The Pathogenesis of Bleomycin-Induced Lung Injury in PDF

Experimental Lung Research, Early Online, 1–17, 2014
Copyright © 2014 Informa Healthcare USA, Inc.

ISSN: 0190-2148 print / 1521-0499 online
DOI: 10.3109/01902148.2014.979516
ORIGINAL ARTICLE
The pathogenesis of bleomycin-induced lung injury in

animals and its applicability to human idiopathic
pulmonary ﬁbrosis
James D. Williamson, Laura R. Sadofsky, and Simon P. Hart
Hull York Medical School, Centre for Cardiovascular and Metabolic Research, Academic Respiratory Medicine, Castle Hill
Hospital, Hull, United Kingdom
Exp Lung Res Downloaded from informahealthcare.com by Universitaet Zuerich on 12/23/14
A B STRA CT
Idiopathic pulmonary fibrosis (IPF) is a devastating disease of unknown etiology, for which there is no curative
pharmacological therapy. Bleomycin, an anti-neoplastic agent that causes lung fibrosis in human patients has
been used extensively in rodent models to mimic IPF. In this review, we compare the pathogenesis and histolog-
ical features of human IPF and bleomycin-induced pulmonary fibrosis (BPF) induced in rodents by intratracheal
delivery. We discuss the current understanding of IPF and BPF disease development, from the contribution of
alveolar epithelial cells and inflammation to the role of fibroblasts and cytokines, and draw conclusions about
what we have learned from the intratracheal bleomycin model of lung fibrosis.
For personal use only.
KEYWORDS bleomycin, idiopathic pulmonary ﬁbrosis, intratracheal, rodent modeling
EXPERIMENTAL MODELING OF terstitial pneumonia (UIP) [4], a patchy interstitial

IDIOPATHIC PULMONARY FIBROSIS fibrosis with areas of established fibrosis and others
of fibroblastic proliferation and ongoing collagenous
Idiopathic pulmonary fibrosis (IPF) is an incurable deposition known as fibroblastic foci [5, 6]. Typically,
chronic progressive lung disease of unknown etiol- the distribution of fibrosis in the lung is basal and sub-
ogy [1]. The median survival time is only 2–3 years pleural [7], often associated with honeycomb change
from diagnosis [2], which is worse than the prog- (dilated small airways giving the appearance of clus-
nosis of many cancers [3]. While IPF patients make ters of cysts 3–10 mm in diameter) observed on high-
up around 20% of adult patients who undergo lung resolution-computed tomography imaging [3, 5].
transplantation, one third of those accepted for trans- International treatment guidelines are clear that
plant die on the waiting list. Patients with IPF suffer there is currently no medical therapy that improves
from progressive disabling breathlessness, and IPF is survival in IPF [8], and it is fair to say that progress in
a cause of significant morbidity and mortality. In ad- developing treatments has been hampered by our lack
vanced stages of the disease, use of primary care re- of understanding of the pathogenesis of pulmonary fi-
sources is high and hospital admissions are common. brosis. Traditionally, it had been believed that fibrosis
IPF is a chronic, fibrosing interstitial pneumonia followed inflammation, and in the past potent anti-
characterized by the histological pattern of usual in- inflammatory and immunosuppressant treatments
such as prednisone were prescribed widely. However,
interim analysis of the PANTHER trial demon-
Received 30 July 2014; accepted 18 October 2014 strated an overall harmful effect from combined
The authors would like to thank the University of Hull, Dr. Assem Allam, and
immunosupression with prednisone, azathioprine,
the Daisy Charity for their continued support and contributions to the research and N-acetylcysteine (NAC) compared with placebo
environment at Castle Hill Hospital. [2], with more patients in the triple therapy harm
Address correspondence to Simon P. Hart, Hull York Medical School, Center
hospitalized and dying as a result of worsening lung
for Cardiovascular and Metabolic Research, Academic Respiratory Medicine,
Castle Hill Hospital, Kingston upon Hull HU16 5JQ, United Kingdom. disease, and so the continued use of these therapeutic
E-mail: S.Hart@hull.ac.uk modalities cannot be recommended. Subsequent
1
2 J. D. Williamson et al.
results from the NAC-only arm showed no improve- this [29, 30]. Many excellent reviews have discussed
ment over placebo [9]. the mechanisms of action of BLM [31–33]. As
However, new treatment modalities are coming BLM does not cross the cellular plasma membrane
to the fore, and the novel therapies pirfenidone and efficiently [34], how sufficient BLM enters the cell to
nintedanib may impact disease progression; phase III induce cytotoxicity is unknown. Potential receptors
trials of pirfenidone have shown a decrease in dis- for the molecule have been identified on hamster
ease progression, as patients receiving pirfenidone lung fibroblasts [34] and BLM-sensitive human head
were found to have a decreased decline in forced and neck cancer cell lines [35], while a potential
vital capacity (FVC), with increased exercise toler- role for the carnitine transporter hCT2 in BLM
ance and longer progression-free survival [10], while uptake has been reported [36]. Within the cell,
nintedanib reduced the decline in FVC but not the BLM is inactivated by bleomycin hydrolase (BH),
time to first acute exacerbation compared to placebo an intracellular cysteine protease, producing less
[11]. Both drugs represent potentially vital treat- active metabolites [37, 38], and the absence of this
ments and may improve the quality and duration of enzyme has been linked to the susceptibility of the
life for the IPF patient, though convincing data on pa- lungs to BLM toxicity in rabbits [38], and humans
tient reported outcomes and mortality are currently show a similar low expression of BH in the lung
lacking. [39]. Moreover, BLM-susceptible cancer cell lines
The primary difficulty in developing efficacious show lower BH expression than BLM-resistant lines
treatments for IPF is that the pathogenesis of the con- [40], and so lack of BH in the lung may explain the
dition is very poorly defined. Accordingly, modeling susceptibility of this organ to injury.
of fibrotic disease has become an area ripe for investi- Despite its existence having been recognized for
gation. A common model used over the last 20 years is many years, surprisingly little is actually known about
the bleomycin (BLM) model, an experimental tech- human BPF. Statistics vary regarding the frequency
nique that involves the instillation of BLM directly of occurrence of bleomycin-induced pulmonary
into the lungs of rodents. There are a range of poten- fibrosis (BPF) in man. A retrospective study of 835
tial dosing routes for dosing BLM in rodents, includ- patients showed BPF incidence rates of 6.8% in
ing intravenous (IV), subcutaneous (SC), intranasal, patients treated with the drug, with 14% of those
and intratracheal (IT), but IT BLM administration dying of BPF [20]. However, other studies state
has been by far the most popular mode of adminis- the incidence of BPF to be up to 46%, with a 3%
tration [12–14]. mortality rate [41]. Radiologically, human BPF is
characterized by interstitial fibrosis and sub-pleural
nodules at the lung bases, and patchy superimposed
BLEOMYCIN: CHEMISTRY AND basilar opacities [14, 41–44]. Analyses of lungs of pa-
MECHANISMS OF ACTION tients who have died from BLM-induced pulmonary
fibrosis have reported alveolar epithelial cell (AEC)
The bleomycins are a group of glycopeptide antibi- type I (AECI) loss, vascular endothelial cell damage,
otics isolated from Streptomyces verticillus [15, 16]. AECII hyperplasia, alveolar immune cell infiltration,
Bleomycin (BLM) has long been used as a treat- intra-alveolar collagen deposition, interstitial edema,
ment for head and neck carcinomas [17], germ-cell and diffuse alveolar and sub-pleural fibrosis [43–45],
tumors [18] and lymphomas [19], and has been fa- with a single report of potential immune cell infiltra-
vored due to its broad activity and limited myelotoxic- tion into the lung [45]. Symptomatically, human BPF
ity [20]. However, BLM-associated pulmonary fibro- is like other pulmonary fibrotic diseases, as patients
sis (BPF), first described as a side-effect in the 1970s present with dyspnea and a non-productive cough
[21, 22], is a use-limiting factor. [42, 44]. Because of the recognition of BPF in man,
BLM effects cell cytotoxicity by induc- BLM has become the agent of choice for modeling
ing single- (SSB) or double-stranded (DSB) pulmonary fibrosis in rodents in an attempt to better
DNA breaks [23], and may cause cell death by understand the pathogenesis of pulmonary fibrosis in
pseudoapoptosis—similar to apoptosis but charac- general.
terized by rapid DNA fragmentation—or mitotic cell
death [24, 25]. As DSB are more intrinsically toxic,
BLM toxicity is thought related to DSB generation THE CONTROVERSY SURROUNDING
[24]. Though oxidant-mediated damage was initially BLM IN IPF MODELING
thought to induce such strand breaks [26, 27], direct
scission by DNA H+ abstraction [28] is the favored While there are some similarities between the two
theory today, with recent work providing support for disease states (Table 1), the ability of IT BLM to
Experimental Lung Research

Bleomycin-Induced Pulmonary Fibrosis and Human IPF 3
TABLE 1. A Comparison of the Features of Human IPF and Rodent BPF Induced by IT BLM.
Human idiopathic pulmonary Pulmonary fibrosis induced by the

fibrosis (IPF) intra-tracheal delivery of BLM Comments
Pattern of disease progression A slowly developing disease A rapidly developing disease (days In this respect, the two diseases
and development. characterized by irreversible to weeks), which appears to do not seem to share similar
progression and periods of progress at a stable rate and is developmental processes.
acute exacerbation [1]. reversible [53, 54]
The contribution of alveolar Alveolar epithelial cell death is a Alveolar epithelial cell death is Alveolar epithelial cell death in
epithelial cell injury to the rarely observed feature of IPF widely described and observed IPF is poorly understood.
pathogenesis of the disease. [52, 89]; histological [50, 55–59], though this may be More research into alveolar
assessment of early stage due to the direct delivery of a epithelial cell death in IPF is
disease is rare. cytotoxic drug to the lung. required.
The contribution of Although inflammation in the Inflammation, the infiltration of Although an improved
inflammation to the disease has been reported, it the lung by a variety of cell understanding of
pathogenesis of the disease. is generally thought that types, and the expression of inflammation in IPF is
inflammation plays a minor pro-inflammatory cytokines is a required, inflammation may
role in IPF development universally accepted feature of play a role—though potentially
[97–102, 125–132]. this disease [12, 50, 90–95, of different magnitude—in

114–116]. both diseases.
The contribution of fibrotic Increased numbers of collagen Increased numbers of fibrotic In this respect, the two diseases
cytokines and myofibroblasts synthesizing myofibroblasts cytokines and collagen are very similar.
to the pathogenesis of the and fibrotic cytokines are synthesizing myofibroblasts are
disease. observed [168–171, seen in rodent BPF [154–166].
196–200].
Fibrosis localization in disease The development of lung The fibrosis shows a more Both diseases result in fibrosis
fibrosis characterized by bronchiolocentric distribution and the development of
basal, sub-pleural distribution [14]. Fibroblastic foci are fibroblastic foci. However, the
with the appearance of present [49, 50]. distribution of fibrosis is
fibroblastic foci [4–7]. different.

Clinical features and Dyspnea and evidence of It is uncertain whether the forced More research is required to
physiology restriction, with decreased vital capacity and lung determine whether these
forced vital capacity and lung compliance increase, are features are similar or differ
compliance [7]. unchanged, or decrease [14, between the two disease states.
51].
mimic IPF, at least from a morphological point of lung compliance. It has been reported that there may
view in vivo, has been disputed for several reasons. be an increase in functional residual capacity and nor-
First, single-dose IT BLM induces reversible fibrosis mal lung compliance as a result of IT BLM [14], al-
[46], while IPF is a progressive irreversible disease. though others state that static and dynamic compli-
As a result, a multi-dose method using multiple IT ance are decreased as lung volume decreases due to
instillations has been developed [47] though it is not IT BLM [51]. By contrast, IPF is a restrictive disease,
frequently used, and whether there are significant dif- and typical characteristics include reduced forced vi-
ferences in the disease resulting from single and mul- tal capacity, total lung capacity, functional residual
tiple doses is debated [48]. capacity, and residual volume as the lung shrinks and
Second, the localization of fibrosis following IT becomes stiffer due to increasing scarring [7]. More
BLM dosing is initially bronchiolocentric. Patchy le- research is required before this aspect of the two dis-
sions and focal, peribronchial interstitial fibrosis are eases may be regarded similar or not.
later noted; this is not representative of the localiza- Fourth and finally, the main criticism of the BLM
tion of fibrosis in IPF [14]. However, hallmark fea- model of lung fibrosis centers around the inflamma-
tures of IPF such as the development of fibroblastic tory features of BLM-induced disease, as BPF is char-
foci have also been reported in the IT BLM mouse acterized by very severe early inflammation [52]. IPF
model [49, 50] but there are few published reports is not regarded as an inflammatory disease, and so
that directly assess the similarities and differences be- the differences between the two conditions appear
tween rodent BPF and human IPF fibrosis localiza- irreconcilable. Here, we appraise evidence for the
tion and distribution. pathogenic processes involved in IPF and BPF devel-
Third, it is uncertain whether the disease induced opment in order to better understand the validity of
by IT BLM delivered to rodents leads to decreased BLM as a modeling agent for IPF.

