Post on 22-Jun-2020
Pathogenesis and treatment of chemical-induced lung injury
Elisabeth Wigenstam
Department of Public Health and Clinical Medicine
Umeå 2012
Responsible publisher under Swedish law: the Dean of the Medical Faculty
This work is protected by the Swedish Copyright Legislation (Act 1960:729)
ISBN: 978-91-7459-337-2
ISSN: 0346-6612
New Series No: 1468
Cover picture: Print & Media
Printed by: Print & Media
Umeå, Sweden 2012
Carpe Crustulorum
TABLE OF CONTENTS
ABSTRACT 7
ABBREVIATIONS 9
ORIGINAL PAPERS 11
INTRODUCTION 13
Sulfur mustard 13
Acute effects 15
Long-term injuries 15
Lung injuries 15
Genetic disorders 16
Eye and skin damage 16
Melphalan 17
The Immune Response 18
Inflammatory cells 18
Neutrophils 19
Macrophages 21
Lymphocytes 21
Cytokines 22
Pharmacological treatment 23
Glucocorticoids 23
Oxidative stress and antioxidants 24
Vitamin E 26
Chitinases and chitinase-like proteins 27
Ym1 and Ym2 27
AIMS 28
MATERIALS AND METHODS 29
Animals 29
Inflammation 29
Melphalan 29
Endotoxin 30
OVA 30
Drug treatment 31
Dexamethasone 31
Vitamin E 31
Respiratory mechanics 32
Preparation of animals 32
Measurements of respiratory mechanics 33
Methacholine delivery 33
Postmortem analysis 34
Bronchoalveolar lavage 34
Cytokine analysis 34
Analysis of edema formation 35
Analysis of lymphocyte subsets 35
2D gel analysis 37
Collagen assay 38
Statistical analys 38
RESULTS AND DISCUSSION 39
Animal models 39
Acute airway inflammation 40
Effects on respiratory physiology 43
Chronic (or sub-acute) inflammation 44
Lung fibrosis 45
Ym1 and Ym2 47
Reactive airways dysfunction syndrome (RADS) 48
FINAL COMMENTS 50
CONCLUSIONS 52
SVENSK SAMMANFATTNING 53
TACK! 55
REFERENCES 57
7
ABSTRACT Inhalation of chemical substances can cause irritation to airways and
in high doses acute airway injury. When mice are exposed to the
alkylating nitrogen mustard analogue melphalan they develop an
acute airway inflammation with a rapid influx of neutrophils to the
lungs. The acute phase is followed by long-term respiratory
complications characterized by bronchitis, lung fibrosis, and airway
hyperreactivity.
In this thesis, a mouse model for chemical airway inflammation was
established and the effects on the lungs in a time span from 6 hours
up to 3 months were investigated in order to study both acute effects
and possible chronic injury. We find that treatment with
corticosteroids, e.g. dexamethasone, effectively blocks the
inflammatory reaction in several ways: Neutrophil influx to the lungs
is diminished, the expression of the proinflammatory cytokines
interleukin (IL) -6 and IL-1is decreased and edema formation as
well as development of lung fibrosis is mitigated. In acute airway
inflammation we show that the antioxidant vitamin E can be used as a
possible complement to corticosteroids but not as a replacement since
it causes insufficient downregulation of the inflammatory response.
We show the importance of the T lymphocytes as they play a
prominent role in the pathogenesis of long-term lung injuries caused
by melphalan. Especially the minor T cell subset is of major
importance orchestrating a number of responses including the acute
cytokine and neutrophil response and late-phase lung fibrosis.
In order to find the critical time for dexamethasone treatment, mice
were exposed to melphalan, treated with dexamethasone at specific
time points and lung physiology and airway reactivity was measured
in anaesthetized, tracheostomized mice using a small animal
ventilator. From these results we conclude that an early treatment, i.e.
within one hour after exposure, with dexamethasone is needed to
prevent chronic lung injury.
This thesis was undertaken with the main goal to better understand
the pathogenesis of melphalan-induced airway inflammation. We
believe that our findings have shed new light in this area of research
and hope that this increased knowledge may be of future clinical use.
8
9
ABBREVIATIONS -toc -tocopherol; Vitamin E
BAL Bronchoalveolar lavage
BALF Bronchoalveolar lavage fluid
CWA Chemical warfare agent
ELISA Enzyme-linked immunosorbent assay
ERS Respiratory elastance
FOT Forced oscillation technique
G Tissue resistance
H Tissue elastance
HBSS Hank’s balanced salt solution
GC Glucocorticoid
IL Interleukin
ICAM-1 Intracellular adhesion molecule 1
i.p. Intraperitoneal
KO Knock-out
LPS Lipopolysaccharide
MCh Methacholine
MHC Major histocompatibility complex
MIC Methyl isocyanate
NM Nitrogen mustard
NK cell Natural killer cell
OVA Ovalbumin
PEEP Positive end-expiratory pressure
RADS Reactive airways dysfunction syndrome
RRS Respiratory resistance
ROS Reactive oxygen species
TCR T cell receptor
SM Sulfur mustard
VCAM-1 Vascular cell adhesion molecule 1
10
11
ORIGINAL PAPERS This thesis is based on the following publications and manuscripts,
referred to in the text by their Roman numerals.
I Elisabeth Wigenstam, David Rocksén, Barbro Ekstrand-
Hammarström and Anders Bucht
Treatment with dexamethasone or liposome-
encapsuled vitamin E provides beneficial effects after
chemical-induced lung injury.
Inhal Toxicol. 2009 Sep;21(11):958-64
II Barbro Ekstrand-Hammarström, Elisabeth Wigenstam and
Anders Bucht
Inhalation of alkylating mustard causes long-term T
cell-dependent inflammation in airways and growth of
connective tissue.
Toxicology. 2011 Feb 27;280(3):88-97. Epub 2010 Dec 1.
III Elisabeth Wigenstam, Sofia Jonasson, Bo Koch and Anders
Bucht
Corticosteroid treatment inhibits airway
hyperreactivity and lung injury in a murine model of
chemical-induced airway inflammation.
Manuscript
IV Linda Elfsmark*, Elisabeth Wigenstam*, Gunnar Peijler and
Anders Bucht
Murine chitinases Ym1 and Ym2 are highly expressed
in allergen-induced eosinophilic lung inflammation
but not in acute neutrophilic airway response
Manuscript (*Authors contributed equally to this work)
Reprints of the published papers were made with permission from the
respective publisher.
12
INTRODUCTION
13
INTRODUCTION Inhalation of chemical irritants at home or at work is a common cause
of illness due to unintentional toxic exposures. There are many
situations where the accidental release of a toxic chemical may cause
severe injury to the lungs, one of the most severe being the Bhopal
tragedy in 1984. In this industrial catastrophy more than 3800 people
died and several were injured when a steel tank with methyl
isocyanate (MIC) exploded. Ten years after the incident, people still
suffer from health effects (1) and this has led to increased research in
the area of chemical-induced lung injuries. Although we still do not
have all the answers, this thesis represents one step on the way
towards better understanding the pathogenesis in these diseases.
Sulfur mustard
Sulfur mustard, or bis (2-chloroethyl) sulphide; SM, is a highly toxic
vesicant that historically has been used as a chemical warfare agent
(CWA). It is easily produced and encountered in a combination of
chemical forms that include liquids and vapors. Toxicity is attributed
to the lipophilic nature of SM which allows it to rapidly penetrate
tissues and alkylate DNA, proteins and lipids resulting in DNA
damage and cytotoxicity (2). Its active intermediate, sulfonium ion,
reacts rapidly with proteins and nucleic acids, alters chemical
function groups such as amines, carboxyls, phosphates, S-H and O-H
groups and produces alkylation products. This process may result in
crosslinking between adjacent strands of DNA, which has been shown
to be extremely lethal to cells (3).
SM gas was first used during the late stages of World War I and has
since been used as a CWA in several major wars. The latest reported
INTRODUCTION
14
use was in the Iran-Iraq war 1980-1988 where Iraqi forces used it
against civilian populations (4, 5). Both war veterans as well as
civilians were victims of these attacks and now suffer from residual
effects.
Figure 1. A Canadian soldier with mustard gas burns ca 1916-1917.
