REVIEW OF LITERATURE

Review of Literature

2. REVIEW OF LITERATURE

1.1. Interstitial lung diseases

Interstitial lung diseases (ILDs) are a diverse collection of disorders that are characterized by
diffuse parenchymal lung infiltrates on radiography, impaired gas exchange and restricted
physiology on lung function testing. The ILDs, show common pathophysiology in their early
stages, with inflammatory cell infiltrate, arteriolar wall thickening, and frequently, a causal
relationship with cigarette smoking. Yet, at some poorly understood point in the process, the
diseases develop into primarily airway, emphysematous, or fibrotic abnormalities. The term
Idiopathic pulmonary fibrosis (IPF) is used to describe interstitial lung disease for which no
obvious cause can be identified.

Pulmonary arterial hypertension frequently complicates ILD with a relatively high prevalence
(30-40%) and has been classified separately as group III in the Dana Point classification, 2008
(Simonneau, 2009). Clinically significant pulmonary hypertension is seen in advanced stages of
ILD, however, it can even be present at the time of initial diagnosis. Patients with this dual
diagnosis often fare worse than those with pulmonary arterial hypertension alone and are
associated with high mortality. Previous studies have suggested that multiple pathophysiologic
mechanisms contribute to the development of pulmonary hypertension in fibrotic lung diseases
(Colombat, 2007).

1.2. Pathophysiology of pulmonary fibrosis and pulmonary

hypertension

For decades, pulmonary fibrosis was believed to develop secondary to an inflammatory

alveolitis, caused by infiltration of macrophages, lymphocytes and neutrophils. The hypoxemia
of pulmonary fibrosis was attributed to the thickened alveolar interstitium forming an anatomic
barrier to the diffusion of oxygen, leading to the development of an “alveolar–capillary block”
(Austrain, 1951). Inflammation led to recruitment of fibroblast/myofibroblasts, to aberrant
collagen formation and irreversible fibrosis. This hypothesis was challenged due to; i) this
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disorder being largely unresponsive to anti-inflammatory, corticosteroids and

immunosuppressant treatment, and ii) failure of clinical measurements of inflammation to
correlate with stage or outcome (Selman et al 2001).

Another hypothesis regarding pulmonary fibrosis was that the disease progressed linearly from
cellular to fibrotic stages, and that monitoring the functional studies could monitor the fibrotic
process over time and would obviate the need for repeated lung biopsies. Therefore structure–
function correlations in pulmonary fibrosis were studied with the clinical rationale that,
physiologic studies reflected the underlying cellular or fibrotic structure and would allow
assessment of prognosis and response to treatment (Crystal, 1976).

The current concept of pulmonary fibrosis is that it is a primary epithelial/fibroblastic disease,

and inflammation is considered to be the secondary and not the primary event (Selman M, 2001).
Repeated alveolar epithelial cell injury may be caused by disequilibrium in oxidant/antioxidant
balance and this ultimately results in an aberrant extracellular matrix remodeling and fibrosis
(Walters et al, 2008).

The mechanisms of development of pulmonary hypertension in the background of pulmonary

fibrosis are as yet unclear. Many mechanisms have been proposed, including, its occurrence
"secondary" to alveolar remodeling and obliteration of alveoli. It has been suggested to be the
result of vascular inflammation, perivascular fibrosis, thrombotic angiopathy and left sided
cardiac disease (Strange, 2005). If it were always a secondary process, then, the severity of
fibrosis and degree of restrictive physiology would correlate with the prevalence and degree of
pulmonary hypertension. However, the onset of pulmonary hypertension does not correlate with
lung volumes and in some cases pulmonary hypertension is known to occur even in the absence
of extensive parenchymal disease (Shigemitsu, 2007). Therefore, the assumption of pulmonary
hypertension being a secondary change is being questioned (Lettieri et al, 2006).

Other possible mechanisms for pulmonary hypertension in pulmonary fibrosis include

pulmonary hypoxic vasoconstriction and vascular remodeling due to overexpression of cytokines
and growth factors (Pitsiou, 2011). These causes are suggestive of an interrelationship between
aberrant vascular and parenchymal remodeling (Tzouvelekis, 2006). This interrelationship needs

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a detailed investigation in order to explain the lack of association between restrictive

pathophysiology and pulmonary hypertension (Nadrous, 2005, Lettieri, 2006).

2.3: Structural changes after bleomycin injury

2.3.1: Bleomycin induced parenchymal remodeling:

The histopathological changes of bleomycin induced lung injury occur in the bronchioles,
interstitium and distal arterioles. Three distinct temporal phases of the response to bleomycin
administration, have been defined: An inflammation phase (days 1–2), an active fibrosis phase
(days 7–14) and a late fibrosis phase (days 21–35) (Peng, 2013). In the inflammation phase: a
parenchymal inflammatory infiltrate comprising of lymphocytes, macrophages and
polymorphonuclear cells is seen as an early manifestation of bleomycin injury (Fleishman 1971,
Thrall, 1979, Hagiwara, 2000). The inflammatory cells (Hensen et al, 1987) produce a variety of
cytokines and growth factors that regulate proliferation, chemotactism and secretary activity of
the fibroblasts (Gurujeyalakshmi, 2000). Parenchymal remodeling begins with proliferation of
type II pneumocytes (Thrall, 1979, Lindenschmidt, 1986) and early collagen deposition on day 7
(Chaudhary, 2006). Parenchymal collagen deposition progressively increases on days 14 and 21
(Brown, 1988, Oury, 2001, Chaudhary, 2006) and is seen as diffuse fibrosis in the subpleural
region on day 28 (Hagiwara, 2000).

2.3.2: Bleomycin induced vascular remodeling:

Hemodynamic forces participate in vascular remodeling by two important mechanisms: a) by

regulation of endothelial cell function and b) by regulation of the organization within the vessel.
Endothelial cell responses stimulated by flow participate in vessel development, remodeling,
tone, and atherosclerosis. Flow alters endothelial cell properties such as orientation in the vessel
wall, distribution of cytoskeletal elements, and expression of genes, including platelet-derived
growth factor (PDGF), as well as release of endothelium-derived relaxing factor (Bryant, 1999).

Vascular structures adapt to changes in blood flow by adjusting their diameter, leading to
vascular remodeling. Vascular remodeling is a response of blood vessels to both physiological
and pathological stimuli, leading to either vessel enlargement (positive or outward remodeling)

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or reduction (negative or inward remodeling) (Bryant, 1999). Vascular remodeling and loss of
pulmonary capillaries have been shown to be the major contributing factors for the pathogenesis
of pulmonary hypertension in IPF. They have been assessed on animal models, employing
hypoxia or monocrotaline (Robbins, 2004) and in bleomycin induced fibrosis in rabbit model
(Gong, 2005).

