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

REFERENCES

57

REFERENCES 1. Broughton, E. 2005. The Bhopal disaster and its aftermath: a review. Environ Health 4:

6.

2. Papirmeister, B., A. J. Feister, S. I. Robinson, and R. D. Ford. 1991. Medical Defense

Against Mustard Gas: Toxic Mechanisms and Pharmacological Implications. CRC

Press, Boca Raton.

3. Balali-Mood, M., and A. Navaeian. 1986. Clinical and paraclinical findings in 233

patients with sulfur mustard poisoning. B. Heyndrickx. Ghent University Press,

Ghent, Belgium, Proceedings of the secondar world congress on new compounds in

biological and chemical warfare: toxicological evaluation. 464-473.

4. UnitedNationsSecurityCouncil. 1987. Report of the mission dispatched by the

secretary-general to investigate allegations of the use of chemical weapons in the

conflict between the islamic Republic of Iran and Iraq., New York: United nations.

5. Sohrabpour, H. 1984. Clinical manifestations of chemical agents on Iranian combatants

during Iran-Iraq conflict. Arch Belg Suppl: 291-297.

6. Geraci, M. J. 2008. Mustard gas: imminent danger or eminent threat? Ann

Pharmacother 42: 237-246.

7. Somani, S., and S. Babu. 1989. Toxicodynamics of sulfur mustard. Int J Clin

Pharmacol Ther Toxicol 27: 419-435.

8. Balali-Mood, M., and M. Hefazi. 2006. Comparison of early and late toxic effects of

sulfur mustard in Iranian veterans. Basic Clin Pharmacol Toxicol 99: 273-282.

9. Kehe, K., H. Thiermann, F. Balszuweit, F. Eyer, D. Steinritz, and T. Zilker. 2009. Acute

effects of sulfur mustard injury--Munich experiences. Toxicology 263: 3-8.

10. Evison, D., D. Hinsley, and P. Rice. 2002. Chemical weapons. BMJ 324: 332-335.

11. Kehe, K., and L. Szinicz. 2005. Medical aspects of sulphur mustard poisoning.

Toxicology 214: 198-209.

12. Ghanei, M., and A. Harandi. 2007. Long term consequences from exposure to sulfur

mustard: a review. Inhal Toxicol 19: 451-456.

13. Balali-Mood, M., M. Hefazi, M. Mahmoudi, E. Jalali, D. Attaran, M. Maleki, M.

Razavi, G. Zare, A. Tabatabaee, and M. Jaafari. 2005. Long-term complications of

sulphur mustard poisoning in severely intoxicated Iranian veterans. Fundam Clin

Pharmacol 19: 713-721.

14. Bijani, K., and A. Moghadamnia. 2002. Long-term effects of chemical weapons on

respiratory tract in Iraq-Iran war victims living in Babol (North of Iran). Ecotoxicol

Environ Saf 53: 422-424.

15. Emad, A., and G. Rezaian. 1997. The diversity of the effects of sulfur mustard gas

inhalation on respiratory system 10 years after a single, heavy exposure: analysis of

197 cases. Chest 112: 734-738.

16. Dompeling, E., Q. Jöbsis, N. Vandevijver, G. Wesseling, and H. Hendriks. 2004.

Chronic bronchiolitis in a 5-yr-old child after exposure to sulphur mustard gas. Eur

Respir J 23: 343-346.

17. Azizi, F., A. Keshavarz, F. Roshanzamir, and M. Nafarabadi. 1995. Reproductive

function in men following exposure to chemical warfare with sulphur mustard. Med

War 11: 34-44.

18. Ghanei, M., M. Rajaee, S. Khateri, F. Alaeddini, and D. Haines. 2004. Assessment of

fertility among mustard-exposed residents of Sardasht, Iran: a historical cohort

study. Reprod Toxicol 18: 635-639.

19. Pour-Jafari, H. 1994. Congenital malformations in the progenies of Iranian chemical

victims. Vet Hum Toxicol 36: 562-563.

20. Abolghasemi, H., M. H. Radfar, M. Rambod, P. Salehi, H. Ghofrani, M. R. Soroush, F.

Falahaty, Y. Tavakolifar, A. Sadaghianifar, S. M. Khademolhosseini, Z.

REFERENCES

58

Kavehmanesh, M. Joffres, F. M. Burkle, and E. J. Mills. 2010. Childhood physical

abnormalities following paternal exposure to sulfur mustard gas in Iran: a case-

control study. Confl Health 4: 13.

21. Solberg, Y., M. Alcalay, and M. Belkin. 1997. Ocular injury by mustard gas. Surv

Ophthalmol 41: 461-466.

22. Ghasemi, H., T. Ghazanfari, M. Babaei, M. R. Soroush, R. Yaraee, M. Ghassemi-

Broumand, M. A. Javadi, A. Foroutan, M. R. Mahdavi, J. Shams, S. Pourfarzam, S.

Moaiedmohseni, M. R. Nadoushan, P. Owlia, Y. Panahi, Z. M. Hassan, and S.