Although sulfur mustard is an old chemical, it is still a threat to
military personnel and civilian populations as well as an
environmental hazard. Sulfur mustard has been manufactured in
large quantities and stored and dumped in seas in many areas causing
accidental leakage and damage to mainly fishermen (6). As soon as
SM was first used as a warfare agent, research has focused on finding
an effective antidote but despite extensive investigation there has
been no success in this area of research. There are currently no good
antidotes other than decontamination and supportive care to treat the
exposed person.
INTRODUCTION
15
Acute effects
Sulfur mustard is absorbed by inhalation, through the skin or through
the anterior surface of the eye. The first signs appear within one hour,
symptoms are ususally pain, photophobia and tearing. It may also be
absorbed through the gastrointestinal tract following consumption of
contaminated food. It is lipophilic and very reactive (7). Short-term
localized effects of inhaled SM include discomfort in the nose and
sinuses, sore throat, coughing, sneezing and respiratory dyspnea (8,
9).
In conflict, SM has been used to incapacitate large numbers of
soldiers. Injuries are seldom fatal (mortality from exposure to SM was
3 % during WWI) (10) but lead to severe injuries demanding long-
term medical support. Death, although uncommon, is usually due to
pulmonary injury and associated infections (11).
Long-term injuries
Ever since the first use of SM in World War I, it has been known that
SM not only causes acute but also chronic diseases. However, it is not
until the last decades it has been possible to perform detailed, clinical
studies. Long-term complications from SM exposure are ususally
located to the lungs but there are also other reported complications,
e.g. eyes and skin may be affected and there is increasing risk of
genetic and psychological effects, as well as effects on the immune
system.
Lung injuries
Respiratory complications are the most common cause of long-term
disability in patients with SM poisoning. Delayed effects of SM
INTRODUCTION
16
inhalation include persistent cough, expectoration and dyspnea
including wheezing and cyanosis (3, 12). The most common
complications are chronic bronchitis, asthma, bronchiectasis and
large airway narrowing (13-15).
In a case-study by Dompeling et al. long term effects on an 5-year-old
child was investigated (16). The boy was exposed to SM during the
Iran-Iraq war and immediately treated for chemical pneumonia but
was considered recovered after 40 days. During the next five years his
condition worsened and treatment with anti-inflammatory drugs
could not improve lung function. His condition was termed chronic
bronchiolitis for which there was no treatment other than lung
transplantation.
Genetic disorders
Several studies have shown long-term genetic damages due to
mustard gas exposure (17-19). In a study by Abolghasemi et al. (20) it
is described how the effect of mustard gas can affect unborn children.
Among children born in a village where the men had been exposed to
SM it was more common with respiratory injuries and congenital
malformations compared to children whose fathers were not exposed
to SM. The fertility was also negatively affected by SM; several of the
men exposed were infertile afterwards.
Eye and skin damage
The eyes are the most sensitive organs to SM due to the moist
environment and rapid cell divisions (21). If the healing is not
successful, chronic damage to the eyes occurs. Ocular surface
discomfort (burning, itching, and redness) and in severe cases,
blindness, are the most significant symptoms (22, 23).
INTRODUCTION
17
The characteristic skin lesions of SM burns are erythema followed by
blisters (24, 25). The burns heal more slowly compared to thermal
burns (26) and severe injury can cause areas of necrosis.
Melphalan
During World War II, a military research group investigated the
effects of SM and its nitrogen relatives on rapidly dividing cells. These
effects resulted in medical applications of alkylating mustards (27).
Nitrogen mustard (NM) is closely related chemically and
toxicologically to SM and is currently a useful chemotherapeutic
agent (28-30). The uptake of the NM analogue melphalan occurs by
active amino acid transport systems that differ from the uptake of
other alkylating agents (31). Despite different uptake mechanisms,
the toxic properties of NM and SM are similar although with
variations in potency (32, 33).
Sulfur mustard Nitrogen mustard
Melphalan
Figure 2. Structure of sulfur mustard, nitrogen mustard and melphalan.
INTRODUCTION
18
We have used melphalan in our studies in order to induce airway
inflammation in mice. It is easy to work with when dissolved in fluid
and less toxic than SM but still shares the characteristic cell damaging
properties of SM.
The Immune Response
The cells and molecules that comprise the innate immune responses
are the first line of defense against invading pathogenic organisms
and act in a non-specific manner. This means that the cells of the
innate system recognize and respond to pathogens in a generic way,
but unlike the adaptive immune system, it does not provide for long-
lasting or protective immunity to the host. In those circumstances in
which the effector mechanisms of the innate response are not
sufficient to fully eliminate a pathogen challenge, the same cells and
molecules function to efficiently activate the antigen-specific adaptive
arm of the immune response.
Inflammatory cells
During normal conditions, alveolar macrophages are the dominating
cell type found in the lungs. After exposure to a chemical irritant, an
acute airway inflammation develops and neutrophils gather quickly to
the injured area. Lymphocytes enter later in the inflammatory
process. In the next section these cells will be more thouroghly
described.
INTRODUCTION
19
Figure 3. BAL cells from mouse.
Neutrophils
Innate immunity largely involves granulocytes. Granulocytes
constitute a diverse collection of white blood cells (leukocytes) whose
prominent granula give them their characteristic staining pattern.
Neutrophils are phagocytic granulocytes and constitute up to 50-60%
of the circulating leukocytes and are therefore fundamental in the
first line of defense upon injury or inflammatory stimuli. The
recruitment of circulating neutrophils is crucial to immune defense
and tissue repair. At the beginning of an inflammatory response,
chemotactic factors and proinflammatory cytokines are released,
INTRODUCTION
20
signaling the location of injury. This initiates the multistep process of
transendothelial migration of neutrophils (34, 35) (Figure 4).
Figure 4. Transendothelial migration of neutrophils
In the first step, selectins mediate the loose and reversible adhesion of
circulating neutrophils to the vessel wall of activated endothelial cells.
This interaction cannot anchor the cells against the shearing force of
the blood flow, and instead they roll along the endothelium,
continually making and breaking contact. The second step is the firm
adhesion of the neutrophil to the endothelial surface which is a
requirement for migration across the endothelium and is mediated
via endothelial ICAM-1. When the neutrophil binds to ICAM-1, rolling
is arrested and the cell can adhere tight to the vessel wall. In the third
step, the neutrophil crosses the endothelial wall and penetrates the
basement membrane with the help of proteolytic enzymes. This
movement is called diapedesis and enables cells to enter the
subepithelial tissues. The final step in this process is migration
through the tissues along a concentration gradient of chemochines
Basement
membrane
INTRODUCTION
21
secreted by the cells at the site of injury. The same mechanisms can
be applied to the recruitment of other leukocytes, such as monocytes
and eosinophils. The release of specific and appropriate cytokines,
adhesion factors and proinflammatory mediators offers selective
control of the inflammatory action.
Macrophages
Macrophages are mononuclear phagocytes and exist throughout the
body. They are central in defending the lung against particles and
microbial pathogens and because of their phagocytic properties they
are the major reason to why the lung can stay clean and sterile.
Macrophages exhibit a number of critical functions in host defense
mechanisms. They can initiate and prolong inflammatory responses
by both responding to their micro-environment and controlling the
activities of other cells such as neutrophils and lymphocytes (36).
Once activated, they are endowed with an amazing capacity to express
and release a wide variety of mediators.
Normally, macrophages exist in a resting state but following stimuli
they become activated and immediately start recruiting additional
monocytes. The activated monocytes then mature into macrophages.
Lymphocytes
Lymphocytes are small white blood cells catergorized into B cells, T
cells and natural killer (NK) cells. B cells are associated with
antibody-mediated immunity while T- and NK cells are involved in
the cell-mediated immune response.
T cells can be divided into two classes based on the expression of
surface receptor molecules. Approximately 90 % of all T cells carry a
receptor with and chains, hence termed T cells. The T cells
can be further divided into two subgroups; helper T cells which
INTRODUCTION
22
express the co-receptor molecule CD4 and cytotoxic T cells which
express CD8. γδ T cells constitute only a minor part of T cells and
have a specific T cell receptor (TCR) on the surface consisting of and
glycoprotein chains instead of the more commonand chains.
