TNF- [6, 9, 11]. Epithelial-derived IL-5,
GM-CSF, CCL11 (eotaxin-1), CCL24 (eotaxin-2), and CCL5
(RANTES) attract eosinophils [12].
Airway epithelial cells also induce migration of adaptive im-
mune cells (such as dendritic cells and T cells) into mucosae by
producing chemokines. These include Th1 chemokines CXCL9
(Mig), CXCL10 (IP-10) and CXCL11 (I-TAC), Th2 chemokines
CCL1 (I-309) CCL17 (TARC) and CCL22 (MDC), as well as DC
chemokines CCL20 (MIP-3) [9, 13].
Besides recruitment of inflammatory cells, the airway epithe-
lium also helps regulate the inflammatory response by producing
several pro- and anti-inflammatory cytokines, such as interferons
(IFN), TNF
, IL-1ß, and other interleukins. These cytokines help
balance the inflammatory response in such a way that it is adequate
enough to eradicate the pathogens without any serious damage to
the surrounding tissue [6]. Although the inflammatory response is
tightly regulated in healthy individuals, in certain disease states the
balance is disturbed, leading to an excess production of (pro)-
inflammatory mediators and subsequently to tissue injury, accom-
panied by a fibrotic process.
Cellular Players in Lung Fibrosis
Epithelium and Fibrosis
Lung fibrosis is a complex physiologic process, initiated upon
lung injury, and is the result of repair processes gone awry. It in-
volves massive deposition of matrix by an expanded pool of fibro-
genic cells, disruption of the normal tissue architecture, and paren-
chymal destruction.
It was long thought that the pool of fibrogenic cells, that are
responsible for the fibrotic process, is solely derived from migrating
and subsequently expanding resident fibroblasts in the mesen-
chyme. Recent findings however, prove that other cell types may
also contribute to this pool. Amongst these, epithelial cells have
been shown to undergo profibrotic phenotypic changes, referred to
as epithelial-mesenchymal transition.
Epithelial-mesenchymal Transition
EMT was already described in the early 1980s [14], constituting
part of embryonic development and organogenesis, and was later
confirmed in studies showing that EMT contributes to the formation
of the mesoderm and the neural crest [15, 16]. EMT is a process in
which epithelial cells lose their epithelial phenotype, acquire fibro-
blast-like properties, and display reduced cell adhesion and in-
creased motility. The reduction of adhesion molecules allows the
cells to detach from the epithelial layer and migrate towards the site
of injury or inflammation where they exercise their profibrotic ef-
fects. EMT has gained quite some interest in the past few years as a
potential contributor to fibrotic diseases and has been described in
various organs. In vitro studies, performed in primary human bron-
chial epithelial cells (HBEC) and primary alveolar epithelial cells
(AEC), as well as in lung-derived epithelial carcinoma cells (A549
and BEAS2B) [17], demonstrated that these cells can be triggered
to undergo EMT [17-20]. These studies showed that epithelial cells
undergoing EMT have lower levels of epithelial cell markers (E-
cadherin, cytokeratins and ZO-1) and increased levels of mesen-
chymal cell markers (collagens I and III, N-cadherin, EDA-
fibronectin, vimentin, S100A4). The collagens and fibronectin also
contribute directly to buildup of excessive amounts of extracellular
matrix. Also the myofibroblast marker -smooth muscle actin
(SMA) was shown to be increased in cells that underwent transi-
tion. Besides these cell markers, other proteins that may play a role
in the fibrotic process were shown to be enhanced upon triggering
of EMT in lung epithelial cells. Various studies have demonstrated
increased production of members of the matrix metalloprotease
(MMP) family [21, 22]. These proteases may affect the fibrotic
process in many ways, due to their capacity to cleave and activate
various growth factors and ECM components. An imbalance of
these proteases and their natural inhibitors, called tissue inhibitors
of metalloproteases (TIMP), may have profound effects on the gen-
eration of injured tissue and subsequent fibrotic processes.