Vessel ablation in areas of dense fibrosis and within fibroblastic foci leads to overall reduction in
vessel density, resulting in an elevation of pulmonary vascular resistance and predisposing to the
development of pulmonary hypertension (Renzoni et al, 2003). The vascular remodeling also
results in tissue hypoxia. Lack of oxygen significantly disturbs alveolar epithelial cell function,
triggers apoptosis and promotes inflammation. This is distinguished from idiopathic pulmonary
hypertension where remodeling of several cellular components of the pulmonary vasculature
occurs concurrently and is manifested by excessive proliferation of vascular endothelium,
smooth muscle cells, and fibroblasts, resulting in thickening of the walls of the pulmonary
arterioles and formation of plexiform lesions (Pietra, 1989). Hypoxia-inducible factor (HIF)-1 is
a major regulator of vascular remodeling which is over expressed in vascular smooth muscles
(Ball, 2014) in patients with IPF. The activation of HIF-1 promotes increased arterial wall
thickness and distal muscularization during prolonged hypoxia. The systemic loss of a single
HIF-1α allele has been shown to attenuate hypoxic pulmonary hypertension (Krick, 2005,
Tzouvelekis, 2007).

2.4. Quantification of pulmonary remodeling

2.4.1. Ashcroft’s grading of pulmonary fibrosis

The severity of lung fibrosis has been semi-quantitatively graded, on a scale from 0 to 8
(Ashcroft et al, 1988). Grade 0, normal lung; Grade 1, minimal fibrous thickening of alveolar or
bronchiolar walls; Grade 3, moderate thickening of walls without obvious damage to lung
architecture; Grade 5, increased fibrosis with definite damage to lung structure and formation of
fibrous bands or small fibrous masses; Grade 7, severe distortion of structure and large fibrous
areas; Grade 8, total fibrous obliteration of fields. Grades 2, 4 and 6 were intermediate pictures
between the aforementioned criteria.

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2.4.2. Morphometric evaluation of pulmonary remodeling

The pulmonary parenchymal remodeling after intratracheal bleomycin administration is

morphometrically quantified by assessing the fibrosis fraction and alveolar wall area fraction
(Kremer, 1999, Izbicki et al, 2002). The parenchymal fibrotic area (Mata, 2003) and the total
field area are measured after exclusion of bronchioles and large blood vessel area
(Gurujeyalakshmi, 2000) and expressed as a ratio. Such a measurement, demonstrated a 3-fold
increase in fibrosis fraction and a 1.3-fold increase in wall area fraction in bleomycin-treated
mice on day 14, with no further increase on day 21 (Izbicki et al, 2002). It was also demonstrated
that the most suitable time point for assessing lung fibrosis in this model was 14 days after
intratracheal instillation of bleomycin. Thus, it was proposed that computer-assisted
morphometry provided objective and quantitative measurements and was a useful tool for the
evaluation of bleomycin-induced lung injury.

The pulmonary vascular remodeling in pulmonary fibrosis is histologically characterized by

changes in the tunica intima, media and adventitia. These pathological changes have been
morphometrically quantified (Hislop and Reid, 1976). First, each artery is categorized by its
structure (muscular, partially muscular, or nonmuscular). Then using a computer program, the
mean arterial diameter (between external elastic laminae) and medial thickness (between internal
and external elastic laminae) in completely muscular arteries that accompany the terminal and
respiratory bronchioles is measured. It was observed that morphometric evaluation can help in
establishing correlations between structure and function, related to disease heterogeneity in
pulmonary fibrosis (Borzone, 2001).

2.4.3. Structure-function correlations in pulmonary fibrosis

Since pulmonary fibrosis progresses from the cellular to the fibrotic phase, it was proposed that,
the physiologic studies which reflected the underlying cellular or fibrotic structure would allow
assessment of prognosis and response to treatment (Crystal, 1976). Thus, monitoring of the
functional studies over a period of time would reveal the stages in the fibrotic process and thus
would reduce the need for repeated lung biopsies. However, till to date, the pathophysiological

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events leading to the gradual destruction of lung tissue and the consequent decrease in
pulmonary function remains unclear.

Mechanisms other than fibrosis too have been seen to contribute to the abnormal physiology; for
instance, the abnormal physical properties of surfactant in rats treated with bleomycin, has been
shown to contribute to the increased elastic recoil observed in this model (Horiuchi, 1996). The
number of bleomycin challenges was also thought to make a difference in the development of
fibrosis. However, recent studies have shown that repetitive exposures do not produce a more
substantial or progressive fibrotic response in the lung (Peng, 2013). Presently, bleomycin is
believed to lead to bronchiolocenteric or subpleural fibrosis depending on the route of
administration, whether intratracheal or systemic (Jules-Elysee, 1990, Chau, 2005) and has been
shown to induce molecular changes directly relevant to IPF (Peng, 2013).

2.5. Oxidative stress in pulmonary fibrosis

Excessive reactive oxygen species (ROS) are produced by a variety of exogenous and
endogenous mechanisms (Fig. 1). ROS disrupt essential cellular functions and are implicated in
several human diseases, including cancer, Alzheimer's disease, Parkinson's disease, heart failure
and amyotrophic lateral sclerosis. Recently, oxidant–antioxidant imbalance has been implicated
in IPF (Markart, 2009). However the complex signaling mechanisms by which these diverse
oxidants exert their pathophysiological effects on the lungs are still the subject of debate.

It has been suggested that the superoxide radicals generated by bleomycin directly cause single
and double-strand breaks in cellular DNA leading to genomic instability of damaged cells and
induce apoptosis and senescence in epithelial and non-epithelial cells of the lung (Wang, 2002).
For example, bleomycin induced apoptosis of alveolar macrophage (Zhao, 2004) is associated
with p53 translocation from the cytosol to the nucleus (Davis, 2000). However, bleomycin has
been shown to inhibit myofibroblast apoptosis through the release of insulin-like growth factor-1
by macrophages (Wynes, 2004). Such initial lung injury increases the influx of activated
inflammatory cells into lung parenchyma. The inflammatory cells further produce ROS, and this
cascading reaction, contributes importantly to the pathogenesis of bleomycin induced pulmonary
fibrosis (Wang, 2002).