T cells are not restricted to antigen binding by major
histocompatibility complex (MHC) as opposed to the T cells and
seem to be able to identify whole proteins instead of peptide
fragments. They are mainly located within epithelial tissues of the
skin, lung and intestine (37-39). They have been shown to recruit
inflammatory cells (40, 41) and secrete factors that can influence
epithelial growth and repair (42) suggesting that they have an
important role in surveillance of the neighbouring epithelial cells for
microbial infections and injury.
Cytokines
The inflammatory response is a complex phenomenon in which a
number of cells and molecules are involved. The response involves
cytokine production important in the migration of leukocytes from
the blood stream to the affected tissue. Cytokines can have many roles
in the inflammatory process and affect a number of different cells.
IL-1β is a proinflammatory cytokine involved in the initiation and
persistence of the inflammatory response. A number of human and
animal studies have revealed the presence of IL-1 in chronic
inflamed tissues (43, 44) and inhibition of IL-1 at the beginning of
an inflammatory disease causes attenuation of fibrosis (45)
suggesting a causative link between IL-1 and chronic inflammation.
By inducing the expression of the cell adhesion molecules ICAM-1,
vascular cell adhesion molecule (VCAM)-1, and E-selectin on vascular
endothelial cells, IL-1β participates in the recruitment of leukocytes to
INTRODUCTION
23
the tissue (43). IL-6 is another proinflammatory cytokine required for
the induction of acute-phase reaction and is considered important to
maintain homeostasis (46).
Pharmacological treatment
Glucocorticoids
Glucocorticoids (GCs) are among the most prescribed drugs in clinical
medicine today and are used to treat a variety of inflammatory
disorders (47-50). They have inhibitory effects on the accumulation
and activation of inflammatory cells e.g. eosinophils, lymphocytes and
neutrophils (51). GCs are lipophilic molecules that interact with
specific receptors and as nearly every cell in the body expresses the
GC-specific glucocorticoid receptor, endogenous and synthetic GCs
affect virtually every major organ system. In humans, GCs inhibit the
production of several pro-inflammatory cytokines which is mediated
by the activated glucocorticoid receptor, such as IL-1, IL-2, IL-4, IL-5,
IL-6, IL-8, IL-11, IL-12, IFN-, TNF- and GM-CSF (52-57). Other
effects from GC treatment are down-modulation of cell adhesion
molecules and apoptosis in T-cells, monocytes and eosinophils.
Corticosteroids are a family of drugs related to steroids naturally
produced in the adrenal cortex, such as cortisone. Corticosteroids can
kill lymphocytes by inducing apoptotic cell death. They are useful
anti-inflammatory agents but can also be immuosuppressive which
means that the efficacy of the immune system is reduced, thereby
increasing the risk of catching infections. This implicates a need for
alternative of complementary therapeutic drugs.
INTRODUCTION
24
Oxidative stress and antioxidants
Oxidative stress is defined as an imbalance between the production of
reactive oxygen species (ROS) and antioxidant defences that can lead
to cellular and tissue damage (58). ROS can both prevent disease by
assisting the immune system, mediating cell signaling (59, 60) and
playing an important role in apoptosis (61), but when produced in
large amounts, they can be harmful to cells. It is mainly the
macromolecules, such as lipids, DNA and proteins that are subjected
to damage (62). The state of oxidative stress is thought to contribute
to the pathogenesis in a number of diseases including inflammatory
diseases in the lung.
When the lungs are exposed to melphalan, this leads to activation of
an acute systemic inflammatory response. The first events include
activation of pulmonary endothelium and macrophages, upregulation
of adhesion molecules and production of cytokines and chemokines
inducing a massive accumulation of neutrophils to the affected tissue
(63) (Figure 5). The neutrophils adhere to vessel walls and
transmigrate across the endothelium and epithelium and release a
variety of cytotoxic and pro-inflamamtory compunds in the alveolar
space, such as proteolytic enzymes, ROS and nitrogen species (64,
65). This prolongs a vicious cycle by recruitment of additional
inflammatory cells that in turn produce more cytotoxic mediators
leading to extensive injury if the cascade is not broken. Some of the
most common ROS include superoxide anion radical (O2-), hydrogen
peroxide (H2O2), hydroxyl radical (OH-) and hypohalous acids such
as HOCl. ROS can lead to cell injury by a number of mechanisms,
including: (a) direct damage to DNA resulting in strand breaks and
point mutations; (b) lipid peroxidation; (c) oxidation of proteins that
INTRODUCTION
25
alter protein activity (66, 67) and (d) alteration of transcription
factors leading to increased expression of pro-inflammatory genes
(68, 69).
Antioxidants are compunds characterized by their ability to be
oxidized in place of other compounds present. The cells of the body
have their own system to neutralize free radicals by expressing
endogenous antioxidants e.g. superoxide dismutase, catalase and
glutathione peroxidase (70).
Figure 5. Illustration of one of the mechanisms of lung injury.
INTRODUCTION
26
Vitamin E
Vitamin E is an essential, fat soluble antioxidant. It is present in all
cell membranes at low concentrations and is important to maintain
the integrity of long-chain polyunsaturated fatty acids in the
membranes of cells and thus maintain their bioactivity. In nature,
eight substances have been found to have vitamin E activity: - -,-
and -tocopherol; and -, -, - and -tocotrienol. The most common
is -tocopherol (-toc) and it is considered the most important
lipophilic antioxidant in tissues in vivo (71).
Vitamin E does not only exert antioxidant properties. In Figure 5, the
green stars mark where it may exert its effects on preventing acute
lung injury in mice.
1) By affecting the surface adhesion molecules on either the
neutrophil or the endothelium, vitamin E may inhibit
neutrophil adhesion (72-75).
2) Inhibited binding of the neutrophil to the endothelium
Vitamin E acts by inhibiting neutrophil recruitment into the
lung which could be a result shown by other groups (76-78).
3) Vitamin E can change the membrane stability of either the
neutrophil or endothelial and epithelial cells making it more
difficult for neutrophils to pass into the alveolar space (79).
4) Vitamin E interrupts the destructive reaction cascade initiated
by ROS by donating hydrogen to either the lipid or to the
peroxyl radical (80, 81).
INTRODUCTION
27
Chitinases and chitinase-like proteins
Chitin is a common element in organisms including parasites, fungi
and bacteria but is not found in mammals (82, 83). It functions as
protection against the harsh environment outside the organism and
its biosynthesis is regulated by specific enzymes, chitinases. Recently,
chitinases and chitinase-like proteins have been discovered in
mammalian tissue, despite the lack of chitin production (84-86). The
role of chitinases in mammals is unclear but there are theories
suggesting a role in anti-parasitic functions in invertebrates (87, 88).
It has also been hypothetized that chitinases are involved in the
specific T helper 2 (Th2) immune response in asthma and allergy
(89).
Ym1 and Ym2
Ym1 and Ym2 are homologous to chitinases and have chitinase
activity (85, 86). They are both expressed during Th2-type
inflammation in mice (90-92). Ym1 is synthesized and secreted by
activated macrophages during inflammation and was originally
identified as a 45-kDa protein produced in parasitic infections (93).
Ym1 and Ym2 are highly homologous with each other and are rodent
specific. Ym 2 was identified as a molecule whose production is
elevated in BALF in an allergy model of mice (94).
In order to determine the specificity of Ym1 and Ym2 in allergic
airway models, the expression of Ym1 and Ym2 in three different
models of airway inflammation was compared and analyzed (Paper
IV).
28
AIMS The overall aim of this thesis was to develop a mouse model for
exposure of toxic chemicals to the lungs and to use this model for
studies of disease mechanisms and pharmaceutical intervention. The
specific aims were to:
Study both the acute and delayed toxic effects of melphalan in
terms of cytokine expression, cell infiltration, lung edema and
fibrosis.
Investigate the therapeutic effects of dexamethasone and
vitamin E in mice exposed to the nitrogen mustard analogue
melphalan.
Examine the role of the and T cell subsets on both the
acute and long-term response.
Evaluate the time window for early corticosteroide treatment
in chemical-induced lung injury.
Investigate effects on respiratory physiology in exposed
animals and compare the effects of increasing concentration
methacholine with healthy controls and dexamethasone-
treated animals.
