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ROLE OF NEUTROPHILIC INFLAMMATION IN OZONE-INDUCED EPITHELIAL
ALTERATIONS IN THE NASAL AIRWAYS OF RATS

presented by

Hye Youn Cho

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1M animus-m4

ROLE OF NEUTROPHILIC INFLAMMATION IN OZONE-INDUCED EPITHELIAL
ALTERATIONS IN THE NASAL AIRWAYS OF RATS

By

Hye Youn Cho

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Pharmacology and Toxicology
Institute of Environmental Toxicology

1998

ABSTRACT

ROLE OF NEUTROPHILIC INFLAMMATION IN OZONE-INDUCED EPITHELIAL
ALTERATIONS IN THE NASAL AIRWAYS OF RATS

By

Hye Youn Cho

Ozone (03) is a principal oxidant air pollutant in photochemical smog. More than
50% of the US. population live in regions where the atmospheric ozone concentrations
exceed the concurrent National Ambient Air Quality Standard for this pollutant.
Epithelial cells lining the centriacinar region of lung and the proximal aspects of nasal
passage are primary target sites for ozone-induced injury in laboratory animals. Acute
exposure of rats to high ambient concentrations of ozone (e.g., 0.5 ppm) results in
transient neutrophilic inflammation, epithelial proliferation and mucous cell metaplasia
(MCM) in the nasal transitional epithelium (NTE) lining the proximal nasal airways. The
principal purpose of the present study was to investigate the role of pre-metaplastic
cellular responses, especially neutrophilic inflammation, in the pathogenesis of ozone-
induced MCM in the NTE of rats. For this purpose, three specific hypotheses-based
whole-animal inhalation studies were conducted. Male F344/N rats were exposed in
whole-body inhalation chambers to 0 (filtered air) or 0.5 ppm ozone for l - 3 days (8
h/day). Histochemical, immunochemical, molecular and morphometric techniques were
used to investigate the ozone-induced cellular and molecular events in the NTE. In
addition, two in vitro studies, using explants of microdissected maxilloturbinates, were

also conducted to examine the effects of ozone-inducible cytokines (i.e., tumor necrosis

factor-alpha; TNF-a, and interleukin-6; IL-6) on mucin gene (rMuc-SAC) expression.
Ozone induced a rapid increase of rMuc-SAC mRNA in nasal tissues within hours after
the start of exposure. It preceded the appearance of MCM by 3 days, and persisted with
MCM for 2 days. Ozone-induced neutrophilic inflammation accompanied the mucin gene
upregulation at l, 2, and 3 days of exposure, but was resolved when MCM first appeared
in the NTE at 1 day after 3 days of exposure. Antibody-mediated depletion of circulating
neutrophils attenuated ozone-induced MCM, although it did not affect the ozone-induced
epithelial hyperplasia and mucin mRNA upregulation. In another study, it was found that
pre-existing neutrophilic rhinitis induced by endotoxin augmented the ozone-induced
MCM. However, pre-existing rhinitis did not alter the severity of ozone-induced
epithelial hyperplasia and mucin gene upregulation. Ozone also induced transient
increases in TNF-a and IL-6 mRNA expression concurrently with the initiation of mucin
mRN A upregulation in nasal tissues. In addition, exogenous TNF-a and IL-6 induced
increases in mucin mRNA levels in microdissected maxilloturbinates in vitro, in the
absence of neutrophils. In conclusion, ozone-induced MCM is, at least in part, neutrophil-
dependent. Though ozone alone is sufficient to induce epithelial proliferation and mucin
gene upregulation which are early NTE cell events prior to the development of MCM,
neutrophil-mediated inflammatory responses are essential for full phenotypic expression
of MCM. Pro-inflammatory cytokines (i. e., TNF-a and IL-6) may be putative mediators
of the ozone-induced upregulation of mucin mRNA in the NTE. The results from these
series of studies have provided a better understanding of important molecular and cellular

mechanisms involved in the pathogenesis of ozone-induced MCM in rat nasal airways.

To my parents
Jung S00 Cho and Moon Ja Lee
for their unconditional love and unending sacrifice

iv

ACKNOWLEDGMENTS

I would like to thank many people who have greatly influenced my intellectual and
personal development leading to this milestone. My first thanks go to Dr. Jack Harkema,
my research advisor, for his steadfast encouragement and financial support throughout the
graduate work. His masterful guidance allowed me the latitude and responsibility to
ultimately determine the right direction and scope of my research. The degree and
dissertation would have been beyond my reach without his patience and effort. I am
especially grateful to Dr. Robert Roth, my academic advisor, who strongly facilitated my
progression throughout the degree process. He gave generously of his stimulating ideas
and shared his expertise in my dissertation project. My sincere appreciation is extended
to Dr. Jon Hotchkiss, who provided me with excellent insights into every step and detail
of this project. It was a pleasure to have the benefit of his conscientious guidance. I
express my deep appreciation to Dr. Norbert Kaminski for his generous support and
advice at critical stages of the research, which made this thesis a better quality research
product. I am truly grateful to Dr. Patricia Ganey, who offered invaluable suggestions
and criticism but always had a word of encouragement during my graduate work. I will
never forget Dr. Jay Goodman who contributed to my intellectual growth and important
decisions fi'om the very start at Michigan State University. I wish to thank in particular
Dr. Byung Kak Kim, my advisor at Seoul National University, for his encouragement and
stimulation. I was particularly fortunate to meet and work with an extremely capable
research assistant, Ms. Catherine Bennett, who shared pleasure and adversity with me

throughout the years. I gratefully acknowledge her perfect and sincere assistance.

My time at Michigan State University was also highlighted by many friends who
shared of information, expertise and friendship throughout the years - especially, my
fellow graduate students, Xinguang Li and Rebecca Marcus, and senior students, Dr. Julia
Pearson, Kenneth Hentschel, Dr. Frederic Moulin, Amy Herring, Courtney Sulentic, Dr.
Rosie Sneed, and Dr. Jim Wagner at Department of Pharmacology & Toxicology, Dr.
Michelle Fannuchi in our laboratory, and Mi Young Jang at Department of Human
Nutrition. I extend my appreciation to Ms. Kathy Campbell and Ms. Amy Porter at
Clinical Center Laboratory, Mr. Ralph Common and Ms. Donna Craft at Department of
Pathology, and Mr. Bob Crawford and Ms. Therese Schmidt at Department of
Pharmacology & Toxicology for their cheerful technical assistance.

This dissertation is the end of results of my dad and mom who have inspired and
supported me - emotionally, intellectually, and financially - with love, faith and wisdom
in this and every other endeavor I have undertaken. They are the most difficult to thank,
as words are inadequate to convey the strength of my love and depth of my gratitude to
them. I am grateful to my mother-in law for her understanding and patience. I especially
thank my brother and sister-in law for love and encouragement they provided throughout
this process.

Last but most heartfelt appreciation must go to my husband, Min Chang. His
constant love, support, understanding, and burden-sharing have made my degree possible.
He has served as a steady presence during difficult times, and has gone an extra 10 miles
to help me even at busy times in his dissertation study. I owe my lovely 20 month-old
son, Justin (Seung Bin), for his patience with all the days that he has been staying far

apart from mom. He, himself, was the greatest encouragement to attain this goal.

vi

TABLE OF CONTENTS

LIST OF TABLES ................................................................................ ix

LIST OF FIGURES .............................................................................. x
CHAPTER 1

INTRODUCTION ............................................................................... 1

Ozone in the Atmosphere ................................................................... 2

Tropospheric Ozone ......................................................................... 3

Reactions of Ozone in Biologic Systems ................................................. 7

Human Health Effects of ozone ........................................................... 9

Field and Epidemiological Studies ............................................. 9

Controlled Human Exposure Studies ......................................... 10

Exacerbation of Respiratory Disease ......................................... 14

Factors Modifying Responsiveness to Ozone ............................... 15

Morphological Changes Induced by Ozone in Laboratory Animals ................. 16

Primary Sites of Injury in Respiratory Tract ................................ 16

Centriacinar Region of the Lung ............................................... 17

Nasal Airways and Mucous Cell Metaplasia ................................ 19

Mucus in Airways ........................................................................... 25

Functions of Mucus ............................................................. 25

Structure and Synthesis of Mucin .............................................. 27

Tissue Specificity and Multiplicity of Mucin Expression .................. 30

Mucin in Pathologic Conditions ............................................... 32

Regulation of Mucin Gene Expression ....................................... 33

Specific Aims of Thesis .................................................................... 35
CHAPTER 2

INFLAMMATORY AND EPITHELIAL RESPONSES DURING THE
DEVELOPMENT OF OZONE-INDUCED MUCOUS CELL

METAPLASIA IN THE NASAL EPITHELIUM OF RATS ............................... 40
Abstract ...................................................................................... 41
Introduction .................................................................................. 42
Materials and Methods ..................................................................... 45
Results ........................................................................................ 56
Discussion ................................................................................... 72

CHAPTER 3

NEUTROPHIL-DEPENDENT AND -INDEPENDENT ALTERATIONS

IN THE NASAL EPITHELIUM OF OZONE-EXPOSED RATS ........................ 79
Abstract ....................................................................................... 80
Introduction ................................................................................... 81

vii

Materials and Methods ..................................................................... 83

Results ........................................................................................ 90
Discussion .................................................................................. 106
CHAPTER 4
EFFECTS OF PRE-EXISTING RHINITIS ON THE OZONE-INDUCED
MUCOUS CELL METAPLASIA IN RAT NASAL EPITHELIUM ................... 112
Abstract ..................................................................................... 113
Introduction ................................................................................ 1 14
Materials and Methods .................................................................... 116
Results ...................................................................................... 121
Discussion ................................................................................... 135
CHAPTER 5
OZONE-INDUCIBLE CYTOKINES AND THEIR ROLE IN
MUCIN GENE EXPRESSION IN RAT NASAL TISSUES ............................. 139
Introduction ................................................................................. 140

Stuoy I .' T hue-Dependent Changes of TNF-a and IL-6 mRNA Levels
in Nasal Airways afier Single and Repeated Ozone Exposure . . . . . . ..l42

Materials and Methods .................................................................... 142
Results ....................................................................................... 144
Discussion .................................................................................. 147
Study 2: Efi'ects of Soluble TNF-a and IL-6 on Rat MUC-5AC mRNA
Expression ........................................................................... 149
Materials and Methods .................................................................... 149
Results ....................................................................................... 156
Discussion ................................................................................... 162
CHAPTER 6
SUMMARY AND CONCLUSIONS ........................................................ 164
REFERENCES .................................................................................. 171

viii

LIST OF TABLES

Table 3-1. Experimental groups and animal assignment ................................... 84

ix

Figure 1-1.

Figure 1-2.

Figure 1-3.

Figure 1-4.

Figure 1-5.

Figure 1-6.

Figure 2-1.

Figure 2-2.

Figure 2-3.

Figure 2-4.

Figure 2-5.

LIST OF FIGURES

Generation of tropospheric ozone in unpolluted and
polluted atmospheres ........................................................... 6

Reaction of ozone with unsaturated fatty acid in the
presence or absence of water .................................................. 8

Anatomic location of the main injury site in the nasal
airways of rats exposed to ozone ............................................ 22

The mucous (goblet) cells in airway epithelium
responsible for the synthesis, storage, and secretion of
mucous glycoproteins (mucins) .............................................. 26

Structure of secretory mucin monomer ..................................... 31

Hypothetical pathogenesis of ozone-induced mucous cell
metaplasia. ...................................................................... 36

Anatomic location of nasal tissues selected for morphometric
and RT-PCR analyses ........................................................ 48

Immunostaining of BrdU using avidin-biotin complex method to

determine cells undergoing DNA synthesis in the S-phase of
the cell cycle .................................................................... 51

Light photomicrographs of maxilloturbinates from rats killed

after 7-day exposure to O-ppm ozone (filtered air) (A), or

from rats killed 2 h afier 1-day exposure (B), 2 h after 3-day

exposure (C), or 4 days after 3-day exposure (D) to 0.5-ppm

ozone. Tissues were stained with H & E ................................... 61

Light photomicrographs of maxilloturbinates from rats killed
afier 7-day exposure to O-ppm ozone (filtered air) (A), or
from rats killed 2 h after 3-day exposure (B), or 4 days after 3-day

exposure (C) to 0.5-ppm ozone. Tissues were stained with
AB/PAS ......................................................................... 63

Time-dependent changes in the number of intraepithelial
neutrophils in the NTE ........................................................ 65

Figure 2-6.

Figure 2-7.

Figure 2-8.

Figure 2-9.

Figure 2-10.

Figure 2-11.

Figure 3-1.

Figure 3-2.

Figure 3-3.

Figure 3-4.

Figure 3-5.

Figure 3-6.

Time-dependent changes in the BrdU-labeling index (L1) in
the NTE .......................................................................... 66

Time-dependent changes in the epithelial cell numeric density
in the NTE ....................................................................... 67

Time-dependent changes in the amount of stored
mucosubstances in the NTE .................................................. 68

Time-dependent changes in the number of mucous cells in the
NTE .............................................................................. 69

Digitized image of an ethidium bromide-stained agarose
gel with representative RT-PCR cDNA bands for
rMuc-SAC and cyclophilin from each exposure group .................... 70

Time-dependent changes in the abundance of rMuc-SAC
mRNA in maxilloturbinates .................................................. 71

Effect of antiserum treatment on the number of circulating
blood neutrophils ............................................................... 95

Light photomicrographs of maxilloturbinates from rats treated
with control serum and killed 4 days after 3—day exposure to
0-ppm ozone (filtered air) (A), or rats treated with control
senun (B) or antiserum (C) and killed 4 days after 3 day-

exposure to 0.5 ppm ozone. Tissues were stained with
H & E ............................................................................. 96

Light photomicrographs of maxilloturbinates from rats treated
with control serum and killed 4 days afier 3-day exposure to
O-ppm ozone (filtered air) (A), or rats treated with control
serum (B) or antiserum (C) and killed 4 days after 3 day-

exposure to 0.5 ppm ozone. Tissues were stained with
AB/PAS .......................................................................... 98

Effect of antiserum treatment on the number of intraepithelial
neutrophils in the NTE ........................................................ 100

Effect of antiserum treatment on the BrdU-labeling index
(Ll, %) in theNTE ............................................................ 101

Effect of antiserum treatment on the epithelial cell numeric
density in the NTE ............................................................ 102

xi

Figure 3-7.

Figure 3-8.

Figure 3-9.

Figure 4-1.

Figure 4-2.

Figure 4-3.

Figure 4-4.

Figure 4-5.

Figure 4-6.

Figure 4-7.

Figure 4-8.

Effect of antiserum treatment on the amount of stored
intraepithelial mucosubstances in the NTE ................................. 103

Effect of antiserum treatment on the mucous cell numeric
density in the NTE ........................................................... 104

Effect of antiserum treatment on the ozone-induced
rMuc-SAC mRNA upregulation in maxilloturbinates .................... 105

Light photomicrographs of maxilloturbinates from rats

exposed to (A) saline and filtered air (0 ppm ozone), (B)

100-ug endotoxin and filtered air, (C) saline and 0.5-ppm

ozone, or (D) 100-ug endotoxin and 0.5-ppm ozone and

killed 2 h after the end of the 3-day inhalation exposure.

Tissues were stained with toluidin blue ..................................... 125

Light photomicrographs of maxilloturbinates from rats

exposed to (A) saline and filtered air (0 ppm ozone), (B)

lOO-ug endotoxin and filtered air, (C) saline and 0.5-ppm

ozone, or (D) 100-ug endotoxin and 0.5-ppm ozone and

killed 4 days after the end of the 3-day inhalation exposure.

Tissues were stained with AB/PAS .......................................... 127

Effect of pre-existing rhinitis on ozone-induced neutrophilic
influx in the NTE .............................................................. 129

Effect of pre-existing rhinitis on ozone-induced epithelial
hyperplasia in the NTE ....................................................... 130

Effect of pre—existing rhinitis on ozone-induced increase in
the stored mucosubstances in the NTE ..................................... 131

Effect of pre-existing rhinitis on ozone-induced increase in
the mucous cells in the NTE ................................................. 132

Digitized image of an ethidium bromide-stained agarose
gel with representative RT-PCR cDNA bands for
rMuc-SAC and cyclophilin from each exposure group

(n = 8/group) .................................................................... 133

Effect of pre-existing rhinitis on ozone-induced rMuc-SAC
mRNA upregulation in maxilloturbinates .................................. 134

xii

Figure 5-1.