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ENDOGENOUS SOURCES EXOGENOUS SOURCES

Mitochondrial Electron Transport Chain Radiation (UV Light, X-Rays, Gamma Rays)

Autoxidation Reactions

Redox Cycles

Cytochrome P450 ROS Chemicals, Drugs

Reactions
Environmental Toxins
Xanthine Oxidase

Diet
NADPH
Oxidases, Lipoxygenases,
Prostaglandin
Synthase Smog
Transition Metals
Inflammation/ Oxidative Reactions

Fig. 1. Exogenous and endogenous mechanisms of production of reactive oxygen species

2.5.1. Exogenous ROS

Exogenous ROS is produced from pollutants, tobacco, smoke, drugs, xenobiotics, or radiation
(Fig.1). These injurious agents generate damaging intermediates through their interaction with
water present in the human body. In this process, water loses an electron and through a three-step
chain reaction, it is sequentially converted to hydroxyl radical (-OH), hydrogen peroxide (H2O2),
superoxide radical (O2-) and ultimately oxygen (O2) (Fig. 2). The hydroxyl radical in turn,
immediately removes electrons from any molecule in its path, turning that molecule into a free
radical and so propagating a chain reaction. Hydrogen peroxide, formed, is actually more
damaging to DNA than hydroxyl radical since the lower reactivity of hydrogen peroxide
provides enough time for the molecule to travel into the nucleus of the cell, subsequently
damaging the DNA.

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e- e- e- e-
O2 O-2 H2O2 OH. H2O
Oxygen Superoxide Hydrogen Hydroxyl Water
Anion Peroxide Radical

Superoxide dismutase Glutathione peroxidase

Catalase

Fig. 2. Illustrates the formation and elimination of reactive oxygen species (ROS)

2.5.2. Endogenous ROS

Endogenous ROS are produced intracellularly, in cell membranes, mitochondria, peroxisomes,

and endoplasmic reticulum (Han et al, 2001) when stimulated by hypoxia, serum deprivation,
integrin, tumour necrosis factor (TNF-α) signaling, p53 signaling etc. Mitochondrial ROS is
generated at the electron transport chain. Electrons leaking from the electron transport chain
directly act on oxygen, producing free radicals such as superoxide anion (O2−). O2– is then
converted to nonradical derivatives such as hydrogen peroxide (H2O2) either spontaneously or
catalyzed by superoxide dismutase (Li, 2013) (Fig. 2).

Cytosolic ROS is produced by enzymes such as nicotinamide adenine dinucleotide phosphate

(NADPH) oxidases and xanthine oxidases (Finkel, 2000). The NADPH oxidase enzymes
produce superoxide from oxygen using electrons from NADPH (Quinn, 2006) and plays
essential roles in innate immunity and host defense mechanism against pathogens. Xanthine
oxidase catalyses vitamin A (retinol) oxidation to retinoic acid, with ROS-generation in vitro.
Exogenous xanthine oxidase induces the activation of caspase-3 and reduces the expression of
the anti-apoptosis protein Bcl-2, thereby reducing endothelial cell viability, while endogenous
xanthine oxidase is necessary for maintaining endothelial cell survival (Kou, 2008). Xanthine
oxidase modulation of cell survival signaling pathways is therefore an important contributor to
endothelial mechanisms for microvascular rarefaction.

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2.6. Consequences of Oxidative stress

Lipid Peroxidation

Oxidative attack primarily leads to the oxidative degeneration of lipids and the accumulation of
lipid peroxidation (LPO) products (Sies, 1991, 1997); oxysterols, hydroperoxides and
endoperoxides. The latter undergo fragmentation to produce a broad range of reactive carbonyl
intermediates such as α,β-unsaturated aldehydes [4-HNE and acrolein], di-aldehydes [MDA and
glyoxal] and keto-aldehydes [4-oxo-trans-2-nonenal (ONE) and isoketals]. In this process, free
radicals "steal" electrons from the lipids in cell membranes, resulting in cell damage.

The reaction consists of three major steps: initiation, propagation, and termination. Initiation:
occurs when ROS bind to unsaturated lipids and produce fatty acid radicals. Propagation occurs
when the fatty acid radical reacts with molecular oxygen, creating a peroxyl-fatty acid radical.
This unstable radical then reacts with another free fatty acid, producing a different fatty acid
radical and a lipid peroxide, or a cyclic peroxide if it had reacted with itself. This cycle continues
in a "chain reaction mechanism". The non-charged structure of aldehydes generated, allows them
to migrate with relative ease through hydrophobic membranes and hydrophilic cytosolic media,
making these carbonyl compounds far more destructive than ROS and having far-reaching
damaging effects on target sites. At the target sites, they react with nucleophilic groups in
macromolecules like proteins, DNA and aminophospholipids, resulting in their chemical, non-
enzymatic and irreversible modification (Schaur, 2005, Negre-Salvayre, 2008). Termination of
the radical reaction occurs when two radicals react and produce a non-radical species. This
happens when i) the concentration of radical species is high increasing the probability of
collision of two radicals and also when ii) the antioxidant molecules and enzymes such as
vitamin E, superoxide dismutase, catalase, and peroxidase speed up termination by catching free
radicals.

The end products of lipid peroxidation are reactive aldehydes, such as malondialdehyde (MDA)
and 4-hydroxynonenal (HNE). These act as secondary messenger of free radicals as their
biological activities resemble activities of ROS. If the oxidative stress is not terminated fast
enough, these end products are thought to activate several genes related to onset of the innate

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immune response, cell growth, and fibroblast proliferation, subsequently leading to pathological
changes such as damage to the cell membrane, cell death, tissue injury, apoptosis and repair.
Oxidative damage to the DNA of human airway epithelial cells may be a potential mechanism
for the increased incidence of cancer in persons exposed to occupational and environmental air
pollutants (Prahalad, 2000, Bauer, 2004). Another mechanism by which ROS can exert their
pathological effects is through the modification of proteins. ROS act directly or indirectly on
proteins to cause oxidation of the polypeptide backbone, peptide bond cleavage, protein-protein
crosslinking or amino acid side chain modification (Kelly, 2003).

2.7. Oxidative stress induced cell death

Oxidative stress can induce cell death in two ways: by apoptosis and by autophagy.

2.7.1. Apoptosis

The apoptotic cells undergo a series of morphological changes, including cell shrinkage,
membrane blebbing and cleavage of chromosomal DNA leading to the release of the membrane-
bound apoptotic bodies. This type of cell death involves two pathways; the extrinsic and the
intrinsic and two phases: i) the initiation phase, which leads to caspase activation; and ii) the
execution phase. Excessive ROS induces apoptosis through both the extrinsic and intrinsic
pathways (Ozben, 2007).