It is well established that TGFß is a key player in the process of
EMT. TGFß, elicited predominantly by activated inflammatory
cells, including macrophages that are attracted to the site of injury,
binds the TGFß type 2 receptor (TGFBR2), which then forms a
complex with TGFß type 1 receptor (TGFBR1). This complex initi-
ates a signaling cascade in which a complex of Smad2 and Smad3
and subsequently Smad4 is activated. The activated complex of
Smads translocates to the nucleus and induces gene transcription.
Although TGFß appears to be essential for EMT, other factors may
influence this process. Inflammatory cytokines, such as IL-1ß and
TNF [21, 22], and also bacteria [23] and viruses [24] were shown
to enhance TGFß-induced markers of EMT, even though the cyto-
kines themselves do not have an EMT inducing capacity. The pre-
cise mechanisms of this enhancement need to be elucidated yet.
EMT in Disease
As already indicated, EMT is a normal physiologic process
during embryogenesis. Whether EMT really contributes to the gen-
eration of fibroblasts and fibrosis is currently intensely debated in
Current Pharmaceutical Design, 2012, Vol. 18, No. 27 4095
the fields of lung, kidney, and liver fibrosis. Using in vivo tracking
studies, conflicting results have been reported for EMT in kidney
fibrosis. Early studies delivered evidence for a crucial role of EMT
in renal fibrogenesis [25-27]. Later studies in kidney showed that
there is no strong evidence for EMT contributing to the formation
of interstitial myofibroblasts and that interstitial pericytes may have
a more important role [28, 29]. However, in an elegant approach,
Kim et al. demonstrated that in mice alveolar epithelial cells, ex-
pressing transgenic lung specific -galactosidase as marker, ac-
quired a mesenchymal phenotype when the animals were exposed
to adenovirally expressed TGF. The cells acquired vimentin and

SMA and were localized in fibronectin rich areas [30]. This dem-
onstrates that fibroblasts indeed can be derived from epithelial cells
through EMT and may contribute to fibrosis. Interestingly, alveolar
type II cells derived from IPF patients express higher levels of
mRNA for profibrotic proteins and when cultured on fibronectin
express
SMA and vimentin in a TGF-dependent manner [31].
Other Pro-fibrotic Effects of Lung Epithelium
The contribution of lung epithelial cells is not restricted to their
property to undergo EMT. Upon challenge, epithelial cells produce
many factors that either have a direct or indirect effect on the fi-
brotic process. Warshamana et al. demonstrated that isolated mur-
ine bronchiolar-alveolar epithelial cells produce factors including
platelet-derived growth factor (PDGF) isoforms A and B and TGFß
[32]. Other studies have confirmed the expression of TGFß in lung-
derived epithelial cells [33, 34]. Here, we will discuss how these
various factors, that are produced by epithelial cells, contribute to
fibrosis.
TGFß is a pleiotropic cytokine that has many versatile effects
on the fibrogenic process in the lung, including but not restricted to
the already discussed potentiation of EMT. TGFß isoforms have
also been shown to regulate deposition of many components of the
ECM components [35] and affect the migratory and differentiation
potential of fibroblasts towards myofibroblasts [36]. TGFß may
also affect the epithelial cell fate itself by promoting epithelial cell
apoptosis [37], as well as migration [30], possibly through a TGFß-
mediated autocrine loop.
PDGF serves as a growth factor for mesenchymal cells and was
shown to be essential in myofibroblast development as PDGF-A
knock-out mice were devoid of alveolar myofibroblast progenitors
[38], while on the other hand, overexpression of PDGF-A, driven
by the surfactant protein C (SPC) promoter in lung epithelium of
transgenic mice, is associated with mesenchymal hyperplasia [39].
In addition, PGDF has the potency to affect fibrogenesis by induc-
ing mesenchymal cell proliferation and migration, and by enhanc-
ing collagen and MMP production [40].
The connective tissue growth factor (CTGF) is another factor
that is produced by lung-derived epithelial cells and which has been
implicated in lung fibrogenesis. Kasai et al. have demonstrated that
TGFß-induced EMT in the lung carcinoma cell line A549 is ac-
companied by increased production of CTGF in cells undergoing
EMT [19]. Also CTGF is considered a profibrotic marker. Various
studies have demonstrated its role in ECM production in a lung
fibrosis model [41], in human renal cells [42], and by gingival fi-
broblasts [43]. Furthermore, CTGF has been shown to promote
fibroblast cell migration, contraction, adhesion, and proliferation
[44-48].