C 2014 Informa Healthcare USA, Inc.
FIGURE 1. A schematic representation of the pathology of BPF. IT BLM administration results in an initial
inflammatory response characterized by the influx of neutrophils and mononuclear cells to the lung.
Pro-inflammatory factors expressed by neutrophils and macrophages, as well as alveolar epithelial cells (AEC) and
alveolar macrophages (AM) exposed to BLM, induce a pro-inflammatory environment leading to AEC injury and
further leukocyte recruitment. Factors known to be expressed include monocyte chemoattractant protein 1
(MCP-1), macrophage inflammatory protein 1α (MIP-1α), tumor necrosis factor α (TNF-α), and interleukin 1β
(IL-1β). Reactive oxygen species (ROS) and matrix metalloproteinases (MMPs) expressed by infiltrating
leukocytes may contribute to AEC damage. Other factors expressed by AECs, AMs, neutrophils, and exudate
macrophages, such as platelet-derived growth factor (PDGF), endothelin-1 (ET-1), or transforming growth factor
β (TGF-β), induce the transdifferentiation of resident mesenchymal cells into a contractile phenotype—the
myofibroblast—which synthesizes extracellular matrix.
THE PRIMARY INJURY IN RODENT 14 days, which then continues to worsen prior to res-
BPF—ALVEOLAR EPITHELIAL CELL olution [12, 54] (Figure 2). The pattern of disease
(AEC) INJURY development, however, differs slightly depending on
the route of administration of BLM, as recently out-
Rodent BPF begins with an acute inflammatory lined by Rydell-Törmänen et al. [54].
stage, involving AEC damage, inflammatory cell re- In fibrosis induced by IT BLM in rodents, alve-
cruitment, and pro-inflammatory mediator release. olar epithelial cell (AEC) damage is a widely iden-
A sub-acute stage follows, with pro-fibrotic cytokine tified characteristic. AECI loss has been reported to
expression and fibroblast proliferation and differen- appear early in rodent BPF [55], and may occur to a
tiation around the sites of injury. The final stage is significant extent prior to the onset of fibrosis [56].
characterized by increased collagen deposition and fi- This is followed by the appearance of hyperplastic
brosis [53] (Figure 1). The development of the dis- AECII cells [50, 55, 57], which are considered to
ease is rapid, with inflammation peaking at around result in the inappropriate repair of the epithelium
7 days following BLM administration, before giv- [55, 57–59]. One current paradigm suggests BPF
ing way to collagen deposition and fibrosis onset at stems from inappropriate AEC death and subsequent

Currently, it is unknown how AEC injury and

death occurs in rodent BPF, but both angiotensin
II and Fas signaling are associated with AEC death
in the BLM-treated lung [68–70]. Blockade of AEC
apoptosis using captopril, which blocks angiotensin
converting enzyme and Fas-Fas-L signaling, amelio-
rates fibrosis development and decreases lung col-
lagen content [60]. Meanwhile, studies that have
induced AECII apoptosis in mice using inhaled
aerosolized anti-Fas antibody (which mimics Fas lig-
ation by FasL) [71], or prevented bronchiolar epithe-
lial cell apoptosis using a Fas-FasL ligation block-
ing antibody [72], provide evidence that Fas may
play an important role in AEC death. Interestingly,
the apoptosis-inducing Fas ligating antibody induced
AEC TGF-β expression [71], which itself augmented

Fas-induced epithelial cell apoptosis [72]. It was also
demonstrated that this AEC apoptosis results in fibro-
sis [71, 72]. That the transplantation of AECII into
BLM-treated rodents appears to ameliorate fibrosis
[73, 74] lends this theory some weight.
One would expect to see AEC injury in response to
BLM delivered directly into the lung. Perhaps the fi-
brosis induced by BLM does not have to start with the
death of AEC, but rather, it happens because these
FIGURE 2. A diagrammatic representation of the time-course

of rodent BPF development following instillation of BLM are the first cells to contact BLM, a notion echoed by
intratracheally. Type I alveolar epithelial cell (AECI) death many groups over the years [14, 54, 75]. However,
occurs rapidly and is followed by inflammation, which peaks by the few studies using non-IT systemic BLM dosing
the seventh day. Hereafter, inflammation tapers off and fibrotic
regimens (either intraperitoneal or intravenous) have
processes begin, with fibrosis development occurring noticeably
from day 14 onward. Fibrosis continues to worsen until disease also observed AEC death and hyperplasia [76–78].
resolution or animal sacrifice. Therefore, while it is possible that BLM has gained
access to the lung, it is also possible that AEC death
repair attempts, which may eliminate anti-fibrotic ep- may occur due to BLM without initial direct exposure
ithelial functions and allow a pro-fibrotic environ- being required.
ment to develop [60]. Indeed, epithelial loss with Due to the results of work using BLM, AEC in-
concurrent fibroblastic growth has been reported in jury, apoptosis, loss, and hyperplasia are now often
vivo [61]. How this may occur in BPF is unresolved, regarded as key in mechanisms of IPF development
though injured AEC may express factors that medi- [7, 52, 65]. Indeed, Selman and Pardo [52] sug-
ate mesenchymal proliferation such as TGF-β [62] gested IPF occurred in the absence of inflammation
and endothelin-1 [63], while mesenchymal cells may as a result of repeated microscopic injury leading to
express factors that induce AEC injury, suggesting a AEC damage and activation, which leads to hyper-
complex and as yet incompletely resolved interplay plastic AECII cells that fail to transform appropri-
between AECs and fibroblasts in the development ately into AECI cells. These “incorrect” AECII cells,
of BPF. Moreover, epithelial anti-fibrotic functions which are abundant in the lungs of IPF patients [65],
are mediated by AEC-synthesized factors such as express cytokines to induce fibroblast proliferation,
prostaglandin E2 [64, 65], though the role this factor transdifferentiation, and collagen expression, leading
plays in BPF is unresolved as results are conflicting; to fibroblastic focus formation around sites of AEC
some studies suggest decreased, and others increased, death and resulting in fibrosis via aberrant epithelial-
lung PGE2 expression following BLM delivery [66, mesenchymal cross-talk [7, 52, 65] (Figure 3).
67]. Thus, how AEC death results in a fibrotic envi- Although the cause of the proposed AEC injury in
ronment in BPF remains unclear, and to our knowl- IPF is unknown, it has been reported that AEC dam-
edge, there is no direct evidence that epithelial in- age may be due to Fas-Fas-L, as both Fas and Fas-L,
tegrity modulates fibroblast proliferation in rodent and Fas-associated signaling molecules are present at
BPF, save a few works correlating AEC death with higher levels in the lungs of IPF patients [79, 80]. An-
fibrogenesis. giotensinogen overexpression and mesenchymal cell

FIGURE 3. A hypothesis of the skewed axis of epithelial–mesenchymal interaction states that in response to an
unknown injury, death and activation of type I and type II alveolar epithelial cells (AECI and AECII) results in the
expression of pro-fibrotic growth factors leading to fibroblast transdifferentiation. Myofibroblasts then express
angiotensin or transforming growth factor β (TGF-β), which induces alveolar epithelial cell apoptosis and
mesenchymal apoptosis resistance while also potentially leading to further mesenchymal transdifferentiation.
Excess collagen synthesis by myofibroblasts causes the formation of fibroblastic foci, the hallmark of IPF.
angiotensin expression in the fibrotic lung [81–83], broblastic focus formation, from which mesenchy-
which could feasibly lead to alveolar injury, can also mal cells may express factors such as angiotensin to
be found in the lungs of rodents with BPF. However, perpetuate AEC death, in a cycle of dysregulated
it has been reported that angiotensin converting en- cross-talk and perpetual injury. Indeed, fibroblasts
zyme (ACE) inhibitors do not increase the survival from both humans with IPF and rats with paraquat-
time in patients with IPF [84], and so this may not induced fibrosis were reported to induce AEC apop-
play a role in human IPF development. tosis [88], and if this results in aberrant AECII in
The aberrant AECII, which may proliferate in an an attempt to repair lost AEC, then the skewed
attempt to repair the injury but are of as yet undefined epithelial-mesenchymal axis may be perpetuated to
provenance (though several putative pathways result- cause fibrosis.
ing in aberrant AECII were alluded to by King et al. While the absence of AECI and the presence of
[85], including Wnt and Shh) are also activated, ex- aberrant AECII are often said to be features of IPF,
pressing increased levels of fibroblast-activating and evidence demonstrating early AEC injury or death
AEC-destroying TGF-β [86], pro-fibrotic plasmino- occurring away from areas of advanced fibrotic dis-
gen activation inhibitor [87], and other growth fac- ease and honeycomb changes in human IPF is scant.
tors. It is proposed that these factors promote fi- Post-mortem immunohistochemistry of IPF lung

tissue has shown AEC abnormality in areas of hon- natively activated macrophages, are involved in this
eycomb change [52], while one retrospective analysis interaction [96].
of IPF patient lung biopsies demonstrated AECII There have also been reports of increased levels
apoptosis even in areas of otherwise normal alveoli, of pro-inflammatory cytokines in the bronchoalve-
as determined by TUNEL and electron microscopy, olar lavage (BAL) fluid of IPF patients, includ-
which was absent in control lung biopsies [89]. ing the chemoattractant chemokines IL-8 (a potent
Though this work suggests AEC apoptosis may occur neutrophil chemoattractant), MCP-1, and MIP-1α,
prior to fibrotic onset, it is not possible to glean from likely expressed by alveolar macrophages [97–99].
such works just when in the pathogenic process of AMs are present in greater numbers in IPF lungs
IPF such abnormality and apoptosis occurs; is it the [100], and may also produce TNF-α [101] and in-
first injury prior to the development of any fibrosis, or duce the expression of IL-4 and IL-5 by lymphocytes
does this injury become evident as fibrosis becomes when grown in co-culture, suggesting that IPF AMs
more widespread? Moreover, sub-clinical disease is promote a Th2 inflammatory environment [102]. In-
rarely studied and patients with early disease infre- terestingly, alternatively activated AMs account for
quently undergo lung biopsy, so whether AEC injury up to 90% of AMs in IPF patients [96], though their
and subsequent hyperplasia is the initial, causative role remains relatively undefined. Unlike work assess-
injury is unresolved. A lack of human studies investi- ing cytokine release following BLM stimulation, there
gating the temporal development of IPF means that has been little work investigating pro-inflammatory
discerning whether AECII hyperplasia indeed follows cytokine release by AECs in IPF, though there are
AECI death is impossible. Disordered AECII lining reports of increased TNF-α mRNA in AEC in IPF
the alveoli and airspaces may merely be a character- lungs [103], and of TNF-α and IL-1β in the AECII
istic of end-stage disease, characterized by interstitial of IPF patients in areas of current fibrotic remodel-
fibrosis and areas of honeycomb change [65]. ing [104]. Other studies have reported the overex-
pression of IL-4, known to induce fibroblast colla-
gen synthesis and inflammatory cytokine expression,
THE SECOND INJURY IN RODENT and IL-5, an eosinophil chemoattractant [105, 106].
BPF—PRO-INFLAMMATORY CYTOKINE That many of these cytokines are seen in both hu-
RELEASE AND INFLAMMATORY CELL man IPF and rodent BPF suggests potentially anal-
INFLUX ogous roles, though one must be wary in inferring
such associations; cytokine release may be reflective
The inflammatory response—so widely observed in of damage within the lung and may not be causative of
BPF—is often used as an argument that BPF in fibrosis
rodents does not mimic IPF in human. However, While the above cytokines and chemokines are
while anti-inflammatory therapies for human IPF mostly involved in inflammation and immune cell re-
have proven ineffective at best, there is evidence for cruitment, some may also have roles in fibrotic de-
inflammatory cytokine production and immune cell velopment. TNF-α and IL-1β have been proposed
infiltration in IPF. However, there has been little work to induce fibroblast collagen synthesis and prolifer-
directly comparing the inflammatory processes in ro- ation [107, 108], although this has been challenged
dent BPF and human IPF. [109–111], while MCP-1 may induce fibroblast col-
The expression of pro-inflammatory cytokines in lagen synthesis via autocrine fibroblast TGF-β up-
the lung is a widely described feature of BPF induced regulation and prostaglandin E2 suppression [112,
by IT instillation. Activated alveolar macrophages 113]. However, roles for these mediators in fibrosis
(AMs) and epithelial cells are the primary source development are poorly defined.
of such mediators, with AECs reportedly express- Immune cell infiltration is a major feature of
ing TNF-α, IL-6, and IL-1β at increased levels BPF in rodents treated with IT BLM. Neutrophils,
following BLM stimulation in vitro and in vivo macrophages, eosinophils, and lymphocytes may all
[90–92], and AMs releasing TNF-α, MIP-1α, MCP- infiltrate [12, 50, 94, 114–116]. Although neutrophil
1, and IL-1β [91, 93–95]. All of these cytokines infiltration is reported in early BPF [50, 116], it
are involved in recruitment of immune cells to is uncertain the role this cell type has in BPF and
sites of injury including the up-regulation of vas- it has been stated that their transient presence has
cular endothelial cell adhesion molecules, which no direct contribution to fibrogenesis [50]. Dif-
may tether immune cells (in the case of TNF- ferent issues have been put forward regarding the
α and IL-1β). Some current paradigms suggest role of macrophages; while some state that exudate
epithelial–mesenchymal interactions alone cause IPF, macrophage numbers increase in rodent BPF, other
others suggest lung macrophages, particularly alter- state there is a decrease [50, 94], though it does