Faghihzadeh. 2008. Long-term ocular complications of sulfur mustard in the

civilian victims of Sardasht, Iran. Cutan Ocul Toxicol 27: 317-326.

23. Javadi, M. A., S. Yazdani, H. Sajjadi, K. Jadidi, F. Karimian, B. Einollahi, M. R.

Ja'farinasab, and M. Zare. 2005. Chronic and delayed-onset mustard gas keratitis:

report of 48 patients and review of literature. Ophthalmology 112: 617-625.

24. Smith, W. J., and M. A. Dunn. 1991. Medical defense against blistering chemical

warfare agents. Arch Dermatol 127: 1207-1213.

25. Momeni, A. Z., S. Enshaeih, M. Meghdadi, and M. Amindjavaheri. 1992. Skin

manifestations of mustard gas. A clinical study of 535 patients exposed to mustard

gas. Arch Dermatol 128: 775-780.

26. Mellor, S. G., P. Rice, and G. J. Cooper. 1991. Vesicant burns. Br J Plast Surg 44: 434-

437.

27. Goodman, L. S., M. M. Wintrobe, W. Dameshek, M. J. Goodman, A. Gilman, and M.

T. McLennan. 1984. Landmark article Sept. 21, 1946: Nitrogen mustard therapy.

Use of methyl-bis(beta-chloroethyl)amine hydrochloride and tris(beta-

chloroethyl)amine hydrochloride for Hodgkin's disease, lymphosarcoma, leukemia

and certain allied and miscellaneous disorders. By Louis S. Goodman, Maxwell M.

Wintrobe, William Dameshek, Morton J. Goodman, Alfred Gilman and Margaret T.

McLennan. JAMA 251: 2255-2261.

28. Palumbo, A., S. Bringhen, T. Caravita, E. Merla, V. Capparella, V. Callea, C.

Cangialosi, M. Grasso, F. Rossini, M. Galli, L. Catalano, E. Zamagni, M. T.

Petrucci, V. De Stefano, M. Ceccarelli, M. T. Ambrosini, I. Avonto, P. Falco, G.

Ciccone, A. M. Liberati, P. Musto, M. Boccadoro, and G. I. M. E. Italian Multiple

Myeloma Network. 2006. Oral melphalan and prednisone chemotherapy plus

thalidomide compared with melphalan and prednisone alone in elderly patients with

multiple myeloma: randomised controlled trial. Lancet 367: 825-831.

29. Palumbo, A., S. Bringhen, A. M. Liberati, T. Caravita, A. Falcone, V. Callea, M.

Montanaro, R. Ria, A. Capaldi, R. Zambello, G. Benevolo, D. Derudas, F. Dore, F.

Cavallo, F. Gay, P. Falco, G. Ciccone, P. Musto, M. Cavo, and M. Boccadoro.

2008. Oral melphalan, prednisone, and thalidomide in elderly patients with multiple

myeloma: updated results of a randomized controlled trial. Blood 112: 3107-3114.

30. Berenson, J. R., R. Boccia, D. Siegel, M. Bozdech, A. Bessudo, E. Stadtmauer, J.

Talisman Pomeroy, R. Steis, M. Flam, J. Lutzky, S. Jilani, J. Volk, S. F. Wong, R.

Moss, R. Patel, D. Ferretti, K. Russell, R. Louie, H. S. Yeh, and R. A. Swift. 2006.

Efficacy and safety of melphalan, arsenic trioxide and ascorbic acid combination

therapy in patients with relapsed or refractory multiple myeloma: a prospective,

multicentre, phase II, single-arm study. Br J Haematol 135: 174-183.

31. Goldenberg, G., H. Lam, and A. Begleiter. 1979. Active carrier-mediated transport of

melphalan by two separate amino acid transport systems in LPC-1 plasmacytoma

cells in vitro. J Biol Chem 254: 1057-1064.

32. Calvet, J., J. Gascard, S. Delamanche, and C. Brink. 1999. Airway epithelial damage

and release of inflammatory mediators in human lung parenchyma after sulfur

mustard exposure. Hum Exp Toxicol 18: 77-81.

33. Smith, K., W. Smith, T. Hamilton, H. Skelton, J. Graham, C. Okerberg, R. Moeller, and

B. J. Hackley. 1998. Histopathologic and immunohistochemical features in human

skin after exposure to nitrogen and sulfur mustard. Am J Dermatopathol 20: 22-28.

REFERENCES

59

34. Springer, T. A. 1994. Traffic signals for lymphocyte recirculation and leukocyte

emigration: the multistep paradigm. Cell 76: 301-314.

35. Carlos, T. M., and J. M. Harlan. 1994. Leukocyte-endothelial adhesion molecules.

Blood 84: 2068-2101.

36. Brain, J. D. 1992. Mechanisms, measurement, and significance of lung macrophage

function. Environ Health Perspect 97: 5-10.

37. Chen, Y., K. Chou, E. Fuchs, W. Havran, and R. Boismenu. 2002. Protection of the

intestinal mucosa by intraepithelial gamma delta T cells. Proc Natl Acad Sci U S A

99: 14338-14343.