Figure 5-2.

Figure 5-3.

Figure 5-4.

Figure s-s.

Figure 5-6.

Figure 5-7.

Figure 5-8.

Figure 5-9.

Figure 6-1.

Time-dependent changes in ozone-induced TNF-a mRN A
expression in maxilloturbinates ............................................. 145

Time-dependent changes in ozone-induced IL-6 mRN A
expression in maxilloturbinates ............................................. 146

Culture of microdissected nasal tissue ...................................... 150

Design of a rat Muc-SAC internal standard for use in a
quantitative RT-PCR assay to measure steady-state levels
of rMuc-AC mRNA .......................................................... 155

Digitized images of representative agarose gels and

standard plots for quantitation of rMuc-SAC mRNA

molecules in maxilloturbinates using a rMuc-SAC-specific

internal standard (IS) rcRNA ................................................ 157

Effects of TNF-a on rMuc-SAC mRNA expression in
explants of microdissected maxilloturbinates ............................. 158

Cytotoxicity of TNF-a to maxilloturbinate explants
indicated as % LDH release compared to vehicle-exposed
controls .......................................................................... 159

Effects of IL-6 on rMuc-SAC mRNA expression in
explants of microdissected maxilloturbinates ............................. 160

Cytotoxicity of IL-6 to maxilloturbinate explants
indicated as % LDH release compared to vehicle-exposed
controls ......................................................................... 161

Summarized diagram of results .............................................. 170

xiii

CHAPTER 1

Introduction

Ozone in the Atmosphere

Ozone (03) is a naturally occuning atmospheric gas composed of three atoms of
oxygen. It is found both in the stratosphere (atmosphere at altitudes between 20 and 50
km above the earth) and troposphere (atmosphere between the earth’s surface and the
tropopause, at about 10 to 18 km altitude) of the earth’s atmosphere. In the stratosphere,
concentration of ozone is high, normally 10 parts per million (ppm), compared to lower
concentrations in the troposphere, usually around 20 parts per billion (ppb). The high
concentrations of ozone in the stratosphere serve as a natural protective shield for filtering
out dangerous levels of ultraviolet radiation from the sun that could cause profound acute
and chronic injury to the skin (e. g., sunburn, skin cancer). At high altitudes, ozone is
generated by the photochemical reaction of a molecular oxygen with an oxygen atom
produced by photodissociation of molecular oxygen by deep ultraviolet radiation (U .8.
Environmental Protection Agency (EPA), 1996). Depletion of stratospheric ozone by
chlorofluorocarbons generated from refrigerators, solvents or other human activities
allows shorter wavelength ultraviolet radiation to be transmitted through the stratosphere
and into the troposphere. Therefore, it is currently a major environmental concern and an
area of active research. The problems associated with depletion of stratospheric ozone
should not, however, be confused with the health and environmental problems associated
with elevated levels of ozone in the troposphere where it is a major component of
photochemical smog. The primary focus of the present thesis pertains to the health
effects of tropospheric ozone and specifically to the potential adverse effects of ambient

levels of ozone on the airway epithelial cells lining the upper respiratory tract.

Tropospheric Ozone

Ozone is a highly reactive gas and the principal oxidant pollutant in photochemical
smog found in many urban centers throughout the world. It is currently one of the most
pervasive problems to human health among the major air pollutants, identified by the
US. EPA.

To protect human public health, a primary National Ambient Air Quality Standard
(NAAQS) for ozone was first designated under the Clean Air Act established by the EPA
and passed by the US. Congress in 1971. The recently revised NAAQS (July, 1997)
states that an ambient air quality monitoring site would be in compliance if the 3-year
average of the annual fourth-highest daily maximum 8—hour average ozone concentration
does not exceed 0.08 ppm (U .S. EPA, 1997).

It has been estimated by the US. EPA that in 1991, 69 million people in the United
States lived in areas that were not in compliance with NAAQS for ozone (U .S.EPA,
1991). The episodic high concentrations of ozone, especially during the summer in large
urban areas like Los Angeles, where the ambient ozone can reach concentrations as high
as 0.2 to 0.3 ppm, poses significant threats to the health of its inhabitants. Even higher
ambient levels of ozone have been reported in other metropolitan areas such as Mexico
City where 1-h maximum ozone concentrations are often twice as high as those reported

in Los Angeles (Calderon Garciduenas et al., 1992, 1995).

The naturally occurring tropospheric ozone is a key intermediate in the degradation of
volatile organic compounds (V OCs) emitted in the troposphere from biogenic sources.
Though ozone at low concentrations is an important factor in maintaining a clean
troposphere, at high concentrations, this irritating compound has the potential to
compromise human health. Tropospheric ozone generation is dependent on three
principal but complex processes : (l) the emission of nitrogen oxides (NO,) and VOCs
into the atmosphere from anthropogenic and natural sources, (2) the transport of these
emissions and their reaction products, and (3) the chemical reactions occurring in the
ambient air concurrent with the transport and the emissions (U .S.EPA, 1996).

The principal chemical reaction producing atmospheric ozone in the troposphere as
well as in the stratosphere is that between atomic and molecular oxygen (Seinfeld, 1989).

02+O+M—>O3+M (1)
where M is any third body (e.g., N2) that removes the energy of the reaction and stabilizes
ozone. In the troposphere, the oxygen atoms are produced by photodissociation of
nitrogen dioxide (N 02):

N02 + hv—> NO + O (2)
where the photon (hv) has a wavelength between 280 and 430 nm. The nitric oxide (NO)
produced in this reaction reacts rapidly with ozone to regenerate N02:

NO + 03 —) NO2 + 02 (3)

The above three reactions occur rapidly, establishing a steady-state ozone concentration

according to the photostationary state relation:

JIN02]
k [N0]

 

[03 I = (4)

where k is the rate constant for reaction 3 and JlNOz] is the photolysis rate of NO2
(reaction 2). Therefore, at a ratio of [NOz]/[NO] = 1, [O3 ] predicted by the
photostationary state at solar noon in US. latitudes is about 20 ppb by volume. Ozone
concentrations in unpolluted tropospheric air vary between 20 and 50 ppb. However, in
some polluted urban areas, levels as high as 500 ppb (0.5 ppm) are reported (Calderon
Garciduenas et al., 1992, 1995).

In polluted urban centers, the steady-state level of ozone is disturbed by the heavy
emissions of VOCs as sources of hydrocarbons (RH, where R is an alkyl group) derived
mainly from motor vehicles and fuels (Peden et al., 1995). Oxidation of hydrocarbon
molecules by hydroxyl radicals produce peroxyradicals (R02) that react with NO to form
N02. Therefore, the one ozone molecule needed to convert NO to NO2 (reaction 3) is no

longer needed in the polluted atmosphere, and this results in a build up of ozone in the

ambient air. The net process is :

 

no2 + NO —) No2 + R0 (5)
NO2 + hv —> NO + o (6)
o+o,+M—> 03 + M (7)

Net: R0, + 02 + hv - R0 + 03 (8)

Conclusively, the rate of ozone generation is related closely to the rate of R02 production

in polluted urban areas. The overall process is summarized in Figure 1-1.

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Noz +0: /. oz 0
o + NoiVmc K +

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Reaction of Ozone in Biologic Systems

Ozone is a potent oxidant. In biological systems, it can react with a variety of
macromolecules that are susceptible to electrophilic attack. These include biomolecules
containing thiol or amine groups (e.g., proteins) or unsaturated carbon-carbon bonds (e. g.,
unsaturated fatty acids) (Peden et al., 1995; Pryor, 1992; U.S.EPA, 1996). Because it is
such a highly reactive molecule, ozone cannot penetrate the airway lining fluids (mucus
and surfactant) and the apical membrane of the surface epithelial cells without reacting
with biomolecules present in this air/tissue boundary. Though ozone can directly react
with these target molecules, the major effects of ozone in airway tissues must be exerted
through toxic products generated from these reactions (Pryor, 1992).

Ozone reaction (ozonolysis) products formed in the body by interaction with lipids or
proteins, etc., are a complex array of compounds. Unsaturated fatty acids in the
membrane lipid bilayers or in the fluids are known as primary targets for reactions with
inhaled ozone. Criegee has proposed that a transient intermediate (carbonyl oxide) is
formed during early stage of ozonolysis with the unsaturated fatty acids and that
subsequent reactions of this compound determined the final products, depending on the
presence or absence of water (Criegee, 1975; Pryor and Church, 1991). In a lipophilic
environment, Criegee ozonation leads to the production of ozonides. In the aqueous
environment, the carbonyl oxide intermediate generates an aldehyde and a
hydroxyhydroperoxide compound, which splits out a hydrogen peroxide to form a second

molecule of aldehyde. Figure 1-2 summarizes the chemical reactions of ozone with

unsaturated fatty acids.

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The ozonolysis products are more stable than ozone itself and able to diffuse further
into tissue. These secondary products have the potential to injure resident lung cells such
as macrophages and epithelial cells, and could produce tertiary product molecules. Each
level of products that is formed in this cascade would have its own characteristic types of
biological activity, and relay the effects of ozone increasingly fiirther from the air/tissue
barrier potentially resulting in lung damage, inflammation, and changes in host defense

capability (Leikauf et al. , 1995; Peden et al., 1995).

Human Health Effects of Ozone

Field and Epidemiological Studies

Ozone concentrations at ground level have a wide range of potential adverse effects
in respiratory airways of exposed human populations. Recent epidemiological studies
addressing the acute effects of ambient ozone have yielded significant associations with
health outcomes, including decreases in lung function, aggravation of pre-existing
respiratory disease, and increases in daily hospital admissions especially during the most
polluted days of the summer (Bumet et al., 1994; Higgins et al., 1990; Spektor et al.,
1988, 1991). In most of these studies, the responses of healthy children are similar to
those seen in adults.

A number of studies have also addressed the chronic effects of ambient ozone
concentrations on the respiratory system. Recent studies of the human health effects in

highly air polluted regions of Mexico City have reported significant nasal pathological

changes in the residents. Nasal biopsies from these people often exhibit a wide range of
histopathological alterations including marked decreases in the number of ciliated cells,
basal cell hyperplasia, squamous cell metaplasia, epithelial dysplasia, submucosal gland
proliferation, and mild-to-moderate chronic inflammatory cell infiltration (Calderon
Garciduenas et al., 1992, 1995). In epidemiological studies, it is hard to disentangle the
effects of ozone from those of other air pollutants (e.g., acid aerosols, particulate matter).
Therefore, data from these studies are not enough to definitely conclude that chronic

ozone exposure causes significant long-terrn changes to the human respiratory tract.

Controlled Human Exposure Studies

0 Physiological Responses to Ozone Exposure

The physiological responses induced by short-term ozone exposure are separated into
respiratory symptoms (e.g., cough, dyspnea), measured lung function responses (e.g.,
changes in lung volume or airway resistance), and airway responsiveness. In healthy
human subjects, the pulmonary responses induced by short-term inhalation exposure to
controlled ambient concentrations of ozone (i.e., _<_ 0.3 ppm) consist of decreased
inspiratory capacity, mild bronchoconstriction, rapid shallow breathing pattern
(ozone-induced tachypnea) during exercise, and accompanying symptoms of cough,
airway irritation, and chest discomfort associated with deep inspiration (Avol et al. , 1983;

F olinsbee et al., 1994; Gong, Jr. et al., 1986). Many studies have addressed decrements

10

in forced expiratory volume (F EV), which reflects decrements in forced vital capacity
(F VC) and increases in central and peripheral airway resistance (RN) in the lungs.
Neurogenic inhibition of maximal inspiration by stimulation of C-fiber afferents, either
directly or from ozone-induced inflammatory products, is believed to be a possible
mechanism leading to the observed pulmonary responses. The responses of healthy
children to acute ozone exposure are similar, in most studies, to those seen in adults.
Exposure to acute higher concentrations of ozone (0.3 - 0.5 ppm, 2 - 3 h) in exercising or
resting healthy adult subjects also induced decrements in the FEV and flows as well as
increases in airway responsiveness (Folinsbee et al., 1978; Horvath et al., 1979). The
ozone concentration appears to make greater impact on the pulmonary function
responses, while mean ventilation and exposure duration serve as secondary determinants
of the response at any given ozone concentration (Adams et al. , 1981). Similar responses
have been seen with prolonged exposures (4 - 8 h) to lower concentrations of ozone (0.08
- 0.16 ppm) (Hazucha et al., 1992). A rapid recovery or attenuation of ozone-induced
spirometry (i. e., changes in lung volume) and symptom responses followed the repeated
exposure (Hazucha et al., 1992; Horvath et al., 1979). Significant reduction in exercise
performance has been also observed in athletes exposed to ozone while they perform high
intensity exercise (Schelegle and Adams, 1986; Spektor et al., 1988).

In addition to functional responses, ozone exposure causes airway
hyperresponsiveness as demonstrated by an increased physiological response to
nonspecific subsequent stimuli such as SO2 or specific allergens (Golden et al., 1978). It
suggests that ozone-exposed airways are predisposed to narrowing of respiratory airways

after secondary exposure to a variety of stimuli (Spektor et al. , 1988). Changes in airway

ll

responsiveness appear to be resolved but more slowly than are changes in the FEV
(Folinsbee et al., 1984). The mechanism underlying the increases of airway
responsiveness is only partially understood. Epithelial damages may direct the access of
inflammatory mediators (e.g., cytokines, eicosanoids or neuropeptides) to the smooth
muscle in airways and result in the increases of airway responsiveness (Hazucha et al.,

1996; Kleeberger and Hudak, 1992; O'Byme et al., 1984).

o Inflammatory Responses to Ozone Exposure

The physiological responses to ozone are accompanied by cellular and biochemical
changes in the airways. Short-terrn exposure to ozone causes acute inflammatory changes
throughout the respiratory tract, including the nose. A number of studies have analyzed
bronchoalveolar lavage fluid of humans exposed to a single acute ozone (0.2 - 0.6 ppm, 1
- 4 h), which has been used as a useful tool to assess its constitutive elements (e.g., cells,
proteins) and the extent and course of inflammation in the lung (Aris et al., 1993; Devlin
et al., 1995; Kehrl et al., 1987; Koren et al., 1989a,b; McGee et al., 1990; Schelegle et
al., 1991; Seltzer et al., 1986). The analyses of lavage have indicated increases in
inflammatory cells (e.g., neutrophils), epithelial cell damage, altered epithelial
permeability (i. e., increased serum proteins such as albumin) and production of
proinflammatory cytokines including tumor necrosis factor-alpha (TNF-a), interleukin-
lbeta (IL-113), interleukin-6 (IL-6), as well as eicosanoids (e.g., prostaglandin E2). These

responses are detectable as early as 1 h after exposure. The 4 - S-fold increases in the

12

number of neutrophils by ozone exposure (0.4 - 0.6 ppm) equal or exceed those found in
the bronchoalveolar lavage fluid from individuals exposed to other airway irritants (e. g.,
asbestos or silica) or from individuals with airway disorders such as pulmonary fibrosis
or connective tissue disorders (Chemiak et al. , 1990). The increased levels of neutrophils
and mediators in the lavage fluid persist at least for 18 h after the end of exposure (Koren
et al., 1989a,b). The persistent presence of these mediators suggests that they play an
important role in resolving inflammation and injury. The time-response profiles vary for
different mediators and inflammatory cells (Koren et al., 1991; Schelegle et al., 1991).
Another study indicates that these inflammatory responses can occur after acute exposure
to lower ambient concentrations of ozone (0.08 - 0.1 ppm, 6.6 h) (Devlin et al., 1991).

Acute ozone exposure causes inflammation and increased permeability even in the
nasal passages as indicated by increased levels of neutrophil and albumin in nasal lavage
fluid (Bascom et al., 1990; Graham et al., 1988; Graham and Koren, 1990; Henderson et
al., 1988; McBride et al., 1994). A recent study in children has presented evidence of a
possible relationship between nasal inflammation and measured ambient ozone
concentrations (Calderon Garciduenas et al., 1995).