Compare the expression of inflammatory biomarkers in our
mouse model with the expression in two other models of
induced lung inflammation.
MATERIALS AND METHODS
29
MATERIALS AND METHODS Animals
Female mice (Charles River, Germany, Bar Harbor, USA and Taconic
(M&B), Denmark) were used in these studies. All animals were of
C57BL/6 origin, a strain well characterized and used in murine
models of airway inflammation. The mice were fed with standard food
and water ad libitum and kept at least one hour before the
experiments started in the laboratory room to become acclimatized.
Their care and the experimental protocols were approved by the
Regional Animal Research Ethics Committee in Umeå, Sweden.
Inflammation
Melphalan
Melphalan (4-[bis(2-chlorethyl)amino]-L-phenylalanine) (Sigma, St
Louis, USA) was dissolved in acidic ethanol to a concentration of 100
mg/ml and further diluted in phosphate-buffered saline (PBS) at least
250 times to final concentrations just before administration. To
determine the optimal concentration of melphalan to induce airway
inflammation, a dose-response titration was performed. Groups of
mice were exposed to final concentrations of 0.04 mg/ml-0.4 mg/ml
melphalan. The mice were briefly anaesthetized with Isofluran and
melphalan was administrated by tracheal instillation in a volume of
50 l (0.1 mg/kg-1 mg/kg). Control mice receiving only the solvent
did not show any response different from that of naïve mice.
MATERIALS AND METHODS
30
Endotoxin
Inhalation of bacterial endotoxin (lipopolysaccharide, LPS) evokes a
well-defined acute airway inflammation (95) and has been used in
Paper IV in our animal model. Mice were exposed to an aerosol of
LPS (Escherichia coli O128:B12; Sigma, St Louis, MO) for 15 min
using a nose-only Battelle exposure chamber. Control mice were
exposed to an aerosol of solvent alone.
OVA
Ovalbumin (OVA) was used in Paper IV to induce airway
hyperreactivity in mice. This model is widely used as a model for
asthma because the pathology observed in the mouse model
resembles that of human disease (96-98). Mice were sensitized with
OVA adsorbed to aluminum hydroxide gel (1:3) by intraperitoneal
(i.p.) injection at day 0 and 14. On days 29, 32 and 34 mice were
challenged with an aerosol of OVA using a nose-only Battelle
exposure chamber (99). Control mice were given no other exposure
than aerosolized OVA at day 29, 32 and 34.
Figure 6. Schedule of OVA exposure
MATERIALS AND METHODS
31
Drug treatment
Dexamethasone
The synthetic glucocorticoid dexamethasone was administered i.p. at
different time points (1, 2 or 6 hours) after tracheal instillation of
melphalan to yield a dose of 10 mg/kg bodyweight (Paper I and III).
This dose was determined from a previous study where dose-response
effects were evaluated (100). Control animals were treated with PBS.
Vitamin E
To administer vitamin E in order to ensure a satisfactory cellular
uptake, we first needed to prepare liposomes as carriers (Paper I). A
mixture of the lipid dipalmitoylphosphatidylcholine (Sigma) and
vitamin E (Sigma) was dissolved in a solvent consisting of chloroform
and methanol. The solvent was removed in an evaporator and the
lipid film was dissolved in PBS to a concentration of 10 mg/ml.
Liposomes were formed prior to use by sonicating the lipid solution.
One hour after melphalan exposure, the liposomes were injected i.p.
(50 mg/kg bodyweight). The dose was determined from previous
experiments (78, 101)
MATERIALS AND METHODS
32
Respiratory mechanics
Preparation of animals
Animals were weighed and anesthetized with pentobarbital sodium
(68 mg/kg i.p. from local suppliers)(102). Mice were tracheostomized
with an 18-gauge cannula and mechanically ventilated in a quasi-
sinusoidal manner with a small animal ventilator at a frequency of 3.0
Hz and a tidal volume of 12 ml/kg body weight. Positive end-
expiratory pressure (PEEP) of 3 cmH2O was applied by submerging
the expiratory line in water. A warming pad prevented cooling of the
animal. After ensuring a good ventilation of the animal, the muscle
relaxant pancuronium bromide (Pavulon, 0.1 mg/kg, i.p., from local
suppliers) was given to fascilitate the mechanichal ventilation. To
establish stable baseline lung mechanics and to ensure a similar
volume history before the experiments, four sigh maneuvers at three
times the tidal volume was performed. The mice were then allowed a
two-minute resting period before start of the experiment.
Figure 7. The animal ventilator (flexiVent, SCIREQ, Montreal, PQ, Canada)
used to measure respiratory mechanics in mice.
MATERIALS AND METHODS
33
Measurements of respiratory mechanics
The ventilator is built around a computer-controlled precision piston
pump that can combine mechanical ventilation with a variety of
volume and pressure controlled maneouvres to obtain reproducible
measurement of respiratory mechanics. During a perturbation the
piston stops and a predetermined volume of air is sent into the
airways. Via a nebulizer, it is also possible to administer inhaled
substances, e.g. MCh directly to the airways.
Dynamic lung mechanics was measured by applying a sinusoidal
standardized breath and the resulting data was analyzed using the
single compartment model and multiple linear regression in order to
give respiratory resistance (RRS) and respiratory elastance (ERS) (103).
The forced oscillation technique (FOT) was used to analyze tissue
resistance (G) and tissue elastance (H) (102, 104-106). All parameters
were measured continuously using a standardized script and the
maximum response to a given concentration of MCh was reported.
Methacholine delivery
A standard procedure to measure airway reactivity in mice is to give
them incremental doses of the airway-constricting agonist
methacholine and then record the response of the lung to each dose
(107). To investigate the effect of increasing doses of inhaled (i.h.)
MCh delivery into the mouse trachea, doses of MCh (0=saline, 25, 50
and 100 mg/ml) were given at five-minute intervals (Paper III). MCh
was diluted in NaCl and a volume of 20 l was given in an aerosol
during approximately ten seconds.
MATERIALS AND METHODS
34
Postmortem analysis
Bronchoalveolar lavage
The mice were sacrificed by cervical dislocation at specified time
points after melphalan instillation and the trachea was cannulated
with polyethylene tubing. Bronchoalveolar lavage was performed
using 1-ml aliquots of ice-cold Hank’s balanced salt solution (HBSS)
to a total volume of 4 ml. The bronchoalveolar lavage fluid (BALF)
was centrifuged at 4C for 10 min at 500 x g and supernatant from
each animal was frozen in aliquots at -80 C for future analyses.
Cytokine analysis
The concentration of IL-1, IL-6 and IL-23 in tissue, serum and BALF
was measured using enzyme-linked immunosorbent assay (ELISA).
Briefly, capture antibodies were incubated in microtiter 96 wells
ELISA plates (Nunc-Immuno Plate with Max Sorb surface, Tamro
MedLab AB, Mölndal, Sweden) at 4°C over night. The plates were
washed (0.05 % Tween in PBS) and non-specific binding was blocked
with a blocking buffer consisting of bovine serum albumin (1 %) and
sucrose (5 %) in PBS. One hour later 100 l of BALF was added to the
wells. After two hours of incubation, bound cytokine was detected
using a biotin-coupled detection antibody which was quantified using
a streptavidin-peroxidase conjugate. After repeated washing, a ready-
to-use peroxidase substrate (R&D Systems) was used. The plates were
incubated at room temperature for 20 minutes and terminated by
adding 50 l of 1 M H2SO4 to each well. The plate was read at 450 nm
in a Labsystems iEMS ELISA reader (Vantaa, Finland).
MATERIALS AND METHODS
35
Analysis of edema formation
Mice were sacrificed by cervical dislocation 18 hrs after drug
treatment. The whole lung package was dissected and washed in
HBSS after the heart and trachea were removed. Excessive fluid was
carefully removed by drying the lung with a piece of tissue paper. The
lung was put on a piece of aluminium folio and weighed (wet weight)
immediately. The lungs were dried over night in an oven at 50 ºC and
weighed again (dry weight). The ratio between wet and dry weight is a
marker for edema formation and was calculated and plotted in a
diagram.