38. Hayday, A. C., S. Roberts, and E. Ramsburg. 2000. gammadelta cells and the regulation

of mucosal immune responses. Am J Respir Crit Care Med 162: S161-163.

39. Braun, R., C. Ferrick, P. Neubauer, M. Sjoding, A. Sterner-Kock, M. Kock, L. Putney,

D. Ferrick, D. Hyde, and R. Love. 2008. IL-17 producing gammadelta T cells are

required for a controlled inflammatory response after bleomycin-induced lung

injury. Inflammation 31: 167-179.

40. Boismenu, R., L. Feng, Y. Y. Xia, J. C. Chang, and W. L. Havran. 1996. Chemokine

expression by intraepithelial gamma delta T cells. Implications for the recruitment

of inflammatory cells to damaged epithelia. J Immunol 157: 985-992.

41. DiTirro, J., E. R. Rhoades, A. D. Roberts, J. M. Burke, A. Mukasa, A. M. Cooper, A. A.

Frank, W. K. Born, and I. M. Orme. 1998. Disruption of the cellular inflammatory

response to Listeria monocytogenes infection in mice with disruptions in targeted

genes. Infect Immun 66: 2284-2289.

42. Boismenu, R., and W. L. Havran. 1994. Modulation of epithelial cell growth by

intraepithelial gamma delta T cells. Science 266: 1253-1255.

43. Dinarello, C. 2009. Immunological and inflammatory functions of the interleukin-1

family. Annu Rev Immunol 27: 519-550.

44. Watkins, L. R., M. K. Hansen, K. T. Nguyen, J. E. Lee, and S. F. Maier. 1999. Dynamic

regulation of the proinflammatory cytokine, interleukin-1beta: molecular biology

for non-molecular biologists. Life Sci 65: 449-481.

45. Piguet, P. F., C. Vesin, G. E. Grau, and R. C. Thompson. 1993. Interleukin 1 receptor

antagonist (IL-1ra) prevents or cures pulmonary fibrosis elicited in mice by

bleomycin or silica. Cytokine 5: 57-61.

46. Kishimoto, T., S. Akira, and T. Taga. 1992. Interleukin-6 and its receptor: a paradigm

for cytokines. Science 258: 593-597.

47. af Klint, E., C. Grundtman, M. Engström, A. I. Catrina, D. Makrygiannakis, L.

Klareskog, U. Andersson, and A. K. Ulfgren. 2005. Intraarticular glucocorticoid

treatment reduces inflammation in synovial cell infiltrations more efficiently than in

synovial blood vessels. Arthritis Rheum 52: 3880-3889.

48. Meduri, G. U., A. S. Headley, E. Golden, S. J. Carson, R. A. Umberger, T. Kelso, and

E. A. Tolley. 1998. Effect of prolonged methylprednisolone therapy in unresolving

acute respiratory distress syndrome: a randomized controlled trial. JAMA 280: 159-

165.

49. Busse, W. W., C. Lenfant, and R. F. Lemanske. 2002. Asthma guidelines: a changing

paradigm to improve asthma care. J Allergy Clin Immunol 110: 703-705.

50. Scadding, G. K. 2001. Corticosteroids in the treatment of pediatric allergic rhinitis. J

Allergy Clin Immunol 108: S59-64.

51. Georén, S., P. Ahnblad, P. Stjärne, A. C. Wikström, and P. Stierna. 2005. Significance

of endogenous glucocorticoid sensitivity for airway eosinophilia in a murine model

of allergy. Acta Otolaryngol 125: 378-385.

52. Kunicka, J. E., M. A. Talle, G. H. Denhardt, M. Brown, L. A. Prince, and G. Goldstein.

1993. Immunosuppression by glucocorticoids: inhibition of production of multiple

lymphokines by in vivo administration of dexamethasone. Cell Immunol 149: 39-

49.

REFERENCES

60

53. Tobler, A., R. Meier, M. Seitz, B. Dewald, M. Baggiolini, and M. F. Fey. 1992.

Glucocorticoids downregulate gene expression of GM-CSF, NAP-1/IL-8, and IL-6,

but not of M-CSF in human fibroblasts. Blood 79: 45-51.

54. Haynesworth, S. E., M. A. Baber, and A. I. Caplan. 1996. Cytokine expression by

human marrow-derived mesenchymal progenitor cells in vitro: effects of

dexamethasone and IL-1 alpha. J Cell Physiol 166: 585-592.

55. Knudsen, P. J., C. A. Dinarello, and T. B. Strom. 1987. Glucocorticoids inhibit

transcriptional and post-transcriptional expression of interleukin 1 in U937 cells. J

Immunol 139: 4129-4134.

56. Mori, A., O. Kaminuma, M. Suko, S. Inoue, T. Ohmura, A. Hoshino, Y. Asakura, K.

Miyazawa, T. Yokota, Y. Okumura, K. Ito, and H. Okudaira. 1997. Two distinct

pathways of interleukin-5 synthesis in allergen-specific human T-cell clones are

suppressed by glucocorticoids. Blood 89: 2891-2900.