Caspases (cystein-aspartase) are cystein proteases which are found in their inactive pro-form in
living cells. Following cleavage, they acquire their protease activity. Activated caspases cleave
specific substrates which in turn transmit either the apoptotic signal to the nucleus or
mitochondria (caspase-3, 6, 7) or interfere with the anti-apoptotic protection (cellular FLICE-like
inhibitory protein cFLIP). The caspase cascade thus induces DNA fragmentation and cell death
(Drakopanagiotakis, 2008).

The extrinsic apoptotic pathway

In the extrinsic pathway, ROS is an upstream event for Fas activation via phosphorylation, that
results in the recruitment of Fas-associated protein with death domain (FADD), caspase 8 and
induction of apoptosis (Gupta, 2012). The death ligands such as tumor necrosis factor (TNF-α),
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Fas ligand (FasL), and TNF-related apoptosis-inducing ligand (TRAIL) trigger the extrinsic
pathway through their respective receptors (Morissette, 2009). The extrinsic pathway is
inhibited by three mechanisms: i) FLIP (Fas-activated death domain (FADD)-like interleukin-1
converting enzyme (FLICE)-like inhibitor of apoptosis protein), which binds to procaspase-8
without activating it. ii) Decoy receptors that bind to and antagonize membrane death receptors
for death ligand binding and iii) Heat shock proteins (Drakopanagiotakis, 2008).

During normal wound healing, fibroblast number is reduced by apoptosis (Desmouliere, 1995).
However in a profibrotic milieu, the fibroblasts, become resistant to Fas-mediated
apoptosis (Moodley, 2004) due to; i) an increased expression of X-linked inhibitor of apoptosis
and FLIP proteins (Tanaka, 2002), and ii) decreased levels of surface Fas and increased levels of
soluble Fas (Buhling, 2005). These fibroblasts undergo activation and transition to
myofibroblasts and further synthesize and deposit collagen and other extracellular matrix
components within the fibrotic lesions.

The intrinsic apoptotic pathway

The intrinsic pathway of apoptotic cell death, is mainly triggered by cellular stresses like
oxidative stress or UV light that cause mitochondrial DNA damage (Roos, 2006). In the intrinsic
pathway, ROS facilitate cytochrome c release by activating pore-stabilizing proteins (Bcl-2 and
Bcl-xL) (Martindale, 2002). The Bcl-2 proteins layered on the surface of the mitochondria,
detect damage, and activate Bax proteins, which punch holes in the mitochondrial membrane,
causing cytochrome c to leak out. This cytochrome c binds to Apaf-1, or apoptotic protease
activating factor-1, which is free-floating in the cell's cytoplasm. Using energy from the ATPs in
the mitochondrion, cytochrome c joins Apaf-1 and caspase-9 to form the apoptosome (activated
caspase-9) that activates caspase-3. The caspase-3 then cleaves the proteins of the mitochondrial
membrane, causing it to break down and starts a chain reaction of protein denaturation and
eventually phagocytosis of the cell.

2.7.2. Autophagy

Autophagy (the Greek term meaning “self-eating,”) is a self-catabolic process involving

sequestration of cytoplasmic contents (exhausted organelles and protein aggregates) for

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degradation in lysosomes (Shrivastava, 2011). It contributes to cellular homeostasis in both

health and disease conditions by providing (a) an alternative source of metabolic fuel, (b) by
removing damaged cellular components which are toxic to the cell, and (c) by promoting cell
death (Haspel, 2011). Recently, ROS induced autophagy has been implicated in the pathogenesis
of IPF (Patel, 2012, Araya, 2013).

Autophagic cell death does not involve caspase activation and thus differs from apoptotic cell-
death. In the setting of IPF, elevations in enoplasmic reticulum stress (Korfei, 2008), oxidative
stress (Kliment, 2010), and HIF-1α (Tzouvelekis, 2007), may induce autophagy. However, this
needs further confirmation because in vitro studies have shown that TGF-β inhibits autophagy in
fibroblasts, thereby suggesting that elevated TGF-β activity in the IPF lung may account for this
disconnect (Patel, 2012). Insufficient autophagy may be potentially involved in driving
accelerated epithelial cell senescence and myofibroblast differentiation in IPF (Araya, 2013).

2.8. Pathogenesis of pulmonary fibrosis

2.8.1. Stage 1: Cellular injury: Role of Damage-associated molecular patterns

The first stage is the least understood. The onset of tissue inflammation is believed to start at the
sites of epithelial injury. Cell injury exposes nuclear components such as DNA, histones, and
other nuclear proteins which are called as Damage-associated molecular patterns (DAMPs or
alarmins). The up-regulated DAMPs, stimulate a highly conserved innate cellular response
which causes either mild acute inflammation and healing, or a robust chronic inflammation and
autoimmunity, or subchronic inflammation and fibrosis. The nature and course of inflammatory
process is determined by the type of cellular debris, the cellular activation pathways that are used
to recognize these basic DAMP signals and the surrounding cytokine and growth factor milieu in
which they are found. Since, the inflammatory process commonly precedes and accompanies
fibrosis in IPF, therefore initial research focused on the potential role of inflammatory
mechanisms driving fibrosis (Luziana, 2008). However, there is enough evidence to suggest that
fibrosis may occur independently of inflammation (Schwarz, 2003). Thus, the study of cellular
players and cytokines released in the epithelial mesenchymal trophic unit gains importance.

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Stage 1: Cellular injury Stage 4: Impaired reepithelialisation

Release of DAMPs Protease –antiprotease imbalance
Aberrant remodeling, Fibrosis

Oxidative injury

Stage 2: Basement membrane disruption Stage 3: Epithelial myoepithelial transition

Fibroblast migration and proliferation Myofibroblast foci
Basement membrane disruption Angiogenesis

Fig. 3. Illustrates the various stages of pulmonary fibrosis

2.8.2. Stage 2: Propagation of lesion: Role of cytokines

In this stage, it is seen that the inflammatory response and repair of damaged tissue is regulated
by the interactions between the epithelial and mesenchymal cells, the cytokines released, and the
extracellular matrix. These include the Transforming growth factor (TGF-β), Platelet derived
growth factor (PDGF), Tumor necrosis factor (TNF-α) etc (Clark, 2007). Overall, it remains to
be clarified, whether fibrosis is a direct consequence of inflammation, or that inflammation and
fibrosis are causally non connected co-findings, or that inflammation and fibrosis develop
through relatively independent yet mutually interacting mechanisms (Luziana, 2008).