These studies are indicative for the regulatory role of lung
epithelial cells in the fibrogenic process. It has to be noted that the
production of the profibrotic molecules listed above is not solely
produced by epithelial cells. As also discussed elsewhere in this
review, mesenchymal and inflammatory cells may significantly
contribute to their production. The lung epithelium however may
also affect the fibrogenic process indirectly through recruitment
and/or activation of these cells.
4096 Current Pharmaceutical Design, 2012, Vol. 18, No. 27
Fibroblasts
Unlike the tightly organized airway epithelium, the underlying
connective tissue (lamina propria) consists of mesenchymal cells,
bathing in a mesh of ECM. The two layers are separated by a dense
cell free layer of ECM, called basement membrane. The fibroblasts
residing in this layer and the epithelium are in close contact. It is
well established that the two cell types interact in a bidirectional
fashion by producing many factors.
The fibroblast is considered as the classical cell type involved
in fibrosis. Fibroblasts isolated from IPF patients indeed show a
more profibrotic secretory phenotype and fail to invade the ECM,
suggesting that intrinsic defects in the fibroblasts may underlie the
pathogenesis of pulmonary fibrosis [49, 50]. In the presence of
profibrotic stimuli, such as TGFß and ECM components (e.g. colla-
gen I and fibronectin), these cells differentiate into a myofibroblast
phenotype, characterized by expression of SMA, secretion of col-
lagen type I and III, and increased migration and contractility [51].
Fibroblasts from IPF patients behave differently in various assays,
compared to cells from normal donors. For instance, IPF fibroblasts
are relatively resistant to oxidative stress and Fas-induced apoptosis
and this correlates with the expression of anti-apoptotic factors [52,
53]. The group of Riches demonstrated that TNF
sensitizes pri-
mary human lung fibroblasts from IPF patients to FAS-induced
apoptosis [54, 55]. IL-6 enhanced apoptosis in normal fibroblasts
but inhibited apoptosis in IPF cells [56]. Interestingly, PGE2 defi-
ciency appears to protect human primary fibroblasts against apopto-
sis, while enhancing apoptosis in alveolar epithelial cells [57]. Cai
et al. studied motility of human primary fibroblasts from IPF pa-
tients and demonstrated that IPF fibroblasts have slightly enhanced
migratory capacity compared to normal donors which may be due
to the lower levels of FRNK, an inhibitor of migratory capacity
[58]. Interestingly, FRNK is decreased in IPF patients and is also
involved in controlling fibroblast to myofibroblast conversion [59].
Comparing human primary fibroblasts from IPF patients versus
those from normal donors, Xia et al. showed that IPF fibroblasts
have enhanced proliferative capacity compared to normal cells.
Moreover, in patient cells proliferation is not inhibited by the inter-
action of integrin with polymerized collagen in contrast to the pro-
liferative restraint in normal cells under these conditions [60].
It is commonly believed that the fibroblast is the major con-
tributor to the pool of myofibroblasts in fibrogenic foci. A prerequi-
site for this contribution is the migration of fibroblasts towards foci,
and subsequent expansion and differentiation. Many factors have
been identified over the years that activate the fibroblast and
thereby enhance their migratory and/or proliferative capacity. These
include the already discussed growth factors PDGF, CTGF, and
TGFß, but also chemokines such as CCL11 [61], CCL21 [62],
CCL24 [63], and CCL26 [63].
Fibrocytes
Fibrocytes are a unique cell population that have been identified
and characterized to play a role in wound healing [64-66]. The
presence of the fibrocytes appears to be a characteristic of fibrotic
diseases, from lung, heart, skin, kidney to liver [64] [67-69] [70].
One of the unique features of these cells is their ability to home
from the blood stream to sites of tissue damage. As a consequence,
fibrocytes might also contribute to the increasing number of fibro-
blasts in fibrotic lesions. Over the last couple of years, research on
fibrocytes has intensified by investigating the origin of these cells,
as well as its contribution to fibrotic lesions [71, 72].