appear that blocking macrophage influx ameliorates tive inflammation in IPF lungs having been reported.
BPF [117]. Both cells, however, produce cytokines These aggregates were, at the time, proposed to con-
implicated in BPF, such as TGF-β and MMPs in tribute to IPF development [137, 138]. Again, this
the case of neutrophils [116, 118] and in the case of idea was largely ignored until Xue et al. [139] also re-
macrophages, MMPs, TGF-β1, endothelin-1, TNF- ported such aggregates in IPF lungs, with increased
α, and IL-1β [63, 116, 117, 119, 120]. circulating B-lymphocyte activating factor correlat-
Whether lymphocytes and eosinophils contribute ing with worse outcomes. Further, potentially B-cell-
to BPF is similarly questioned; eosinophil blockade generated immune complexes were found near to
has been seen to both ameliorate BPF and to have no the lung vasculature, as noted by Schwartz et al.
effect [121, 122], and while Schrier et al. [123] stated [140], and may act as neutrophil chemoattractants.
athymic, T-cell deficient mice were protected against Earlier, Wallace et al. [141] had identified an IgG
BPF, other work using nude mice noted BPF devel- autoantibody in IPF patients capable of binding an
opment in the absence of T-lymphocytes [115]. Thus, AECII antigen, which may account for such com-
the contribution of lymphocytes and eosinophils re- plexes. Thus, B-cells may have a greater role in IPF
mains obscure. than currently appreciated.
There have been many reports demonstrating that Bringardner et al. [1] postulated several poten-
immune cells are present in the lungs of IPF patients; tial roles for inflammation in the development of
eosinophils, T and B lymphocytes, neutrophils, and IPF including the direct inflammatory hypothesis,
macrophages have all been observed. While increased in which the action of immune cells leads to stan-
eosinophil numbers are noted in the bronchoalveo- dard inflammation resulting in excess repair pro-
lar fluid of IPF patients [124, 125], and eosinophilic cesses; the matrix hypothesis, in which pro-fibrotic
infiltration is associated with more progressive dis- and pro-inflammatory mediators become matrix-
ease [126], no unequivocal contribution of this cell embedded and promote excess collagen deposition;
type to IPF has been suggested. Neutrophils, while the plasticity hypothesis, which suggests inflamma-
associated with IPF in earlier works [127, 128], ap- tory cells become cells that promote or become ac-
pear present in the IPF lung only in limited numbers tively involved in repair; and the vascular hypoth-
[129], and some works have suggested that neutrophil esis, in which microvascular injury or endothelial
counts are only slighter higher in BALF from IPF pa- necrosis—potentially due to endothelial antibody de-
tients than in controls [125]. Nevertheless, high lev- position and the presence of anti-endothelial antibod-
els of neutrophil elastase are seen in the parenchyma ies (as seen in IPF and UIP)—allows cytokine traf-
of IPF patients [130], though it has been suggested ficking, fibrotic phenotype generation, or antibody
that increased neutrophil counts in the lung of IPF deposition in the lung, which may drive the survival
patients may in fact be a favorable prognostic indica- and activation of macrophages and the expression of
tor in early-stage disease patients [131]. However, the cytokines implicated in IPF.
full role of neutrophils in IPF is unresolved. Before any true parallels can be drawn between the
In contrast, pulmonary infiltration by lymphocytes inflammatory processes involved in IPF and BPF, ad-
is better described. Increased T-cell infiltration of the ditional work must be carried out to determine the
lung was noted in IPF [132], and IPF was accordingly full cytokine and inflammatory cell profiles of the
regarded a “T-cell alveolitis” [133]. Interestingly, T- lung in these diseases, the contribution of these to
cells from IPF patients may induce fibroblast prolif- the condition, and whether immune cell infiltration
eration and collagen synthesis in vitro [134], which contributes to IPF, or is an incidental finding. How-
may of course contribute to IPF, but little further in- ever, there are many who would debate a contribution
vestigation was carried out until recently when Daniil to IPF of inflammatory cells at all, and while various
and colleagues [129] reported that lymphocytes ac- studies have suggested that the blockade of immune
counted for 58% of the immune cells in IPF lungs, cell infiltration may ameliorate fibrotic development
and that increased CD8+ lymphocyte infiltration cor- in rodents [142], it has of course been impossible to
related with decreased FVC and increased dyspnea, a determine whether immune cell influx blockade has
notion later reported by Papiris et al. [135] who sug- a similar effect in IPF patients. It is also worth con-
gested that CD8+ T-cells are associated with ongoing sidering that, characterized as it is by mass immune
progressive fibrosis. That such cells are more com- cell influx into the lung, perhaps rodent BPF more
mon in the lung than often supposed [136] suggests closely mimic human ARDS, a condition in which
a potential role in IPF pathogenesis. neutrophilic infiltration of the lung precedes alveo-
Interestingly, B-cell infiltration has also been lar damage and respiratory failure [143]. However, to
noted, with the development of aggregates composed our knowledge, the bleomycin model has never before
mostly of B-cells, with some T-cells, in areas of ac- been used to model ARDS in rodents.

THE “FINAL” INJURY—PRO-FIBROTIC IT BLM [157], or up-regulated expression of throm-

CYTOKINE RELEASE AND FIBROBLAST bospondin, also seen in the lung after BLM admin-
PROLIFERATION istration [165]. Therefore, the axis of TGF-β syn-
thesis and activation appears to be skewed in favor
One aspect of fibrotic lung disease development, of mesenchymal proliferation and transdifferentiation
which appears common to both IPF and BPF is in BPF. That CTGF—a cytokine synthesized by and
the increased expression of pro-fibrotic cytokines in assisting in the functions of TGF-β—has also been
the lungs, and the most widely implicated factor is detected in the lungs of rodents given intratracheal
TGF-β, a potent inducer of fibroblast proliferation, BLM, but not in the lungs of untreated animals, sug-
α-smooth muscle actin expression and differentia- gests that CTGF too may contribute to fibrosis as
tion, and collagen synthesis [144] via pathways such induced by BLM instillation [166], potentially via
as Smad signaling increasing collagen expression via this skewed pro-fibrotic axis. However, such is the
stimulation of COLIA2 gene transcription [145], and multi-functionality of TGF-β that a complete block
α-SMA gene expression via Smad3 [146] and its in- of this cytokine may result in potentially dangerous
teraction with Smad 4 [147]. TGF-β1 requires acti- side-effects [167].
vation to induce its pro-fibrotic effects, mediated by In IPF also, Khalil and colleagues [168] found in-
many ligands including the epithelia-specific α V β 6 in- tense staining for TGF-β in the AECs of patients with
tegrin [148] and thrombospondin-1 [149], which re- advanced IPF, as well as in areas of extracellular ma-
sults in the cleavage of the molecule from latency as- trix deposition and hyperplastic AECs. Similar results
sociated peptides, rending the cytokine active. Active were reported by Kapanci et al. [169], who noted re-
TGF-β may also act by inducing fibroblast synthesis generative AECII, fibroblasts, and endothelial cells
of, and responsiveness to, connective tissue growth expressing high levels of TGF-β in IPF patients. Cells
factor (CTGF) [150–153] inducing transdifferentia- in areas of severe fibrosis also showed high TGF-β
tion and collagen synthesis via autocrine CTGF sig- levels. Similarly, increased TGF-β was found in BAL
naling [150], resulting in differentiated, highly syn- from IPF patients [170], though this was not univer-
thetic mesenchymal cells known as myofibroblasts. sal, but higher TGF-β levels, though characteristic
Several early works implicated TGF-β, particularly of less restrictive disease, correlated with decreased
TGF-β1, in the pathogenesis of BPF. Vyalov et al. survival times. Later work by Khalil and colleagues
[154], noted increased numbers of myofibroblasts at [171] found that latent TGF-β was found in all lung
24 hours post-BLM located in areas of excess col- cells from IPF patients except subepithelial fibrob-
lagen, and this appeared associated with TGF-β. It lasts, and that AMs from IPF patients expressed sig-
was later found that gene expression of TGF-β1 is nificantly more active TGF-β than AMs from con-
increased in the lung following BLM insult [155], trol patients. There was also more activated TGF-β1
and increased TGF-β mRNA and protein are seen in BAL fluid. As with all TGF-β activation, this may
in both the inflammatory stage and the reparative be associated with the α V β 6 integrin or TSP-1—α V β 6
stage, particularly by parenchymal cells and alveolar has been seen to be expressed by AECs of patients
macrophages, and by bronchial epithelia, respectively with IPF [172, 173]—which today is being explored
[156]. Subsequently, increased TGF-β expression by as a potential avenue for therapy. TGF-β may it-
both AEC and AM has been reported in BPF [62, self induce α V β 6 expression, suggesting that TGF-
157]. These works, together with a report of temporal β may perpetuate its own activation in the IPF lung
and spatial concordance between maximum TGF-β [172].
expression in the lung and maximum collagen pro- In addition to roles in fibroblast proliferation and
duction, and between TGF-β localization and areas transdifferentiation, TGF-β is a chemoattractant for
of fibrotic repair [158], suggested at the time that monocytes [174], and may act upon lung epithelial
TGF-β was a strong candidate cytokine in the devel- cells. Interestingly, the expression of TGF-β has been
opment of IT-BLM mediated fibrosis. This has been the partial basis for theories that interaction between
supported more recently by work showing that block- AECs and fibroblasts may be the primary contribu-
ade of TGF-β signaling ameliorates fibrosis [159, tory factor to the development of IPF. While the loss
160], as does the administration of TGF-β peptide of AECII may tip the balance in favor of fibroblastic
inhibitors and soluble receptors [161, 162], result- proliferation, the expression of TGF-β by AECs
ing in fewer myofibroblasts and decreased collagen may result in increased fibroblastic activity while also
synthesis. Further, increased levels of active TGF- preventing re-epithelialization [52]. In addition to
β have been reported in the BPF lung in rodents preventing the proliferation of epithelial cells, includ-
[163, 164], which may be associated with increased ing AECII [175–177], which could potentiate an en-
α V β 6 expression in the lungs of rodents treated with vironment of constant repair attempts, TGF-β may