2.8.2.1: Tumor necrosis factor (TNF-α)

TNF-α is a pleiotropic inflammatory cytokine that disrupts epithelial intercellular junctions and
elevates transepithelial permeability, leading to tissue injury and cell death by apoptosis
(Waheeda, 2013). It is generated in a precursor form called transmembrane TNF-α (TmTNF-α).
The soluble form of TNF-α (sTNF α), is released, after being processed by metalloproteinases,
such as TNF-α-converting enzyme (TACE, ADAM -17). sTNF α mediates its biological
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activities through two receptors: Type 1, TNF-R1 and Type 2, TNF-R2 (CD120a and CD120b,
respectively) (Horiuchi, 2010). Both sTNF-α and TmTNF-α are involved in the inflammatory
response (Eissner, 2004). TNF–α mediates pleotropic effects including apoptosis, cell
proliferation and further cytokine production (Fig. 4). The TNF–α levels have been shown to
peak on day 7 after bleomycin administration, preceding the increase in collagens I and III
mRNA levels (Phan, 1992).

Macrophage

Endothelial cells TNF monocyte/macrophage

Activated endothelial cells Fibroblast IL-1 T Lymphocyte

IL-8 PGE2, MMPs, IL-6

Leucocyte adhesion and diapedesis Matrix remodeling

Fig. 4. Illustrates the pleiotropic effects of TNF–α

TNF–α, is a death ligand and can trigger the extrinsic apoptosis pathway like Fas ligand (FasL)
and TNF-related apoptosis-inducing ligand (TRAIL) (Morissette, 2009). TNF–α expression is up
regulated in macrophages on day 14 and 21 after bleomycin administration, and correlates with
the appearance of lung parenchymal inflammation (Ortiz, 1998). It is therefore considered that
TNF-α plays a critical role in local inflammation.

2.8.3. Stage 3: Propagation of lesion: Role of various cells

Once initiated the lung pathology is propagated in the epithelial–mesenchymal trophic units by
the alveolar epithelial cells, fibroblasts/myofibroblasts, endothelial cells, neutrophils,
lymphocytes and macrophages. The epithelial–mesenchymal trophic unit consists of opposing

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layers of epithelial and mesenchymal cells. The area between these two cell layers, the basement
membrane zone, contains extracellular matrix and a network of nerve fibers. Recognition of the
attenuated fibroblast sheath as a distinct layer of resident fibroblasts is the key to the concept of
an epithelial–mesenchymal trophic unit (Evans, 1999).

2.8.3.1. Alveolar epithelial cells

The alveolar epithelial cells (AEC) include, the type-I pneumocytes (95% cells) and the type-II
pneumocytes (5% cells). The type II AEC’s are capable of proliferation and generation of both
type-II and type-I cells. They secrete a number of molecules such as surfactant-associated
products, cytokines and growth factors, enzymes and matrix proteins (Drakopanagiotakis, 2008),
which directly interact with signaling molecules and effect diverse signaling pathways regulating
cell proliferation, apoptosis differentiation and growth. Caveolin-1, a scaffold protein of caveolae
is one such protein, which is particularly abundant in alveolar epithelial type I cells, in
endothelial and smooth muscle cells, and in fibroblasts of lung tissue. Caveolin-1 is involved in
bleomycin-induced apoptosis and senescence of normal and lung cancer cells.

Irreversible fibrotic change results when there is ineffective replacement of damaged epithelium
within the pulmonary alveolus. The determinants of type II alveolar epithelial cell behavior
include components of extracellular matrices (ECMs) and a select group of growth factors.
Previously the alveolar epithelial cells have been suggested to interact with the inflammatory
cells, fibroblasts and endothelial cells and contribute to the fibrotic response (Bussolati, 2006).

2.8.3.2. Fibroblast and Myofibroblast

The fibroblasts are the sentinel cells which act as the central effector cells of the progressive
fibrotic process in IPF. The onset of lung fibrosis is controlled by three subsets of fibroblasts: (i)
The fibroblasts that are spatially close to the epithelium and produce differentiation factors, are
likely regulators of local responses (Evans, 1999), (ii) The more distant fibroblasts which
produce factors that stimulate proliferation and iii) The perivascular fibroblasts or fibrocytes
which surround the arterioles (Lin, 2008). The perivascular fibroblasts or adventitial fibroblasts
have been found to be key regulators of pulmonary vascular function and structure from the
“outside-in.” (Stenmark, 2006) (Fig. 5).

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These adventitial fibroblasts are capable of undergoing phenotypic changes, which include
proliferation, differentiation, upregulation of contractile and extracellular matrix proteins, and
release of factors that directly affect medial smooth muscle cell tone and growth and stimulate
recruitment of inflammatory and progenitor cells to the vessel wall.

Fig. 5. Illustrates the interactions between the infiltrating leucocytes- adventitial fibroblast-
myofibroblasts and cytokines mediating tone and structure of the vessel wall (Adapted from
Stenmark KR et al, Role of the adventitia in pulmonary vascular remodeling. Physiol
2006; 21: 134-45)

The adventitial cells have important immunomodulatory functions and some also serve as vessel
wall progenitors (Passman, 2008). These have been suggested to differentiate into mature cells of
the tissue, including vascular smooth muscle, white adipocytes, and possibly neurons.
Comparative study of the genomic phenotype of pulmonary fibroblasts from the lungs of
advanced IPF patients and normal controls has revealed altered expression in proteasomal
constituents, ubiquitination-mediators, Wnt, apoptosis, vitamin metabolic pathways and cell
cycle regulators, in IPF fibroblasts (Emblom-Callahan et al, 2010). This comprehensive study
provides a foundation for future research into this devastating disease.

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2.8.3.3. Endothelial cell

The endothelial cells (ECs) form the inner lining of the capillaries, arteries and veins. In the
capillaries, the endothelial cells are ensheathed by pericytes, whereas in arteries, arterioles,
venules and veins the smooth muscle cells (SMCs) surround ECs. During inflammation,
alteration of EC function occurs following release of TNF-α or interleukins from macrophages.
These result in activation of the EC by the nuclear factor-κB (NF-κB) pathway (Jeon, 2003).
Adhesion molecules, such as selectins, integrins, intercellular adhesion molecule 1(ICAM1) and
vascular cell-adhesion molecule 1 (VCAM1) are then expressed on the EC. These adhesion
molecules exert chemoattractive functions. A cascade of events then follows, that leads to the
tethering to and rolling of leukocytes on the EC surface, their firm adhesion and subsequently
their transmigration.