Analysis of lymphocyte subsets
Subpopulation of lymphocytes in BAL fluid was further determined in
Paper II by flow cytometry using monoclonal antibodies against CD3,
CD4, CD8, TCR, TCR, B220, CD25 and NK cells. Isotype-matched
antibodies were used as negative controls. In order to obtain enough
cells for analysis, BAL cells from 5-6 animals in each group was
pooled. For staining, 200.000 cells were added into tubes, washed in
ice-cold PBS containing 3% fetal bovin serum (FCS) and blocked for
non-specific binding using 4 l Fc Block (anti-CD16/CD32,
Pharmingen Becton Dickinson, San Jose, CA, USA) and 10 l rat
serum for 5 min at 4°C. Saturated concentrations of specific
antibodies were added to the cells and allowed to bind for 45 min at
4°C in darkness. After staining, cells were washed in ice-cold 3 %
FCS-PBS and red blood cells were lysed. The remaining cells were
washed in ice-cold PBS and fixed before analysis with a FACSort
flowcytometer (Becton Dickinson). At least 30.000 total events were
collected per sample. A lymphocyte gate was set in a region according
MATERIALS AND METHODS
36
to lymphocyte population characteristic forward scatter (FCS) and
side scatter (SSC) profile. To obtain NK cells and NK/T cells, the cells
were stained with anti-mouse NK1.1–PE, anti-mouse CD3-FTIC and
B22O-Chr in the same tube. To obtain TCR and TCR, cells were
stained with anti-mouse CD3-FITC, anti-mouse TCR-CyChr and
anti-mouse TCR-PE in the same tube. To detect activation markers,
cells were stained with anti-mouse CD4-PE and anti-mouse CD25-
Chr in the same test tube. Total numbers of CD3, CD4, CD8, B and
NK cells in BALF and percentage of TCR and TCR cells was
determined.
Analysis of mRNA expression
BALF cells and lung tissue was collected 20 hrs after last
exposure/challenge, frozen in liquid nitrogen and stored at –80°C
until mRNA analysis. The cell samples were homogenized and total
cytoplasmic RNA was then isolated using RNeasy® mini kit
according to manufacturer’s instructions (Qiagen GmbH, Hilden,
Germany). A rotor-stator homogenizer was used for homogenization
of lung tissue and total cytoplasmic RNA was isolated with an
RNeasy® midi kit. The purified RNA was quantified
spectrofotometrically and 4 µg of RNA was used for synthesizing
complementary DNA using oligo(dT) primers according to a
previously described protocol (108). The specific gene expression of
Ym1, Ym2, ICAM-1, VCAM-1 and the house keeping gene Hprt was
analyzed using TaqMan® Gene expression assays (Applied
Biosystems, Carlsbad, CA) according to manufacturer’s instructions.
Amplification of cDNA was performed by a two-step PCR protocol
(95°C, 10 min, followed by 50 cycles of 95°C for 15 sec and 60°C for
60 sec) using iCycler real-time PCR detection system (Bio-Rad
MATERIALS AND METHODS
37
Laboratories, Hercules, CA). The number of cycles needed to increase
the fluorescence intensity above the background threshold (cycle
threshold: CT) was used for calculation of the relative expression of
genes. The levels of target mRNA was normalized to the house
keeping gene using the ΔΔCT method (109), correcting for differences
in PCR amplification efficiency of each primer and probe set.
2D gel analysis
In Paper IV, the protein levels of Ym2 in lung tissue from OVA-
endotoxin- and melphalan exposed animals were analyzed.
Homogenates of entire lungs were centrifuged and supernatants were
recovered and stored at –80°C. From each lung extract sample, 750
µg protein was applied to nonlinear pH 3–10 immobilized pH
gradient strips. Isoelectric focusing was performed and strips were
then equilibrated at room temperature for 15 min in SDS-
equilibration buffer and for another 15 min with SDS-equilibration
buffer supplemented with 2.5 % (w/v) iodoacetamide. After
equilibration, strips were applied to 10 % SDS-PAGE gels.
Electrophoresis was carried out at 2.5 W per gel during the first 30
min followed by 17 W per gel until complete. Gels were stained using
the colloidal Coomassie procedure (110) and were stored at 4°C in 25
% ammonium sulfate. The gels were scanned at high resolution and
qualitative analysis of protein spots was performed by digital image
analysis with ImageJ 1.43u (U.S. National Institutes of Health,
Bethesda, MD).
MATERIALS AND METHODS
38
Collagen assay
Mice were sacrificed 2 (Paper III), 4 (Paper I), 8 and 12 weeks (Paper
II) after melphalan instillation. Lungs were quickly removed, rinsed
in PBS and frozen in liquid nitrogen. The lungs were later thawed on
ice, cut in small pieces and incubated with vigorous stirring at 4C in
10 ml 0.5 M acetic acid with pepsin (ratio of 1:10 pepsin:tissue wet
weight). After 24 hours incubation the solution was centrifuged at
850 x g for 15 minutes and the collagen content in the clear solution
was subsequently determined using the colorimetric Sircol Collagen
assay kit (Biocolor Ltd., Belfast, UK) according to the manufacturer’s
description. The assay was performed in duplicate. Results are
expressed as g collagen/lung calculated as the difference between
soluble collagen content in the lungs of animals exposed to melphalan
and control animals that received solvent (Paper II). In Paper III, the
total amount of collagen is presented.
Statistical analys
All statistical calculations were performed using GraphPad Prism 4
(GraphPad Software, Inc., San Diego, CA). Results are expressed as
mean values and standard deviations (Paper I) or standard error of
means (Paper II-IV). Statistical comparisons were determined by
unpaired Student’s t-test. If more than two groups were compared,
analysis of variances (ANOVA) was performed followed by Dunnett’s
multiple comparison test when comparisons were performed versus a
single control group and Bonferroni’s post hoc test when comparisons
were performed between all. Analysis of correlation (Paper IV) was
performed using the non-parametric Spearman rank test (two-tailed).
For each analysis, P values less than 0.05 were regarded as
significant.
RESULTS AND DISCUSSION
39
RESULTS AND DISCUSSION Animal models
There are both pros and cons of using animal models instead of
clinical studies. Mouse models have the advantage of short
experimental cycles and it is easy to study them over time in a
controlled environment. They allow for convenient specimen
collection and there is a broad range of different transgenic mouse
models that allow for investigation of mechanistic pathways for many
diseases. Another advantage is that they are easy to breed and house.
A disadvantage is that it is difficult to make direct comparisons with
the human disease, i.e. there are significant differences in sensitivity
between mice and humans.
We must bear in mind the limitations of murine models of chemical
airway inflammation. The mouse lung is considerably different in
structure from the human lung. The airways of humas are unique,
with more branches than in other species, especially in mice, and the
outcome of specific treatments in mouse is not necessarily the same
in human. However, the obvious advantages of being able to exam
systemic effects have driven us to further develop the methods and
protocols to improve the similarities to human diseases.
Inbread mouse strains are generated by sister-brother mating for 20
or more generations. We chose to work with the C57BL/6 strain
which is the most widely used mouse inbred strain, established in the
1930s. It is robust and easy to breed and commonly used to generate
genetically modified strains.
RESULTS AND DISCUSSION
40
Acute airway inflammation
When mice are exposed to melphalan, they develop an acute airway
inflammation characterized by fast recruitment of neutrophils to the
lungs with maximum level reached after approximately 24 hrs (Paper
II, Figure 1 B and C). The inflammatory process, however, is activated
immediately after encounter with the toxic chemical. A time kinetic
study of the cytokine secretion in BAL and serum from exposed mice
show that the levels of the pro-inflammatory cytokines IL-1 and IL-6
peak at 6 hrs after exposure (Paper II, Figure 2). These cytokines play
a major role in the acute-phase response (111). Thus, it is not
surprising to find IL-1 and IL-6 in the lavage fluid and serum at an
early time point. In Paper I, the cytokine analysis was performed on
BAL 6 hrs after exposure based upon the time-kinetics study (Figure
3). Our results show a clear-cut reduction of both cytokines by
dexamethasone treatment one hour after instillation of melphalan.