induce the apoptosis of lung epithelial cells via ized to areas of active fibrosis in rodent BPF [194],
caspase-3 activation [72]. Therefore, should the and may themselves synthesize TGF-β [195], result-
theory of AECII death and mesenchymal imbalance ing in self-perpetuating fibrotic cycles.
be proven true, TGF-β may prove a vital cytokine In IPF, contractile, collagen-producing myofibrob-
in IPF and rodent BLM-induced fibrosis, being able lasts are very widely regarded as the key effector
to both kill the epithelium and prevent its re-growth, cells of fibrotic development [196, 197], and their
and allow the overgrowth of fibroblasts, skewing presence in fibrotic foci and their excessive colla-
the epithelial-mesenchymal balance in favor of the gen and ECM expression was noted in early work
fibroblast. by Kuhn and McDonald [198]. Fibroblasts and my-
While both ET-1 and platelet-derived growth fac- ofibroblasts represent a critical component of the
tor (PDGF) expression have been noted in the lungs epithelial-mesenchymal hypothesis of IPF develop-
of rodents treated with IT BLM, particularly by AEC, ment. Although the development of fibroblastic foci
but also by AM in the case of PDGF [63, 178, 179], appears self-explanatory—overproduction of TGF-β
these are not widely researched cytokines in this con- and other cytokines induces the transdifferentiation,
dition. In IPF, however, these cytokines have been which then goes un-checked due to the loss and ac-
given far more attention. PDGF is a potent fibrob- tivation of AECII and may indeed induce further
lastic mitogen, inducing the survival, proliferation, AEC loss due to apoptotic factor expression—why
and migration of myofibroblasts. It may also induce these foci remain in IPF and do not resolve is un-
myofibroblast collagen expression, as reviewed by known. Accordingly, there have been suggestions that
Bonner [180]. Furthermore, the concomitant expres- mesenchymal cells—particularly myofibroblasts—are
sion of PDGF and TGF-β has been reported in fi- more resistant to apoptosis in IPF lungs that in nor-
brotic disease in humans [181], and the expression of mal lungs [197, 199, 200], though the evidence is far
PDGF-B mRNA has been seen in both IPF and inter- from conclusive.
stitial lung disease patients [182–184], with excess ex-
pression of PDGF noted by alveolar macrophages ob-
tained from IPF patients [184]. Meanwhile, ET-1 is THE CONTRIBUTION OF THE IT BLM
induced by many factors including TGF-β [185], and MODEL OF FIBROSIS TO OUR
mediates fibroblast chemotaxis, proliferation [186], UNDERSTANDING OF IPF
and transdifferentiation [187, 188]. Roles for ET-1
in fibrosis are poorly defined, though lung tissue and So, what has the rodent model of BPF informed us
BAL samples from IPF patients may have increased about the pathogenesis of human IPF? Certainly,
levels of ET-1 [189, 190], and once again, in IPF by assessing the response to BLM in rodents when
patients, neutrophils and macrophages may be ET-1 the drug is delivered intratracheally and comparing
sources [189]. Both of these cytokines could become the resultant disease and its histological features
foci for future research as more is learned about their to IPF, the model has aided the development of
role in IPF and may even become a focus of potential theories of pathogenesis, though whether these
drug therapies as, unlike TGF-β, their functionality hypotheses are correct or not remains to be seen.
in the lung is more restricted, though results from the Nowhere is this more evident than the theory of
BUILD-3 trial of the ET-1 receptor blocker bosen- AEC damage and aberrant wound healing leading
tan showed no effect of the drug on time to a no- to fibrosis. Associated studies, such as those that
ticeable worsening of IPF, defined as a confirmed de- have identified AEC death leading to fibrosis, the
crease from baseline in FVC or diffusing capacity of expression of angiotensin and Fas/Fas-L in the lung,
the lung for carbon monoxide of ≥10% and ≥15%, and the transplantation of healthy AECs or AEC
respectively; acute exacerbation of IPF or death; or apoptosis blockade preventing fibrosis, have aided
patient quality of life or dyspnea [191]. in the development of the epithelial-mesenchymal
ET-1, PDGF, and TGF-β may induce the trans- cross-talk theory of IPF development. However, this
differentiation of fibroblasts into myofibroblasts, the may have resulted in a culture whereby alternative
prominent collagen-producing cells in both IPF and theories are overlooked, potentially to the detriment
BPF. The origin of the excess fibroblastic cells in IPF of progression of our understanding of IPF.
remains uncertain and has been the focus of many re- With regards to the expression of fibrotic cytokines
views. Increased numbers of these cells have been re- and the effects that these mediators have on the fi-
ported in all fibrotic conditions, and accordingly, my- broblast, it is notable that while similar cytokines
ofibroblasts are present in greater numbers in rodent have been noted in both rodent BPF and human IPF,
BPF lungs [192]. The main source of procollagen I much of this work has occurred concurrently. There-
mRNA expression, [193], myofibroblasts are local- fore, while the modeling of IPF using IT BLM may

not have contributed anything new to theories of IPF BLM is given to both dogs and mice [77, 142, 203,
progression, it has reinforced the roles of TGF-β and 204]. As IV BLM results in a disease which may
the fibroblast in IPF. Development of pirfenidone, more closely model IPF, it may be suggested that
which may act by preventing fibroblast proliferation re-discovering a model that has not been routinely
and collagen production via mesenchymal cell TGF- used for over 20 years should in fact the next step.
β blockade [201] and which has proven effective in Finally, it goes without saying that mice are not
the treatment of IPF [202], may have been made pos- humans. Seok et al. [205] examined a variety of in-
sible due to contributions from research investigating flammatory diseases and found that while the ge-
both human IPF and rodent BPF; in this case the two nomic alterations between conditions such as burns
models have complimented one another. Also poten- and trauma are broadly similar in humans, the ge-
tially useful are the identification of TGF-β activating nomic alterations in mice are very different from hu-
peptides in the BLM-infused mouse, such as the α V β 6 mans but also from each other—mice exposed to the
integrin or TSP-1. α V β 6 is now being identified as ex- same injurious stimulus demonstrated different alter-
pressed at higher levels in the lungs of IPF patients as ations. This also appears to be the case with rodent
well as BLM-treated rodents, and investigated as a BPF and human IPF. Peng et al. [48] saw parallels
potential therapeutic target [172, 173]. However, the between gene enrichment in lung tissue samples from
intratracheal BLM model has proven a hindrance to IT BLM-treated mice undergoing the active fibrotic
our understanding of IPF in other respects. Hundreds phase and patients with rapidly progressing disease
of studies have reported amelioration of BPF develop- (but not the more common slowly progressing dis-
ment in BLM-treated rodents by genetic manipula- ease), but these parallels were not seen at other stages.
tions such as the knock-out of particular cytokines or Perhaps we have failed to focus on the salient features
the knock-down of associated proteins, or the block- of BPF, which are analogous with IPF? Or maybe fu-
ade of pro-fibrotic or pro-inflammatory factors us- ture therapies for IPF will result not from manipula-
ing experimental pharmaceutical agents, yet none of tion of the final common pathway of fibrosis, but from
these (save pirfenidone and nintedanib) have reached looking upstream at what triggers the initial lung in-
the clinic. The use of IT BLM as a modeling tool may jury in IPF. In that circumstance the bleomycin ani-
therefore have failed to translate into a clinical benefit mal model will truly be redundant.
in man.
It may be suggested that as BPF induced in ro- Declaration of interest: The authors report no
dents via IT BLM distillation is inherently reversible, conflicts of interest. The authors alone are respon-
while IPF is not, then perhaps IT-BLM-mediated sible for the content and writing of the article. SH
BPF is more inherently treatable? It appears clear has received financial and non-financial support from
that rodent BPF is not a progressive condition, and Beohringer Ingelheim and Intermune.
is therefore perhaps more susceptible to treatment JW is funded by the University of Hull.
than the slow, perpetual disease that IPF represents.
Perhaps a better knowledge of the processes involved
in the resolution of the rodent disease would assist in REFERENCES
the development of drugs, which may assist a similar
process in man; to our knowledge, there are no works [1] Bringardner BD, Baran CP, Eubank TD, Marsh CB: The role
assessing this stage of rodent BPF, and this would, of inflammation in the pathogenesis of idiopathic pulmonary
at least, be an interesting addition to our knowledge fibrosis. Antioxid Redox Sign. 2008;10(2):287–301.
[2] McGrath EE, Millar AB: Hot off the breath: triple therapy for
of the condition and could perhaps shed more light idiopathic pulmonary fibrosis–hear the PANTHER roar. Tho-
on the suitability, or lack thereof, of this model in rax. 2012;67(2):97–98.
mimicking IPF. [3] Adamali HI, Maher TM: Current and novel drug thera-
Where can research in this field go from here? It is pies for idiopathic pulmonary fibrosis. Drug Des Dev Ther.
possible that the BLM model may still be of use; BLM 2012;6:261–272.
[4] Nalysnyk L, Cid-Ruzafa J, Rotella P, Esser D: Incidence
administered by systemic routes may more accurately and prevalence of idiopathic pulmonary fibrosis: review of
mimic fibrotic disease [54]. IV administration of the literature. Eur Respir Rev: Official J Eur Resp Soc.
BLM is a rare dosing method in animal studies today, 2012;21(126):355–361.
though earlier work using this method produced [5] Dempsey OJ, Kerr KM, Gomersall L, Remmen H, Currie GP:
disease more reminiscent of IPF than that following Idiopathic pulmonary fibrosis: an update. QJM: Monthly J As-
soc Phys. 2006;99(10):643–654.
IT dosing. Reports of AEC injury and hyperplasia are [6] Nicholson AG, Fulford LG, Colby TV, du Bois RM, Hansell
noted in these early works [77, 78], as is infiltration DM, Wells AU: The relationship between individual histologic
of the lung by immune cells [203, 204]. Pulmonary features and disease progression in idiopathic pulmonary fibro-
fibrosis tends to be diffuse and sub-pleural when IV sis. Am J Respir Crit Care Med. 2002;166(2):173–177.

[7] Harari S, Caminati A: Idiopathic pulmonary fibrosis. Allergy. solute values determine cell death pathway. Brit J Cancer.
2005;60(4):421–435. 2001;84(9):1272–1279.
[8] Raghu G, Collard HR, Egan JJ, Martinez FJ, Behr J, Brown [25] Tounekti O, Pron G, Belehradek J, Jr., Mir LM: Bleomycin,
KK, et al.: An official ATS/ERS/JRS/ALAT statement: id- an apoptosis-mimetic drug that induces two types of cell death
iopathic pulmonary fibrosis: evidence-based guidelines for depending on the number of molecules internalized. Cancer
diagnosis and management. Am J Respir Crit care Med. Res. 1993;53(22):5462–5469.
2011;183(6):788–824. [26] Sugiura Y, Kikuchi T: Formation of superoxide and hy-
[9] Idiopathic Pulmonary Fibrosis Clinical Research Network, droxy radicals in iron(II)-bleomycin-oxygen system: elec-
Martinez FJ, de Andrade JA, Anstrom KJ, King TE, Jr., Raghu tron spin resonance detection by spin trapping. J Antibiot.
G: Randomized trial of acetylcysteine in idiopathic pulmonary 1978;31(12):1310–1312.
fibrosis. N Engl J Med. 2014;370(22):2093–2101. [27] Sausville EA, Peisach J, Horwitz SB: Effect of chelating agents
[10] King TE, Jr., Bradford WZ, Castro-Bernardini S, Fagan EA, and metal ions on the degradation of DNA by bleomycin. Bio-
Glaspole I, Glassberg MK, et al.: A phase 3 trial of pirfenidone chemistry. 1978;17(14):2740–2746.
in patients with idiopathic pulmonary fibrosis. N Engl J Med. [28] Rodriguez LO, Hecht SM: Iron(II)-bleomycin. Biochemical
2014;370(22):2083–2092. and spectral properties in the presence of radical scavengers.
[11] Richeldi L, du Bois RM, Raghu G, Azuma A, Brown KK, Biochem Biophys Res Commun. 1982;104(4):1470–1476.
Costabel U, et al.: Efficacy and safety of nintedanib in idio- [29] Liu LV, Bell CB, 3rd, Wong SD, Wilson SA, Kwak Y, Chow
pathic pulmonary fibrosis. N Engl J Med. 2014;370(22):2071– MS, et al.: Definition of the intermediates and mechanism of
2082. the anticancer drug bleomycin using nuclear resonance vibra-