Origin and Characterization of Fibrocytes
Fibrocytes were first identified in 1994 as circulating cells that
homed and extravasated into sites of tissue injury contributing to
wound healing [73]. Fibrocytes are bone marrow-derived circulat-
ing cells that express markers for both hematopoietic cells (CD34,
CD43, and CD45), as well as markers linked to stromal cells, colla-
Lekkerkerker et al.
gen I and III [74]. They most likely derive from a common myeloid
precursor similar to dendritic cells and macrophages and can be
distinguished from monocytes, macrophages, and fibroblasts
through a combination of markers [75]. Expression of chemokine
receptors CCR2, CXCR4, and CCR7 enables them to migrate to
sites of injury, where especially CXCR4 is expressed on 90% of the
circulating fibrocytes [76, 77].
Recently, it was described that fibrocytes, recruited from the
circulation into tissues, can differentiate into various cell types
depending on the tissue niche [78]. During the fibrocytes' journey
through the tissue, hematopoietic surface receptors CD34, CD45,
and CXCR4 on the fibrocytes gradually decrease, whereas the ex-
pression of the mesenchymal markers, such as collagen, fibronectin,
and proteoglycans gradually increases. Within the lung, the re-
cruited fibrocytes may further differentiate towards a myofibroblast
phenotype with the expression of
SMA [79].
Culture of Fibrocytes
To enable more in-depth research of fibrocytes, over the last
years multiple culture protocols have been set up using different
cellular sources. Early studies on culture of fibrocytes made use of
a CD14+ enriched mononuclear cell population in contact with T
cells. It was also shown that especially TGF induced the differen-
tiation and functional activity of these cultured fibrocytes. These
differentiated, cultured fibrocytes expressed SMA and contracted
collagen gels in vitro, two hallmarks of wound-healing myofibro-
blasts [76]. This was substantiated by a study demonstrating that
increased TGF in serum stimulates the differentiation of the
CD14+ cells, isolated from peripheral blood of burn patients, into
collagen-producing cells [80].
Current protocols mostly use either peripheral blood mononu-
clear cells or purified CD14+ monocytes for fibrocyte differentia-
tion in vitro [79]. However, other studies suggest that fibrocytes
may also differentiate from a population of bone-marrow derived
CD45+ CXCR4+ cells found in peripheral blood [74].
In the presence of serum, fibrocytes require up to 2 weeks to
differentiate, whereas in the absence of serum this process is accel-
erated with cells appearing in culture after only a few days. A num-
ber of other molecules can influence the differentiation of fibro-
cytes in vitro, and presumably in vivo. TGF, IL-4, and IL-13 all
promote fibrocyte differentiation, where specifically TGF appears
to enhance kinetics of fibrocyte differentiation [81] [74]. It is cur-
rently assumed that fibrocytes that differentiate in the presence or
absence of serum reflect the same cell type, differing only in their
kinetics of generation.
In addition, TLR2 agonists indirectly inhibit fibrocyte differen-
tiation and cause some other cell type in the PBMC population to
inhibit monocytes from differentiating into fibrocytes [82]. A very
recent study [83] shows opposing effects of two different forms of
hyaluronic acid on fibrocyte differentiation from purified mono-
cytes in serum free conditions. High molecular weight hyaluronic
acid potentiated fibrocyte differentiation, while low molecular
weight hyaluronic acid inhibited fibrocyte differentiation.
The fibrocyte culture protocols have been explored in more
detail by Pilling et al. [84], identifying conditions that affect this
differentiation, including the choice of anti-coagulant, media and
media supplements, culture substrates, and cell density.
Role of Fibrocytes in Lung Fibrosis In vivo
In vitro studies provide evidence for differentiation of fibro-
cytes into fibroblasts and eventually myofibroblasts, indeed sug-
gesting that fibrocytes could play an important role in fibrosis.