This study together with other (100, 112) proves that i.p. treatment
with dexamethasone is highly efficient when it comes to inhibition of
the early cytokine cascade. We also noted a reduction of cytokine
levels following treatment with Vitamin E also, but the effects were
weaker and did not reach statistically significant levels.
The aim in Paper I was to compare the therapeutic effects of
dexamethasone and vitamin E on melphalan-induced lung injury. Six
and 18 hrs after exposure, mice were sacrificed and BAL was
withdrawn (Figure 1). Differential cell count showed a strong
reduction of neutrophils both 6 and 18 hrs after exposure in the group
treated with dexamethasone proving its powerful anti-inflammatory
features. Vitamin E given after 6 hrs showed tendencies towards
decreased number of inflammatory cells and after 18 hrs there was a
RESULTS AND DISCUSSION
41
significant decrease of neutrophils when compared to animals
receiving only PBS (Figure 1 B). Edema formation at 18 hrs was also
examined and our results show that dexamethasone but not vitamin E
reduced lung edema (Figure 2).
For humans, the common ways to administer corticosteroids when
treating e.g. asthma, is systemically (orally and intravenous) or
topically (by inhalation). Since it is easier, faster and less stressful for
the animals, we chose to give i.p. injections of dexamethasone in our
studies (Paper I and III). Asides from the practical benefits, it makes
it easier to make comparisons between other studies since this is the
most common administration route of dexamethasone in murine
models.
In Paper III we wanted to compare the therapeutic effects of
dexamethasone when administered at different time points (Figure
3). The results indicated that dexamethasone, when given 1 or 2 hrs
after exposure, efficiently reduces the neutrophil influx to the lungs
and the total leukocyte number. When given after 6 hrs, there was no
significant effect on neutrophils or other cells in BAL.
Based on the results presented in this thesis, it can be stated that
dexamethasone and vitamin E exert their anti-inflammatory
properties through different mechanisms. Dexamethasone is fast-
acting and inhibits the transcription and release of pro-inflammatory
cytokines by inducing gene transcription of IB-, an inhibitor of NF-
B, thereby blocking the translocation of NF-B into the cell nucleus
(113, 114). NF-B is very important for the induction of genes
involved early in the inflammatory process e.g. IL-1 and IL-6.
Dexamethasone is also capable of down-modulating adhesion
RESULTS AND DISCUSSION
42
molecules, such as selectins (115, 116) which prevents the neutrophils
and leukocytes from crossing the endothelium and entering the area
of inflammation. Vitamin E, on the other hand, does not possess the
same anti-inflammatory properties as the corticosteroids. Human
trials with vitamin E supplementation have been contradictory. Some
studies show that high dose of vitamin E can exert protective effects
on cardiovascular diseases (117, 118) and different forms of cancer
(119, 120) while other groups show evidence that vitamin E
supplementation has no beneficial effect on these diseases (121-123).
However, in murine models of induced lung injury, the positive
effects of vitamin E have been determined (78, 101, 124) and we
considered it a possible candidate for treatment in our model of
chemical-induced lung injury. As has been stated in the introduction,
vitamin E can act on many pathways in the acute inflammatory
response. Vitamin E acts as an antioxidant preventing lipid
peroxidation and donating hydrogen to ROS but it can also inhibit
neutrophil migration which reduces the inflammatory response
further. Vitamin E can also act by inhibiting protein kinase C (PKC)
activity in cells, thereby reducing cytokine production (125-127).
Based on our results in Paper I and results from others (128) we
hypothesize that vitamin E can be used as a complement to other
anti-inflammatory drugs such as corticosteroids and that the
combined effect of dexamethasone and vitamin E would yield
beneficial results on treatment of melphalan-induced lung
inflammation.
In Paper II, we examined the roles of the different T lymphocyte
subsets on inflammation by using knock-out (KO) mice lacking either
T cells (TCR–/–), T cells (TCR–/–) and mice completely
deficient in T cell types (TCR–/––/–) and compared the
RESULTS AND DISCUSSION
43
responsiveness with that of wild-type (wt) mice and double KO mice
completely deficient in T cells. The acute airway neutrophilia was
significantly reduced in all three groups when compared to wt (Paper
II, Figure 4). The strongest response was observed for the TCR–/–
and TCR–/––/– indicating that the T cells are somewhat more
important in the neutrophilic response to chemical lung damage than
the T cells (Paper II, Figure 4). The expression of early cytokine
response was also investigated and results show that the expression of
IL-1 in BALF was reduced in all groups 6 hrs after exposure (Paper
II, Figure 5) whereas only T cells contributed to the induction of
IL-6. These cytokines may in concert with IL-23 promote recruitment
of neutrophils by directly affecting the strong neutrophil
chemoattractant IL-17. IL-23 is identified as the key mediator in the
development of the IL-17 producing T cell subset (129-131). We
therefore investigated the capability of T cell deficient mice to
produce IL-23 and found that the T cells are more important for
production of IL-23 than the T cells, since the TCR–/– mice show
significantly decreased levels of IL-23 (Paper II, Figure 6B).
Effects on respiratory physiology
To further evaluate the pathogenesis of melphalan-induced lung
injury, we characterized the effects on respiratory physiology in more
detail. Mice were mechanically ventilated with a small animal
ventilator and given increasing concentrations of MCh and data was
analyzed using both single compartment model and FOT 20 hrs after
exposure. Our results showed a significant MCh-induced increase in
respiratory resistance (RRS), respiratory elastance (ERS), tissue
resistance (G) and tissue elastance (H) indicating that both the
central and peripheral airways are affected by the exposure of
RESULTS AND DISCUSSION
44
melphalan (Paper III, Figure 2). The effect on central airways is
probably due to the fact that melphalan causes an extensive
inflammation with resulting lung edema (Paper I, Figure 2) and the
effect on peripheral parameters indicates a spread of the
inflammation to the peripheral airways.
Dexamethasone was administered 1 and 6 hrs after exposure to the
alkylating mustard and lung mechanics was performed at 20 hrs
revealing significant downregulating effects on all four parameters
measured (Paper I, Figure 2).
Chronic (or sub-acute) inflammation
Studies conducted on people who were exposed to SM during the
Iran–Iraq war show that the chronic lung injuries are the most
difficult complications to treat (8, 15, 16). Therefore, we considered it
important to set up a chronic model of chemical-induced lung injury
in mice in order to investigate the pathogenesis of the disease and to
examine the treatment effects of dexamethasone and vitamin E.
Mice were subjected to melphalan exposure and sacrificed at different
time points between 1 and 90 days post exposure. Differential cell
count analysis showed that three days after exposure to melphalan,
lymphocytes were recruited to the damaged area and continue to
gather until they reach their maximum level in BALF after 14 days
(Paper II, Figure 3). At this time-point lymphocytes constituted
approximately 20% of the total cell population. A detailed analysis of
lymphocyte subpopulations showed a predominance of T cells with
only a small proportion of B cells and NK cells when compared to
unexposed control animals (Paper II, Table 3). The T cells were both
of the helper type (CD4+) and cytotoxic (CD8+) phenotypes with a
RESULTS AND DISCUSSION
45
predominance of T cells. Such pattern has also been reported in
humans exposed to SM (132-134). The consequences of accumulating
cytotoxic T cells in chronic inflammation is not fully elucidated,
however, there are evidence suggesting a role in collagen production
(135).
Treatment with dexamethasone 1 hr after exposure to melphalan
resulted in a significant inhibition of lymphocyte recruitment at day
14 (Paper I, Figure 4 and Paper III, Figure 4). Treatment after 2 and
6 hrs was also significant (Paper III, Figure 4) but not as efficient as
the 1 hr treatment. Thus, it seems that inhibition of the early
inflammatory response is important also forprevention of the delayed
response.
Lung fibrosis
A key feature in tissue repair is the production of collagen in injured
tissue. Fibroblasts enter the location of injury and produce collagen
which is necessary to replace lost tissue during destructive
inflammatory responses. Overabundance of collagen production may
lead to scarring that can cause permanent distortion of the tissue, a
condition known as fibrosis. Therefore, a common way of examining
long-term fibrotic effects is by measuring the amount of collagen
content in injured tissue.
RESULTS AND DISCUSSION
46
Figure 8. Possible outcomes after exposure to alkylating mustard.