The circulating endothelial progenitor cells (EPCs) mediate intimal repair and stimulate
angiogenesis. IPF patients show a marked EPC depletion, which is more severe in the presence
of pulmonary hypertension. Depletion of EPCs therefore may play an important role in linking
pulmonary fibrosis and pulmonary hypertension. The inability to compensate for the decrease in
EPCs could be the expression of the secondary marrow failure which often develops in patients
with pulmonary hypertension. Therapeutic modulation of EPCs, by statins, bosentan and
pirfenidone have been used to prevent the development of pulmonary hypertension in patients
with IPF. The role of antioxidants in modulating the EPCs in IPF and pulmonary hypertension
needs to be further investigated.

2.8.3.4. Neutrophils

Neutrophilia is a common feature of pulmonary fibrosis (Kinder et al., 2008). Neutrophilia is a

reliable predictor of early mortality in IPF patients (Kinder, 2008) and is associated with dismal
prognosis (Turner-Warwick, 1987). However, others have reported that neutrophilia is not
associated with poorer clinical course and that bleomycin-induced fibrosis is enhanced in the
absence of neutrophils (Thrall, 1981, Watters, 1987). The role of neutrophilsas to whether it is a
primary pathogenic cause in acute exacerbations of IPF or it is acting as an epiphenomenon of
advanced fibrotic disease remains an uncertainty to date.

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Neutrophil recruitment occurs at the sites of inflammation in IPF, due to: i) Induction of CXC
chemokines and interleukins like IL-17A (Laan, 1999, Miyamoto et al., 2003), ii) Significant
upregulation of the anti-apoptotic protein Bcl-2 in the neutrophils (Mermigkis, 2001) and iii)
Sub-clinical infection in destroyed lung or the fibrogenetic secretion of chemotactic factors, in
advanced fibrotic disease. The exaggerated neutrophil responses may contribute to the
development of fibrosis by triggering vascular endothelial cell apoptosis (Zhu, 2011).
Corticosteroids, although minimally active as a therapeutic option in IPF, have been shown to
significantly increase the rate of neutrophil apoptosis, while simultaneously reducing the rate of
alveolitis and subsequent fibrosis in bleomycin model (Li, 2004).

2.8.3.5. Lymphocytes

The CD4 and CD8 T lymphocytes commonly infiltrate the lung parenchyma in patients with
pulmonary fibrosis and in animal models (Luzina, 2008). T lymphocytes modulate the
inflammatory and healing responses in the lungs in a profibrotic or antifibrotic manner,
depending on their phenotype. However, T lymphocytic infiltration per se is not likely to be the
central driving force in most cases of pulmonary fibrosis. Also their role in regulating the
accumulation of extracellular matrix, particularly collagen, is not completely understood. CD8 T
cells, predominate and are distributed diffusely throughout the lung interstitium, whereas CD4
cells tend to be localized in lymphoid follicles (Kradin, 1986). A systematic review of the
phenotypes of pulmonary T lymphocytes and related profibrotic and antifibrotic mechanisms is
therefore required.

The CD4 Th cells produce cytokines that act profibrotically or antifibrotically and include, IL-4,
IL-13, TGF-β, oncostatin M, IFN-γ, TNF-α (Atamas, 2003). The CD4+ T helper 17 (Th17) cell
subset that expresses the proinflammatory cytokine IL-17A is emerging as an important initiator
of fibrosis. IL-17A expression has also been implicated in the pathogenesis of pulmonary fibrosis
(Joshi 2009, Wilson, 2010) and myocardial fibrosis (Feng, 2009). Cytotoxic (CD8) T cells
eliminate virus-infected and tumor cells through cytotoxic mechanisms, which may also
contribute to regulation of fibroblast activities. Two major pathways of T cell-mediated
cytotoxicity are the perforin/granzyme pathway, which is predominant in CD8 T cells, and the
Fas-mediated pathway, which is particularly important for IFN-γ-producing CD4 T cells. Thus,

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phenotypic reshaping, rather than elimination of the infiltrating pulmonary T lymphocytes, may
be a promising approach to improving outcomes in patients with pulmonary fibrosis.

2.8.3.6. Macrophages

Monocytes are recruited to sites of tissue injury and they differentiate into distinct specialized
effector macrophage populations, depending on the extracellular milieu present at the site of
injury. Each different macrophage cell population has a dramatically different impact on fibrosis
initiation, propagation, and resolution. Type I, Monocytes, promote the initiation of fibrosis by
releasing cytokines, ROS, and promote myofibroblast resistance to apoptosis.

Type 2, Monocytes promote the resolution of fibrotic disease through differentiation into
regulatory macrophages (Mreg) that inactivate myofibroblasts and inhibit type 1 and type 2
macrophages through local production of interleukin (IL)-10 and/or Arginase-1. Type 3,
Monocytes promotes the progression of fibrotic disease through differentiation into profibrotic
macrophages and fibrocytes that produce various fibroblast stimulatory growth factors and
cytokines (Duffield, 2013).

2.8.4. Stage 4: Epithelial mesenchymal transition

In this stage, epithelial mesenchymal transition progresses under the influence of polypeptide
cytokine mediators and growth factors including; transforming growth factor (TGFβ), vascular
endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) (Lappi-Blanco, 2002),
platelet-derived growth factor (PDGF), connective tissue growth factor (CTGF) (Lasky, 1998),
insulin-like growth factor (IGF) (Krein, 2002), epidermal growth factor (Prasse, 2006) and
endothelin (ET)-1 (Ogata, 2004).

Amongst these, TGF-β is a well-studied critical mediator of fibrogenesis having direct effects on
fibroblast proliferation and the differentiation of fibroblasts into myofibroblasts and thereby
affecting the synthesis of extracellular matrix (Coker, 1997, Hetzel, 2005). TGF-ß1 also mediates
the proapoptotic effects on epithelial cells and antiapoptotic effects on myofibroblasts (Victor,
2006).TGF-β1, activates two important anti-apoptotic pathways in lung fibroblasts: i) focal
adhesion kinase (FAK) pathway by activation of Smad3 and ii) Phosphatidyl-inositol 3 kinase

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(PI3K)/Akt pathway via activation of p38 mitogen-activated protein kinase (Horowitz, 2007). In
animal models, inhibition of TGF-β1-signaling has shown to attenuate lung fibrosis (Bonniaud,
2005) suggesting that drug-targeting of the TGF-β pathway could provide a useful therapeutic
intervention in fibrotic diseases. However it may result in a number of unacceptable adverse
effects particularly, tumour promotion (Wakefield, 2002).