However, evidence for fibrocyte into myofibroblast differentiation
in vivo has been lacking until recently. A recent study in a mouse
model of bleomycin-induced pulmonary fibrosis signifies a mecha-
nistic role of fibrocytes in IPF [85]. Fibrocytes expressing both
CD45 and collagen I traffic to the lungs and differentiate into myo-
Cellular Players in Lung Fibrosis
fibroblasts [85] [77]. In addition, this study elegantly showed that
the chemokine receptor ligand pair CXCR4-CXCL12 plays a part in
homing of the fibrocytes to the fibrotic lesions. Fibrosis was en-
hanced by intravenous injection of in vitro-generated human fibro-
cytes and the cells were recruited to the lungs via their expression
of CXCR4. In an intratracheal FITC-induced pulmonary fibrosis
model, fibrocytes use the CCR2-CCL12 axis for their recruitment.
Adoptive transfer of CCR2-expressing fibrocytes augments FITC-
induced fibrosis. Both CCL2 and CCL12 are chemotactic for fi-
brocytes, and neutralization of CCL12 significantly protected from
FITC-induced fibrosis. Therefore, CCL12 is probably the CCR2
ligand responsible for driving fibroblast proliferation in the mouse
[86].
Induction of lung fibrosis in IL-10 overexpressing mice is also
associated with an increase of CD45 and collagen I expressing fi-
brocytes. In addition, CCR2 and its ligand, CCL2, are highly
upregulated in these mice, suggesting that IL-10-induced fibrocyte
recruitment is CCR2-CCL2 specific [87]. In conclusion, various
mouse models for pulmonary fibrosis all point toward a role for
fibrocytes.
Several lines of evidence also imply a role for fibrocytes in
human lung fibrosis ([65, 74, 88, 89]. Early evidence for a role of
fibrocytes in fibrosis stemmed from an asthma patient study, where
they detected fibrocyte-like cells in the bronchial mucosa of pa-
tients with allergic asthma. The number of these cells increased
during an asthmatic reaction induced by allergen exposure, suggest-
ing that circulating fibrocytes may function as precursors of bron-
chial myofibroblasts and may contribute to the onset of fibrosis in
asthma [90]. A study of Mehrad et al. demonstrated that fibrotic
interstitial lung diseases have enhanced expression of CXCL12 in
lungs and plasma of patients. The increased CXCL12 levels were
associated with higher number of circulating fibrocytes in the pe-
ripheral blood [88].
This observation was confirmed in IPF patients where fibro-
cytes were identified in the lung parenchyma. CXCL12, the specific
ligand for CXCR4, was elevated in bronchoalveolar lavage, indicat-
ing that also in IPF patients CXCR4-positive fibrocytes may mi-
grate into the IPF lungs contributing to myofibroblast expansion
[65]. Recently the same group proposed the circulating fibrocyte as
a novel biomarker for bronchiolitis obliterans syndrome (BOS)
development in lung transplant patients, a fibrotic process resulting
in progressive narrowing of bronchiolar lumens and airflow ob-
struction. Also in these patients, increased circulating fibrocyte
levels correlated with development of BOS after lung transplanta-
tion [91].
Moreover, a study in IPF patients comparing the presence of
fibrocytes in blood of IPF patients vs healthy controls clearly
showed a higher number of fibrocytes characterized by CD34+
CD45+ in peripheral blood for the IPF patients. A further increase
of fibrocytes was observed during acute disease exacerbation
and these cells were also observed in fibrotic lesions of IPF pa-
tients, corroborating the concept that fibrocytes contribute to the
increased numbers of fibroblasts in fibrotic lesions [89]. Taken
together, these data suggest that fibrocytes are involved in the
pathogenesis of human lung fibrosis.
Macrophages
Macrophages are responsible for immune surveillance and tis-
sue homeostasis. They are able to engulf pathogens using a broad
repertoire of pathogen recognition receptors (PRRs) and destroy
them via degradation within phagolysosomes. Within the process of
tissue homeostasis, macrophages play an essential role in removing
dead and dying cells and toxic materials. Furthermore, macro-
phages are crucial in the orchestration of the wound healing proc-
ess. To perform these important functions, macrophages consist of
different subpopulations which are strategically positioned through-
out the body [92]. These specialized macrophages have specific
Current Pharmaceutical Design, 2012, Vol. 18, No. 27 4097
characteristics facilitating their function specific for that anatomical
location. For example, the liver contains specialized macrophages
called Kupffer cells and the lungs contain alveolar macrophages.