[12] Thrall RS, McCormick JR, Jack RM, McReynolds RA, Ward tional spectroscopy and related methods. Proc Natl Acad Sci
PA: Bleomycin-induced pulmonary fibrosis in the rat: in- USA. 2010;107(52):22419–22424.
hibition by indomethacin. Am J Pathol. 1979;95(1):117– [30] Decker A, Chow MS, Kemsley JN, Lehnert N, Solomon
130. EI: Direct hydrogen-atom abstraction by activated bleomycin:
[13] Snider GL, Celli BR, Goldstein RH, O’Brien JJ, Lucey EC: an experimental and computational study. J Am Chem Soc.
Chronic interstitial pulmonary fibrosis produced in hamsters 2006;128(14):4719–4733.
by endotracheal bleomycin. Lung volumes, volume-pressure [31] Hecht SM: Bleomycin: new perspectives on the mechanism of
relations, carbon monoxide uptake, and arterial blood gas action. J Nat Prod. 2000;63(1):158–168.
studied. Am Rev Respir Dis. 1978;117(2):289–297. [32] Pogozelski WK, Tullius TD: Oxidative strand scission of nu-
[14] Borzone G, Moreno R, Urrea R, Meneses M, Oyarzun M, Lis- cleic acids: routes initiated by hydrogen abstraction from the
boa C: Bleomycin-induced chronic lung damage does not re- sugar moiety. Chem Rev. 1998;98(3):1089–1108.
semble human idiopathic pulmonary fibrosis. Am J Respir Crit [33] Burger RM: Cleavage of nucleic acids by bleomycin. Chem
Care Med. 2001;163(7):1648–1653. Rev. 1998;98(3):1153–1170.
[15] Umezawa H, Maeda K, Takeuchi T, Okami Y: New antibi- [34] Pron G, Belehradek J, Jr., Mir LM: Identification of a plasma
otics, bleomycin A and B. J Antibiot. 1966;19(5):200–209. membrane protein that specifically binds bleomycin. Biochem
[16] Umezawa H, Takeuchi T, Hori S, Sawa T, Ishizuka M: Stud- Biophys Res Commun. 1993;194(1):333–337.
ies on the mechanism of antitumor effect of bleomycin of squa- [35] Pron G, Mahrour N, Orlowski S, Tounekti O, Pod-
mous cell carcinoma. J Antibiot. 1972;25(7):409–420. devin B, Belehradek J, Jr., et al.: Internalisation of the
[17] Suzuki Y, Miyake H, Sakai M, Inuyama Y, Matsukawa J: bleomycin molecules responsible for bleomycin toxicity: a
Bleomycin in malignant tumors of head and neck. Keio J Med. receptor-mediated endocytosis mechanism. Biochem Pharma-
1969;18(3):153–162. col. 1999;57(1):45–56.
[18] Samuels ML, Johnson DE, Holoye PY: Continuous in- [36] Aouida M, Poulin R, Ramotar D: The human carnitine trans-
travenous bleomycin (NSC-125066) therapy with vinblas- porter SLC22A16 mediates high affinity uptake of the an-
tine (NSC-49842) in stage III testicular neoplasia. Cancer ticancer polyamine analogue bleomycin-A5. J Biol Chem.
Chemother Rep Part 1. 1975;59(3):563–570. 2010;285(9):6275–6284.
[19] Schein PS, DeVita VT, Jr., Hubbard S, Chabner BA, Canel- [37] Lefterov IM, Koldamova RP, King J, Lazo JS: The C-terminus
los GP, Berard C, et al.: Bleomycin, adriamycin, cyclophos- of human bleomycin hydrolase is required for protection
phamide, vincristine, and prednisone (BACOP) combination against bleomycin-induced chromosomal damage. Mutat Res.
chemotherapy in the treatment of advanced diffuse histiocytic 1998;421(1):1–7.
lymphoma. Ann Intern Med. 1976;85(4):417–422. [38] Lazo JS, Humphreys CJ: Lack of metabolism as the biochem-
[20] O’Sullivan JM, Huddart RA, Norman AR, Nicholls J, Dear- ical basis of bleomycin-induced pulmonary toxicity. Proc Natl
naley DP, Horwich A: Predicting the risk of bleomycin lung Acad Sci USA. 1983;80(10):3064–3068.
toxicity in patients with germ-cell tumours. Ann Oncol: Offic [39] Bromme D, Rossi AB, Smeekens SP, Anderson DC, Payan
J Eur Soc Med Oncol/ESMO. 2003;14(1):91–96. DG: Human bleomycin hydrolase: molecular cloning, se-
[21] Cancer CSC-OGotEOfRotTo: Study of the clinical ef- quencing, functional expression, and enzymatic characteriza-
ficiency of bleomycin in human cancer. Brit Med J. tion. Biochemistry. 1996;35(21):6706–6714.
1970;2(5710):643–645. [40] Ferrando AA, Velasco G, Campo E, Lopez-Otin C: Cloning
[22] Halnan KE, Bleehen NM, Brewin TB, Deeley TJ, Harri- and expression analysis of human bleomycin hydrolase, a cys-
son DF, Howland C, et al.: Early clinical experience with teine proteinase involved in chemotherapy resistance. Cancer
bleomycin in the United Kingdom in series of 105 patients. Res. 1996;56(8):1746–1750.
Brit Med J. 1972;4(5841):635–638. [41] Sleijfer S: Bleomycin-induced pneumonitis. Chest. 2001;
[23] Huang CH, Mirabelli CK, Jan Y, Crooke ST: Single- 120(2):617–624.
strand and double-strand deoxyribonucleic acid breaks [42] Blum RH, Carter SK, Agre K: A clinical review
produced by several bleomycin analogues. Biochemistry. of bleomycin—a new antineoplastic agent. Cancer.
1981;20(2):233–238. 1973;31(4):903–914.
[24] Tounekti O, Kenani A, Foray N, Orlowski S, Mir LM: The [43] Jones AW: Bleomycin lung damage: the pathology and nature
ratio of single- to double-strand DNA breaks and their ab- of the lesion. Brit J Dis Chest. 1978;72(4):321–326.

[44] Bedrossian CW, Luna MA, Mackay B, Lichtiger B: Ul- [62] Azuma A, Li YJ, Abe S, Usuki J, Matsuda K, Henmi S,
trastructure of pulmonary bleomycin toxicity. Cancer. et al.: Interferon-{beta} inhibits bleomycin-induced lung fi-
1973;32(1):44–51. brosis by decreasing transforming growth factor-{beta} and
[45] Simpson AB, Paul J, Graham J, Kaye SB: Fatal bleomycin thrombospondin. Am J Respir Cell Mol Biol. 2005;32(2):
pulmonary toxicity in the west of Scotland 1991–95: a re- 93–98.
view of patients with germ cell tumours. Brit J Cancer. [63] Mutsaers SE, Foster ML, Chambers RC, Laurent GJ, McAn-
1998;78(8):1061–1066. ulty RJ: Increased endothelin-1 and its localization during the
[46] Brown RF, Drawbaugh RB, Marrs TC: An investigation of development of bleomycin-induced pulmonary fibrosis in rats.
possible models for the production of progressive pulmonary Am J Respir Cell Mol Biol. 1998;18(5):611–619.
fibrosis in the rat. The effects of repeated intratracheal instilla- [64] Lama V, Moore BB, Christensen P, Toews GB, Peters-Golden
tion of bleomycin. Toxicology. 1988;51(1):101–110. M: Prostaglandin E2 synthesis and suppression of fibroblast
[47] Degryse AL, Tanjore H, Xu XC, Polosukhin VV, Jones proliferation by alveolar epithelial cells is cyclooxygenase-2-
BR, McMahon FB, et al.: Repetitive intratracheal bleomycin dependent. Am J Respir Cell Mol Biol. 2002;27(6):752–758.
models several features of idiopathic pulmonary fibrosis. [65] Selman M, Pardo A: Role of epithelial cells in idiopathic pul-
Am J Physiol Lung Cell Mol Physiol. 2010;299(4):L442– monary fibrosis: from innocent targets to serial killers. Proc
L452. Am Thoracic Soc. 2006;3(4):364–372.
[48] Peng R, Sridhar S, Tyagi G, Phillips JE, Garrido R, Harris P, [66] Molina-Molina M, Serrano-Mollar A, Bulbena O, Fernandez-
et al.: Bleomycin induces molecular changes directly relevant Zabalegui L, Closa D, Marin-Arguedas A, et al.: Losar-
to idiopathic pulmonary fibrosis: a model for “active” disease. tan attenuates bleomycin induced lung fibrosis by increasing
PloS one. 2013;8(4):e59348. prostaglandin E2 synthesis. Thorax. 2006;61(7):604–610.
[49] Azuma A, Takahashi S, Nose M, Araki K, Araki M, Takahashi [67] Moore BB, Ballinger MN, White ES, Green ME, Herrygers
T, et al.: Role of E-selectin in bleomycin induced lung fibrosis AB, Wilke CA, et al.: Bleomycin-induced E prostanoid recep-
in mice. Thorax. 2000;55(2):147–152. tor changes alter fibroblast responses to prostaglandin E2. J
[50] Izbicki G, Segel MJ, Christensen TG, Conner MW, Breuer Immunol. 2005;174(9):5644–5649.
R: Time course of bleomycin-induced lung fibrosis. Int J Exp [68] Hagimoto N, Kuwano K, Nomoto Y, Kunitake R, Hara
Pathol. 2002;83(3):111–119. N: Apoptosis and expression of Fas/Fas ligand mRNA in
[51] Manali ED, Moschos C, Triantafillidou C, Kotanidou A, Psal- bleomycin-induced pulmonary fibrosis in mice. Am J Respir
lidas I, Karabela SP, et al.: Static and dynamic mechanics of the Cell Mol Biol. 1997;16(1):91–101.
murine lung after intratracheal bleomycin. BMC Pulm Med. [69] Li X, Zhang H, Soledad-Conrad V, Zhuang J, Uhal BD:
2011;11:33. Bleomycin-induced apoptosis of alveolar epithelial cells re-
[52] Selman M, Pardo A: Idiopathic pulmonary fibrosis: an epithe- quires angiotensin synthesis de novo. Am J Physiol Lung Cell
lial/fibroblastic cross-talk disorder. Respir Res. 2002;3:3. Mol Physiol. 2003;284(3):L501–L507.
[53] Iyer SN, Hyde DM, Giri SN: Anti-inflammatory effect of pir- [70] Wang R, Zagariya A, Ibarra-Sunga O, Gidea C, Ang E, Desh-
fenidone in the bleomycin-hamster model of lung inflamma- mukh S, et al.: Angiotensin II induces apoptosis in human
tion. Inflammation. 2000;24(5):477–491. and rat alveolar epithelial cells. Am J Physiol. 1999;276(5 Pt
[54] Rydell-Törmänen K, Andreasson K, Hesselstrand R, Risteli J, 1):L885–L889.
Heinegard D, Saxne T, et al.: Extracellular matrix alterations [71] Hagimoto N, Kuwano K, Miyazaki H, Kunitake R, Fujita M,
and acute inflammation; developing in parallel during early Kawasaki M, et al.: Induction of apoptosis and pulmonary fi-
induction of pulmonary fibrosis. Lab Invest J Tech Method brosis in mice in response to ligation of Fas antigen. Am J
Pathol. 2012;92(6):917–925. Respir Cell Mol Biol. 1997;17(3):272–278.
[55] Kumar RK, Watkins SG, Lykke AW: Pulmonary responses to [72] Hagimoto N, Kuwano K, Inoshima I, Yoshimi M, Naka-
bleomycin-induced injury: an immunomorphologic and elec- mura N, Fujita M, et al.: TGF-beta 1 as an enhancer of
tron microscopic study. Exp Pathol. 1985;28(1):33–43. Fas-mediated apoptosis of lung epithelial cells. J Immunol.
[56] Lee TH, Avraham H, Lee SH, Avraham S: Vascular en- 2002;168(12):6470–6478.
dothelial growth factor modulates neutrophil transendothe- [73] Wang D, Morales JE, Calame DG, Alcorn JL, Wetsel RA:
lial migration via up-regulation of interleukin-8 in hu- Transplantation of human embryonic stem cell-derived alve-
man brain microvascular endothelial cells. J Biol Chem. olar epithelial type II cells abrogates acute lung injury in mice.
2002;277(12):10445–10451. Mol Ther: J Am Soc Gene Ther. 2010;18(3):625–634.
[57] Mishra A, Doyle NA, Martin WJc: Bleomycin-mediated pul- [74] Serrano-Mollar A, Nacher M, Gay-Jordi G, Closa D, Xaubet
monary toxicity: evidence for a p53-mediated response. Am J A, Bulbena O: Intratracheal transplantation of alveolar type
Respir Cell Mol Biol. 2000;22(5):543–549. II cells reverses bleomycin-induced lung fibrosis. Am J Respir
[58] Kawamoto M, Fukuda Y: Cell proliferation during the process Crit Care Med. 2007;176(12):1261–1268.
of bleomycin-induced pulmonary fibrosis in rats. Acta Pathol. [75] Matute-Bello G, Frevert CW, Martin TR: Animal models
Japonica. 1990;40(4):227–238. of acute lung injury. Am J Physiol Lung Cell Mol Physiol.
[59] Sugahara K, Iyama K, Kuroda MJ, Sano K: Double in- 2008;295(3):L379–L399.
tratracheal instillation of keratinocyte growth factor pre- [76] Adamson IY, Bowden DH: The pathogenesis of bloemycin-
vents bleomycin-induced lung fibrosis in rats. J Pathol. induced pulmonary fibrosis in mice. Am J Pathol.
1998;186(1):90–98. 1974;77(2):185–197.
[60] Wang R, Ibarra-Sunga O, Verlinski L, Pick R, Uhal BD: Ab- [77] Adamson IY, Bowden DH: Origin of ciliated alveolar ep-
rogation of bleomycin-induced epithelial apoptosis and lung ithelial cells in bleomycin-induced lung injury. Am J Pathol.
fibrosis by captopril or by a caspase inhibitor. Am J Physiol 1977;87(3):569–580.
Lung Cell Mol Physiol. 2000;279(1):L143–L151. [78] Adamson IY, Bowden DH: Bleomycin-induced injury and
[61] Adamson IY, Young L, Bowden DH: Relationship of alveo- metaplasia of alveolar type 2 cells. Relationship of cellu-
lar epithelial injury and repair to the induction of pulmonary lar responses to drug presence in the lung. Am J Pathol.
fibrosis. Am J Pathol. 1988;130(2):377–383. 1979;96(2):531–544.