In accordance with clinical data, in vitro ozone exposure studies suggest that
pulmonary epithelial cells can directly respond to inhaled ozone. Ozone induces
production of many mediators (e.g., IL-lB, IL-6, TNF-a, IL-8, prostaglandins) from the
epithelial cells as well as from inflammatory cells in culture (e.g., macrophages,

neutrophils) (Beck et al., 1994; Devlin et al. , 1994; McKinnon et al., 1993).

13

Exacerbation of Respiratory Disease

People with pre—existing pulmonary disease may be at increased risk from ozone
exposure. Because of their existing functional limitations, any further decrease in
function would lead to a greater overall functional decline. Furthermore, some
individuals with pulmonary disease may have an inherently greater sensitivity to ozone.
Therefore, studies of the subpopulation with pre-existing impediments in pulmonary
fimction and exercise capacity are of primary concern in evaluating the health effect of
ozone.

Asthmatics, by definition, have inherently increased bronchial responsiveness to
inhaled irritants. People with mild to moderate asthma are sensitive to ozone exposure
causing further increases (2 - 4-fold) in airway responsiveness (Kreit et al., 1989). In
addition, asthmatics exposed to ozone have greater changes in airway resistance and
expiratory flow, while they tend to have similar changes in volume-related responses and
in symptom responses (e.g., cough and short breath) compared to non-exposed asthmatics
(Kreit et al., 1989). McBride et al. (1994) have exposed asthmatics with histories of
allergic rhinitis to ozone (0.24 ppm, 90 min), and observed significant increases in the
numbers of neutrophils and epithelial cells in nasal lavage fluid from the asthmatics
compared to those in healthy subjects. This suggests that the upper airways of asthmatic
individuals are more sensitive to the acute inflammatory effects of ozone than those of
non-asthmatic, healthy subjects. These observations represent a plausible link between
elevated ambient ozone concentration during summer and increased hospital admissions

for asthmatics. A number of epidemiological studies have shown a consistent

l4

relationship between ambient ozone exposure and acute respiratory morbidity in this
population (Krzyzanowski et al., 1992; Lebowitz et al., 1991; Thurston et al., 1997).
Especially in children, small decreases in FEV and increases in respiratory symptoms,
including exacerbation of asthma, occur with increasing ambient ozone concentration
(Koenig et al., 1985).

Increased airway responsiveness to ozone is also reported in subjects with allergic
rhinitis (who do not have asthma-like symptoms) but to a lesser degree than that observed
in asthmatics (McDonnell et al., 1987). Patients with mild to moderate chronic
obstructive pulmonary disease have also shown an alteration in pulmonary responses
characterized by decreases in pulmonary functions (e.g., FEV) after ozone exposure (0.12

- 0.41 ppm, 1 - 3 h) at rest or with exercise (Kehrl et al., 1985).

Factors Modifying Responsiveness to Ozone

Factors such as smoking status, age, gender, race, season, and mode of breathing
during exposure can also influence the airway responses to ozone (U .S.EPA, 1997).
None of these potential influences on the ozone responsiveness has, however, been
thoroughly investigated and adequately addressed in clinical studies to date. The
observations that healthy older adults appear to be less responsive to ozone than young

adults (McDonnell et al., 1993), however, has been confirmed to the point that it can be

considered in risk assessment.

15

Morphological Changes Induced by Ozone in Laboratory Animals

Primary Sites of Injury in the Respiratoty Tract

The respiratory tracts in mammalian species are lined by several morphologically
distinct epithelial cells (Harkema, 1992; Harkema et al., 1991; Plopper et al., 1983). The
epithelial morphology and cell composition vary depending on anatomic regions
examined (e.g., nasal airways versus bronchiolar airways). Due to their luminal location,
airway epithelial cells form a barrier to the external environment. Therefore, they are the
first point of contact for inhaled antigens, particulates or other xenobiotics including
ozone, many of which have the potential to cause epithelial cell injury.

Previous studies have documented the injurious effects of ozone on epithelial cells
lining the upper and lower respiratory airways in various laboratory animals (Boorman et
al., 1980; Plopper et al., 1979). Regional dosirnetry and tissue sensitivity are critical
factors that determine the distribution of the epithelial lesions caused by ozone exposure
in laboratory animals (Kimbell et al., 1993). In these experimental animal models, some
of the largest effective doses of inhaled ozone are known to be delivered to two airway
regions, the nose and the centriacinar pulmonary region (i. e., the junction of conducting
airway and gas exchange region of the lung) (Miller et al., 1985; Oveiton et al., 1987). In
addition, the epithelial cells lining these two district airway regions are believed to be
most vulnerable to the toxic effects of ozone (Boorman et al., 1980; Harkema et al.,

1987; Plopper et al. , 1979).

16

Centriacinar Region of the Lung

The centriacinar region of the lung, the junction of conducting airway and gas
exchange region, is one of the most susceptible airway sites to ozone toxicity. All
mammalian species studied react to inhaled ozone (5 1.0 ppm) in a similar manner, with
species variation in morphological responses depending, in part, on the basic structure of
this pulmonary region (cg, presence or absence of the respiratory bronchioles, the last
conducting airways) and the distribution of sensitive cells.

It has been demonstrated that the intensity of the toxicant—induced lesions in
centriacinar region is directly related to the acinar volume (Mercer et al., 1991). Acini,
the basic structural unit of lung, consist of a terminal bronchus, respiratory bronchioles
(when present), alveolar ducts and alveoli. The acini that do not have respiratory
bronchioles have a smaller volume than those that do contain these alveolarized
bronchioles. In most of the smaller and some of the larger species (e.g., horse, ox, sheep,
pig, rabbit, guinea pig, hamster, rat and mouse), there is a single very short, or absent,
alveolarized bronchiole that directly joins alveolar ducts (i. e., respiratory bronchioles)
(Tyler et al., 1991). In all of these species, the epithelial population is simple cuboidal,
with approximately equal numbers of ciliated and nonciliated or Clara cells (Plopper et
al., 1983, 1989). Humans and a number of species including monkeys, dogs and cats
have centriacinar pulmonary regions characterized by several generations of respiratory
bronchioles. Respiratory bronchioles in these have the luminal surface lined by epithelial
populations characteristic of more proximal conducting airways, i. e., simple cuboidal

epithelium, interrupted by outpoketings lined by Type 1 and Type 2 pneumocytes

l7

characteristic of alveoli in the gas exchange area. Though the precise mechanisms have
not been elucidated, the differences in the basic morphology of centriacinar region and
size of the acinus seem to contribute to the greater responses of nonhuman primates to
ozone (e.g., increases in epithelial thickness and number of cells) compared to those of
rodents (Barry et al., 1985; Harkema et al., 1993; Plopper et al., 1991).

Ozone-induced epithelial degenerative changes in the centriacinar regions occur soon
after exposure. The epithelial cells most damaged by acute or chronic ozone exposure are
ciliated cells and Type I cells (Pino et al., 1992). Both of these cell types have very large
surface areas exposed to inhaled gases relative to their cell volume (U.S.EPA, 1996).
Loss of cilia and necrosis are characteristic features of injury in ciliated cells after acute
ozone exposure (Castleman et al., 1980; Stephens et al., 1974). The type 1 pneumocytes
undergo vacuolization, necrosis, and exfoliation leaving bare areas of basement
membrane and resistant type 2 pneumocytes after ozone exposure (Stephens et al., 1974).
Type 2 cells proliferate and some differentiate into Type 1 cells during the repair process
(Evans et al., 1975; Barry et al., 1983). These epithelial changes are accompanied by an
acute inflammatory response characterized by increased numbers of neutrophils and
alveolar macrophages in the affected centriacinar regions along with hyperemia and
interstitial edema, and fibrinous exudate (Boorman et al., 1980; Evans et al., 1975;
Fujinaka et al., 1985; Stephens et al., 1974). Repeated, chronic exposures to ozone cause
alveolar septal thickening due to (1) increased matrix (i.e., proliferation of fibroblasts and
accumulation of collagen) and (2) thickened alveolar epithelium by proliferation of Type

2 cells (Boorman etal., 1980; Fujinaka et al. , 1985).

18

Several studies have examined the postexposure period (“recovery”) following acute
or chronic ozone exposure. Plopper et. al. (1978) reported that the epithelial cells in the
centriacinar region of rats returned to normal appearance 6 days afier 72 h of exposure,
while incomplete resolution has also been reported in various animals species (Ibrahim et

al., 1979; Moore and Schwartz, 1981).

Nasal Airways and Mucous Cell Metaplasia

The nose conditions inhaled air and serves as an important defense mechanism
(“scrubbing tower”) in the upper respiratory tract against many inhaled pollutants (Eccles,
1982; Geurkink, 1983). The “scrubbing” process reduces inhaled airway concentration of
ozone and protects the lower respiratory tract from injurious levels of ozone. Since the
nose is the port of entry for the respiratory tract, it receives a large dose of inhaled
pollutants. It is, therefore, vulnerable to epithelial cell injury caused by a wide range of
irritating airborne xenobiotics including ozone, formaldehyde, chlorine and cigarette
smoke, and infectious microbial agents (U .S.EPA, 1996).

There is a large range of variation in the structure of the nasal cavity among the
laboratory animals as well as between these animal species and humans (Schreider and
Raabe, 1981). The complexity of the nasal cavity may affect the inter-species differences
in morphological responses to ozone. There is a striking similarity in the nasopharyngeal
cavity between nonhuman primates (e. g., macaque monkey) and humans (Schreider and
Raabe, 1981). Therefore, studies of the aerosol and gas deposition in this region of the

monkey could provide useful information for extrapolation to humans.

19

Besides differences in architecture of the nose among species, there are also species
differences in the distribution of nasal epithelial populations and the type of nasal cells
within these populations. There are, however, four distinct nasal epithelial populations in
most laboratory animal species (Harkema and Hotchkiss, 1994). They include (1) a
stratified squamous epithelium, which is primarily restricted to the nasal vestibule, (2)
ciliated, pseudostratified respiratory epithelitun in the main nasal chamber and
nasopharynx, which consists of six morphologically distinct cell types including
numerous mucous (goblet) and ciliated cells, and overlies approximately 46 % of nasal
cavity in F344 rats, (3) nonciliated or poorly ciliated transitional epithelium with 1 - 2
cells in thickness, lying between squamous epithelium and respiratory epithelium in the
anterior or proximal aspect of the main chamber and consisting of cuboidal, columnar and
basal cells with few mucus-producing goblet cells, and (4) the olfactory nerve epithelitun
located in the dorsal or dorsoposterior aspect of the nasal airways, essential for the sense
of smell.

In various laboratory animals, it has been recognized that considerable amounts of
inhaled ozone (40 - 70 %) can be absorbed by nasal tissues (Miller et al., 1979;
Yokoyama and Frank, 1972). Nasal epithelial cells are known to be principal targets for
ozone-induced injury in the upper airways of both rats and monkeys.

The pathologic effects of ozone on nasal airways of macaque monkeys have been
described by Harkema et al. (1987a,b). Monkeys exposed to acute or chronic ozone
exposures (0.15 or 0.3 ppm, 8 h/day for 6 or 90 days) developed epithelial alterations in
the proximal nasal cavity. The lesions were observed mainly in the nasal transitional

epithelium (NT E) as well as in the respiratory epithelium lining the anterior nasal

20

airways. Nasal epithelial lesions were characterized by neutrophilic inflammation, loss of
ciliated cells, epithelial hyperplasia, and marked increases of mucous cells and the
amount of intraepithelial mucosubstances. An appearance of mucous cells in an
epithelium that is normally devoid of the mucous secretory cells (e.g., NTE) is referred to
as mucous cell metaplasia (MCM), while an increase in mucous cells in a respiratory
epithelium that normally contains some of these secretory cells (e.g., respiratory
epithelium) is referred to as mucous cell hyperplasia.

MCM is the principal pathologic change that occurs in the NTE of F 344/N rats as
well as bonnet monkeys exposed to repeated ozone. Exposure of rats to repeated acute
ozone (0.4 - 0.8 ppm, 6 - 8 h/day for 5 7 days) results in MCM accompanied by epithelial
hyperplasia in the NTE (Henderson et al., 1993; Hotchkiss et al., 1991; Reuzel et al.,
1990). The nasal lesions including MCM are restricted to the NTE lining the lateral
meatus (i.e., maxilloturbinates, lateral sides of nasoturbinates, and lateral walls) in the
proximal aspect of the nasal cavity (Figure 1-3). Acute responses of NTE to ozone
include epithelial cell death, epithelial cell exfoliation, and neutrophilic influx (Harkema
et al., 1989; Hotchkiss and Harkema, 1992; Hotchkiss et al., 1997). NTE cells respond
rapidly with DNA synthesis and subsequent cell proliferation prior to the development of
MCM (Hotchkiss et al., 1997).

Long-term exposures of rats to ozone (0.5 or 1 ppm for 20 months, or 0.25 ppm for 2
years) have more severe and extensive metaplastic and hyperplastic lesions in the nasal
airways (Harkema et al., 1994; Smiler et al., 1988). MCM has also been described in the
distal pulmonary airways of rats chronically exposed to high concentrations of ozone (20

months, 1.0 ppm). The MCM in the pulmonary airways, however, was markedly less

21

Figure 1-3. Anatomic location of the main injury site in the nasal airways of
rats exposed to ozone. (A) Exposed lateral wall of nasal airway.
MT = maxilloturbinate; NT = nasoturbinate; ET = ethmoturbinate;
HP = hard plate; NP = nasopharynx. (B) Anterior face of tissue
block from proximal nasal airway. Gray area = nasal tissues
including septum (S), MT, NT, and HP; Black area = nasal
passage of lateral meatus lined by nasal transitional epithelium
(NTE). (C) Digitized image of the MT in this tissue section. NTE
lines the luminal surface of the turbinate. TB = turbinate bone; LP
= lamina propria containing large blood vessels.

22

 

23

severe than that in the NTE of these same rodents (Plopper et al., 1994). No ozone-
induced changes have been found in the squamous or olfactory epithelium of rat nasal
airways. MCM induced by chronic exposure to ozone (0.5 ppm, for 13 wks) persists for
weeks and even months afier the end of the exposure, depending on the severity of the
initial metaplastic lesions (Harkema et al., 1997). Considering that airway mucus is an
efficient anti-oxidant (Cross et al., 1984), it seems likely that development and
persistence of the MCM is a protective adaptation of the initially injured nasal epithelium
to prevent further damage by ozone.

In addition to ozone, many other inhaled toxicants such as bacterial endotoxin
(Gordon et al., 1996; Harkema et al., 1993), sulfur dioxide (Jany et al., 1991), tobacco
smoke (Lamb and Reid, 1969), acrolein (Borchers and Leikauf, 1997), chlorine (Wolf et
al., 1995), siloxane (Burns-Naas et al., 1998), 3-methylcholanthrene (Rehm and Kelloff,
1991), and machining fluids (Gordon and Harkema, 1995) can induce MCM in airway
epithelium of rats. However, little is known about the specific cellular and molecular
mechanisms underlying the toxicant-induced MCM in the airway epithelium. Recent
studies have investigated the association of inflammatory mediators with the abnormal
increases in the production or secretion of airway mucins in laboratory animals.
Neutrophil elastase is a well-known mucous secretagogue and induces MCM in rodent
airways (Breuer et al., 1985, 1993; Sommerhoff et al., 1990; Jamil et al., 1997; Kim et
al., 1987). Both soluble TNF-ci and IL-6 induce mucin hypersecretion in airway

epithelial cells in vitro at concentrations that also cause mucin gene upregulation (Levine

24

et al., 1994, 1995). Transgenic mice that overexpress IL-4 or IL-5 have MCM (Rankin et
al. , 1996; Jain-Vora et al., 1997; Lee et al. , 1997) and mucin hypersecretion (McBride et

al., 1994) with mucin gene upregulation in tracheobronchial or pulmonary airways.
However, the precise roles of these inflammatory mediators in the mucous

overproduction or hypersecretion are still not clarified.