57. Rolfe, F. G., J. M. Hughes, C. L. Armour, and W. A. Sewell. 1992. Inhibition of

interleukin-5 gene expression by dexamethasone. Immunology 77: 494-499.

58. Sies, H., and E. Cadenas. 1985. Oxidative stress: damage to intact cells and organs.

Philos Trans R Soc Lond B Biol Sci 311: 617-631.

59. Finkel, T. 1998. Oxygen radicals and signaling. Curr Opin Cell Biol 10: 248-253.

60. Rhee, S. G. 1999. Redox signaling: hydrogen peroxide as intracellular messenger. Exp

Mol Med 31: 53-59.

61. Dröge, W. 2002. Free radicals in the physiological control of cell function. Physiol Rev

82: 47-95.

62. Freeman, B., and J. Crapo. 1982. Biology of disease: free radicals and tissue injury. Lab

Invest 47: 412-426.

63. Chow, C. W., M. T. Herrera Abreu, T. Suzuki, and G. P. Downey. 2003. Oxidative

stress and acute lung injury. Am J Respir Cell Mol Biol 29: 427-431.

64. Shasby, D. M., K. M. Vanbenthuysen, R. M. Tate, S. S. Shasby, I. McMurtry, and J. E.

Repine. 1982. Granulocytes mediate acute edematous lung injury in rabbits and in

isolated rabbit lungs perfused with phorbol myristate acetate: role of oxygen

radicals. Am Rev Respir Dis 125: 443-447.

65. Harlan, J. M., P. D. Killen, L. A. Harker, G. E. Striker, and D. G. Wright. 1981.

Neutrophil-mediated endothelial injury in vitro mechanisms of cell detachment. J

Clin Invest 68: 1394-1403.

66. Fialkow, L., C. K. Chan, S. Grinstein, and G. P. Downey. 1993. Regulation of tyrosine

phosphorylation in neutrophils by the NADPH oxidase. Role of reactive oxygen

intermediates. J Biol Chem 268: 17131-17137.

67. Fialkow, L., C. K. Chan, D. Rotin, S. Grinstein, and G. P. Downey. 1994. Activation of

the mitogen-activated protein kinase signaling pathway in neutrophils. Role of

oxidants. J Biol Chem 269: 31234-31242.

68. Haddad, J. J., R. E. Olver, and S. C. Land. 2000. Antioxidant/pro-oxidant equilibrium

regulates HIF-1alpha and NF-kappa B redox sensitivity. Evidence for inhibition by

glutathione oxidation in alveolar epithelial cells. J Biol Chem 275: 21130-21139.

69. Baldwin, A. S. 2001. Series introduction: the transcription factor NF-kappaB and

human disease. J Clin Invest 107: 3-6.

70. Fink, M. P. 2002. Role of reactive oxygen and nitrogen species in acute respiratory

distress syndrome. Curr Opin Crit Care 8: 6-11.

71. De la Fuente, M., and V. M. Victor. 2000. Anti-oxidants as modulators of immune

function. Immunol Cell Biol 78: 49-54.

72. Faruqi, R., C. de la Motte, and P. E. DiCorleto. 1994. Alpha-tocopherol inhibits agonist-

induced monocytic cell adhesion to cultured human endothelial cells. J Clin Invest

94: 592-600.

73. Devaraj, S., D. Li, and I. Jialal. 1996. The effects of alpha tocopherol supplementation

on monocyte function. Decreased lipid oxidation, interleukin 1 beta secretion, and

monocyte adhesion to endothelium. J Clin Invest 98: 756-763.

REFERENCES

61

74. Wu, D., T. Koga, K. R. Martin, and M. Meydani. 1999. Effect of vitamin E on human

aortic endothelial cell production of chemokines and adhesion to monocytes.

Atherosclerosis 147: 297-307.

75. Erl, W., C. Weber, C. Wardemann, and P. C. Weber. 1997. alpha-Tocopheryl succinate

inhibits monocytic cell adhesion to endothelial cells by suppressing NF-kappa B

mobilization. Am J Physiol 273: H634-640.

76. Islam, K. N., S. Devaraj, and I. Jialal. 1998. alpha-Tocopherol enrichment of monocytes

decreases agonist-induced adhesion to human endothelial cells. Circulation 98:

2255-2261.

77. Yoshida, N., T. Yoshikawa, H. Manabe, Y. Terasawa, M. Kondo, N. Noguchi, and E.

Niki. 1999. Vitamin E protects against polymorphonuclear leukocyte-dependent

adhesion to endothelial cells. J Leukoc Biol 65: 757-763.

78. Rocksén, D., B. Ekstrand-Hammarström, L. Johansson, and A. Bucht. 2003. Vitamin E

reduces transendothelial migration of neutrophils and prevents lung injury in

endotoxin-induced airway inflammation. Am J Respir Cell Mol Biol 28: 199-207.

79. Wang, X., and P. J. Quinn. 1999. Vitamin E and its function in membranes. Prog Lipid

Res 38: 309-336.

80. Nachbar, F., and H. C. Korting. 1995. The role of vitamin E in normal and damaged

skin. J Mol Med (Berl) 73: 7-17.