2.8.4.1. Fibroblast growth factors (FGF)

Fibroblast Growth Factors (FGFs) are polypeptides with diverse biological activities. They have
been classified into bFGF (basic FGF) and aFGF (acidic FGF) according to their isoelectric point
(Gambarini, 1982). On the basis of their mechanism of action, the FGFs are classified as
intracellular FGFs (iFGFs), canonical FGFs and hormone-like FGFs (hFGFs) and are referred to
as intracrine, paracrine and endocrine FGFs, respectively. Paracrine FGFs bind to and activate
cell surface tyrosine kinase FGFRs and function in multiple developmental processes including
differentiation, cell proliferation and migration. Endocrine FGFs function over long distances as
endocrine hormones and mediate biological responses in an FGFR-dependent manner. Intracrine
FGFs act as FGFR-independent intracellular molecules that regulate the function of voltage
gated sodium channels (Itoh, 2010).

FGF is immunolocalized to airway epithelial cells, basement membrane, mesenchymal cells and
smooth muscle cells of large airways and vessels (Chernick-Mellins, 2002). FGF acts as mitogen
for epithelial and epidermal cells and stimulates fibroblast and endothelial cell growth and also
induces surfactant protein expression and branching morphogenesis. In the lung, bFGF has been
suggested to be an effector of type II cell proliferation (Leslie, 1990, Sannes, 1994). bFGF is
seen to be present in the alveolar basement membrane bound to heparan sulfate proteoglycan
(Saksela, 1990, Sannes, 1992). The high affinity of bFGF and other FGF for heparan sulfate
proteoglycans on the cell surface and within extracellular matrix (Baird, 1990) could provide an
extracellular storage depot for the growth factors, whose availability to high affinity receptors
may then depend on proteolytic modification of the matrix during the tissue remodeling
(Bashkin, 1989). bFGF is liberated from storage sites by enzymatic activity following cell
damage between days 3 to 7 of bleomycin exposure as evidenced by obvious discontinuities in

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the alveolar basement membrane, and between days 7 and 14, as significant increase in
immunoreactivity for bFGF within the alveolar basement membrane (Sannes, 1996).

2.8.4.2. Vascular endothelial growth factor (VEGF)

Vascular endothelial growth factor (VEGF) is a powerful angiogenic molecule which is found in
great amount in the lung. VEGF is released principally by respiratory epithelial cells and is a key
factor in the maintenance of endothelial cells. VEGF increases cell survival via (PI3K)/Akt
pathway activation leading to caspase-9 and Bcl-2-associated death promoter (Bad) inactivation.
VEGF inhibits endothelial cell apoptosis by increasing the expression of Bcl-2 and surviving
through MAPK/ERK pathway activation and SAPK/JNK pathway inhibition. Thus, decreased
VEGF signaling arising as a result of oxidative stress or other causes results in endothelial cell
apoptosis, migration impairment and general endothelium dysfunction. The capillary bed appears
essential for the growth and maintenance of alveolar septa.

VEGF acts in the adult to stabilize mature vessels (Maharaj, 2006). The levels of VEGF mRNA
expression in pulmonary arterial endothelial cells, has been reported to decrease 2 weeks and 4
weeks after intratracheal bleomycin (Gong, 2005). This was seen to correlate with the evolution
of pulmonary hypertension induced by intratracheal bleomycin in immature rabbits. It has been
suggested that intratracheal bleomycin may induce the pathological changes of pulmonary
arteries which results in pulmonary hypertension. The role of VEGF in parenchymal remodeling
in pulmonary fibrosis remains to be elaborated.

2.8.4.3. Aberrant extracellular matrix deposition

In this stage, aberrant remodeling is seen to be associated with increased extracellular matrix
(ECM) deposition, and with a dysregulation of matrix metalloproteases (MMPs) and their
inhibitors (Antoniou, 2007). In the early phase of bleomycin-induced lung injury, there is
accumulation of hyaluronan in the alveolar space and interstitial compartment (Nettelbladt,
1991). Collagen synthesis reaches its peak during the second week and then gradually subsides
(Phan, 1980). Other components of the extracellular matrix that are increased include
fibronectin, laminin, biglycan, and elastin (Westergren-Thorsson, 1993).

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The collagen–elastin–proteoglycan matrix is believed to play a major role in determining the

viscoelastic behavior of the parenchymal tissues and is potentially responsible for tissue
resistance (Dolhnikoff, 1999). The extracellular matrix also dictates the apoptotic profile of
AECs and fibroblasts (Zhu, 2001, Kim, 2006). The integrity of the basement membrane offers
survival signals for adherent epithelial cells, while disruption of the adhesion leads to apoptotic
cell death.

2.9. Endogenous anti-oxidant defense mechanism

The host cells have relatively high intracellular concentrations of endogenous antioxidant,
glutathione, which provide a strong basal scavenging capacity and restore the balance between
the free radicals produced and their corresponding rates of clearance. The other important
antioxidant defense mechanism in nearly all cells exposed to ROS, are the superoxide
dismutases (SOD). The SOD enzymes catalyze the dismutation of superoxide into oxygen and
hydrogen peroxide. The hydrogen peroxide produced is then degraded by three enzymatic
pathways: (1) Catalase, which is concentrated in peroxisomes located next to mitochondria,
which reacts with the hydrogen peroxide to catalyze the formation of water and oxygen. (2)
Glutathione peroxidase which reduces hydrogen peroxide by transferring the energy of the
reactive peroxides to the small sulfur-containing protein called glutathione. The selenium
contained in these enzymes acts as the reactive center, carrying reactive electrons from the
peroxide to the glutathione and (3) Peroxiredoxins which degrade H2O2, within the mitochondria,
cytosol, and nucleus.

2.9.1. Glutathione redox cycle

Glutathione Peroxidases (GPx) are a family of enzymes that can decompose H2O2 and various
lipid peroxides by the Glutathione redox cycle (Fig. 6) (Kinnula, 2005). In this reaction, reduced
glutathione (GSH) is used as a substrate to metabolise H2O2, resulting in H2O and oxidised
glutathione (GSSG). Oxidised Glutathione (GSSG) can then be reduced back to GSH by the
enzyme GSH reductase. The GSH cycle is a pivotal antioxidant defence mechanism for cells and
prevents the depletion of cellular thiols (Heffner, 1989). The bronchial epithelial cells and
alveolar macrophages synthesize and secrete the intracellular and extracellular GPx.