During an immune response, monocytes are recruited from the
circulation which differentiate into tissue macrophages. Following
tissue damage and/or infection macrophages exhibit primarily a
pro-inflammatory phenotype and secrete pro-inflammatory media-
tors, such as TNF
and IL-1. These pro-inflammatory macrophages
are referred to as classically activated macrophages or M1 macro-
phages. Various chronic inflammatory diseases and autoimmune
diseases, such as rheumatoid arthritis, are associated with activation
of M1 macrophages [93]. These M1 macrophages also produce Th1
and Th17 polarizing cytokines like IL-12 and IL-23 to further drive
and regulate the inflammatory response. Nitric oxide is another
mediator excreted by pro-inflammatory macrophages which is toxic
for microorganisms but also very damaging for healthy neighboring
cells. The macrophages also produce MMPs facilitating tissue ho-
meostasis.
To prevent an exacerbated immune response and collateral
damage to surrounding tissue, the M1 macrophage response needs
to be tightly controlled. Macrophages that play a role in wound
healing are characterized by an alternative activated phenotype and
are designated M2 macrophages. This subset secretes anti-
inflammatory mediators and is strongly associated with Th2 medi-
ated inflammation (e.g. IL-4 production) and antagonizes M1
macrophages to regulate the immune response.
A tightly regulated balance between M1 and M2 macrophages
is thus crucial for maintaining immunological homeostasis and
tissue integrity. Recent studies have shown that M1 macrophages
can convert into M2 macrophages, indicating a dynamic balance
between both macrophage subtypes [94]. A disturbed balance in the
regulation of these macrophage subtypes may result in excessive
inflammation or wound healing and have detrimental effects on
normal function of tissue and organs [94].
Macrophages in Fibrotic Diseases
A major initiator of fibrosis is the persistence of exogenous and
endogenous stimuli of pathogens or tissue injury [95]. Both M1 and
M2 macrophages are involved in the process of fibrosis. Figure 2
gives an overview of these two macrophage types and the factors
produced by macrophages involved in fibrosis. M1 macrophages
produce MMPs, resulting in degradation of ECM and thereby facili-
tating the influx of more inflammatory cells from the circulation
[96]. They furthermore contribute to fibrosis through the production
of TNF
and IL-1
[97, 98]. Nevertheless, M2 macrophages are
considered to be the predominant macrophage subtype contributing
to fibrosis. They play an important role in wound healing and pro-
duce amongst others fibrogenic mediators such as PDGF and TGF

[99, 100]. A strong link between M2 macrophages and fibrosis was
made in human patients, including IPF patients, which exhibit a
highly progressive fibrotic disease in the absence of infection [101].
M2 macrophages are important in wound healing and produce
TIMPs and MMPs to remove apoptotic debris, thereby preventing
an immunological response of tissue damaging M1 macrophage.
Furthermore, tissue macrophages reside in close proximity of
myofibroblasts and are able to interact with these cells in various
ways [102]. M2 macrophages can directly affect fibrosis by the
excretion of profibrotic mediators, such as TIMP, and thereby di-
rectly inhibiting ECM turnover [94, 103, 104]. M2 macrophages
also produce fibronectin, thus directly contributing to the buildup of
excessive ECM [105].
Besides the direct effect of M2 macrophages on fibrosis, M2
macrophages also indirectly contribute to fibrosis through activa-
tion of other cell types such as T cells, fibroblasts, and endothelial
cells and thereby aggravating fibrosis [104]. Macrophages engulf
pathogens and dead cells and subsequently present the antigens to T
cells. M2 macrophages are able to enhance Th2 responses [106] and
4098 Current Pharmaceutical Design, 2012, Vol. 18, No. 27
Monocyte Macrophage
Fig. (2). Overview of the macrophages that play a role in lung fibrosis and the factors that contribute to the direct or indirect induction of fibrosis.
Th2 cytokines in turn can enhance collagen synthesis in fibroblasts
and promote myofibroblast formation [107-109]. Furthermore, by
engulfing dead cells, macrophages produce TGF
which is a strong
profibrotic factor [102, 110, 111].