81. Burton, G. 1994. Vitamin E: molecular and biological function. Proc Nutr Soc 53: 251-

262.

82. Boot, R., A. Bussink, M. Verhoek, P. de Boer, A. Moorman, and J. Aerts. 2005. Marked

differences in tissue-specific expression of chitinases in mouse and man. J

Histochem Cytochem 53: 1283-1292.

83. Shahabuddin, M., T. Toyoshima, M. Aikawa, and D. C. Kaslow. 1993. Transmission-

blocking activity of a chitinase inhibitor and activation of malarial parasite chitinase

by mosquito protease. Proc Natl Acad Sci U S A 90: 4266-4270.

84. Boot, R. G., E. F. Blommaart, E. Swart, K. Ghauharali-van der Vlugt, N. Bijl, C. Moe,

A. Place, and J. M. Aerts. 2001. Identification of a novel acidic mammalian

chitinase distinct from chitotriosidase. J Biol Chem 276: 6770-6778.

85. Jin, H. M., N. G. Copeland, D. J. Gilbert, N. A. Jenkins, R. B. Kirkpatrick, and M.

Rosenberg. 1998. Genetic characterization of the murine Ym1 gene and

identification of a cluster of highly homologous genes. Genomics 54: 316-322.

86. Sun, Y. J., N. C. Chang, S. I. Hung, A. C. Chang, C. C. Chou, and C. D. Hsiao. 2001.

The crystal structure of a novel mammalian lectin, Ym1, suggests a saccharide

binding site. J Biol Chem 276: 17507-17514.

87. Herrera-Estrella, A., and I. Chet. 1999. Chitinases in biological control. EXS 87: 171-

184.

88. Elias, J. A., R. J. Homer, Q. Hamid, and C. G. Lee. 2005. Chitinases and chitinase-like

proteins in T(H)2 inflammation and asthma. J Allergy Clin Immunol 116: 497-500.

89. Holt, P. G. 2000. Parasites, atopy, and the hygiene hypothesis: resolution of a paradox?

Lancet 356: 1699-1701.

90. Fajardo, I., L. Svensson, A. Bucht, and G. Pejler. 2004. Increased levels of hypoxia-

sensitive proteins in allergic airway inflammation. Am J Respir Crit Care Med 170:

477-484.

91. Zhao, J., H. Zhu, C. H. Wong, K. Y. Leung, and W. S. Wong. 2005. Increased lungkine

and chitinase levels in allergic airway inflammation: a proteomics approach.

Proteomics 5: 2799-2807.

92. Sandler, N. G., M. M. Mentink-Kane, A. W. Cheever, and T. A. Wynn. 2003. Global

gene expression profiles during acute pathogen-induced pulmonary inflammation

reveal divergent roles for Th1 and Th2 responses in tissue repair. J Immunol 171:

3655-3667.

REFERENCES

62

93. Chang, N. C., S. I. Hung, K. Y. Hwa, I. Kato, J. E. Chen, C. H. Liu, and A. C. Chang.

2001. A macrophage protein, Ym1, transiently expressed during inflammation is a

novel mammalian lectin. J Biol Chem 276: 17497-17506.

94. Webb, D., A. McKenzie, and P. Foster. 2001. Expression of the Ym2 lectin-binding

protein is dependent on interleukin (IL)-4 and IL-13 signal transduction:

identification of a novel allergy-associated protein. J Biol Chem 276: 41969-41976.

95. Rylander, R., and M. C. Snella. 1983. Endotoxins and the lung: cellular reactions and

risk for disease. Prog Allergy 33: 332-344.

96. Renz, H., H. R. Smith, J. E. Henson, B. S. Ray, C. G. Irvin, and E. W. Gelfand. 1992.

Aerosolized antigen exposure without adjuvant causes increased IgE production and

increased airway responsiveness in the mouse. J Allergy Clin Immunol 89: 1127-

1138.

97. Lack, G., A. Oshiba, K. L. Bradley, J. E. Loader, D. Amran, G. L. Larsen, and E. W.

Gelfand. 1995. Transfer of immediate hypersensitivity and airway

hyperresponsiveness by IgE-positive B cells. Am J Respir Crit Care Med 152:

1765-1773.

98. Eum, S. Y., S. Hailé, J. Lefort, M. Huerre, and B. B. Vargaftig. 1995. Eosinophil

recruitment into the respiratory epithelium following antigenic challenge in hyper-

IgE mice is accompanied by interleukin 5-dependent bronchial

hyperresponsiveness. Proc Natl Acad Sci U S A 92: 12290-12294.

99. Svensson, L., B. Lilliehöök, R. Larsson, and A. Bucht. 2003. gammadelta T cells

contribute to the systemic immunoglobulin E response and local B-cell reactivity in

allergic eosinophilic airway inflammation. Immunology 108: 98-108.

100. Rocksén, D., B. Lilliehöök, R. Larsson, T. Johansson, and A. Bucht. 2000. Differential

anti-inflammatory and anti-oxidative effects of dexamethasone and N-

acetylcysteine in endotoxin-induced lung inflammation. Clin Exp Immunol 122:

249-256.