Mucus in Airways

Function of Mucus

The luminal surfaces of the respiratory, gastrointestinal and reproductive tracts of
mammals is covered by a protective mucous layer that is produced and secreted by
mucous goblet cells (Figure 1-4) in the surface epithelium and submucosal glands.
Mucus is a complex mixture of large glycoproteins (mucins), water, electrolytes, protein,
lipid, DNA, and various xenobiotic materials including bacteria and bacterial products
(Boat et al., 1976a,b). The mucus hydrates and lubricates the epithelium lining the
respiratory tracts from the nasal passages to the respiratory bronchioles in mammals. It
serves as an important physical barrier to protect airway tissues against airborne
toxicants. Foreign substances entrapped in the luminal mucous layer are constantly
removed from the airway by ciliary beating, a process called mucociliary clearance

(Rose, 1992; Van Klinken et al., 1995).

25

 

Secreted
Mucus

Cilia ted Cell

Stored Mucins
(Glycoproteins)

Sero us Cell

  

\ Basal Cell

Mucin mRNA

Mucous (Goblet) Cell

Figure 1-4. The mucous (goblet) cell in airway epithelium responsible
for the synthesis, storage, and secretion of mucous
glycoproteins (mucins)

26

Structure and Synthesis of Mucin

The major macromolecular components of mucus are mucins, which are large
heterogeneous, high-molecular-weight glycoproteins (200 - 15,000 KD). Due to their
large size, high degree of glycosylation, and frequent contamination with non-covalently
associated lipids and peptides (Rose, 1992; Slayter et al., 1984), analysis of the primary
structure of mucin glycoproteins has been difficult using traditional biochemical and
biophysical techniques. Recently, a breakthrough was made by the derivation of
antibodies directly to the deglycosylated protein core of mucins by
trifluoromethanesulfonic acid- (Edge et al., 1981) or HF-mediated (Shekels et al., 1995)
deglycosylation processes. The development of mucin antibodies has led to marked
increases in the knowledge of the core protein structures as well as mucin genes by cDNA
cloning and sequencing (Perini et al., 1989). To date, nine human mucin genes and
encoded mucin proteins (MUC-l, MUC-2, MUC-3, MUC-4, MUC-SAC, MUC-SB,
MUC-6, MUC-7 and MUC-8) have been reported (Gendler and Spicer, 1995; Gum, Jr.,
1992; Van Klinken et al., 1995), and their homologues have been identified in various
animals including rodents (Inatomi et al., 1997; Ohmori et al., 1994; Randell et al., 1996;
Shekels et al., 1995). By convention, human mucin genes are designated by MUC,
mouse by Muc, and rat by rMuc (Gendler and Spicer, 1995).

Mucins exist as a secretory or a membrane-associated form (Gum, Jr., 1992; Rose,
1992). MUC-l is the only membrane-associated mucin, identified to date, containing

hydrophobic membrane-spanning domains. It is ubiquitously and aberrantly expressed

27

by various carcinomas, which makes MUC-l an important marker in malignancy (Braga
et al., 1992; Dahiya et al. , 1993; Gum, Jr., 1992).

The secretory mucins (except MUC-7) contain two distinct domains : (1) a highly
glycosylated core protein region (apomucin), and (2) naked hydrophobic protein flanking
region. The central protein core of each mucin molecule contains extended arrays of
conserved tandemly repeated peptide sequences (~ 20 amino acid repeat units), which
vary between different mucin gene products (Crepin et al., 1990). This apomucin core is
rich in serine, threonine and proline. The mucin protein backbone is assembled from the
mucin mRNA templates in the rough endoplasmic reticulum (Rose, 1992). As newly

synthesized mucin protein moves through the cell from the rough endoplasmic reticulum
toward the Golgi apparatus, branched oligosaccharide side chains with 2 - 22 sugars per
chain are added to serine or threonine residues in the protein core via O-glycosylation (or
rarely N-glycosylation), resulting in 60 - 90 % of mucin mass derived from these
carbohydrate side chains. The sugar constituents are N-acctyl-glucosamine, N-
acetylgalactosamine, galactose, fucose, and sialic acids. N-acetylgalactosaminc is always
the initial sugar unit of the oligosaccharide chains transferred to serine or threonine in the
mucin core by N-acetylgalactosaminetransferase in the rough endoplasmic reticulum
(Rose, 1992). Sequential stepwise O-glycosylation by specific glycosyltransferases leads
to formation of core-type structures, which are elongated to completed oligosaccharides.
The glycoproteins are then further glycosylated within the Golgi apparatus (specifically
in the trans-Golgi compartment) by adding terminal sugar residues such as sialic acids
and galactose, or sulfates, which results in the formation of negatively charged mucin

glycoproteins (Bennett and Wild, 1991). The extremely diverse composition of sugars

28

and the degree of sulfation of the oligosaccharide side chains contribute to the inherent
heterogeneity of mucin. The high proline contents in the protein core may help to
maintain a particular conformation of the apomucin for the close packing of the large
carbohydrate side chains.

The 5’- (amino-terminal) and 3’- (carboxy-terrninal) flanking regions of mucin
protein core contain unique non-repetitive, cysteine-rich sequences. They participate in
polymerization of mucin monomers via disulfide bonds to form a high-molecular-weight
mucin complex. The timing and process of assembly of mucin subunits into polydispersc
macromolecules are not well defined. It has been believed that mucin oligomerization
may occur in the endoplasmic reticulum prior to the elongation of oligosaccharide side
chains (Dekker and Strous, 1990; McCool et al., 1994). Some core O-glycosylation with
the initial sugar unit (i. e., N-acetylgalactosamine) seems to precede the mucin
oligomerization to stabilize the extended conformation of mucin peptide so that N- and
C-terminal domains are kept well separated. Another line of recent studies has
demonstrated that mucin oligomerization takes place downstream to the trans-Golgi
compartment with fully glycosylated mucin subunits (Sheehan et al. , 1996).

The matured mucous glycoproteins of up to at least decamers are stored in secretory
granules (for days in human cells), and then released via periodic exocytosis from the
apical surface (Lundgren and Shelhamer, 1990). Once secreted, mucin molecules form a
gel via hydrophilic non-covalent bonds between the oligosaccharide of the mucin
oligomers to maintain mucous barrier (Strous and Dekkcr, 1992). MUC-7, a salivary

mucin, lacks both a membrane-spanning domain and the cysteine-rich regions, and is the

29

only soluble secreted mucin identified to date. Figure 1-5 illustrates a hypothetical

structure of the secretory mucin monomer.

Tissue-Specificity and Multiplicity of M ucin Expression

Molecular analyses have indicated that expression of secretory mucin is relatively
tissue- and cell-specific (Audie et al., 1993; Bobek et al., 1996; Ho et al., 1995; Keates et
al., 1997; Shankar et al., 1997, Shekels et al., 1998). In brief, MUC-2 is expressed in the
small intestine, colon, and tracheobronchial tissue, and MU C-3 is primarily expressed in
small intestine and colon, and gallbladder. MUC-4 is observed primarily in colon and
bronchial tissue. MUC-SAC is found in tracheobronchial, gastric tissues, gallbladder and
cndocervix, and MUC-SB is also found in gallbladder, tracheobronchial tissue and
endocervix. MUG-6 is primarily noted in stomach, gallbladder, cndocervix, seminal
vesicles, pancreas and Brunner’s glands. MUC-7 was isolated from salivary glands and
MUC-8 encodes a tracheobronchial mucin. However, complete organ distribution studies
of these mucins have not been completed.

There are organs such as the lung in which multiple mucin genes or gene products
are co-expressed (Aubert et al., 1991; Guzman et al., 1996; Reid et al., 1997; Voynow
and Rose, 1994). As indicated above, at least five different mucin genes (MUC-2, MUC-
4, MUC-SAC, MUC-SB, and MUC-8) have been detected in respiratory airways. Recent
studies have indicated that MUC-5AC as well as its rat homologue (rMuc-SAC) is the

major mucin observed in surface epithelium of respiratory airways, while MUC-SB is

30

H ,3 r I, J 4 A D’ . SH
. r . IV ' I ' I a f

l 5 J1 ll fl__|

| Central Tandemly Repetitive Domain]

0 Core protein (apomucin)
0 Abundant O-glycosylation sites (Ser 81 Thr)
0 Amino acid sequences vary from mucin to mucin

| Flanking Regions'

0 Naked protein - no oligosaccharides
0 Cys-rich, unique, non-repetitive sequences
0 Mucin oligomer formation by disulfide linkages

 

Figure 1-5. Structure of secretory mucin monomer

31

mainly expressed in submucosal glands (Bluth et al., 1995; Guzman et al., 1996;
Hovenberg et al., 1996; Mecrzrnan et al., 1994) . Though no precise link has been made
between the mucin gene products observed in airway tissues and mucins present in
airway secretion fluids, it is clear that multiple mucins are responsible for the total airway
mucin secretion (Shankar et al., 1997; Steiger et al. , 1994; Van Klinken et al. , 1995).

It is not clear why the body needs several forms of mucin and how tissue-specific
regulation of mucin is achieved. Further studies on the structure-fimction relationship of
different mucins might give insights to the multiplicity and tissue-specificity of mucin

expression.

Mucin in Pathologic Conditions

Regardless of the beneficial roles of the airway mucus, excess mucus is a frequent
problem in respiratory airways. Overproduction and hypersecretion of mucins
accompanying MCM are important pathologic features of chronic respiratory diseases
such as bronchitis, asthma, rhinitis, and cystic fibrosis (Aikawa et al., 1992; Reid, 1954;
Robbins et al., 1984a,b; Chartrard and Marks, 1994). Excess luminal mucus may restrict
airflow and plug the conducting airways, and may contribute significantly to the
morbidity and mortality associated with these airway chronic respiratory diseases.

In addition to the increased production of mucins, a number of studies have
investigated the influence of diseases on the structure of airway mucins. Glycosylation
including sulfation of mucins appears to be subject to modification under pathologic

conditions (e.g., cystic fibrosis, bronchitis, bronchiectasis). More than 200 types of

32

oligosaccharide chains have been found in the airway mucus of patients with cystic
fibrosis (Lamblin et al., 1991; Rose, 1992). The apparent heterogeneity of mucin
oligosaccharide core structure in pathologic tissues may reflect either disease-related
alterations in parameters affecting glycosylation and other post-translational
modifications (e.g., nucleotide-sugar concentration, altered expression or activity of
specific glycosyltransferases, rates of transport of mucin protein through the endoplasmic
reticulum and Golgi apparatus), or the activation of specific mucin genes that are more

highly expressed in disease states and have different glycosylation patterns.

Regulation of Mucin Gene Expression

Though little is known concerning the underlying mechanisms of accelerated mucin
production, increases in steady-state levels of mucin mRN A as a consequence of
abnormal control of either transcription rate or RNA stability have been considered as
putative regulatory mechanisms of mucin overproduction. Indeed, upregulation of a
specific mucin mRNA (e.g., MUC-2) has been frequently reported in patients with cystic
fibrosis (Li et al., 1997). Observations from rat models of bronchitis induced by
exposure to airway irritants such as ozone, endotoxin, sulfur dioxide, or acrolein, or by
viral infections have also shown that the increases in the number of mucous cells and the
amount of stored mucosubstances are associated with the elevated level of airway-
specific mucin mRNA steady-state (Basbaum et al., 1990; Borchers and Leikauf, 1997;

Jany et al., 1991; Li et al., 1997).

33

Little information has been available on cis- and trans-acting elements or
transcription factors controlling transcription of mucin genes. A recent identification of
Nuclear Factor 1 (NF1)-MUCSB and its binding site in the 3’ region of MUC-SB gene in
difl‘erentiated mucous cells has provided a new paradigm with which to examine the
transcriptional regulation of mucin gene expression (Pigny et al., 1996). In addition,
putative binding sequences for ubiquitous transcriptional factors such as NF-kB and Spl
have been identified in 5’-flanking regions of other mucin genes (i. e., MUC-SAC, MUC-
2, rMuc-2) (Li et al., 1998; Nogami et al., 1997). Receptors and downstream signal
transduction mechanisms related to the airway mucin gene expression have not been
widely studied. However, growth factors such as retinoic acid are well known as positive
regulatory factors of mucin gene (MUC-2, MUC-SAC) expression and mucous cell
differentiation in airway epithelial cells through the retinoic acid receptor-mediated
pathway (Koo et al., 1997). Use of beta-2 receptor agonists indicates that MUC-SAC
mRNA upregulation can also occur via activation of G-protein-coupled cell surface
receptor pathway in airway epithelial cells (Kherallah et al., 1997). It has been suggested
that tyrosine kinase mediates the upregulation of MUC-2 (Li et al., 1997) or MUC-SAC
(V oynow et al., 1997) mRNA induced by inflammatory agents (e.g., endotoxin,
neutrophil elastase) in cultured epithelial cells.

Mucin mRNA stability may also be involved in the regulation of gene-specific
apomucin production. It has been known that several mucin genes (e.g., MUC-2 and
MUC-7, rMuc-2) have one or more destabilizing AU-rich elements (ARES) (Van Klinken
et al., 1995). Both MUC-SAC and MUC—l contain different long-tandem-repeats in the

3’-flanking area which are also suggested as other destabilizing sequences (Van Klinken

34

et al., 1995). Stimulation of the binding of specific nuclear or cytoplasrrric proteins to
these destabilizing sequences may help to prevent the degradation of mucin mRN A and
contributes to the increased steady-state of mucin mRN A levels.

Further elucidation of airway mucin genes and their regulatory mechanisms may aid
our understanding of malfunction of mucous-producing goblet cells. In addition,
inhibition of regulatory pathways of mucin genes by specific antagonists would constitute
a new therapeutic strategy to reduce morbidity and mortality in chronic mucous-

obstructive airway diseases such as chronic bronchitis, cystic fibrosis, and asthma.

Specific Aims of Thesis

The goal of the present thesis study was to understand the pathogenesis of ozone-
induced nasal epithelial cell responses, hyperplasia and MCM, in the NTE of rats.
Specifically, this study was designed to determine the role of neutrophilic inflammation
in the cellular and molecular events involved in (1) the proliferation of resistant epithelial
cells in the NTE of rats by inhaled ozone, and (2) the metaplastic transformation of
nonsecretory epithelium to mucus-secretory epithelium. Based on the previous findings
that transient neutrophilic influx is conspicuous and restricted to the NTE where the
distinctive ozone-induced epithelial morphological changes occur (Harkema et al. , 1989;
Hotchkiss et al., 1991, 1997), we have postulated that the infiltrating neutrophils will
play important roles in ozone-induced epithelial proliferation and mucin gene

upregulation, and ultimately mucous metaplastic transformation in the NTE (Figure 1-6).

35

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Our guiding hypothesis was, therefore, that ozone-induced epithelial hyperplasia and
MCM in the rat NTE are mediated by acute neutrophilic inflammation. The specific aims

were I

Aim 1: To test the hypotheses that (1) acute ozone exposure induces upregulation of an
airway mucin-specific gene (rMuc-SAC) prior to the development of MCM, and (2)
neutrophilic inflammation in the NTE precedes or coincided with epithelial DNA

synthesis and rMUC-SAC mRN A upregulation.

E

' 2: To test the hypothesis that ozone-induced epithelial proliferation (i. e., DNA

synthesis and hyperplasia), mucin gene upregulation and MCM are neutrophil-dependent.

Aim 3: To test the hypothesis that pre-existing neutrophilic rhinitis augments the ozone-

induced epithelial hyperplasia, mucin mRNA upregulation and MCM in the NTE.

_'___4: To test the hypothesis that came-inducible pro-inflammatory cytokines (TNF-a,

IL-6) cause upregulation of mucin mRN A expression in the NTE.

To address these specific aims, we designed a series of studies using a rat (F344/N,
male) model of acute (0.5 ppm, 8 h/day for 3 days) ozone-induced epithelial hyperplasia
and MCM. We focused on the ozone-induced alterations occurring in the NTE lining the

maxilloturbinates located in the proximal nasal airways of rats (Figure l-3C in page 23).

37

We utilized molecular, immunochemical, histocherrrical, morphometric techniques to
characterize mucin mRNA expression, neutrophilic inflammation, epithelial cell injury
and proliferation, and MCM in nasal airways. We also employed an in vitro tissue
culture technique to understand the putative role of individual cytokines on mucin gene
expression in the maxilloturbinates.