Macrophages in IPF
Based on experimental data with the bleomycin-induced fibro-
sis mouse model, alveolar macrophages are thought to produce the
majority of the active TGF
that promotes pulmonary fibrosis
[112]. Interestingly, the primary level of control is not in the regula-
tion of mRNA expression, but in the regulation of both the secre-
tion and activation of latent TGF
1 [113, 114]. Furthermore, acti-
vated alveolar macrophages from rat lungs after bleomycin admini-
stration demonstrated elevated amounts of active TGF
1, as well as
plasmin and thrombosponin-1 (TSP-1) [115]. Plasmin and TSP-1
are tightly involved with the activation of latent TGF
1. TSP-1 is a
glycoprotein which is expressed by various cell types such as
macrophages, fibroblasts and endothelial cells, and is often associ-
ated at sites of inflammation and wound healing [116]. Further-
more, inhibition of TSP-1 with antisense technology inhibits TGF

activation and subsequently fibrosis [117]. Plasmin dependent acti-
vation of TGF
may occur at the cell surface of the activated alveo-
lar macrophages and requires interaction between TSP-1 and CD36
(a receptor for TSP-1) [118]. M2 macrophages are therefore in-
volved in the activation and the production of latent TGF
, thereby
adding another layer of complexity in the process of fibrosis.
PDGF, which stimulates the proliferation, survival, and migra-
tion of myofibroblasts is also expressed by M2 macrophages [119-
121]. Furthermore, the percentage of PDGF-positive macro-
phages in IPF was 3-fold increased in comparison to normal lung,
indicating a strong correlation between PDGF producing macro-
phages and IPF [122].
CCL-18, also known as pulmonary activation-related chemo-
kine (PARC), is highly expressed in alveolar macrophages from
IPF patients [123-125]. CCL-18 production is strongly associated
with activation of M2 macrophages, implicating that alveolar
macrophages have an M2 phenotypes in IPF [126]. CCL18 has
some overlapping functions with TGF
, such as stimulating colla-
gen production by fibroblasts [127, 128]. Furthermore, Prasse et al.
showed that CCL-18 concentration in serum strongly correlates
with severity of IPF and is a predictive value for mortality [129].
Towards New In Vitro Models
In the past decades much effort was put in the development of
in vitro and in vivo models to unravel the molecular mechanisms
regulating fibrotic processes in the lung. Initial studies focused on
various cell lines derived from the lung (e.g. A549, NCI-H292,
Lekkerkerker et al.
Secretion of factors
Inflammatory directly and indirectly
M1 macrophage promoting fibrosis
TNF
IL-1
MMPs
TGF
PDGF
CTGF
CCL18
Fibronectin
Profibrotic TSP1
M2 macrophage
BEAS2B and 16HBE) as an in vitro model for molecular and cellu-
lar processes in lung epithelium, and contributed considerably to
the insight we have nowadays on the signaling pathways epithelial
cells utilize to exercise their effects. However, cell lines may not
always provide the best model for studying molecular processes as
they often carry transforming mutations and have abnormal chro-
mosome copy numbers. In addition, extensive passaging of cells
and varying culture conditions may introduce additional genetic and
post-transcriptional changes affecting molecular and cellular func-
tion and causing inconsistencies between different reports. For ex-
ample, inconsistent characteristics have been reported for A549
cells [130] and Swain et al., using Raman spectroscopy, demon-
strated that A549 cells differ considerably from primary cells [131].
In addition, the expression of various genes involved in cell com-
munication and adhesion are differently regulated in cell lines ver-
sus primary cells [132]. Also at the functional level, lung cell lines
differ considerably from primary lung cells. For example, in con-
trast to human primary lung epithelial cells, the commonly used cell
line BEAS-2B fails to differentiate and form tight junctions when
cultured on air-to-liquid interface [133]. Also, barrier function, cell
spreading, and wound healing differ between primary cells and cell
lines [134]. The use of cell lines may therefore introduce biases
towards certain molecular pathways or the risk that important cellu-
lar processes are overlooked. Employment of primary cells and
preferably those from fibrosis patients will minimize such risks and
provide us with better insights in the molecular processes involved
in fibrotic disease.