101. Suntres, Z., and P. Shek. 1997. Protective effect of liposomal alpha-tocopherol against

bleomycin-induced lung injury. Biomed Environ Sci 10: 47-59.

102. Jonasson, S., G. Hedenstierna, H. Hedenström, and J. Hjoberg. 2009. Comparisons of

effects of intravenous and inhaled methacholine on airway physiology in a murine

asthma model. Respir Physiol Neurobiol 165: 229-236.

103. Irvin, C. G., and J. H. Bates. 2003. Measuring the lung function in the mouse: the

challenge of size. Respir Res 4: 4.

104. Hantos, Z., B. Daróczy, B. Suki, S. Nagy, and J. J. Fredberg. 1992. Input impedance

and peripheral inhomogeneity of dog lungs. J Appl Physiol 72: 168-178.

105. Gomes, R. F., X. Shen, R. Ramchandani, R. S. Tepper, and J. H. Bates. 2000.

Comparative respiratory system mechanics in rodents. J Appl Physiol 89: 908-916.

106. Jonasson, S., G. Hedenstierna, and J. Hjoberg. 2010. Concomitant administration of

nitric oxide and glucocorticoids improves protection against bronchoconstriction in

a murine model of asthma. J Appl Physiol 109: 521-531.

107. Bates, J. H., M. Rincon, and C. G. Irvin. 2009. Animal models of asthma. Am J Physiol

Lung Cell Mol Physiol 297: L401-410.

108. Larsson, R., D. Rocksén, B. Lilliehöök, A. Jonsson, and A. Bucht. 2000. Dose-

dependent activation of lymphocytes in endotoxin-induced airway inflammation.

Infect Immun 68: 6962-6969.

109. Pfaffl, M. W. 2001. A new mathematical model for relative quantification in real-time

RT-PCR. Nucleic Acids Res 29: e45.

110. Neuhoff, V., N. Arold, D. Taube, and W. Ehrhardt. 1988. Improved staining of proteins

in polyacrylamide gels including isoelectric focusing gels with clear background at

nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250.

Electrophoresis 9: 255-262.

111. Baumann, H., and J. Gauldie. 1994. The acute phase response. Immunol Today 15: 74-

80.

REFERENCES

63

112. Barnes, P. J. 1998. Anti-inflammatory actions of glucocorticoids: molecular

mechanisms. Clin Sci (Lond) 94: 557-572.

113. Scheinman, R., P. Cogswell, A. Lofquist, and A. J. Baldwin. 1995. Role of

transcriptional activation of I kappa B alpha in mediation of immunosuppression by

glucocorticoids. Science 270: 283-286.

114. Auphan, N., J. DiDonato, C. Rosette, A. Helmberg, and M. Karin. 1995.

Immunosuppression by glucocorticoids: inhibition of NF-kappa B activity through

induction of I kappa B synthesis. Science 270: 286-290.

115. Jilma, B., J. Voltmann, S. Albinni, P. Stohlawetz, I. Schwarzinger, C. H. Gleiter, A.

Rauch, H. G. Eichler, and O. F. Wagner. 1997. Dexamethasone down-regulates the

expression of L-selectin on the surface of neutrophils and lymphocytes in humans.

Clin Pharmacol Ther 62: 562-568.

116. Nakagawa, M., G. P. Bondy, D. Waisman, D. Minshall, J. C. Hogg, and S. F. van

Eeden. 1999. The effect of glucocorticoids on the expression of L-selectin on

polymorphonuclear leukocyte. Blood 93: 2730-2737.

117. Diaz, M. N., B. Frei, J. A. Vita, and J. F. Keaney. 1997. Antioxidants and

atherosclerotic heart disease. N Engl J Med 337: 408-416.

118. Marchioli, R. 1999. Antioxidant vitamins and prevention of cardiovascular disease:

laboratory, epidemiological and clinical trial data. Pharmacol Res 40: 227-238.

119. Klein, E. A., I. M. Thompson, S. M. Lippman, P. J. Goodman, D. Albanes, P. R. Taylor,

and C. Coltman. 2003. SELECT: the selenium and vitamin E cancer prevention

trial. Urol Oncol 21: 59-65.

120. Ni, J., M. Chen, Y. Zhang, R. Li, J. Huang, and S. Yeh. 2003. Vitamin E succinate

inhibits human prostate cancer cell growth via modulating cell cycle regulatory

machinery. Biochem Biophys Res Commun 300: 357-363.

121. Chiabrando, C., F. Avanzini, C. Rivalta, F. Colombo, R. Fanelli, G. Palumbo, and M.

Roncaglioni. 2002. Long-term vitamin E supplementation fails to reduce lipid

peroxidation in people at cardiovascular risk: analysis of underlying factors. Curr

Control Trials Cardiovasc Med 3: 5.

122. Lonn, E., S. Yusuf, V. Dzavik, C. Doris, Q. Yi, S. Smith, A. Moore-Cox, J. Bosch, W.

Riley, K. Teo, and S. Investigators. 2001. Effects of ramipril and vitamin E on

atherosclerosis: the study to evaluate carotid ultrasound changes in patients treated

with ramipril and vitamin E (SECURE). Circulation 103: 919-925.