Several studies conducted on chemical airway inflammation in
animals are performed using bleomycin, a cell toxic substance that
resembles melphalan in many ways. Bleomycin is clinically used as an
anti-cancer drug but because of its celltoxic properties, it can induce
long-lasting inflammation in lung tissue which in turn may cause
structural changes and lung fibrosis (136-138). In our studies, we
showed evidence of collagen desposition in lungs of melphalan-
exposed mice after 2 weeks (Paper III, Figure 5), 4 weeks (Paper I,
Figure 5) and after 8 and 12 weeks (Paper II, Figure 8). As mentioned
before, collagen is produced when the lung is subjected to injury but
we believe that the accumulation of lymphocytes (mainly T cell) at a
later stage of the inflammation is also of importance in this process.
In mice depleted of T cells, the collagen deposition was diminished
(Paper II, Figure 8D) proving an important role for the T cells in
late-phase fibrosis. These results are consistent with results from
other groups showing the contribution of lymphocytes in collagen
production (135, 139, 140). However, Paper II is to our knowledge the
first study to characterize the contribution of the different subsets of
T lymphocytes on both acute and long-term reponse.
RESULTS AND DISCUSSION
47
Treatment with dexamethasone 1 or 2 hrs post exposure resulted in a
significant decrease in collagen deposition (Paper I, Figure 5 and
Paper III, Figure 5). Effects were observed also for the 6 hrs group
but they were not statistically significant. Vitamin E treatment 1 hr
post exposure efficiently downregulated the collagen amount
supporting the idea that the actions of Vitamin E and dexamethasone
early in the inflammatory process is important also for the prevention
of long-term responses.
Ym1 and Ym2
In Paper IV, we compared the expression of the chitinase-like
proteins Ym1 and Ym2 in three murine models of induced airway
inflammation; LPS-, melphalan- and OVA-induced (Paper IV).
Experimental asthma is characterized by both early- and late-phase
bronchoconstriction, infiltration of inflammatory cells, mucus
production, peripheral blood eosinophilia and airway remodeling.
Many of these findings mimic those found in human asthma, thus
providing an important tool in the study of the disease (141-144).
In most animal models of asthma, mice are sensitized by an i.p.
injection of OVA with a Th2 skewing adjuvant, such as alum (145-
147). Sensitization in itself induces the production of OVA-specific
immunoglobulin (Ig) E. Upon secondary exposure to aerosolized
OVA, sensitized animals then develop airway hyperresponsiveness
and start recruiting eosinophils into the airways. If mice are not first
sensitized with an i.p injection the response is much weaker (148,
149).
RESULTS AND DISCUSSION
48
LPS-induced lung injury is well characterized (100, 108, 150), as is
melphalan-induced inflammation and these models were therefore
used to compare the expression of Ym1 and Ym2 with the expression
in OVA-induced inflammation.
We found that the expression of both Ym1 and Ym2 mRNA in lung
tissue and BALF from OVA-sensitized mice is significantly
upregulated compared to healthy controls (Paper IV, Figure 2 and
Figure 3). On protein level, Ym2 was only detected in OVA-sensitized
animals (Figure 4). There was no expression of Ym2 in either of the
other two models but correlation analysis showed that Ym1 is present
at low levels in all models of airway inflammation while Ym2 is
restricted to OVA-induced injury only. It has been shown by others
that Ym1 is present in unstimulated lung (85, 94), supporting our
results. Taken together, we conclude that Ym2 expression is strongly
associated with experimental asthma but not with melphalan- or LPS-
induced airway inflammation.
Reactive airways dysfunction syndrome (RADS)
In 1985 Brooks et al. described a new condition they entitled RADS
(151). It was defined as asthma-like symptoms arising within 24 hrs
of a single, massive, chemical exposure e.g. a single unexpected
inhalation of high concentrations of irritant fumes, vapors, fog, or
smoke that results in acute cough, dyspnea, and wheezing. Common
agents are chlorine (152-154), acetic acid (155), sulfur dioxide (156),
isocyanates (157) and airborne particulates (158). RADS might be
confused with occupational asthma, where there is a sensitization
period of months or years before symptoms appear, and with
aggravation of underlying asthma. However, RADS refers to the acute
irritant-induced asthma. Bronchial biopsies demonstrate loss of
RESULTS AND DISCUSSION
49
epithelium, subepithelial fibrosis, and infiltrates with lymphocytes
but not eosinophils (as would be characteristic of asthma) (159-162).
The majority of patients with RADS improve over time, although
many continue to have respiratory symptoms for at least a year and
can suffer from bronchial hyperreactivity for several years.
In Paper II we show evidence that many of the symptoms described
above are also found in our model of chemical-induced lung injury.
We exposed mice to a single dose of a NM analogue and results show
acute airway neutrophilia and long-term lymphocyte response
followed by formation of connective tissues. However, there are no
signs of airway hyperreactivity 14 days after exposure which would be
expected in RADS. One possible explanation for this is that mice are
less sensitive than humans to inhaled chemical substances and would
have required a higher dose in order to maintain airway
hyperreactivity over time.
50
FINAL COMMENTS Taken together, the work in this thesis has shed new light on the
pathogenesis and treatment of chemical-induced airway
inflammation. By developing a robust and reliable mouse model, the
consequences of melphalan exposure to the airways have been
thoroughly investigated. The nitrogen mustard analogue melphalan
causes an acute inflammation with a high number of neutrophils,
edema formation and increased levels of proinflammatory cytokines
in the lungs. The long-term consequences are increased levels of T
lymphocytes and collagen formation.
In the case of a chemical disaster, it is important to know how to best
treat exposed persons. The triage is a method well established among
paramedics but it has room for improvement. The concept of triage is
to quickly determine the level of care the exposed individuals require
but it can be difficult to determine the grade of injury if no visible
symptoms appear. We know that symptoms after SM exposure are
not always visible at the acute stage but that they can arise later and
often deteriorate over time. From studies on war veterans it is shown
that the lung injuries are difficult to treat and often need lifetime
medical treatment. The common way to treat patients believed to
have been exposed in a chemical disaster is with anti-inflammatory
drugs, e.g. corticosteroids. In our animal model we found that
therapeutic treatment with the corticosteroid dexamethasone proved
to be an efficient treatment. All of the early and late symptoms were
partly or completely diminished. In order to find the critical time
window for efficient treatment, we administered the drug 1, 2 or 6 hrs
after exposure and found that in order to reduce injury, both acute
and sub-acute, the 1 hr treatment was to prefer. These results indicate
51
the need to quickly categorize and medicate exposed persons in order
to prevent chronic lung injury.
A remarkable finding in this study was discovered when the T
lymphocytes in chronic inflammation were more thoroughly
investigated. The T cells which only constitute approximately 5 %
of the T cells, exerted powerful effects on both early expression of
proinflammatory cytokines but also on the late-phase response.
Apparently, this group of T cells orchestrates more functions than
earlier believed in the lungs immune defense.
52
CONCLUSIONS This thesis has been undertaken with the main goal to better
understand the pathogenesis of melphalan-induced airway
inflammation and to evaluate the efficacy of anti-inflammatory
treatment. From the present experiments the following conclusions
can be drawn:
Our mouse model of a single exposure of chemical irritant
shows similarities with delayed responses observed in humans
with RADS.
T-cells are crucial for the early expression of pro-
inflammatory cytokines but also contribute to the late-phase
expansion of T-cells and formation of lung fibrosis.
Dexamethasone exerts powerful therapeutic properties in
protection against acute lung injury when administered 1 or 2
hrs after exposure to a lung toxic agent. It also protects against
long-term immune activation and lung fibrosis.
Vitamin E down-modulates the recruitment of neutrophils to
the lungs and protects against excessive collagen formation
but is not as efficient as dexamethasone in its anti-
inflammatory properties.
Lung physiology examination demonstrated that lung toxic
chemicals can cause airway hyperreactivity in both central and
peripheral airways. Early treatment with dexamethasone
decreases airway hyperreacticity.