Novel studies are being developed to study the role of epithelial
cells in vitro in even greater detail and under more physiological
conditions. The use of cells of primary origin is thereby indispensa-
ble. In lung biology many researchers groups have put considerable
effort in optimizing protocols for the isolation of primary human
lung-derived cells. Epithelial cell cultures from both bronchial
(HBEC) and alveolar (AEC) origin are widely used for functional
studies. These cells are usually isolated from lung resection or
transplant tissue by enzymatic treatment, and are cultured in serum
free conditions, although culturing procedures differ from one insti-
tution to the other. A major advantage of these cultures is that they
are also available from patients such as IPF, COPD, asthma and
cystic fibrosis, allowing comparisons between patient groups, as
well as with normal individuals.
A review by Van de Bovenkam et al. [135] gives a good over-
view of in vitro cell culture systems for studying the role of primary
hepatic stellate cells in liver fibrosis. In the fields of cystic fibrosis
and asthma, the pulmonary research community frequently includes
human primary cells from patients in their studies. Unfortunately,
the use of human primary lung cells from IPF patients in fibrosis-
Cellular Players in Lung Fibrosis
relevant functional assays is limited. This is mainly due to limited HBEC
availability and logistical, regulatory, and technical issues surround- IFN
ing the access, isolation, and culture of such cells. Recently, the
ECIPF work group reported on those issues [136]. IL
It is, however, important to use these cells under physiological IPF
conditions and in a disease-relevant context, e.g. by culturing M1
epithelial cells on air-to-liquid interface or by setting up co-cultures M2
of fibroblasts and macrophages. The study of cells, derived from
MMP
IPF patients, in functional assays relevant for fibrosis (such as pro-
liferation, apoptosis, (de)differentiation, EMT, motility, oxidative PARC
stress, inflammatory responses), in combination with functional PBMC
genomics, can give invaluable insight in the possible molecular PDGF
mechanisms contributing to fibrosis. Indeed, as described in this
review, cells from IPF patients behave differently in functional PRR
assays, compared to cells from normal donors. Most studies using SMC
IPF patient material so far have focused on lung fibroblasts, as de- SPC
scribed above, and demonstrate that fibroblasts from IPF patients,
in comparison with cells from normal donors, are more active in TGF

various cellular assays, mimicking fibrosis disease processes. These TGF
R
studies demonstrate that fibroblasts from IPF patients behave dif- Th
ferently than cells from healthy donors. Limited experience has
TIMP
been reported on macrophages and epithelial cells from IPF pa-
tients, but also those cells appear to differ in their behavior from TLR
normal cells. Therefore to understand molecular and cellular proc- TNF
esses related to IPF, it is of importance to use IPF derived cells as TSP-1
much as possible. If feasible, the assays should be set up in condi-
tions that mimic the pathophysiology as much as possible, includ- CONFLICT OF INTEREST
ing cultures of epithelial cells on air-to-liquid interface, co-culture
The authors confirm that this article content has no conflicts of
of epithelial cells, or fibroblasts with macrophages, etc.
interest.
CONCLUSIONS
ACKNOWLEDGEMENTS
Fibrosis and especially idiopathic pulmonary fibrosis is a dis-
The authors wish to thank Dr. R. Geels, Galapagos, for genera-
ease that is receiving increasing attention from scientists and the
tion of the figures.
pharmaceutical industry. Unfortunately, little is known about the
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AEC = Alveolar epithelial cells
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CD = Cluster of differentiation [12]
DC = Dendritic cell
[13]
ECM = Extracellular matrix
EMT = Epithelial-mesenchymal transition
FITC = Fluorescein isothiocyanate [14]
Current Pharmaceutical Design, 2012, Vol. 18, No. 27 4099
= Human bronchial epithelial cells
= Interferon
= Interleukin
= Idiopathic pulmonary fibrosis
= Classicaly activated macrophage
= Alternatively activated macrophage
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= Platelet-derived growth factor
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= Transforming growth factor beta
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= Toll-like receptor
= Tumor necrosis factor
= Thrombospondin-1
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Received: March 6, 2012 Accepted: March 14, 2012
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