123. Lee, I. M., N. R. Cook, J. M. Gaziano, D. Gordon, P. M. Ridker, J. E. Manson, C. H.

Hennekens, and J. E. Buring. 2005. Vitamin E in the primary prevention of

cardiovascular disease and cancer: the Women's Health Study: a randomized

controlled trial. JAMA 294: 56-65.

124. Kilinç, C., O. Ozcan, E. Karaöz, K. Sunguroğlu, T. Kutluay, and L. Karaca. 1993.

Vitamin E reduces bleomycin-induced lung fibrosis in mice: biochemical and

morphological studies. J Basic Clin Physiol Pharmacol 4: 249-269.

125. Boscoboinik, D., A. Szewczyk, C. Hensey, and A. Azzi. 1991. Inhibition of cell

proliferation by alpha-tocopherol. Role of protein kinase C. J Biol Chem 266: 6188-

6194.

126. Ricciarelli, R., A. Tasinato, S. Clément, N. Ozer, D. Boscoboinik, and A. Azzi. 1998.

alpha-Tocopherol specifically inactivates cellular protein kinase C alpha by

changing its phosphorylation state. Biochem J 334 ( Pt 1): 243-249.

127. Tasinato, A., D. Boscoboinik, G. Bartoli, P. Maroni, and A. Azzi. 1995. d-alpha-

tocopherol inhibition of vascular smooth muscle cell proliferation occurs at

physiological concentrations, correlates with protein kinase C inhibition, and is

independent of its antioxidant properties. Proc Natl Acad Sci U S A 92: 12190-

12194.

128. Boskabady, M. H., S. Amery, N. Vahedi, and M. R. Khakzad. 2011. The effect of

vitamin E on tracheal responsiveness and lung inflammation in sulfur mustard

exposed guinea pigs. Inhal Toxicol 23: 157-165.

REFERENCES

64

129. Cua, D., J. Sherlock, Y. Chen, C. Murphy, B. Joyce, B. Seymour, L. Lucian, W. To, S.

Kwan, T. Churakova, S. Zurawski, M. Wiekowski, S. Lira, D. Gorman, R.

Kastelein, and J. Sedgwick. 2003. Interleukin-23 rather than interleukin-12 is the

critical cytokine for autoimmune inflammation of the brain. Nature 421: 744-748.

130. Iwakura, Y., and H. Ishigame. 2006. The IL-23/IL-17 axis in inflammation. J Clin

Invest 116: 1218-1222.

131. Langrish, C., Y. Chen, W. Blumenschein, J. Mattson, B. Basham, J. Sedgwick, T.

McClanahan, R. Kastelein, and D. Cua. 2005. IL-23 drives a pathogenic T cell

population that induces autoimmune inflammation. J Exp Med 201: 233-240.

132. Emad, A., and Y. Emad. 2007. Increased in CD8 T lymphocytes in the BAL fluid of

patients with sulfur mustard gas-induced pulmonary fibrosis. Respir Med 101: 786-

792.

133. Chrysofakis, G., N. Tzanakis, D. Kyriakoy, M. Tsoumakidou, I. Tsiligianni, M.

Klimathianaki, and N. Siafakas. 2004. Perforin expression and cytotoxic activity of

sputum CD8+ lymphocytes in patients with COPD. Chest 125: 71-76.

134. Saetta, M., A. Di Stefano, G. Turato, F. Facchini, L. Corbino, C. Mapp, P. Maestrelli,

A. Ciaccia, and L. Fabbri. 1998. CD8+ T-lymphocytes in peripheral airways of

smokers with chronic obstructive pulmonary disease. Am J Respir Crit Care Med

157: 822-826.

135. Rezzonico, R., D. Burger, and J. Dayer. 1998. Direct contact between T lymphocytes

and human dermal fibroblasts or synoviocytes down-regulates types I and III

collagen production via cell-associated cytokines. J Biol Chem 273: 18720-18728.

136. Chandler, D. 1990. Possible mechanisms of bleomycin-induced fibrosis. Clin Chest

Med 11: 21-30.

137. Hay, J., S. Shahzeidi, and G. Laurent. 1991. Mechanisms of bleomycin-induced lung

damage. Arch Toxicol 65: 81-94.

138. Swaisgood, C., E. French, C. Noga, R. Simon, and V. Ploplis. 2000. The development

of bleomycin-induced pulmonary fibrosis in mice deficient for components of the

fibrinolytic system. Am J Pathol 157: 177-187.

139. Luzina, I. G., J. C. Papadimitriou, R. Anderson, K. Pochetuhen, and S. P. Atamas. 2006.

Induction of prolonged infiltration of T lymphocytes and transient T lymphocyte-

dependent collagen deposition in mouse lungs following adenoviral gene transfer of

CCL18. Arthritis Rheum 54: 2643-2655.

140. Huaux, F., T. Liu, B. McGarry, M. Ullenbruch, Z. Xing, and S. Phan. 2003. Eosinophils

and T lymphocytes possess distinct roles in bleomycin-induced lung injury and

fibrosis. J Immunol 171: 5470-5481.