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2 H2O2 2 H2O + O2
(catalase)

2GSH + H2O2 GS–SG + 2 H2O

(glutathione peroxidase)

Fig. 6: Glutathione Redox cycle

2.9.2. Heme oxygenase (HO)-1

Heme oxygenase (HO)-1 is the inducible isoform of heme oxygenase. It is a cytoprotective

enzyme that plays a central cytoprotective role in the defense against oxidative and inflammatory
insults in the lung (Fig. 7 ). HO-1 is a critical arbitrator of cell death pathways in the pulmonary
diseases, such as ARDS, COPD, asthma, pulmonary vascular disease etc. In the lung, HO-1 is
expressed in various cells types, including type II pneumocytes and alveolar macrophages. There
is limited data of its involvement in IPF and lung cancer. HO-1 has been shown to be
upregulated in alveolar macrophages of patients with interstitial lung disease.

The pharmacologic inhibition of HO-1 has been shown to abrogate bleomycin induced fibrosis.
The mechanisms of action of HO-1 are pleiotropic and include; i) Anti-inflammatory action
inhibiting leukocyte infiltration. HO-1 catalyzes the degradation of heme, a potent oxidant, into
biliverdin, iron, and CO. ii) Promotion of VEGF-driven noninflammatory angiogenesis, which
facilitates tissue repair. Iii) Serving as an endogenous ligand for TLR-4 and thus affect innate
immune response.

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Free Heme catalyzes the production of free radicals through the Fenton reaction. Under normal
homeostasis, this prooxidant effect is tightly controlled by insertion of heme into the heme
pockets of hemoproteins. Under oxidative stress, however, some hemoproteins release their
heme groups, producing free heme that can catalyze the production of free radicals and lead to
cell and tissue injury. The host cells protect themselves from free heme by rapid induction of the
HO-1 isoenzyme which increases the rate of heme catabolism and catalyzes the degradation of
heme, into biliverdin, iron, and carbon monoxide (CO) (Fig. 7). These downstream products of
heme catabolism have recently been found to mediate the antioxidant, antiapoptotic,
antiproliferative, vasodilatory, and anti-inflammatory properties of HO-1. While mounting
evidence supports a protective role for HO-1 in pulmonary fibrosis, the mechanisms underlying
its beneficial effect are as yet unclear and require further investigation.

anti-inflammatory
anti-apoptotic
anti-coagulation
anti-proliferative
Vasodilation

NADPH CO

Heme Heme Billverdin–Ixα billverdin Billrubin-IXα

Oxidant oxygenase reductase anti-oxidant
O2 Fe2+ anti-inflammatory
anti-proliferative
anti-oxidant
Ferritin

Fig.7. Protective role of Hemoxygenase in pulmonary fibrosis

HO-1 has been suggested to mediate protection against pulmonary vascular disease. This
cytoprotective effect of HO-1 against TNF mediated apoptosis requires the activation of NFkB
(Gozzelino, 2010). An increase in lung HO-1 mRNA and protein expression in small pulmonary
arteries has been shown during hypoxia (Carraway, 2002). Some proposed mechanisms include:
1) Protection against apoptosis in several cell types by HO-1 (Zhang, 2004), 2) Attenuation of
proliferation of vascular smooth muscle cells by HO-I (Choi, 2004), 3 Beneficial vasodilatory

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 Review of Literature

and antiproliferative effects of CO (Ryter, 2007). The effect of HO-1 modulation on

angiogenesis and on pulmonary hypertension in ILD needs to be elaborated.

2.10. Role of N-acetylcysteine on pulmonary fibrosis and

hypertension

NAC the pre-acetylysed form of the simple amino acid cysteine, is a synthetic precursor of
cysteine and reduced glutathione (Ramen, 2015). NAC has been used in the clinical practice for
more than 40 years for its mucolytic and anti-oxidant effects. It is generally well tolerated,
except for gastrointestinal side effects due to breakdown of gastric mucus in some patients
receiving oral NAC. NAC, given at a daily dose of 1800 mg, has been shown to restore depleted
pulmonary glutathione levels and to result in a statistically significant improvement in lung
function in patients with fibrosing alveolitis after 12 weeks of treatment. NAC given 600 mg/day
for 5 days has been shown to transiently increase the reduced glutathione concentrations in
plasma and BALF (Bridgeman, 1991). The favourable effects of NAC on the lung function of
patients with IPF (Behr, 1997, Demedts, 2005, Behr, 2009) have led to its use as a combination
drug in the three drug regimen for IPF, comprising of Azathioprine, Prednisone and N-
acetylcysteine. However, results of a recent trial showed some conflicting results which need
further careful investigation (Raghu, 2012).

The mechanisms of action of NAC include: (1) Production of intracellular glutathione which
protects cells from oxidant injury, (2) Direct scavenging of ROS (Aruoma, 1989, De Vries,
1993) thereby reducing hydroxyl radicals and H2O2 levels, (3) Increasing the activity of reduced
glutathione level, (4) Blocking the activation of signal regulated kinases such as p44/42 (Erk1/2),
JNK, p38MAPK, by end products of lipid peroxidation (Ciencewicki, 2008) and (5) Preventing
apoptosis caused by oxidative stress and promoting cell survival by activating signal regulating
pathways (Zafarullah, 2003).

Cortijo et al, (2001) demonstrated that oral NAC given 7 days prior to intratracheal instillation
of bleomycin reduced lung damage in rats by increasing total glutathione and taurine levels.
NAC stimulates glutathione synthesis by enhancing glutathione-S–transferase activity (De Vries,
1993). However, NAC administration from 7 days after bleomycin administration failed to

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inhibit the pulmonary fibrosis. In lung tissues of rats pretreated with NAC, an associated increase
in the activity of GPx, suggests the role of GPx in increasing activities of reduced glutathione
(Yildirima, 2005).

Serrano Mollar et al, (2003) showed that NAC was able to prevent the upregulation of P-selectin,
which is required for the leucocyte migration into the inflammed areas. Additionally it prevented
myeloperoxidase activity, which is a marker of neutrophil accumulation. Treatment with NAC
reduced the lung hydroxyproline by 50% (Cortijo et al, 2001) and lessened the fibrotic area
assessed by morphometric analysis.

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