[79] Kuwano K, Hagimoto N, Kawasaki M, Yatomi T, Nakamura expression of monocyte chemoattractant protein-1 (MCP-1)
N, Nagata S, et al.: Essential roles of the Fas-Fas ligand path- in bleomycin-induced lung injury of rats studied by a novel
way in the development of pulmonary fibrosis. J Clin Invest. monoclonal antibody against rat MCP-1. J Leukocyte Biol.
1999;104(1):13–19. 1994;56(6):741–750.
[80] Maeyama T, Kuwano K, Kawasaki M, Kunitake R, Hagimoto [95] Gasse P, Riteau N, Charron S, Girre S, Fick L, Petrilli V, et al.:
N, Matsuba T, et al.: Upregulation of Fas-signalling molecules Uric acid is a danger signal activating NALP3 inflammasome
in lung epithelial cells from patients with idiopathic pulmonary in lung injury inflammation and fibrosis. Am J Respir Crit Care
fibrosis. Eur Respir J: Official J Eur Soc Clin Resp Physiol. Med. 2009;179(10):903–913.
2001;17(2):180–189. [96] Gibbons MA, MacKinnon AC, Ramachandran P, Dhaliwal
[81] Wang R, Ramos C, Joshi I, Zagariya A, Pardo A, Selman M, K, Duffin R, Phythian-Adams AT, et al.: Ly6Chi mono-
et al.: Human lung myofibroblast-derived inducers of alveo- cytes direct alternatively activated profibrotic macrophage
lar epithelial apoptosis identified as angiotensin peptides. Am regulation of lung fibrosis. Am J Respir Crit Care Med.
J Physiol. 1999;277(6 Pt 1):L1158–L1164. 2011;184(5):569–581.
[82] Uhal BD, Kim JK, Li X, Molina-Molina M: Angiotensin- [97] Car BD, Meloni F, Luisetti M, Semenzato G, Gialdroni-Grassi
TGF-beta 1 crosstalk in human idiopathic pulmonary fibro- G, Walz A: Elevated IL-8 and MCP-1 in the bronchoalveo-
sis: autocrine mechanisms in myofibroblasts and macrophages. lar lavage fluid of patients with idiopathic pulmonary fibro-
Curr Pharm Design. 2007;13(12):1247–1256. sis and pulmonary sarcoidosis. Am J Respir Crit Care Med.
[83] Molina-Molina M, Xaubet A, Li X, Abdul-Hafez A, Friderici 1994;149(3 Pt 1):655–659.
K, Jernigan K, et al.: Angiotensinogen gene G-6A polymor- [98] Carre PC, Mortenson RL, King TE, Jr., Noble PW, Sable CL,
phism influences idiopathic pulmonary fibrosis disease pro- Riches DW: Increased expression of the interleukin-8 gene by
gression. Eur Respir J: Official J Eur Soc Clin Respir Physiol. alveolar macrophages in idiopathic pulmonary fibrosis. A po-
2008;32(4):1004–1008. tential mechanism for the recruitment and activation of neu-
[84] Nadrous HF, Ryu JH, Douglas WW, Decker PA, Olson trophils in lung fibrosis. J Clin Invest. 1991;88(6):1802–1810.
EJ: Impact of angiotensin-converting enzyme inhibitors and [99] Standiford TJ, Rolfe MW, Kunkel SL, Lynch JP, 3rd, Burdick
statins on survival in idiopathic pulmonary fibrosis. Chest. MD, Gilbert AR, et al.: Macrophage inflammatory protein-
2004;126(2):438–446. 1 alpha expression in interstitial lung disease. J Immunol.
[85] King TE, Jr., Pardo A, Selman M: Idiopathic pulmonary fibro- 1993;151(5):2852–2863.
sis. Lancet. 2011;378(9807):1949–1961. [100] Schwartz DA, Helmers RA, Dayton CS, Merchant RK, Hun-
[86] Khalil N, O’Connor RN, Flanders KC, Unruh H: TGF- ninghake GW: Determinants of bronchoalveolar lavage cel-
beta 1, but not TGF-beta 2 or TGF-beta 3, is differentially lularity in idiopathic pulmonary fibrosis. J Appl Physiol.
present in epithelial cells of advanced pulmonary fibrosis: 1991;71(5):1688–1693.

an immunohistochemical study. Am J Respir Cell Mol Biol. [101] Zhang Y, Lee TC, Guillemin B, Yu MC, Rom WN: En-
1996;14(2):131–138. hanced IL-1 beta and tumor necrosis factor-alpha release and
[87] Fujii M, Hayakawa H, Urano T, Sato A, Chida K, Naka- messenger RNA expression in macrophages from idiopathic
mura H, et al.: Relevance of tissue factor and tissue fac- pulmonary fibrosis or after asbestos exposure. J Immunol.
tor pathway inhibitor for hypercoagulable state in the lungs 1993;150(9):4188–4196.
of patients with idiopathic pulmonary fibrosis. Thromb Res. [102] Furuie H, Yamasaki H, Suga M, Ando M: Altered accessory
2000;99(2):111–117. cell function of alveolar macrophages: a possible mechanism
[88] Uhal BD, Joshi I, True AL, Mundle S, Raza A, Pardo A, et al.: for induction of Th2 secretory profile in idiopathic pulmonary
Fibroblasts isolated after fibrotic lung injury induce apoptosis fibrosis. Eur Respir J: Official J Eur Soc Clin Respir Physiol.
of alveolar epithelial cells in vitro. Am J Physiol. 1995;269(6 Pt 1997;10(4):787–794.
1):L819–L828. [103] Piguet PF, Ribaux C, Karpuz V, Grau GE, Kapanci Y: Ex-
[89] Barbas-Filho JV, Ferreira MA, Sesso A, Kairalla RA, Car- pression and localization of tumor necrosis factor-alpha and
valho CR, Capelozzi VL: Evidence of type II pneumocyte its mRNA in idiopathic pulmonary fibrosis. Am J Pathol.
apoptosis in the pathogenesis of idiopathic pulmonary fibro- 1993;143(3):651–655.
sis (IFP)/usual interstitial pneumonia (UIP). J Clin Pathol. [104] Pan LH, Ohtani H, Yamauchi K, Nagura H: Co-expression
2001;54(2):132–138. of TNF alpha and IL-1 beta in human acute pulmonary fi-
[90] Song M, He B, Qiu Z: Expressions of TNF alpha, PDGF in brotic diseases: an immunohistochemical analysis. Pathol Int.
alveolar type II epithelial cells of rats with bleomycin-induced 1996;46(2):91–99.
pulmonary fibrosis. Zhonghua jie he he hu xi za zhi (Chinese J [105] Gharaee-Kermani M, Phan SH: Lung interleukin-5 expression
Tuberculosis Respir Dis.) 1998;21(4):221–223. in murine bleomycin-induced pulmonary fibrosis. Am J Respir
[91] Cavarra E, Carraro F, Fineschi S, Naldini A, Bartalesi B, Pucci Cell Mol Biol. 1997;16(4):438–447.
A, et al.: Early response to bleomycin is characterized by dif- [106] Gharaee-Kermani M, Nozaki Y, Hatano K, Phan SH: Lung
ferent cytokine and cytokine receptor profiles in lungs. Am J interleukin-4 gene expression in a murine model of bleomycin-
Physiol Lung Cell Mol Physiol. 2004;287(6):L1186–L1192. induced pulmonary fibrosis. Cytokine. 2001;15(3):138–147.
[92] Aumiller V, Balsara N, Wilhelm J, Gunther A, Konigshoff M: [107] Piguet PF, Collart MA, Grau GE, Kapanci Y, Vassalli
WNT/beta-catenin signaling induces IL-1beta expression by P: Tumor necrosis factor/cachectin plays a key role in
alveolar epithelial cells in pulmonary fibrosis. Am J Respir Cell bleomycin-induced pneumopathy and fibrosis. J Exp Med.
Mol Biol. 2013;49(1):96–104. 1989;170(3):655–663.
[93] Everson MP, Chandler DB: Changes in distribution, mor- [108] Vesey DA, Cheung C, Cuttle L, Endre Z, Gobe G, Johnson
phology, and tumor necrosis factor-alpha secretion of alve- DW: Interleukin-1 beta stimulates human renal fibroblast pro-
olar macrophage subpopulations during the development liferation and matrix protein production by means of a trans-
of bleomycin-induced pulmonary fibrosis. Am J Pathol. forming growth factor-beta-dependent mechanism. J Lab Clin
1992;140(2):503–512. Med. 2002;140(5):342–350.
[94] Sakanashi Y, Takeya M, Yoshimura T, Feng L, Morioka T, [109] Tufvesson E, Westergren-Thorsson G: Alteration of pro-
Takahashi K: Kinetics of macrophage subpopulations and teoglycan synthesis in human lung fibroblasts induced by