141. Gil, F. R., and A. M. Lauzon. 2007. Smooth muscle molecular mechanics in airway

hyperresponsiveness and asthma. Can J Physiol Pharmacol 85: 133-140.

142. Bochner, B. S., and W. W. Busse. 2005. Allergy and asthma. J Allergy Clin Immunol

115: 953-959.

143. Bousquet, J., P. K. Jeffery, W. W. Busse, M. Johnson, and A. M. Vignola. 2000.

Asthma. From bronchoconstriction to airways inflammation and remodeling. Am J

Respir Crit Care Med 161: 1720-1745.

144. Fanta, C. H. 2009. Asthma. N Engl J Med 360: 1002-1014.

145. Wilder, J. A., D. D. Collie, B. S. Wilson, D. E. Bice, C. R. Lyons, and M. F. Lipscomb.

1999. Dissociation of airway hyperresponsiveness from immunoglobulin E and

airway eosinophilia in a murine model of allergic asthma. Am J Respir Cell Mol

Biol 20: 1326-1334.

146. Yu, M., M. Tsai, S. Y. Tam, C. Jones, J. Zehnder, and S. J. Galli. 2006. Mast cells can

promote the development of multiple features of chronic asthma in mice. J Clin

Invest 116: 1633-1641.

147. Kumar, R. K., C. Herbert, and P. S. Foster. 2008. The "classical" ovalbumin challenge

model of asthma in mice. Curr Drug Targets 9: 485-494.

REFERENCES

65

148. Williams, C. M., and S. J. Galli. 2000. Mast cells can amplify airway reactivity and

features of chronic inflammation in an asthma model in mice. J Exp Med 192: 455-

462.

149. Mayr, S. I., R. I. Zuberi, M. Zhang, J. de Sousa-Hitzler, K. Ngo, Y. Kuwabara, L. Yu,

W. P. Fung-Leung, and F. T. Liu. 2002. IgE-dependent mast cell activation

potentiates airway responses in murine asthma models. J Immunol 169: 2061-2068.

150. Haglund, L., S. M. Bernier, P. Onnerfjord, and A. D. Recklies. 2008. Proteomic

analysis of the LPS-induced stress response in rat chondrocytes reveals induction of

innate immune response components in articular cartilage. Matrix Biol 27: 107-118.

151. Brooks, S., M. Weiss, and I. Bernstein. 1985. Reactive airways dysfunction syndrome

(RADS). Persistent asthma syndrome after high level irritant exposures. Chest 88:

376-384.

152. Leroyer, C., J. L. Malo, D. Girard, J. G. Dufour, and D. Gautrin. 1999. Chronic rhinitis

in workers at risk of reactive airways dysfunction syndrome due to exposure to

chlorine. Occup Environ Med 56: 334-338.

153. Moore, B., and M. Sherman. 1991. Chronic reactive airway disease following acute

chlorine gas exposure in an asymptomatic atopic patient. Chest 100: 855-856.

154. Donnelly, S., and M. FitzGerald. 1990. Reactive airways dysfunction syndrome

(RADS) due to chlorine gas exposure. Ir J Med Sci 159: 275-276; discussion 276-

277.

155. Kern, D. G. 1991. Outbreak of the reactive airways dysfunction syndrome after a spill

of glacial acetic acid. Am Rev Respir Dis 144: 1058-1064.

156. Piirilä, P. L., H. Nordman, O. S. Korhonen, and I. Winblad. 1996. A thirteen-year

follow-up of respiratory effects of acute exposure to sulfur dioxide. Scand J Work

Environ Health 22: 191-196.

157. Perfetti, L., B. Bramé, M. Ferrari, and G. Moscato. 2003. Occupational asthma (OA)

with sensitization to diphenylmethane diisocyanate (MDI) presenting at the onset

like a reactive airways dysfunction syndrome (RADS). Am J Ind Med 44: 325-328.

158. Banauch, G., D. Alleyne, R. Sanchez, K. Olender, H. Cohen, M. Weiden, K. Kelly, and

D. Prezant. 2003. Persistent hyperreactivity and reactive airway dysfunction in

firefighters at the World Trade Center. Am J Respir Crit Care Med 168: 54-62.

159. Lemière, C., J. L. Malo, and M. Boutet. 1997. Reactive airways dysfunction syndrome

due to chlorine: sequential bronchial biopsies and functional assessment. Eur Respir

J 10: 241-244.

160. Boulet, L., M. Laviolette, H. Turcotte, A. Cartier, M. Dugas, J. Malo, and M. Boutet.

1997. Bronchial subepithelial fibrosis correlates with airway responsiveness to

methacholine. Chest 112: 45-52.

161. Meggs, W. J. 1994. RADS and RUDS--the toxic induction of asthma and rhinitis. J

Toxicol Clin Toxicol 32: 487-501.

162. Bardana, E. J. 1999. Reactive airways dysfunction syndrome (RADS): guidelines for

diagnosis and treatment and insight into likely prognosis. Ann Allergy Asthma

Immunol 83: 583-586.