The chitinase-like protein Ym2 is a biomarker for allergic
airway inflammation but not for chemical-induced
neutrophilic inflammation in airways
53
SVENSK SAMMANFATTNING Inhalation av kemiska ämnen kan orsaka irritation i luftvägarna och i
högre doser även akut lungskada. Det finns många tillfällen då vi
riskerar att utsättas för toxiska kemikalier, t.ex. under arbete eller vid
en eventuell olycka där det läcker ut giftiga ämnen. Bhopal-olyckan
1984 är ett uppmärksammat fall där flera tusen människor omkom
efter att en tank med metylisocyanat exploderat. Årtionden efteråt
lider människor fortfarande av kroniska lungsjukdomar som följd av
exponering för det toxiska ämnet i hög dos. Andra ämnen som kan
orsaka förödande skador även lång tid efter exponering är olika
senapsgaser.
I första världskriget användes senapsgas för första gången i större
mängder och senare även i Iran-Irakkriget under mitten av 1980-
talet. Senapsgas är ett blåsbildande ämne som orsakar direkta skador
på huden i form av vätskefyllda blåsor och även skador på ögonen
men de allvarligaste sjukdomarna är lungrelaterade. Vi ville i denna
avhandling försöka ta reda på mer om både akuta effekter av kemiska
ämnen på lungorna men även undersöka de kroniska skadorna mer
ingående.
När möss exponeras för en analog till senapsgas (melfalan) utvecklar
de en akut luftvägsinflammation då inflammatoriska celler snabbt
ansamlas i lungorna. Den akuta fasen följs av kroniska respiratoriska
skador som karakteriseras av bronkit, lungfibros och hyperreaktivitet
i luftvägarna.
I denna avhandling har vi satt upp en musmodell för kemiskt
inducerad lungskada och undersökt effekterna på lungorna i ett
tidsintervall från 6 timmar upp till 3 månader för att kunna
undersöka både akuta och kroniska skador. Vi fann att behandling
med kortikosteroider, dexametason i detta fall, effektivt blockerade
den inflammatoriska reaktionen på flera sätt: Neutrofilinflödet till
lungorna minskade, uttryck av proinflammatoriska cytokiner (IL-
1och IL-6) reducerades och ödembildning och fibrosbildning blev
mindre omfattande.
54
I akut luftvägsinflammation visade vi att antioxidanten vitamin E kan
användas som ett möjligt komplement till kortikosteroider vid
behandling men inte ersätta dem helt eftersom vi endast observerade
en begränsad behandlingseffekt då endast vitamin E administrerades.
Vi visade även betydelsen av T-lymfocyter i patogenesen av kroniska
lungskador orsakade av lungtoxiska ämnen och betydelsen av T-
cellerna, som trots sitt låga antal, reglerar det medfödda
immunförsvaret vid kemisk lungskada.
I avseende att hitta den kritiska tidpunkten för dexametason-
behandling, exponerades möss för melfalan och behandlades med
dexametason vid olika tidpunkter. Därefter mätte vi luftvägs-
reaktiviteten, inflammationssvar och bindvävstillväxt i lungor. Från
dessa forsök drog vi slutsatsen att tidig behandling med
kortikosteroider, helst inom en timme efter exponering, är viktig för
att förhindra akuta och kroniska lungskador.
Syftet med denna avhandling var att undersöka hur lungorna hos
möss påverkas av toxiska kemikalier samt att utvärdera medicinska
behandlingsmetoder. Vi hoppas att vårt arbete ska ligga till grund för
fortsatt tillämpad forskning som kan leda till förbättrat medicinskt
omhändertagande av människor som blivit exponerade för toxiska
kemikalier.
55
TACK! Sådär ja – nu har ni kommit till den (för många) intressantaste delen i avhandlingen! Det har varit ett privilegium att få jobba med detta projekt men jag hade inte klarat det utan hjälp. Jag har så många jag vill tacka som gjort det möjligt för mig att genomföra detta. Först, Anders Bucht, min huvudhandledare på FOI som alltid haft tid över att diskutera resultat och som stöttat och väglett hela vägen trots att du själv haft många bollar i luften. Jag har även fått möjlighet att jobba i lite andra projekt parallellt vilket har varit väldigt givande. Thomas Sandström, min biträdande handledare på lungkliniken. Tack vare dig och dina kollegor har jag fått en djupare kunskap i den kliniska delen av mitt arbete. Tack också för ditt uppriktiga intresse i mitt arbete. Ett stort tack till alla mina kollegor i tox-gruppen! Min “coach” och handledare på lab, Barbro Ekstrand-Hammarström! Du har lärt mig allt jag kan och jag är så tacksam för all hjälp och praktisk handledning under åren. Min vapendragare och vän Camilla Österlund som jag delat denna doktorandtid med. Det har varit bitvis rätt stressigt på vårt skrivrum i år men nu är vi båda äntligen i hamn – lycka till med nya bebisen! David Rocksén som introducerade mig på lab och varit till stor hjälp under skrivandet av mitt första arbete. Nu får du äntligen se vad som blev av mig. Linda Elfsmark, medförfattare på arbete IV och en härlig reskompis att ha med till Uppsala där vi körde geler in på sena natten. Äntligen är du tillbaks på jobbet, vi har saknat dig! Åsa Gustafsson, Emelie Näslund Salomonsson och Mona Koch som båda ingått i ”måndagslunch”-gänget och levererat både god mat och trevliga pratstunder (och den senare har även stått för leveranserna av kvalitetsplast till hemmet ) Bo Koch, som med fantastiskt tålamod lärt mig hantera flexiventen även om vi inte alltid var överens om färgerna. Christine Akfur och Lina Ågren– tack för ovärdelig hjälp med allt från cellräkning till mikroskopering. Sofia Jonasson som introducerat mig för nya metoder och tillbringat ett antal timmar med mig i ”tick-tackrummet”. Tack för ett värdefullt arbete med input och kommentarer på mitt skrivarbete. Vilket bra manus vi fick ihop i slutändan! Lina Thors - den hundrädda konståkerskan med konstig (=dålig?) musiksmak som kommit med pepp och glada tillrop hela tiden. Kommer att sakna att ha dig vägg-i-vägg nu när du flyttat till ”Sibirien”.
Djurhuspersonalen – Karin, Lotta och Lena. Era insatser har varit mycket värdefulla!
56
De administrativa stöttepelerna Kerstin och Lena på Universitetet. Ni har sannerligen underlättat mitt jobb. Tack även till bibliotekarierna Kjerstin och Maria på FOI som fått jobba hårt mot slutet av skrivarbetet med att plocka fram pek.
Tack även till all övrig personal på FOI som förgyllt luncher och fikaraster med vardagliga ting. Ingen nämnd, ingen glömd. Stort tack till mina barndomsvänner (och fireflies) Carro, Lollo och Jenny som stöttat och peppat genom hela den här tiden. Ni är de bästa av vänner och betyder massor för mig. Jenny, snart står du också här… Mina vänner i Sävar som jag lärt känna på senare år. Stina, Cissi, Veronica och Emelie. Bättre grannar går inte att få, vi hade verkligen tur! Särskilt tack till Stina som lurat med mig ut i löparspåret och fått mig att inse att även jag kan springa (om än inte särskilt fort). Nu väntar Portugal! Pappa och Mamma som har varit med och format mig till den människa jag är idag. Mamma, jag hoppas du ser mig från himlen och är stolt över mig. Wiveca, du är en fantastisk mormor till våra barn och jag är så tacksam för att du finns i deras och våra liv. Min käre storebror Tomas med familj – vi ses alldeles för sällan men när vi gör det är det värt all väntan (och restid). Nog för att det är vackert på Frösön men jag hade gärna haft er lite närmare. ”Svärfamiljen” (eller familjer snarare) i Glommers – vårt andra hem. Det är fantastiskt skönt att åka upp till stugan och känna hur stressen bara rinner av en. Tack för att ni alltid finns där för oss och ställer upp när det behövs.
Sist men absolute inte minst vill jag tacka min underbara familj för allt stöd och all kärlek. Min man Anders, klippan jag lutar mig mot när det stormar omkring mig. Tack för att du alltid står vid min sida! Mina fantastiska barn Elsa och Sixten som ger mig så mycket glädje och kärlek. Jag älskar er.
/Elisabeth
This work was supported by grants from the National Board of Health and Welfare, the Swedish Ministry of Defence and the Swedish Armed Forces
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