interleukin-1 beta and tumor necrosis factor-alpha. J Cell [126] Peterson MW, Monick M, Hunninghake GW: Prog-
Biochem. 2000;77(2):298–309. nostic role of eosinophils in pulmonary fibrosis. Chest.
[110] Solis-Herruzo JA, Brenner DA, Chojkier M: Tumor necro- 1987;92(1):51–56.
sis factor-alpha inhibits collagen gene-transcription and [127] Haslam PL, Turton CWG, Lukoszek A, Salsbury AJ, Dewar
collagen-synthesis in cultured human-fibroblasts. J Biol Chem. A, Collins JV, et al.: Bronchoalveolar lavage fluid cell counts
1988;263(12):5841–5845. in cryptogenic fibrosing alveolitis and their relation to therapy.
[111] Siwik DA, Chang DLF, Colucci WS: Interleukin-1 beta and Thorax. 1980;35(5):328–339.
tumor necrosis factor-alpha decrease collagen synthesis and in- [128] Reynolds HY, Fulmer JD, Kazmierowski JA, Roberts WC,
crease matrix metalloproteinase activity in cardiac fibroblasts Frank MM, Crystal RG: Analysis of cellular and protein-
in vitro. Circ Res. 2000;86(12):1259–1265. content of broncho-alveolar lavage fluid from patients with id-
[112] Gharaee-Kermani M, Denholm EM, Phan SH: Costimulation iopathic pulmonary fibrosis and chronic hypersensitivity pneu-
of fibroblast collagen and transforming growth factor beta(1) monitis. J Clin Invest. 1977;59(1):165–175.
gene expression by monocyte chemoattractant protein-1 via [129] Daniil Z, Kitsanta P, Kapotsis G, Mathioudaki M, Kollintza A,
specific receptors. J Biol Chem. 1996;271(30):17779–17784. Karatza M, et al.: CD8+ T lymphocytes in lung tissue from pa-
[113] Moore BB, Peters-Golden M, Christensen PJ, Lama V, Kuziel tients with idiopathic pulmonary fibrosis. Respir Res. 2005;6.
WA, Paine R, et al.: Alveolar epithelial cell inhibition of fibrob- [130] Obayashi Y, Yamadori I, Fujita J, Yoshinouchi T, Ueda
last proliferation is regulated by MCP-1/CCR2 and mediated N, Takahara J: The role of neutrophils in the pathogenesis
by PGE2. Am J Physiol-Lung C. 2003;284(2):L342–L349. of idiopathic pulmonary fibrosis. Chest. 1997;112(5):1338–
[114] Giri SN, Hyde DM, Nakashima JM: Analysis of bronchoalve- 1343.
olar lavage fluid from bleomycin-induced pulmonary fibrosis [131] Tabuena RP, Nagai S, Tsutsumi T, Handa T, Minoru T,
in hamsters. Toxicol Pathol. 1986;14(2):149–157. Mikuniya T, et al.: Cell profiles of bronchoalveolar lavage
[115] Helene M, Lake-Bullock V, Zhu J, Hao H, Cohen DA, Kaplan fluid as prognosticators of idiopathic pulmonary fibrosis/usual
AM: T cell independence of bleomycin-induced pulmonary fi- interstitial pneumonia among Japanese patients. Respiration.
brosis. J Leukocyte Biol. 1999;65(2):187–195. 2005;72(5):490–498.
[116] Izumo T, Kondo M, Nagai A: Effects of a leukotriene B4 [132] Hunninghake GW, Kawanami O, Ferrans VJ, Young RC, Jr.,
receptor antagonist on bleomycin-induced pulmonary fibro- Roberts WC, Crystal RG: Characterization of the inflamma-
sis. Eur Respir J: Official J Eur Soc Clin Respir Physiol. tory and immune effector cells in the lung parenchyma of
2009;34(6):1444–1451. patients with interstitial lung disease. Am Rev Respir Dis.
[117] Okuma T, Terasaki Y, Kaikita K, Kobayashi H, Kuziel WA, 1981;123(4 Pt 1):407–412.
Kawasuji M, et al.: C-C chemokine receptor 2 (CCR2) de- [133] Kradin RL, Divertie MB, Colvin RB, Ramirez J, Ryu J, Car-
ficiency improves bleomycin-induced pulmonary fibrosis by penter HA, et al.: Usual interstitial pneumonitis is a T-cell alve-
attenuation of both macrophage infiltration and production olitis. Clin Immunol Immunopathol. 1986;40(2):224–235.
of macrophage-derived matrix metalloproteinases. J Pathol. [134] Selman M, Gonzalez G, Bravo M, Sullivan-Lopez J, Ramos C,
2004;204(5):594–604. Montano M, et al.: Effect of lung T lymphocytes on fibroblasts
[118] Kim JY, Choeng HC, Ahn C, Cho SH: Early and late changes in idiopathic pulmonary fibrosis and extrinsic allergic alveolitis.
of MMP-2 and MMP-9 in bleomycin-induced pulmonary fi- Thorax. 1990;45(6):451–455.
brosis. Yonsei Med J. 2009;50(1):68–77. [135] Papiris SA, Kollintza A, Karatza M, Manali ED, Sotiropoulou
[119] Baer M, Dillner A, Schwartz RC, Sedon C, Nedospasov C, Milic-Emili J, et al.: CD8 +T lymphocytes in bron-
S, Johnson PF: Tumor necrosis factor alpha transcription choalveolar lavage in idiopathic pulmonary fibrosis. J Inflamm.
in macrophages is attenuated by an autocrine factor that 2007;4:14.
preferentially induces NF-kappa B p50. Mol Cell Biol. [136] Papiris SA, Kollintza A, Kitsanta P, Kapotsis G, Karatza M,
1998;18(10):5678–5689. Milic-Emili J, et al.: Relationship of BAL and lung tissue
[120] Gasse P, Mary C, Guenon I, Noulin N, Charron S, Schnyder- CD4+ and CD8+ T lymphocytes, and their ratio in idiopathic
Candrian S, et al.: IL-1R1/MyD88 signaling and the inflam- pulmonary fibrosis. Chest. 2005;128(4):2971–2977.
masome are essential in pulmonary inflammation and fibrosis [137] Campbell DA, Poulter LW, Janossy G, du Bois RM: Immuno-
in mice. J Clin Invest. 2007;117(12):3786–3799. histological analysis of lung tissue from patients with crypto-
[121] Gharaee-Kermani M, McGarry B, Lukacs N, Huffnagle G, genic fibrosing alveolitis suggesting local expression of immune
Egan RW, Phan SH: The role of IL-5 in bleomycin-induced hypersensitivity. Thorax. 1985;40(6):405–411.
pulmonary fibrosis. J Leukocyte Biol. 1998;64(5):657–666. [138] Wallace WA, Howie SE, Krajewski AS, Lamb D: The immuno-
[122] Hao H, Cohen DA, Jennings CD, Bryson JS, Kaplan AM: logical architecture of B-lymphocyte aggregates in cryptogenic
Bleomycin-induced pulmonary fibrosis is independent of fibrosing alveolitis. J Pathol. 1996;178(3):323–329.
eosinophils. J Leukocyte Biol. 2000;68(4):515–521. [139] Xue J, Kass DJ, Bon J, Vuga L, Tan J, Csizmadia E,
[123] Schrier DJ, Phan SH, Mcgarry BM: The effects of the et al.: Plasma B lymphocyte stimulator and B cell differen-
nude (Nu Nu) mutation on bleomycin-induced pulmonary tiation in idiopathic pulmonary fibrosis patients. J Immunol.
fibrosis - a biochemical evaluation. Am Rev Respir Dis. 2013;191(5):2089–2095.
1983;127(5):614–617. [140] Schwartz MI, Dreisin RB, Pratt DS, Stanford RE: Immunoflu-
[124] Boomars KA, Schweizer RC, Zanen P, van den Bosch JMM, orescent patterns in the idiopathic interstitial pneumonias. J
Lammers JWJ, Koenderman L: Eosinophil chemotactic activ- Lab Clin Med. 1978;91(6):929–938.
ity in bronchoalveolar lavage from idiopathic pulmonary fibro- [141] Wallace WA, Schofield JA, Lamb D, Howie SE: Localisation
sis is dependent on cytokine priming of eosinophils. Eur Respir of a pulmonary autoantigen in cryptogenic fibrosing alveolitis.
J. 1998;11(5):1009–1014. Thorax. 1994;49(11):1139–1145.
[125] Yasuoka S, Nakayama T, Kawano T, Ogushi F, Doi H, [142] Matsuse T, Teramoto S, Katayama H, Sudo E, Ekimoto H,
Hayashi H, et al.: Comparison of cell profiles of bronchial and Mitsuhashi H, et al.: ICAM-1 mediates lung leukocyte re-
bronchoalveolar lavage fluids between normal subjects and pa- cruitment but not pulmonary fibrosis in a murine model of
tients with idiopathic pulmonary fibrosis. Tohoku J Exp Med. bleomycin-induced lung injury. Eur Respir J: Official J Eur Soc
1985;146(1):33–45. Clin Respir Physiol. 1999;13(1):71–77.

[143] Williams AE, Chambers RC: The mercurial nature of neu- collagen synthesis in chronic pulmonary inflammation. J Exp
trophils: still an enigma in ARDS? Am J Physiol Lung Cell Mol Med. 1989;170(3):727–737.
Physiol. 2014;306(3):L217–L230. [159] Daniels CE, Wilkes MC, Edens M, Kottom TJ, Murphy SJ,
[144] Desmouliere A, Geinoz A, Gabbiani F, Gabbiani G: Trans- Limper AH, et al.: Imatinib mesylate inhibits the profibrogenic
forming growth factor-beta 1 induces alpha-smooth mus- activity of TGF-beta and prevents bleomycin-mediated lung
cle actin expression in granulation tissue myofibroblasts and fibrosis. J Clin Invest. 2004;114(9):1308–1316.
in quiescent and growing cultured fibroblasts. J Cell Biol. [160] Kurotani R, Okumura S, Matsubara T, Yokoyama U,
1993;122(1):103–111. Buckley JR, Tomita T, et al.: Secretoglobin 3A2 sup-
[145] Cutroneo KR, White SL, Phan SH, Ehrlich HP: Therapies presses bleomycin-induced pulmonary fibrosis by transform-
for bleomycin induced lung fibrosis through regulation of ing growth factor beta signaling down-regulation. J Biol Chem.
TGF-beta1 induced collagen gene expression. J Cell Physiol. 2011;286(22):19682–19692.
2007;211(3):585–589. [161] Wang Q, Wang Y, Hyde DM, Gotwals PJ, Koteliansky VE,
[146] Hu B, Wu Z, Phan SH: Smad3 mediates transforming growth Ryan ST, et al.: Reduction of bleomycin induced lung fibrosis
factor-beta-induced alpha-smooth muscle actin expression. by transforming growth factor beta soluble receptor in ham-
Am J Respir Cell Mol Biol. 2003;29(3):397–404. sters. Thorax. 1999;54(9):805–812.
[147] Ponticos M, Holmes AM, Xu SW, Leoni P, Khan K, Rajkumar [162] Arribillaga L, Dotor J, Basagoiti M, Riezu-Boj JI, Borras-
VS, et al.: Pivotal role of connective tissue growth factor in lung Cuesta F, Lasarte JJ, et al.: Therapeutic effect of a pep-
fibrosis MAPK-dependent transcriptional activation of type I tide inhibitor of TGF-beta on pulmonary fibrosis. Cytokine.
collagen. Arthritis Rheum. 2009;60(7):2142–2155. 2011;53(3):327–333.

[148] Annes JP, Chen Y, Munger JS, Rifkin DB: Integrin [163] Russo RC, Guabiraba R, Garcia CC, Barcelos LS, Roffe E,
alpha(v)beta(6)-mediated activation of latent TGF-beta re- Souza AL, et al.: Role of the chemokine receptor CXCR2 in
quires the latent TGF-beta binding protein-1. J Cell Biol. bleomycin-induced pulmonary inflammation and fibrosis. Am
2004;165(5):723–734. J Respir Cell Mol Biol. 2009;40(4):410–421.
[149] Schultz-Cherry S, Murphy-Ullrich JE: Thrombospondin [164] Atzori L, Chua F, Dunsmore SE, Willis D, Barbarisi M,
causes activation of latent transforming growth factor-beta se- McAnulty RJ, et al.: Attenuation of bleomycin induced
creted by endothelial cells by a novel mechanism. J Cell Biol. pulmonary fibrosis in mice using the heme oxygenase in-
1993;122(4):923–932. hibitor Zn-deuteroporphyrin IX-2,4-bisethylene glycol. Tho-
[150] Grotendorst GR, Duncan MR: Individual domains of connec- rax. 2004;59(3):217–223.
tive tissue growth factor regulate fibroblast proliferation and [165] Yehualaeshet T, O’Connor R, Green-Johnson J, Mai S, Sil-
myofibroblast differentiation. FASEB J: Official Pub Federat verstein R, Murphy-Ullrich JE, et al.: Activation of rat alve-
Am Soc Exp Biol. 2005;19(7):729–738. olar macrophage-derived latent transforming growth factor
[151] Grotendorst GR, Rahmanie H, Duncan MR: Combinatorial beta-1 by plasmin requires interaction with thrombospondin-
signaling pathways determine fibroblast proliferation and my- 1 and its cell surface receptor, CD36. Am J Pathol.
ofibroblast differentiation. FASEB J: Official Publ Federat Am 1999;155(3):841–851.
Soc Exp Biol. 2004;18(3):469–479. [166] Zhang L, Lu HP, Lai GX: A preliminary study of CTGF
[152] Duncan MR, Frazier KS, Abramson S, Williams S, Klapper in bleomycin-induced mouse pulmonary fibrosis. Xi Bao yu
H, Huang X, et al.: Connective tissue growth factor medi- fen zi mian yi xue za zhi (Chinese J Cell Mol Immunol.)
ates transforming growth factor beta-induced collagen synthe- 2005;21(3):290–292.
sis: down-regulation by cAMP. FASEB J: Official Publ Federat [167] Datta A, Scotton CJ, Chambers RC: Novel therapeu-
Am Soc Exp Biol. 1999;13(13):1774–1786. tic approaches for pulmonary fibrosis. Brit J Pharmacol.
[153] Kothapalli D, Frazier KS, Welply A, Segarini PR, Grotendorst 2011;163(1):141–172.
GR: Transforming growth factor beta induces anchorage- [168] Khalil N, O’Connor RN, Unruh HW, Warren PW, Flanders
independent growth of NRK fibroblasts via a connective tis- KC, Kemp A, et al.: Increased production and immunohis-
sue growth factor-dependent signaling pathway. Cell Growth tochemical localization of transforming growth factor-beta in
Differ: Mol Biol J Am Assoc Cancer Res. 1997;8(1):61–68. idiopathic pulmonary fibrosis. Am J Respir Cell Mol Biol.
[154] Vyalov SL, Gabbiani G, Kapanci Y: Rat alveolar myofi- 1991;5(2):155–162.
broblasts acquire alpha-smooth muscle actin expression dur- [169] Kapanci Y, Desmouliere A, Pache JC, Redard M, Gabbiani
ing bleomycin-induced pulmonary fibrosis. Am J Pathol. G: Cytoskeletal protein modulation in pulmonary alveolar
1993;143(6):1754–1765. myofibroblasts during idiopathic pulmonary fibrosis. Possi-
[155] Coker RK, Laurent GJ, Shahzeidi S, Lympany PA, du Bois ble role of transforming growth factor beta and tumor necro-
RM, Jeffery PK, et al.: Transforming growth factors-beta 1, sis factor alpha. Am J Respir Crit Care Med. 1995;152(6 Pt
-beta 2, and -beta 3 stimulate fibroblast procollagen produc- 1):2163–2169.
tion in vitro but are differentially expressed during bleomycin- [170] Hiwatari N, Shimura S, Yamauchi K, Nara M, Hida W,
induced lung fibrosis. Am J Pathol. 1997;150(3):981–991. Shirato K: Significance of elevated procollagen-III-peptide
[156] Santana A, Saxena B, Noble NA, Gold LI, Marshall BC: In- and transforming growth factor-beta levels of bronchoalveo-
creased expression of transforming growth factor beta isoforms lar lavage fluids from idiopathic pulmonary fibrosis patients.
(beta 1, beta 2, beta 3) in bleomycin-induced pulmonary fibro- Tohoku J Exp Med. 1997;181(2):285–295.
sis. Am J Respir Cell Mol Biol. 1995;13(1):34–44. [171] Khalil N, Parekh TV, O’Connor R, Antman N, Kepron W,
[157] Nakagome K, Dohi M, Okunishi K, Tanaka R, Miyazaki Yehaulaeshet T, et al.: Regulation of the effects of TGF-beta
J, Yamamoto K: In vivo IL-10 gene delivery attenuates 1 by activation of latent TGF-beta 1 and differential expres-
bleomycin induced pulmonary fibrosis by inhibiting the pro- sion of TGF-beta receptors (T beta R-I and T beta R-II)
duction and activation of TGF-beta in the lung. Thorax. in idiopathic pulmonary fibrosis. Thorax. 2001;56(12):907–
2006;61(10):886–894. 915.
[158] Khalil N, Bereznay O, Sporn M, Greenberg AH: Macrophage [172] Tatler AL, Saini G, Goodwin A, Gbolohan O, Clifford
production of transforming growth factor beta and fibroblast RL, Al’Hourani M, et al.: Transcriptional Mechanisms