In the first study, we investigated the effect of acute ozone exposure on nasal mucin
mRNA expression, a possible molecular indicator of the following MCM. We also
examined the time-dependent changes of ozone-induced inflammatory and epithelial
responses in the NTE to determine the correlations between pre-mctaplastic responses
(i. e., neutrophilic inflammation, rMuc-SAC mRNA expression, epithelial proliferation)
and the metaplastic response (MCM).

To determine the contribution of neutrophils in ozone-induced epithelial responses,
we depleted rats of circulating blood neutrophils prior to ozone exposure. Systemic
administration of a rabbit anti-rat neutrophil antiserrun was used for this purpose. We
examined the effects of antiserum treatment on the ozone-induced nasal inflammation
(i. e., rhinitis), epithelial proliferation, mucin mRNA upregulation, and MCM in the NTE.

To understand further contribution of neutrophilic inflammatory events on the
epithelial alterations, we investigated the effect of pre-existing neutrophilic inflammation
on the severity of ozone-induced epithelial alterations. For this purpose, we intranasally

exposed rats to a strong pro-inflammatory agent, bacterial endotoxin, to induce

neutrophilic rhinitis prior to ozone exposure.

38

In subsequent in vitro studies, we investigated the role of soluble mediators (i. e.,
TNF-a and IL-6), which can be derived from airway cells by ozone exposure, in the
mucin gene expression of nasal tissues. First, we examined the time-dependent changes
of TNF-a and IL-6 mRNA levels in the nasal tissues of rats exposed to ozone. Then we
investigated the effects of the soluble form of these ozone-inducible cytokines on mucin
mRNA expressions in explants of microdissected maxilloturbinates, in the absence of
neutrophils.

The results of these thesis studies provided new insights into the cellular and
molecular mechanisms underlying MCM, a common epithelial alteration in many chronic
airway diseases (e.g., chronic bronchitis, asthma, cystic fibrosis and rhinitis) besides the

airway alterations induced by air pollutants like ozone.

39

CHAPTER 2

Inflammatory and Epithelial Responses During the Development of
Ozone-Induced Mucous Cell Metaplasia in the Nasal Epithelium of Rats

This study was supported by NHLBI Grant 5R01 HL51712-13.

Manuscripts submitted to Toxicological Sciences.

40

Abstract

Rats repeatedly exposed to high ambient concentrations of ozone develop mucous
cell metaplasia (MCM) in the nasal transitional epithelium (N TE). The present study was
designed to determine the temporal relationships of ozone-induced inflammatory and
epithelial responses and their correlation with subsequent MCM in the NTE of rats. Male
F344/N rats were exposed to 0.5 ppm ozone, 8 h/day for 1, 2, or 3 days. Two h prior to
sacrifice, all the rats were injected intraperitoneally with 5’-bromo-2—deoxyuridine
(BrdU) to label epithelial cells undergoing DNA synthesis. Rat exposed to ozone for 1 or
2 days were killed 2 h after the exposure. Rats exposed to ozone for 3 days were killed 2
h or 1, 2 or 4 days afier the exposure. Control rats were killed after 7-day exposure to
filtered air. One nasal passage from the anterior nasal cavity of each rat was fixed and
processed for light microscopy to morphometrically determine the numeric cell densities
of epithelial cells, neutrophils, and mucous cells, and the amount of intraepithelial
mucosubstances in the NTE. The maxilloturbinate from the other nasal passage was
processed for analysis of an airway mucin-specific gene (i.e., rMuc-SAC mRNA). Acute
ozone exposure induced a rapid increase in rMuc-SAC mRN A levels prior to the onset of
MCM, and the increased levels of rMuc-SAC mRNA persisted with MCM. Neutrophilic
inflammation coincided with epithelial DNA synthesis and upregulation of rMuc-SAC,
but was resolved when MCM first appeared in the NTE. The results of the present study
suggest that upregulation of mucin mRNA by acute ozone exposure are associated with

the concurrent neutrophilic inflammation and epithelial hyperplasia in the NTE, and that

41

ozone-induced MCM may be dependent on these important pre-metaplastic responses

(i.e., mucin mRN A upregulation, neutrophilic inflammation and epithelial proliferation).

Introduction

Ozone (03) is an irritating oxidant gas in photochemical smog, and one of the
regulated criteria air pollutants for which National Air Quality Standards have been
designated under the Clean Air Act (Steinfeld, 1991). Controlled inhalation studies have
demonstrated that acute ozone exposure induces cellular and biochemical changes in the
pulmonary airways of human subjects (Schelegle et al., 1991; Seltzer et al., 1986).
Ozone-induced morphologic changes in the distal centriacinar regions of the lung in
laboratory animals have been well docmnented in numerous studies (Castleman et al. ,
1980; Dungworth, 1989; Stephens et al., 1974).

The airways of the upper respiratory tract, specifically the nose, are also susceptible
to ozone toxicity. Nasal inflammation has been induced in human volunteers acutely
exposed to high ambient concentrations (0.4 - 0.5 ppm for 2 - 4 h) of ozone (Graham et
al., 1988; McBride et al., 1994; Bascom et al., 1990). In addition, nasal epithelial lesions
thought to be related to exposure to air pollution have been described in people living in
ozone-polluted atmospheres of southwest metropolitan Mexico City (Calderon
Garciduenas et al., 1992, 1995). Marked inflammatory and epithelial responses to near
ambient concentrations of ozone have also been demonstrated in the nasal mucosa of both

monkeys and rats (Harkema et al, 1987a,b, 1993; Hotchkiss et al. , 1989).

42

We have previously reported that acute or chronic exposures of 0.5 - 1.0 ppm ozone
causes epithelial proliferation and marked mucous cell metaplasia (MCM) in surface
epithelium lining the lateral meatus of the proximal nasal airways (i.e., nasal transitional
epithelium; NTE) in F344/N rats (Harkema et al., 1989, 1992, 1997; Hotchkiss et al.,
1991). The ozone-induced MCM in rat nasal epithelium was similar in character to nasal
epithelial changes previously reported by Harkema et al. (1987b) in macaque monkeys
repeatedly exposed to 0.15 or 0.3 ppm ozone for 6 days or 13 wks (6 h/day, 5 days/wk).

Though the ozone-induced morphological changes in the nasal epithelium have been
well characterized, the cellular and molecular events preceding the onset of MCM and
epithelial hyperplasia have not been thoroughly investigated. Previous studies have
demonstrated that a transient neutrophilic inflammation is conspicuous in the nasal
epithelium prior to the development of MCM (Hotchkiss et al., 1989, 1997). However,
little is known about the relationship of the neutrophilic inflammation with the ozone-
induced epithelial alterations in the nasal airway. In addition, the effects of ozone
exposure on mucin gene expression as well as its relationship to ozone-induced MCM in
the NTE have not been previously investigated. To understand further how and when
ozone induces nasal cell injury and reparative and adaptive changes (i.e., epithelial
proliferation and MCM) in the NTE of rats, it is first important to determine clearly the
temporal relationship of ozone-induced epithelial and inflammatory responses that occur
early after the start of exposure and during the development of the mucous metaplastic
changes.

Therefore, the present study was designed to test the hypotheses that (l) acute ozone

exposure induces upregulation of mucin gene expression prior to the development of

43

MCM, and (2) neutrophilic inflammation precedes, or is concurrent with, mucin gene
overexpression and other pre-metaplastic events (e.g., hyperplasia). For this purpose, rats
were exposed to 0.5 ppm ozone for l - 3 days (8 h/day). Some of the 3-day-exposed rats
were held in air for an additional 1 - 4 days. We determined the time-dependent
inflammatory and epithelial cell responses in the nasal epithelium of the ozone-exposed
rats. We also determined the temporal expression of rMuc-SAC mRNA in the nasal
tissues during and after ozone exposure. In addition, the temporal relationship of mucin
gene upregulation with (1) neutrophilic inflammation, (2) epithelial proliferation (i. e.,
DNA synthesis and numeric density), and (3) onset of MCM was investigated. A better
understanding of these exposure-related cellular and molecular events provides new

insights into the pathogenesis of MCM caused by repeated ozone exposure.

44

Materials and Methods

Animals and exposure. Fifty-six male F344/N rats (Harlan Sprague-Dawley,
Indianapolis, IN), 10 - 12 weeks of age, were randomly assigned to one of 7 exposure
groups (n = 8/group). Male rats were chosen for all experiments to avoid hormonal
changes during the estrous cycle. Estrous cycle-related changes have been shown to alter
secretory cell proliferation in rodent airways (Hayashi et al. , 1979).

Rats were housed two per cage in polycarbonate shoebox-type cages with Cell-Sorb
Plus bedding (A&W Products, Inc., Cincinnati, OH) and filter caps. Water and food (Tek
Lab 1640; Harlan Sprague Dawley, Indianapolis, IN) were available ad Iibitum. The rats
were maintained on a 12-h light/dark cycle beginning at 6:00 am. under controlled
temperature (16 - 25°C) and humidity (40 - 70%).

Prior to the start of the inhalation exposure, rats were conditioned in whole-body
exposure chambers (HG-1000, Lab Products, Maywood, NJ) supplied with filtered air for
1 day. The rats were individually housed in rack-mounted stainless-steel wire cages with
free access to food and water prior to exposure. The chamber temperature and relative
humidity as well as room light setting were maintained as described above.

Rats in one exposure group were exposed to 0 ppm ozone (filtered air) for 7 days
(controls, n = 8). Rats in the other seven exposure groups (n = 8/group) were exposed to
0.5 ppm ozone, 8 h/day, for 1, 2, or 3 days. The rats were exposed to ozone or filtered air
in the whole-body exposure chambers from 6 am to 2 pm in the Inhalation Toxicology

Exposure Laboratory housed in the University Research Containment Facility at

45

Michigan State University. Though food was removed, animals had free access to water
during the exposure. Ozone was generated with an OREC Model O3VI-O ozonizer
(Ozone Research and Equipment Corp., Phoenix, AZ) using compressed air (AGA Gas,
Lansing, MI) as a source of oxygen. No NOx gases have been detected by this method of
generation which uses UV. light to convert oxygen to ozone (Sun et al., 1988). Dilution
air was mixed with ozone and delivered to the chambers using teflon tubing. The total
airflow through the exposure chambers was maintained at approximately 250 L/min (15
chamber air changes/h). The chamber temperature and relative humidity during the
exposure remained the same as those during the animal conditioning period. The
chamber ozone concentration was controlled by adjusting the intensity of UV. radiation
within the ozonizer. It was monitored throughout the exposure with Dasibi 1003 AH
ozone monitors (Dasibi Environment Corp., Glendale, CA), and recorded by Linear 0141
strip chart recorders (Linear Instrument Corp., Reno, NV). The exposure-atmosphere
sampling probes were positioned in the breathing zone of the rats within the middle cage
rack of the HC-1000 chambers. The chamber ozone concentrations (mean 1 standard
deviation) during the 3-day exposure to 0.5 ppm-ozone were 0.523 _t 0.006. The chamber

ozone concentrations during 7-day exposures to filtered air were maintained less than

0.05 ppm.

Tissue selection and preparation for analyses. Two hours prior to the designated
sacrifice, each rat was treated intraperitoneally (ip) with 5’-bromo-2-deoxyuridine (BrdU;
50 mg/Kg body wt.) to label cells undergoing DNA synthesis in the S-phase of the cell

cycle. The rats exposed to ozone for 1 or 2 days were killed 2 h afier the end of exposure.

46

The rats exposed to ozone for 3 days were killed 2 h or 1, 2, or 4 days after the end of
exposure. Control rats were sacrificed after 7 days of exposure to filtered air. Rats were
deeply anesthetized using 4% halothane in oxygen and killed by exsanguination via the
abdominal aorta.

Immediately after death, the head of each rat was removed from the carcass. After
the eyes, lower jaw, skin and musculature were removed from head, the nasal airways
were opened by splitting the nose in a sagittal plane adjacent to the nridline. The
maxilloturbinate (Figure 2-1A) from one nasal passage was excised by microdissection
and immediately homogenized in Tri-Reagent (Molecular Research Center, Cincinnati,
OH). The homogenate was snap frozen in liquid nitrogen and stored at -80°C until
processed for isolation of total RNA and analysis of mucin mRNA.

The other nasal passage was immersed in a large volume of zinc formalin (Anatech,
Ltd., Battle Creek, MI) for at least 24 h. The zinc formalin-fixed nasal tissues were
decalcified in 13% formic acid for 4 days, and then rinsed in tap water at least 2 h as
previously described by Harkema et al. (1988). A tissue block was removed fi'om the
proximal aspect of the nasal cavity by making two transverse cuts perpendicular to the
hard plate. The first cut was immediately posterior to the upper incisor tooth (Figure 2-
1A), and the second cut was at the level of the incisive papilla. The tissue block was
excised, embedded in paraffin, and 5 um-thick sections were cut from the anterior face of
the tissue block. One nasal tissue section from each animal was histochemically stained
with hematoxylin and eosin for morphological identification of epithelial cells. Another

tissue section from each animal was immunohistochemically stained with anti-BrdU

47

Figure 2-1. Anatomic location of nasal tissues selected for morphometric
and RT-PCR analyses. (A) Exposed lateral wall of nasal
airway. Shaded area indicates the maxilloturbinate (MT) in a
nasal passage microdissected for mucin mRNA analysis.
The vertical line indicates anterior surface of the transverse
block used for morphometric analysis. 11 = naris; NT =
nasoturbinate; ET = ethmoturbinate; HP = hard plate; NP =
nasopharynx; b = brain. (B) Anterior face of tissue block from
one proximal nasal airway. S = nasal septum. (C) Enlarged
views of the maxilloturbinate in B illustrating the major
turbinate tissue compartments, TB = turbinate bone; LP =
lamina propria; e = surface epithelium (NTE). (D) Enlarged
view of the NTE lining the maxilloturbinates of a normal
(control) rat. The NTE is a nonciliated cuboidal epithelium, 1 -
2 cell layers thick, with no mucous secretory cells. (E) Enlarged
view of ozone-exposed NTE with ozone-induced MCM. Note
the appearance of numerous mucous cells (arrows) within the
exposed epithelium.

48

 

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antibody (Becton Dickinson Immunocytometry Systems, San Jose, CA) to detect BrdU-
labeled nuclei (Johnson et al., 1990) (Figure 2-2), and counterstained with hematoxylin
(Gill 3; Ricca Chem. Co., Arlington, TX). A third tissue section from the same block was
stained with Alcian Blue (pH 2.5)/Periodic Acid-Schiff‘s sequence (AB/PAS) to identify

acidic and neutral mucosubstances in the surface epithelium.

Morphometly of neutrophilic inflammation, epithelial cell numeric density and DNA
synthesis. The NTE lining the maxilloturbinate of each animal was analyzed using
computerized image analysis and standard morphometric techniques (Hotchkiss and
Harkema, 1992; Hotchkiss et al., 1991). The neutrophil numeric density was determined
by quantitating the number of nuclear profiles of neutrophils in the surface epithelium
lining the maxilloturbinates (i. e., NTE), and dividing the number by the total length of
the basal lamina underlying this epithelium (i.e., intraepithelial neutrophils/mm basal
lamina). Neutrophils were identified by morphologic characteristics that included their
size, darkly stained multi-lobed nucleus, and clear cytoplasm with dust-like granules.
The length of the basal lamina underlying the surface epithelium was calculated from the
contour length of the digitized image of the basal lamina using a Power Macintosh
7100/66 computer and the public domain image analysis software (NIH Image; written by
Wayne Rasband at the US. National Institutes of Health and available on the Internet at
http://rsb.info.nih.gov/nih-image/). The epithelial cell numeric density (i. e., epithelial
nuclei/mm of basal lamina) was determined by counting the total number of epithelial

cell nuclear profiles present in the NTE covering the maxilloturbinate, and dividing this

50

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number by the length of basal lamina. The epithelial cell labeling index (LI) was
determined as an indicator of the epithelial DNA synthesis. The number of BrdU-labeled
NTE cell nuclei was counted, divided by the total number of epithelial cell nuclei and

multiplied by 100 (i.e., % BrdU-labeled epithelial cell nuclei).

Morphometty of stored intraepithelial mucosubstances and mucous cells. To estimate
the amount of the intraepithelial mucosubstances in NTE lining the maxilloturbinates, the
volume density (Vs) of AB/PAS-stained mucosubstances was quantified using
computerized image analysis and standard morphometric techniques. The area of the
AB/PAS-stained intraepithelial mucosubstances was calculated by the image analysis
software program from the automatically circumscribed perimeter of the stained material.
The length of the basal lamina underlying the surface epithelium was determined as
described above. The volume of stored mucosubstances per unit of surface area of
epithelial basal lamina was estimated using the method previously described in detail by
Harkema et al. (1989, 1987b), and expressed as nL/mm2 basal lamina.

The numeric cell densities of mucous cells (epithelial cells containing AB/PAS-
stained mucosubstances) in the NTE lining the maxilloturbinates were also
morphometrically determined. Only AB/PAS-positive epithelial cells with a nuclear

profile were counted, and the data were expressed as the number of mucous cell

nuclei/mm of basal lamina.

52

Analysis for mucin mRNA in maxilloturbinates. Total cellular RNA was isolated
from the maxilloturbinate homogenate according to the method of Chomczynski et al. as
outlined in the Tri-reagent product literature (Chomczynski and Sacchi, 1987). To avoid
DNA contamination, the isolated RNA pellet was resuspended in nuclease-free water and
treated with 10 units RNase-free DNasc I (Boehringer Mannheim GmbH, Germany) in
5X Transcription buffer (Promega, Madison, WI) at 37°C for 30 min. The RNA was
extracted sequentially with equal volumes of phenol/chloroform/isoamyl alcohol mixture
(25:24:1) and chloroform/isoamyl alcohol mixture (24:1), and precipitated. The final
pellet was washed with 75% ethanol, air dried, resuspended in nuclease-free water
containing rRNasin (40 units/100 ul), and the concentration of RNA was determined by
measuring absorbances at 260 nm. All the RNA samples were stored at -80°C.

The RNA was analyzed to determine the steady-state levels of rMuc-SAC mRNA by
quantitating the amount of rMuc-SAC cDNA produced by reverse transcriptase
polymerase chain reaction (RT-PCR). Cyclophilin is an abundant and ubiquitous cellular
protein well known as a major intracellular receptor for the immunosuppresant
cyclosporin A, and considered as a putative molecular chaperone (Matouschek et al. ,
1995; Kern et al., 1994). Because cyclophilin mRNA expression was similar in all
experimental groups, this housekeeping gene was used as an internal standard in this
semi-quantitative RT-PCR analysis.

Primers specific for rat rMuc-SAC cDNA and all-species cyclophilin cDNA
sequences were synthesized and purified by the Macromolecular Structure Facility at

Michigan State University. The sequences of the forward and reverse primers for

53

cyclophilin are 5’- CTT GTC CAT GGC AAA TGC TG-3’ and 5’-GTG ATC TIC TTG
CTG GTC TTG-3’, respectively. The sequences of the rat Muc-5AC forward and reverse
primers are 5’-CAT CAT TCC TGT AGC AGT AGT GAG G-3’ and 5’-GGT ACC CAG
GTC TAC ACC TAC TCC G-3’, respectively. The predicted amplified sizes of the
cyclophilin cDNA and rMuc-SAC cDNA products were ~l90 bp and ~320 bp,
respectively.

A 50 ng/ul working solution of each RNA sample was prepared, and RT-PCR was
performed in 9600 Perkin Ehner Thennocycler. Two pl (100 ng) aliquots were reverse
transcribed into cDNA in a volume of 20 1.11, each, containing PCR buffer (166 mM
(NH4)ZSO4, 50 mM B—mercaptoethanol, 67 uM EDTA, 0.67 M Tris, pH 8.8, 0.8 mg/ml
BSA) plus 5 mM MgC12, 1 mM each dNTP, 10 writs rRNasin, 125 ng oligo(dT),, and 50
units of Moloney Murine Leukemia Virus reverse transcriptase (M-MLV- RT; Gibco
BRL, Gaithersburg, MD). A PCR master mix consisting of PCR buffer, 4 mM MgC12, 6
pmol of each forward and reverse primers of MU C-5 mRNA and cyclophilin mRNA, and
1.25 units Taq DNA polymerase was added to each cDNA sample for a final volume of
50 ul. The Taq was added after the PCR master mix was heated to 85°C for 5 min to
minimize primer dimer formation. PCR amplification was started with 3 min incubation
at 95°C followed by a three step temperature cycle; denaturation at 95°C for 30 sec,
annealing at 56°C for 1 min and extension at 72°C for 1 min for 25 cycles. A final
extension step at 72°C for 10 min was included after the final cycle to complete
polymerization. The number of cycles was chosen to ensure that amplification of

cyclophilin (most abundant gene) did not reach a plateau level.

54

The abundance of mRN A was semi-quantitatively determined by densitometric
analysis of ethidium bromide-stained agarose gel (3%, Nusievezagarose = 3:1) using the
Gel Doc 1000 analysis system (BioRad Laboratories, Inc., Herculese, CA) and Molecular
Analyst Software Version 2.1 on a Power Macintosh 7100/80. The volume of the rMuc-
SAC cDNA band was divided by the volume of the cyclophilin cDNA band. To
minimize the variability of analysis, the quantitation for all the samples was performed at

the same time.

Statistical analyses. All data were expressed as the mean group value :I: the standard

error of the mean (SEM). The natural logarithms of the data were used for statistical
analysis to make the variances approximately equal for all groups. The data were first
analyzed using one-way analysis of variance (ANOVA) to identify the time-response of
inflammation and epithelial events induced by ozone exposure. Significant differences
between air control group and ozone-exposed groups were evaluated using Dunnett’s
Method. Statistical analyses were performed using a commercial statistical analysis
package (SigmaStat; Jandel Scientific Software, San Rafael, CA). The level of statistical

significance was set at p 5 0.05.

55

Results

Histopathology of nasal mucosa

Exposure-related nasal lesions were present only in rats exposed to 0.5 ppm ozone.
No lesions were observed in the nasal airways of filtered air (0 ppm ozone)-exposed rats
(controls). In all of the rats exposed to 0.5 ppm ozone, regardless of the duration of the
exposure, the nasal lesions were restricted to the mucosa containing the NTE that lined
the lateral meatus in the proximal nasal cavity. The character and severity of these site-
specific mucosal lesions, however, varied according to the number of ozone exposures
and the length of time post-exposure. Figure 23 contains light photomicrographs of
representative maxilloturbinates that depict time-dependent changes of inflammatory and
epithelial cell responses in the NTE. Time-dependent progression of MCM in the NTE is
illustrated in Figure 2-4 that contains representative maxilloturbinates stained with
AB/PAS for identification of intraepithelially stored mucosubstances.

After one day of exposure to 0.5 ppm ozone, the principal morphologic alterations in
the NTE were epithelial degeneration and atrophy due to individual cell necrosis and
exfoliation. These ozone-induced alterations in the NTE were most noticeable in the
dorsal and medial aspect of the maxilloturbinate, the lateral ridge of the nasoturbinate and
the dorsal recess of the lateral wall. Concurrent with the epithelial lesions was a mild-
moderate inflammatory response in the affected mucosa, characterized by endothelial

margination of neutrophils in the large capacitance vessels of the lamina propria and an

56

influx of neutrophils in the adjacent interstitial tissue of the lamina propria and also
extending into the NTE.

After 2 days of ozone exposure, the neutrophilic inflammatory response in the nasal
mucosa was similar to that observed after one day of exposure to ozone with conspicuous
accumulations of neutrophils in both the lamina propria and NTE. However, the
epithelial degeneration and atrophy of the NTE observed in the one day-exposed rats was
replaced by a regenerative, basophilic epithelirun in the rats exposed to ozone for 2
consecutive days. The principal features of the regenerative NTE were mild hyperplasia
of basal cells and the appearance of widely scattered mitotic figures. In addition, necrotic
epithelial cells, a common feature in the NTE of the one day-ozone exposed animals,
were infrequently observed in the NTE of the 2-day exposed rats.

Epithelial hyperplasia was the principal feature in the NTE of rats exposed for 3
consecutive days and sacrificed at the end of the exposure. The hyperplastic NTE in
these ozone-exposed rats was approximately 3 - 4 cells in thickness compared to 1 - 2
cells in the NTE of control rats exposed only to filtered air. Only a mild influx of
neutrophils was present in the nasal mucosa containing hyperplastic NTE after 3 days of
ozone exposure.

In rats exposed to 0.5 ppm ozone for 3 days and sacrificed one day after the end of
the exposure, the NTE remained hyperplastic but there were also a few widely scattered
AB/PAS-stained, mucous cells in the NTE indicating the onset of a rrrild MCM. Only a
few widely scattered neutrophils were present in the NTE or lamina propria of these

repeatedly exposed rats.

57

Ozone-induced MCM in the NTE was even more marked in rats exposed to 0.5 ppm
ozone for 3 days and killed 2 or 4 days postexposure. The 3-day-ozone-exposed rats that
were killed 4 days after the end of the exposure had the most severe MCM compared to
the other 3-day-ozone-exposed rats killed 1 or 2 days postexposure. Epithelial
hyperplasia was also a conspicuous feature of the NTE in rats killed 2 or 4 days
postexposure. However, no associated inflammatory cell influx was present in the nasal
mucosa with the hyperplastic and metaplastic NTE of the 3-day-ozone-exposed rats killed

2 or 4 days after the end of the exposure.

Morphometric quantitation

Neutrophilic influx. Exposure to 0.5 ppm ozone resulted in a transient influx of
neutrophils within the NTE lining the maxilloturbinates (Figure 2-5). Significant
increases in the number of intraepithelial neutrophils were evident in rats exposed to 0.5
ppm ozone for 1 and 2 days (3 8- and 47-times greater than air-exposed controls,
respectively). The number of neutrophils in the NTE was attenuated (~ 45%) in rats
exposed to ozone for 3 days, compared to those exposed to ozone for 1 or 2 days. At one
day after 3 days of ozone exposure, the number of intraepithelial neutrophils was 10-fold
greater than air-exposed controls. However, the intraepithelial neutrophil numbers

returned to control levels in rats killed 2 and 4 days postexposure.

58

Epithelial cell DNA synthesis. Only a few widely scattered BrdU-labeled NTE cells
were present in air-exposed control rats (Figure 2-6). There was a marked, but transient,
increase in the number of BrdU-labeled cells in rats exposed to ozone for 2 and 3 days,
with 112-fold and l43-fold increases, compared to the controls, respectively. The
numbers of BrdU-labeled cells in rats exposed to 0.5 ppm ozone and killed 1, 2, or 4 days

postexposure were similar to those of air-exposed control rats.

NT E cell numeric density. Ozone exposure altered the number of NTE cells lining the
maxilloturbinates (Figure 2-7). Rats exposed to ozone for 1 day had 9% fewer NTE cells
than air-exposed control rats. However, rats exposed to ozone for 2 days had NTE cell
numeric densities that were not significantly different fi'om those of controls. After 3
days of ozone exposure, rats had significantly more NTE cells (36%), compared to air-
exposed controls. This ozone-induced NTE cell hyperplasia persisted in rats killed 2 and

4 days postexposure (i.e., 52% and 37% more cells than those in controls, respectively).

Amount of stored intraepithelial mucosubstance. Little AB/PAS-stained intraepithelial
mucosubstances were present in the NTE of control rats or rats exposed to ozone for 1 or
2 days (Figure 2-8). An ozone-induced increase in the amount of stored mucosubstances
was first detected in the NTE of rats exposed to ozone for 3 days and sacrificed 2 h later.
A significant increase (24-fold, compared to controls) in the amount of the intraepithelial
mucosubstances was observed in rats sacrificed 1 day after the 3-day-ozone exposure.

The amount of intraepithelial mucosubstances was increased with time postexposure.

59

Thirty and fifty fold more Vs, compared to that of controls, were observed in rats killed 2

and 4 days postexposure, respectively.

Mucous cell numeric density. Control animals exposed to filtered air (0 ppm ozone) had
no or only a few mucous cells in the NTE. A significant increase (22-fold more than air-
exposed controls) in the number of mucous cells was first detected in 3-day-ozone-
exposed rats killed 1 day postexposure (Figure 2-9). The numeric density of mucous
cells in the NTE of ozone-exposed animals increased with time postexposure. Rats killed

4 days after the 3-day-ozone exposure had 43-fold more mucous cells than air controls.

Mucin mRNA (rMuc-5A C) abundance

Figure 2-10 depicts an agarose gel with representative RT-PCR cDNA products from
each exposure group indicating the abundance of rMuc-SAC and cyclophilin mRN A.
Maxilloturbinates of control animals which normally have few mucous cells had
detectable, but low levels of rMuc-SAC mRNA. Exposure to ozone resulted in marked
increases of rMuc-SAC mRNA in these nasal tissues (Figure 2-11). There were 32% (1
day), 140% (2 day) or 126% (3 day) more rMuc-SAC mRNA in ozone-exposed rats,
compared to air-exposed controls. The elevation of rMuc-SAC mRN A persisted in rats
killed 2 days following the end of the 3-day-ozone exposure (70% more, compared to

controls).

60

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Discussion

The results of the present study demonstrate that a single 8-h exposure to 0.5 ppm
ozone rapidly induces an increase in rMuc-SAC mRN A in the nasal mucosa of rats. This
ozone-induced increase in rMuc-SAC mRNA preceded the morphologic appearance of
increased number of mucous cells in the NTE by three days. In addition, the rMuc-SAC
mRNA level remained elevated for two days after the end of 3 days of ozone exposure
when MCM with copious amounts of intraepithelial mucosubstances was microscopically
evident in the nasal epithelium. A conspicuous neutrophilic inflammatory response
accompanied the ozone-induced increase in rMuc-SAC mRNA in the NTE after 1, 2, or 3
days of exposure. The ozone-induced neutrophilic inflammation was markedly
attenuated 1 day after the end of the 3 days of exposure, and continued to decline during
the postexposure period. In contrast, the severity of the ozone-induced mucous
metaplastic changes increased during this same postexposure period. There was a time-
dependent increase in the amount of intraepithelial mucosubstances and the number of
mucous cells during the four days postexposure. This is the first report, to our
knowledge, indicating that a single or repeated inhalation exposure to a high ambient
concentration of ozone can induce an increase in the steady-state levels of an airway
mucin-specific mRNA in the nasal airways of a laboratory animal. In addition, this study
is the first to demonstrate that this ozone-induced increase in rMuc-SAC mRN A

precedes, by several days, the onset of MCM in the affected nasal epithelium.

72

MCM in rat airway epithelium is also a prominent morphologic response to exposure
to other airway irritants such as sulfur dioxide (Jany et al., 1991), tobacco smoke (Lamb
and Reid, 1969), bacterial endotoxin (Harkema and Hotchkiss, 1993; Shimizu et al.,
1996; Gordon et al., 1996), 3-methylcholanthrene (Rehm and Kelloff, 1991), acrolein
(Borchers and Leikauf, 1997), siloxane (Burns-Naas et al., 1998), and chlorine (Wolf et
al., 1995). However, only a few previous studies have demonstrated irritant-induced
alterations in mucin gene expression associated with MCM in airway epithelium of rats.
Repeated exposure to sulfur dioxide induced early upregulation of mucin mRN A that
persisted throughout the development of MCM in rat tracheobronchial epithelium (J any
et al., 1991). Elevated rMuc-2 mRNA expression concurrent with MCM was also
reported in tracheobronchial and pulmonary airway epithelium of rats after repeated
exposure to acrolein (Borchers and Leikauf, 1997). These previous studies have reported
elevated airway mucin mRNA levels in rat airways after repeated long-term (i.e., >1 wk)
inhalation exposure. The results of the present study suggest that ozone-induced
alteration in mucin mRN A abundance is an early molecular predictor of mucous
metaplastic changes, and probably plays a crucial role in the development of the
phenotypic expression of mucous (goblet) cells in the nasal epithelium (i. e., MCM).