Regulating Expression of the Avb6 Integrin in Ipf. Thorax. [188] Ross B, D’Orleans-Juste P, Giaid A: Potential role of
2012;67:A34–A35. endothelin-1 in pulmonary fibrosis: from the bench to the
[173] John AE, Luckett JC, Tatler AL, Awais RO, Desai A, Hab- clinic. Am J Respir Cell Mol Biol. 2010;42(1):16–20.
good A, et al.: Preclinical SPECT/CT imaging of alphav- [189] Saleh D, Furukawa K, Tsao MS, Maghazachi A, Corrin B,
beta6 integrins for molecular stratification of idiopathic pul- Yanagisawa M, et al.: Elevated expression of endothelin-1 and
monary fibrosis. J Nucl Med: Official Publ, Soc Nucl Med. endothelin-converting enzyme-1 in idiopathic pulmonary fi-
2013;54(12):2146–2152. brosis: possible involvement of proinflammatory cytokines. Am
[174] Reibman J, Meixler S, Lee TC, Gold LI, Cronstein BN, J Respir Cell Mol Biol. 1997;16(2):187–193.
Haines KA, et al.: Transforming growth factor beta 1, a po- [190] Reichenberger F, Schauer J, Kellner K, Sack U, Stiehl P,
tent chemoattractant for human neutrophils, bypasses clas- Winkler J: Different expression of endothelin in the bron-
sic signal-transduction pathways. Proc Natl Acad Sci USA. choalveolar lavage in patients with pulmonary diseases. Lung.
1991;88(15):6805–6809. 2001;179(3):163–174.
[175] Khalil N, O’Connor RN, Flanders KC, Shing W, Whitman [191] King TE, Jr., Brown KK, Raghu G, du Bois RM, Lynch DA,
CI: Regulation of type II alveolar epithelial cell proliferation by Martinez F, et al.: BUILD-3: a randomized, controlled trial
TGF-beta during bleomycin-induced lung injury in rats. Am J of bosentan in idiopathic pulmonary fibrosis. Am J Respir Crit
Physiol. 1994;267(5 Pt 1):L498–L507. Care Med. 2011;184(1):92–99.
[176] Zhang F, Nielsen LD, Lucas JJ, Mason RJ: Transforming [192] Zhang HY, Gharaee-Kermani M, Zhang K, Karmiol S, Phan
growth factor-beta antagonizes alveolar type II cell prolifera- SH: Lung fibroblast alpha-smooth muscle actin expression and
tion induced by keratinocyte growth factor. Am J Respir Cell contractile phenotype in bleomycin-induced pulmonary fibro-
Mol Biol. 2004;31(6):679–686. sis. Am J Pathol. 1996;148(2):527–537.
[177] Yue J, Mulder KM: Transforming growth factor-beta sig- [193] Zhang K, Rekhter MD, Gordon D, Phan SH: Myofibroblasts
nal transduction in epithelial cells. Pharmacol Therapeut. and their role in lung collagen gene expression during pul-
2001;91(1):1–34. monary fibrosis. A combined immunohistochemical and in situ
[178] Park SH, Saleh D, Giaid A, Michel RP: Increased endothelin-1 hybridization study. Am J Pathol. 1994;145(1):114–125.
in bleomycin-induced pulmonary fibrosis and the effect of an [194] Phan SH. The myofibroblast in pulmonary fibrosis. Chest.
endothelin receptor antagonist. Am J Respir Crit Care Med. 2002;122(6 Suppl):286S–289S.
1997;156(2 Pt 1):600–608. [195] Zhang K, Flanders KC, Phan SH: Cellular localiza-
[179] Gurujeyalakshmi G, Hollinger MA, Giri SN: Pirfenidone in- tion of transforming growth factor-beta expression in
hibits PDGF isoforms in bleomycin hamster model of lung fi- bleomycin-induced pulmonary fibrosis. Am J Pathol.
brosis at the translational level. Am J Physiol. 1999;276(2 Pt 1995;147(2):352–361.
1):L311–L318. [196] Scotton CJ, Chambers RC: Molecular targets in pul-

[180] Bonner JC: Regulation of PDGF and its receptors in fi- monary fibrosis: the myofibroblast in focus. Chest.
brotic diseases. Cytokine Growth Factor Rev. 2004;15(4):255– 2007;132(4):1311–1321.
273. [197] Pardo A, Selman M: Idiopathic pulmonary fibrosis:
[181] Bergmann M, Tiroke A, Schafer H, Barth J, Haverich A: Gene new insights in its pathogenesis. Int J Biochem Cell B.
expression of profibrotic mediators in bronchiolitis obliterans 2002;34(12):1534–1538.
syndrome after lung transplantation. Scand Cardiovasc J: SCJ. [198] Kuhn C, McDonald JA: The roles of the myofibroblast in id-
1998;32(2):97–103. iopathic pulmonary fibrosis. Ultrastructural and immunohis-
[182] Antoniades HN, Bravo MA, Avila RE, Galanopoulos T, tochemical features of sites of active extracellular matrix syn-
Neville-Golden J, Maxwell M, et al.: Platelet-derived growth thesis. Am J Pathol. 1991;138(5):1257–1265.
factor in idiopathic pulmonary fibrosis. J Clin Invest. [199] Thannickal VJ, Horowitz JC: Evolving concepts of apopto-
1990;86(4):1055–1064. sis in idiopathic pulmonary fibrosis. Proc Am Thoracic Soc.
[183] Nagaoka I, Trapnell BC, Crystal RG: Upregulation of platelet- 2006;3(4):350–356.
derived growth factor-A and -B gene expression in alveolar [200] Vuga LJ, Ben-Yehudah A, Kovkarova-Naumovski E, Oriss T,
macrophages of individuals with idiopathic pulmonary fibro- Gibson KF, Feghali-Bostwick C, et al.: WNT5A is a regula-
sis. J Clin Invest. 1990;85(6):2023–2027. tor of fibroblast proliferation and resistance to apoptosis. Am J
[184] Martinet Y, Rom WN, Grotendorst GR, Martin GR, Respir Cell Mol Biol. 2009;41(5):583–589.
Crystal RG: Exaggerated spontaneous release of platelet- [201] Macias-Barragan J, Sandoval-Rodriguez A, Navarro-Partida J,
derived growth factor by alveolar macrophages from pa- Armendariz-Borunda J: The multifaceted role of pirfenidone
tients with idiopathic pulmonary fibrosis. N Engl J Med. and its novel targets. Fibrogenesis Tissue Repair. 2010;3:16.
1987;317(4):202–209. [202] Jenkins G: Pirfenidone should be prescribed for patients with
[185] Fonseca C, Abraham D, Renzoni EA: Endothelin in pul- idiopathic pulmonary fibrosis. Thorax. 2013;68(7):603–605.
monary fibrosis. Am J Respir Cell Mol Biol. 2011;44(1): [203] Fleischman RW, Baker JR, Thompson GR, Schaeppi UH, Il-
1–10. lievski VR, Cooney DA, et al.: Bleomycin-induced interstitial
[186] Peacock AJ, Dawes KE, Shock A, Gray AJ, Reeves JT, Lau- pneumonia in dogs. Thorax. 1971;26(6):675–682.
rent GJ: Endothelin-1 and Endothelin-3 induce chemotaxis [204] Hagiwara SI, Ishii Y, Kitamura S: Aerosolized adminis-
and replication of pulmonary-artery fibroblasts. Am J Respir tration of N-acetylcysteine attenuates lung fibrosis induced
Cell Mol Biol. 1992;7(5):492–499. by bleomycin in mice. Am J Respir Crit Care Med.
[187] Shi-wen X, Kennedy L, Renzoni EA, Bou-Gharios G, du 2000;162(1):225–231.
Bois RM, Black CM, et al.: Endothelin is a downstream [205] Seok J, Warren HS, Cuenca AG, Mindrinos MN, Baker HV,
mediator of profibrotic responses to transforming growth Xu W, et al.: Genomic responses in mouse models poorly
factor beta in human lung fibroblasts. Arthritis Rheum. mimic human inflammatory diseases. Proc Natl Acad Sci USA.
2007;56(12):4189–4194. 2013;110(9):3507–3512.


The Pathogenesis of Bleomycin-Induced Lung Injury in PDF

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

The Pathogenesis of Bleomycin-Induced Lung Injury in PDF

Uploaded by

Copyright:

Available Formats

Experimental Lung Research, Early Online, 1–17, 2014

Copyright © 2014 Informa Healthcare USA, Inc.

The pathogenesis of bleomycin-induced lung injury in

KEYWORDS bleomycin, idiopathic pulmonary ﬁbrosis, intratracheal, rodent modeling

EXPERIMENTAL MODELING OF terstitial pneumonia (UIP) [4], a patchy interstitial

Experimental Lung Research

Human idiopathic pulmonary Pulmonary fibrosis induced by the

[97–102, 125–132]. this disease [12, 50, 90–95, of different magnitude—in

fibroblastic foci [4–7]. different.

Experimental Lung Research

Currently, it is unknown how AEC injury and

AEC TGF-β expression [71], which itself augmented

FIGURE 2. A diagrammatic representation of the time-course

Experimental Lung Research

Experimental Lung Research

THE “FINAL” INJURY—PRO-FIBROTIC IT BLM [157], or up-regulated expression of throm-

Experimental Lung Research

2082. the anticancer drug bleomycin using nuclear resonance vibra-

Experimental Lung Research

present in epithelial cells of advanced pulmonary fibrosis: 1991;71(5):1688–1693.

Experimental Lung Research

collagen. Arthritis Rheum. 2009;60(7):2142–2155. 2011;53(3):327–333.

Experimental Lung Research

1):L311–L318. [196] Scotton CJ, Chambers RC: Molecular targets in pul-

You might also like