Our present observations are supported by studies conducted in vitro which examined
mucous differentiation of airway epithelial cells (RTE) induced by retinoic acid, a major
regulator of mucous differentiation, (Guzman et al. , 1996) or colonic epithelial cells (HT-
29) induced by methotrexate, an inhibitor of nucleic acid metabolism (Lesuffleur et al.,
1993, 1990). The results from these studies have demonstrated that mucin messages were

strongly expressed only in cultures that had undergone mucous cell differentiation. In

73

addition, there was a time-lag (2 - 7 days) between the first detection of mucin gene
expression and that of mucus production during the in vitro mucous cell differentiation.
Little is known about the kinetics of mucous biosynthesis in either normal or metaplastic
mucous cells in airway epithelium. However, it is assumed that during the ozone-induced
mucous metaplastic differentiation in the nasal epithelium, the mucin gene is activated in
premetaplastic cells, and it takes time for (1) the synthesis of mucin core protein from the
abundant mucin mRNA, (2) its glycosylation, and (3) the storage of the glycosylated
mucin molecules into secretory granules of fully differentiated mucous cells. Further
studies designed to determine the kinetics of rMuc-SAC mRN A accumulation, apomucin
(protein core) synthesis and glycosylation are needed. Results from such studies will
further our understanding of the underlying molecular mechanisms of ozone-induced
MCM.

In the present study, we determined the time-dependent relationships of pre-
metaplastic inflammatory and epithelial events (i.e., neutrophilic inflammation, epithelial
injury, regeneration and proliferation) in the nasal epithelium induced by single and
repeated exposures to ozone. We have described how these temporal changes in the NTE
correlate with the changes in mucin gene expression and the development of MCM. The
coincidence of the onset of increased steady-state rMuc-SAC mRNA levels and the
transient neutrophilic influx into the NTE, prior to the development of MCM, suggests
that the early neutrophilic inflammatory response may be involved in the upregulation of
mucin mRNA levels in the NTE and the initiation of MCM. Neutrophils, as well as
airway epithelial cells, are significant sources of soluble mediators that can initiate or

amplify inflammatory responses in airway tissues. We hypothesize that neutrophils play

74

an essential role in the ozone-induced mucin gene upregulation and ultimately in the
pathogenesis of the MCM by releasing distinctive soluble mediators or by stimulating
other resident cells (e.g., epithelial cells) to release inflammatory mediators. It has been
known that soluble inflammatory mediators can rapidly modulate various cellular genes
(e.g., genes for secondary mediators like cytokines) during airway injury and repair
induced by inhaled toxicants, including ozone (Leikauf et al., 1995; Levine, 1995;
Nakamura et al., 1992; Warner et al., 1987; Marini et al., 1992). Recently, several
studies have focused on the investigation of the role of inflammatory mediators in the
expression of airway mucin-specific genes. Cytokines such as TNF-a (Levine et al.,
1995), IL-6 (Levine et al., 1994) and IL-4 (Temann et al., 1997; Rankin et al., 1996), or
neutrophil proteases, specifically elastases (V oynow et al., 1997), have been reported to
induce mucin mRNA upregulation in airway epithelial cells in vivo or in vitro.
Neutrophil elastase is a well-known mucous secretagogue and induces MCM in the
airways of laboratory animals (Breuer et al., 1985, 1993; Sommerhoff et al., 1990; Jamil
et al., 1997; Kim et al., 1987). Both soluble TNF-a and IL-6 induce mucin
hypersecretion in airway epithelial cells in vitro at concentrations that also cause mucin
gene upregulation (Levine et al., 1994, 1995). Transgenic mice that overexpress IL-4 or
IL-5 have MCM (Rankin et al., 1996; Jain-Vora et al., 1997; Lee et al., 1997) and mucin
hypersecretion (McBride et al., 1994) with mucin gene upregulation in tracheobronchial
or pulmonary airways. However, the precise roles of these inflammatory mediators in

mucin gene expression or mucous cell function are still not clarified. Furthermore, few

75

studies have investigated the dependency of mediator-induced mucous differentiation or
mucous overproduction on the activation of mucin genes.

Recently we demonstrated that an anti-inflammatory steroid, fluticasone propionate,
decreased neutrophilic inflammation and MCM in the nasal epithelium of rats exposed to
ozone (Hotchkiss et al., 1998). Similarly, another steroid, dexamethasone has been
shown to attenuate rat tracheal MCM induced by neutrophil lysates or elastase (Lundgren
et al., 1988). In addition, Kai et al. (1996) reported that dexamethasone suppressed the
mucin mRN A expression and stored mucous product in airway epithelial cells in culture.
These studies suggest a putative role of inflammatory cells in the induction of MCM, the
upregulation of airway mucin genes, and the overproduction of mucins in airway
epithelium. At present, steroids are among the most efficacious treatments for asthma
(Barnes and Pedersen, 1993). However, only a few studies have documented the benefits
of steroid therapy in alleviating the excessive production of airway mucus (Marom et al. ,
1984; Shimura et al., 1990; Lundgren et al., 1988). In addition, it is not certain whether
or not steroids modulate the mucin gene expression directly by acting on glucocorticoid
regulatory elements present in the mucin gene or indirectly through other anti-
inflammatory mechanisms in airway tissues.

The present study was also designed to examine the kinetics of epithelial injury,
regeneration and proliferative adaptation (i.e., hyperplasia), and especially the time-
dependent relationships of these ozone-induced epithelial changes with mucin gene
expression and MCM. Interestingly, the severity and temporal pattern of nasal epithelial
cell loss, subsequent burst of DNA synthesis, and cell proliferation leading to epithelial

repair during single and repeated daily exposure to ozone were similar to those

76

determined after a single exposure in a previous study reported by Hotchkiss et a1.
(1997). In that study, rats were exposed to 0.5 ppm ozone once for 8 h, and the epithelial
responses were examined 2 - 36 h postexposure. Even though rats in our study received
repeated ozone exposures, temporal relationship of epithelial DNA synthesis and the
proliferation of injured NTE cells observed after 2 and 3 days of exposure were similar to
those observed in the previous study at 24 and 36 h after the single exposure to ozone.
The results from both studies indicate that the induced cellular renewal mechanisms in
the NTE following the first day of ozone exposure are not significantly affected by
subsequent ozone exposure. The epithelial regeneration after 2 days of exposure and
subsequent hyperproliferative response (i.e., epithelial hyperplasia) were concurrent with
increased levels of rMuc-SAC mRNA in the NTE. It is plausible that new epithelial cells
with abundant mucin message repopulate the injured epithelium and are responsible for
the observed increase in mucin mRN A in the regenerative and hyperplastic epithelium. It
is also possible that NTE cells which survive the initial ozone exposure are stimulated by
ozone to upregulate their normally low-constitutive levels of mucin mRN A. The exact
cellular mechanisms responsible for ozone-induced upregulation of rMuc-SAC mRNA in
the NTE cannot be determined from the results of our present study. Further studies,
using in situ hybridization and immunohistochemistry techniques, are needed to identify
the NTE cells that express rMuc-SAC mRNA and produce mucin protein during
regeneration and hyperproliferation after ozone exposure.

Because the onset of epithelial proliferation preceded the appearance of increased
numbers of mucous (goblet) cells, it is possible that epithelial cell proliferation is a

prerequisite for MCM. However, the dependency of MCM on the preceding epithelial

77

proliferative responses cannot be determined from our present results. In our study,
neutrophilic inflammation preceded both the hyperplastic and metaplastic responses in
the ozone-exposed NTE. Though the role of neutrophils in the repair and
hyperproliferation of NTE is unknown, other studies in the literature suggest that these
inflammatory cells are important in airway epithelial repair following ozone-induced
injury in the lungs of laboratory animals (Pino et al., 1992; Hyde et al., 1992).

In conclusion, acute ozone exposure induced increased levels of rMuc-SAC mRNA
in the NTE within hours after the start of exposure. This ozone-induced upregulation of
the airway mucin gene was observed several days before the phenotypic expression and
the intraepithelial production and storage of mucosubstances (i. e., MCM). Mucin gene
upregulation occurred concurrently with ozone-induced neutrophilic inflammation in the
NTE but was maintained even after the initial neutrophilic inflammation was resolved 2
days later. Although temporal correlations of epithelial and inflammatory responses in
the present study do not prove causality, our results indicate that (l) upregulation of
mucin mRN A by acute ozone exposure is associated with the concurrent neutrophilic
inflammation and epithelial hyperplasia in the NTE, and that (2) ozone-induced MCM
may be dependent on these important pre-metaplastic responses (i. e., mucin mRN A

upregulation, neutrophilic inflammation and epithelial proliferation).

78

CHAPTER 3

Neutrophil-Dependent and -Independent Alterations
in the Nasal Epithelium of Ozone-Exposed Rats.

This study was supported by NHLBI Grant HL51712.

Manuscripts submitted to American Journal of Respiratory and Critical Care Medicine.

79

Abstract

Ozone induces epithelial hyperplasia and mucous cell metaplasia (MCM) in nasal
transitional epithelium (NTE) of rats. A transient neutrophilic influx accompanies
upregulation of mucin mRNA prior to the onset of MCM. The present study was
designed to examine the role of neutrophils in ozone-induced epithelial changes in the
NTE of rats. Fourteen h prior to inhalation exposure, male F344/N rats were treated ip
with anti-rat neutrophil antiserum or control serum. For morphometric analyses,
antiserum- or control serum-treated rats were exposed to O (filtered air) or 0.5 ppm ozone
for 3 days (8 h/day). At the end of exposure, rats were treated ip with 5’-bromo-2-
deoxyuridine (BrdU) to label epithelial cells undergoing DNA synthesis, and killed 2 h or
4 days later. Nasal tissues were processed to morphometrically determine the BrdU-
labeling index, the numeric densities of neutrophils, total epithelial cells and mucous
cells, and the amount of intraepithelial mucosubstances (IM) in the NTE. For rMuc-SAC
mRNA analysis, antiserum- or control serum-treated rats were exposure to O or 0.5 ppm
ozone for 1 or 3 days (8 h/day). Rats were killed immediately afier I or 3 days of
exposure, or 4 days after 3 days of exposure, and RNA was isolated from microdissected
maxilloturbinates. At 2 h after 3-day exposure, rats treated with antiserum had ~ 90%
fewer circulating neutrophils than rats treated with control serum. Antiserum-
treated/ozone-exposed rats had 87% less infiltrating neutrophils than control serum-
treated/ozone-exposed rats. At 4 days after 3-day exposure, antiserum-treated/ozone-
exposed rats had 66% less IM and 58% fewer mucous cells in the NTE than did control

serum-treated/ozone-exposed rats. Antiserum treatment had no effects on ozone-induced

80

epithelial cell proliferation and mucin mRNA upregulation. The results of this study
indicated that ozone-induced MCM was neutrophil-dependent, while ozone-induced

epithelial cell proliferation and mucin gene upregulation were neutrophil-independent.

Introduction

Both cellular inflammation and overproduction/hypersecretion of airway mucus are
thought to be important factors in the pathogenesis of many obstructive pulmonary
disorders, including acute and chronic bronchitis (Reid, 1954; Robbins et al., 1984),
asthma (Aikawa et al., 1992), cystic fibrosis (Chartrard and Marks, 1994), and upper
respiratory tract disorders such as allergic rhinitis (Robbins et al., 1984). Similar changes
have also been induced in airway mucosa of laboratory animals by inhaled irritants such
as sulfur dioxide, cigarette smoke, and bacterial endotoxin (Harkema and Hotchkiss,
1993; Lamb and Reid, 1969; Jany et al., 1991).

Ozone, the major oxidant air pollutant in photochemical smog, causes inflammation
and tissue damage in human airways, including the nose (Calderon Garciduenas et al. ,
1992; Koren et al., 1990; Peden et al., 1995). In F344/N rats, we have demonstrated that
short-term (i.e., days) (Hotchkiss et al., 1989; Harkema et al., 1989) or long-term (i. e.,
weeks or months) (Harkema et al., 1994, 1997) exposures to high ambient concentrations
of ozone (0.5 - 1.0 ppm) induce marked mucous cell metaplasia (MCM) with

accompanying increases in stored intraepithelial mucosubstances and number of

81

epithelial cells (i.e., epithelial hyperplasia) in nasal airways. These ozone-induced
epithelial alterations were restricted to the nasal transitional epithelium (NTE), which is
normally devoid of mucous cells, lining the lateral meatus of the proximal aspect of the
nasal cavity (Harkema et al., 1989, 1992; Hotchkiss et al. , 1991). A marked and transient
neutrophilic influx into the NTE preceded the epithelial hyperplasia and MCM induced
by repeated ozone exposure (Harkema et al., 1989; Hotchkiss et al., 1997). More
recently, we have also demonstrated that acute ozone exposure results in an increase in
the steady-state level of an airway mucin-specific (rMuc-SAC) mRNA prior to the onset
of the MCM in rat NTE (Cho et al., 1997). In addition, the mucin gene upregulation (a
putative molecular predictor of MCM) as well as the burst of epithelial DNA synthesis (a
marker of epithelial cell injury and proliferation) coincide with the neutrophilic
inflammation in the NTE (Hotchkiss et aI. , 1997; Cho et al., 1997).

Neutrophils have been implicated as a cause of tissue damage in a number of airway
inflammatory diseases. Activation of neutrophils results in the release of powerful
inflammatory mediators that may damage both cellular and extracellular tissue
components and amplify the inflammatory response (Weiss, 1989; Okrent et al., 1990;
Sibille and Reynolds, 1990). The involvement of neutrophils in ozone-induced injury
and repair has been investigated in distal airways of several laboratory animal species
(Pino et al., 1992; Hyde et al., 1992; Salmon et al., 1998). However, the role of
neutrophils in the pathogenesis of ozone-induced nasal airway lesions in rats has not been
investigated.

The present study was designed to test the hypothesis that neutrophilic inflammation

plays an important role in ozone-induced epithelial alterations (i. e., hyperplasia and

82

MCM) as well as mucin gene upregulation in rat nasal airways. For this purpose, we
depleted rats of their circulating neutrophils using an anti-rat neutrophil antiserum prior
to repeated, acute, ozone exposure. By removing the circulating pool of neutrophils, we
were able to examine the contribution of these inflammatory cells to the pathogenesis of
ozone-induced proliferative and metaplastic alterations in the NTE. The results of this
study provided new insights into the underlying mechanisms of ozone-induced injury,

adaptation, and repair of airway epithelium after short-term exposure to ozone.

Materials and Methods

Animals, neutrophil depletion, and exposure. One hundred and twenty male F3 44/N
rats (Harlan Sprague-Dawley, Indianapolis, IN), 10 - 12 week old, were used in this
study. To morphometrically determine the effect of neutrophil-depletion on ozone-
induced epithelial proliferation and MCM, 48 rats were randomly assigned into one of 8
experimental groups (n = 6/group) (Table 3-lA). To determine the effect of neutrophil-
depletion on ozone-induced increases in the steady-state levels of rMuc-SAC mRNA, 72
rats were randomly divided into one of 12 experimental groups (n = 6/ group) (Table 3-
18). Rats were housed two per cage in polycarbonate shoebox-type cages with Cell-Sorb
Plus bedding (A&W Products, Inc., Cincinnati, OH) and filter caps. Water and food (Tek

Lab 1640; Harlan Sprague Dawley, Indianapolis, IN) were available ad libitum. Rats

83

Table 3-1. Experimental groups and animal assignment.

A. Morphometric Analyses

 

 

 

0 ppm (Filtered Air) 0.5 ppm
Postexposure Time Control Serum Antiserum Control Serum Antiserum
2 h afier 3-day exposure 6' 6 6 6
4 days after 3-day exposure 6 6 6 6

 

B. Mucin (rMuc-SAC) mRNA Analysis

 

 

 

0 ppm (Filtered Air) 0.5 ppm
Postexposure Time Control Serum Antiserum Control Serum Antiserum
2 h after l-day exposure 6 6 6 6
2 h after 3-day exposure 6 6 6 6
4 days after 3-day exposure 6 6 6 6

 

' Animal number.

84

were maintained on a 12-h light/dark cycle beginning at 6:00 am. under controlled
temperature (16 - 25°C) and humidity (40 - 70%). Rats were conditioned in whole-body
exposure chambers (HC-lOOO, Lab Products, Maywood, NJ) supplied with filtered air for
1 day prior to the start of the inhalation exposure as described in Chapter 2.

Fourteen h prior to inhalation exposure, the rats were briefly anesthetized with 4%
halothane in oxygen, and half of the rats were depleted of circulating neutrophils using an
intraperitoneal (ip) injection of 1 ml rabbit anti-rat neutrophil antiserum (antiserum;
Accurate Scientific Corp., Westbury, NY). In normal rats, a single ip injection of this
antiserum is known to deplete the number of circulating blood neutrophils to below 1%
of normal levels in 12 h, and the depletion persists for up to 5 days post-injection (Snipes
et al., 1995). The antiserum is specific for mature neutrophils without damaging cellular
precursors in the bone marrow or other blood components such as red blood cells (Snipes
et al., 1995; Davis et al., 1969). The remaining rats were treated ip with 1 ml of normal
rabbit serum (control serum; Accurate Scientific Corp., Westbury, NY).

The control serum- or antiserum-treated rats designated for morphometric analyses
were exposed to either 0.5 ppm ozone or filtered air (0 ppm), 8 h/day, for 3 days. The
other rats designated for mucin-specific mRNA analysis were exposed to either 0.5 ppm
ozone or filtered air (0 ppm) for 1, 3, or 7 days. All the rats were exposed daily to ozone
or filtered air in the whole-body exposure chambers from 6 am to 2 pm. Ozone was
generated with an OREC Model O3VI-O ozonizer (Ozone Research and Equipment
Corp., Phoenix, AZ) as explained in detail in Chapter 2. The chamber ozone

concentrations (mean 1 standard deviation) for 1 - 3 days of exposure to 0.5 ppm-ozone

8S

were 0.500 j; 0.008. The chamber ozone concentrations during exposures to filtered air

were maintained less than 0.05 ppm.

Blood collection and assessment of circulating neutrophils. At the end of 3 days of
exposure, all the rats exposed for the morphometric analyses were treated ip with 5’-
bromo-2»deoxyuridine (BrdU; 50 mg/Kg body wt.) to label cells undergoing DNA
synthesis in the S-phase of the cell cycle. At 2 h or 4 days later, rats were anesthetized by
4% halothane in oxygen, and approximately 2-ml of blood were drawn fi'om the vena
cava or the left ventricle of the heart of each rat to assess the number of circulating
neutrophils. Blood was collected in evacuated blood collection tubes (Becton Dickenson,
Rutherford, NJ) containing K3-ethylenediaminetetraacetic acid (EDTA). The number of
nucleated cells per cubic millimeter of blood was measured with a Serono-Baker System
9000 automated cell counter (Serono-Baker Diagnostics, Allentown, PA). Differential
counts of leukocytes were determined by counting 100 nucleated white blood cells from
blood smears stained with Wright-Giemsa stain (Diff-Quik; Baxter, McGaw Park, IL).
The total number of neutrophils per cubic millimeter of blood was determined by
multiplying the percent occurrence of neutrophils (i. e., the number of neutrophils per 100
white blood cells) by the total number of nucleated white blood cells per cubic millimeter
of blood. After collecting the blood, rats were killed by exsanguination via the

abdominal aorta.

86

Necropsy and tissue preparation for morphometric analyses. After death, the head of
each rat was removed from the carcass, and the nasal airways were flushed retrograde
through the nasopharyngeal orifice with 5 ml of zinc formalin (Anatech, Ltd.,
Kalamazoo, MI). Afier the eyes, lower jaw, skin and musculature were removed from the
head, the nasal tissues were stored in a large volume of the same fixative for a minimum
of 48 h.

The zinc formalin-fixed nasal tissues were decalcified in 13% formic acid for 4 days,
and then rinsed in tap water for 2 h as previously described by Harkema et al. (1988). A
tissue block was removed from the proximal aspect of the nasal cavity by making two
transverse cuts perpendicular to the hard plate. The first cut was immediately posterior to
the upper incisor tooth (Figure 2-1A in page 49), and the second cut was at the level of
the incisive papilla. The tissue block was excised, embedded in paraffin, and 5 rim-thick
sections were cut from the anterior face of the tissue block.

Nasal tissue sections from each tissue block were histochemically stained with
hematoxylin and eosin for morphological identification of epithelial cells or Alcian Blue
(pH 2.5)/Periodic Acid-Schifl’s sequence (AB/PAS) to identify acidic and neutral
mucosubstances in the surface epithelium, and immunohistochemically stained with anti-
BrdU antibody (Becton Dickinson Immunocytometry Systems, San Jose, CA) to detect

BrdU-labeled nuclei (Johnson et al. , 1990).

87

Morphometry of neutrophilic inflammation, epithelial cell numeric density and DNA
synthesis. The NTE overlying the maxilloturbinate of each animal was analyzed using
computerized image analysis and standard morphometric techniques (Hotchkiss and
Harkema, 1992; Hotchkiss et al., 1991). Neutrophilic inflammation (intraepithelial
neutrophils/mm basal lamina), epithelial cell labeling index (Ll; % BrdU-labeled
epithelial cell nuclei) and epithelial cell numeric density (epithelial nuclei/mm of basal
lamina) were determined using a Power Macintosh 7100/66 computer and the public
domain image analysis software (NIH Image; written by Wayne Rasband at the US.
National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-

image/) as described in detail in Chapter 2.

Morphometry of stored intraepithelial mucosubstances and mucous cells. The
volume density (Vs) of AB/PAS-stained mucosubstances and the numeric cell densities
of mucous cells (epithelial cells containing AB/PAS-stained mucosubstances) in the NTE
lining the maxilloturbinates were determined using computerized image analysis and

standard morphometric techniques as explained in Chapter 2.

Necropsy and microdissection of tissues for mucin mRNA analysis. For mucin mRN A
analysis, rats exposed to ozone or filtered air for 1 day were killed immediately after the
end of exposure. Rats exposed to ozone or filtered air for 3 days were killed immediately
or 4 days after the end of exposure. We chose these three time points for rMuc-SAC
mRNA analysis based on our previous findings that ozone induces mucin mRNA

upregulation after 1 day of ozone exposure (8 h, 0.5 ppm) and this increased mucin

88

mRNA level persists 2 days postexposure (Cho et al., 1997). The head of each rat was
removed and the nasal airways were opened by splitting the nose in a sagittal plane
adjacent to the midline. Maxilloturbinates were excised by microdissection from both
nasal passages of each head (Figure 2-1A in page 49) as described in Chapter 2, and
immediately homogenized in Tri-Reagent (Molecular Research Center, Cincinnati, OH)

to isolate total RNA and analyze the abundance of mucin (rMuc-SAC) mRNA.

Analysis for mucin mRNA in nasal tissues. Total cellular RNA was isolated from the
maxilloturbinate homogenate according to the method of Chomczynski et al. (1987)
following the procedure in Chapter 2. The RNA was then analyzed by reverse
transcriptase polymerase chain reaction (RT-PCR) with rat MU C-SAC-specific primers
to determine the steady-state levels of rMuc-SAC mRNA in maxilloturbinates following
the procedures described in Chapter 2. Cyclophilin mRNA was used as an internal
standard in this semi-quantitative RT-PCR analysis. The abundance of mucin mRNA

was determined by densitometric analysis as described in Chapter 2.

Statistical analyses. All data were expressed as the mean group value :1: the standard
error of the mean (SEM). The data were log transformed to make the variances
approximately equal for all groups. The data from morphometric analyses were analyzed
by three-way analysis of variance (ANOVA) to determine the potential effects of
exposure atmosphere (filtered air or ozone), type of serum injection (control serum or

antiserum), and postexposure time (2 h or 4 days) on circulating neutrophils, neutrophilic

89

influx, epithelial proliferation, and MCM. Student-Newman-Keuls Method, an all
pairwise multiple comparison procedure, was then used to determine the significant
differences in group mean values. The data from rMuc-SAC mRNA analysis were first
analyzed by three-way AN OVA as described above. Only the exposure atmosphere and
the type of serum injection were identified as factors contributing to variances in group
mean mRNA levels. Therefore, data from similarly exposed and injected experimental
groups (e.g., antiserum-treated/air-exposed) that were sacrificed at different times were
combined. The pooled data were then analyzed by Analysis of Contrasts to determine the
differences in the mean rMuc-SAC mRN A levels among the combined experimental
groups. Statistical analyses were performed using a commercial statistical analysis
package (SigmaStat; Jandel Scientific Software, San Rafael, CA). The criterion for

statistical significance was set at p S 0.05 at all analyses.

Results

Number of circulating blood neutrophils. The ip injection of anti-neutrophil
antiserum dramatically decreased the numbers of circulating neutrophils to 12% or 6% of
those in control serum-treated rats exposed to air or ozone, respectively, at 2 h after 3
days of exposure (i.e., 4 days after antiserum treatment) (Figure 3-1). At 4 days
postexposure (i.e., 8 days after antisenun treatment), the number of circulating

neutrophils in antiserum-treated/air—exposed rats was not significantly different from that

90

in air-exposed/control serum-treated rats. However, there was a slight increase (18%) in
the number of circulating neutrophils in antiserum-treated/ozone-exposed rats killed at 4
days postexposure, compared to control serum-treated/ozone-exposed rats. There were
no significant differences in the numbers of circulating neutrophils in air- or ozone-

exposed rats treated with control serum at either postexposure time.

Nasal histopathology. No exposure-related lesions were observed microscopically in
the nasal mucosa of rats treated with either control serum or antiserum and exposed to
filtered air (0 ppm ozone). The nasal lesions in rats exposed to 0.5 ppm ozone were
restricted to the mucosa containing the NTE that lined the lateral meatus in the proximal
nasal cavity of both antiserum- and control serum-treated rats.

Two h after 3 consecutive days of exposure to 0.5 ppm ozone, the principal feature in
the NTE of control serum-treated rats was the appearance of widely scattered mitotic
figures and epithelial hyperplasia. These rats had NTE that was approximately 3 - 4 cells
in thickness. In contrast, control serum-treated/air-exposed rats had NTE that was 1 - 2
cells in thickness. Concurrent with the ozone-induced epithelial hyperplasia was a mild
inflammatory response in the nasal mucosa that was characterized by endothelial
margination of neutrophils in the large capacitance vessels of the lamina propria and an
influx of neutrophils in both the lamina propria and NTE. These ozone-induced
alterations in the NTE were most noticeable in the dorsal and medial aspects of the
maxilloturbinate, the lateral ridge of the nasoturbinate and the dorsal recess of the lateral
wall. Rats in the antiserum-treated/ozone-exposed group also had conspicuous mitotic

figures and epithelial hyperplasia (3 - 4 cells in thickness) in the NTE like those in the

91

control serum-treated/ozone-exposed rats. However, there was no associated
inflammatory cell influx in the hyperplastic NTE of the antiserum-treated/ozone-exposed
rats.

At 4 days after 3 days of ozone exposure, the principal feature in the rats treated with
control serum was MCM characterized by copious AB/PAS-stained mucosubstances in
the mucous cells in the NTE. The ozone-induced MCM in the NTE was most severe in
the dorsal and medial aspects of the maxilloturbinates. Hyperplasia was still evident in
the NTE of these rats. In contrast, antiserum-treated/ozone-exposed rats had only a few
scattered AB/PAS-stained mucous cells in the hyperplastic NTE. One or two rat(s) in
this exposure group had no AB/PAS-positive mucous cells in the NTE lining one
maxilloturbinate. No intraepithelial neutrophils were evident in the NTE of all the rats
killed 4 days postexposure. Figures 3-2 and 3-3 illustrate morphologic similarities and
differences in the NTE lining the maxilloturbinates from air-control, ozone-
exposed/control serum-treated and ozone-exposed/antiserum-treated rats killed at 4 days

postexposure.

Neutrophilic inflammation. Figure 34 illustrates the number of intraepithelial
neutrophils in the NTE 2 h or 4 days after the end of 3 days of exposure to filtered air or
0.5 ppm ozone. At 2 h postexposure, control serum-treated/ozone-exposed rats had
significantly more intraepithelial neutrophils in the NTE, compared to control serum-
treated/air-exposed rats (16-times greater). At the same time point, antiserum-
treated/ozone-exposed rats had markedly fewer intraepithelial neutrophils (87% less),

compared to control serum-treated/ozone-exposed rats. The numeric density of

92

intraepithelial neutrophils was, however, still significantly greater (3.5-times) than that of
antiserum-treated/air-exposed rats. Four days following the last exposure, there was no
difference in the numbers of intraepithelial neutrophils in all rats, independent of
inhalation exposure and type of serum treated. All air-exposed rats had few neutrophils

in the NTE at both postexposure times.

Epithelial cell proliferation. Ozone induced significant increases in the NTE cell
labeling index in both control serum-treated and antiserum-treated rats (20- and 2.8-times
greater than that in corresponding air-exposed controls, respectively) sacrificed 2 h after
the end of exposure (Figure 3-5). There was, however, no significant difference between
the NTE cell labeling index of ozone-exposed rats treated with control serum and
antiserum at this time point. Four days after the end of exposure, the NTE cell labeling
index of the ozone-exposed rats was not significantly different from that of air-exposed
control rats (0 - 2 labeled cells/animal), regardless of the type of serum injected. Air-
exposed/antiserum-treated rats sacrificed 2 h after the end of exposure had significantly
increased NTE labeling index (6-times) than air-exposed/control serum-treated controls.
Two h after the end of exposure, ozone induced a significant increase in the number
of NTE cells in rats treated with control serum (31%), but not in antiserum-treated rats,
compared to air-exposed rats treated with same type of serum (Figure 3-6). At 4 days
postexposure, ozone-exposed rats treated with either control serum or antiserum had
significantly more NTE cells (35% and 31%, respectively), compared to air-exposed rats

treated with same type of serum. There was no significant difference in the number of

93

NTE cells between air-exposed rats sacrificed at either time point, regardless of the type

of serum injected.

Mucous cell metaplasia. At 4 days postexposure, ozone-exposed/control serum-treated
rats had significantly more intraepithelially stored mucosubstances (100-fold) and
mucous cells (18-fold) in the NTE than did air-exposed/control serum-treated rats
(Figures 3-7 and 3-8). Ozone-exposed/antiserum-treated rats had significantly less
intraepithelial mucosubstances and fewer mucous cells in the NTE (66% and 58%,
respectively) than ozone-exposed/control serum-treated rats sacrificed at the same
postexposure time. However, these ozone-exposed/antisenun-treated rats still had
significantly more intraepithelial mucosubstances (35-fold) and mucous cells (7-fold)
than air-exposed/antiserum-treated rats. Air-exposed rats injected with either control
serum or antiserum had few mucous cells and little intraepithelial mucosubstances in the
NTE. At 2 h postexposure, little stored intraepithelial mucosubstances and few mucous

cells were detected in any experimental group.

rMuc-5AC mRNA expression. Ozone exposure induced a significant increase in
steady-state levels of rMuc-SAC mRNA in both control serum-treated (141%) and
antiserum-treated (5 8%) rats, compared to air-exposed control rats treated with the same
type of serum (Figure 3-9). Air-exposed rats treated with antiserum had 2-fold greater

steady-state rMuc-SAC mRN A levels than air-exposed rats treated with control serum.

94

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104

(a) Control Serum Anflserum
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mRNA upregulation in maxilloturbinates. (a) Digitized
image of representative RT-PCR cDNA bands from each
pooled experimental group on an agarose gel stained with
ethidium bromide. cDNA products for rMuc-5 AC and
cyclophilin were produced by reverse transcription of RNA
from maxilloturbinates and PCR-amplification using primers
specific for rat Muc-SAC and cyclophilin cDNA sequences.
(b) Bars represent the pooled group mean _+_- SEM (n = 18/
group). *Significntly different from control serum-treated!
air-exposed rats (p 5 0.05). #Significantly different from
air-exposed rats injected with the same type of serum (p 5
0.05).

105

Discussion

The results of the present study indicate that ozone-induced MCM is, at least in part,
neutrophil-dependent, while the induced epithelial proliferation (i. e., epithelial DNA
synthesis and hyperplasia) and the increase in mucin-specific (rMuc-SAC) mRNA levels
are independent of the ozone-induced neutrophilic influx in the NTE of rats. To the best
of our knowledge, this is the first study which reports the contribution of neutrophils in
ozone-induced nasal epithelial alterations in the NTE of rats. Treatment with anti-
neutrophil serum depleted the circulating pool of neutrophils and markedly attenuated the
ozone-induced neutrophilic influx into the NTE (~ 90% fewer than control serurn-
treated/ozone-exposed rats), by 2 h after 3 days of exposure. At four days afier exposure,
antiserum-treated animals had 60 - 70% less ozone-induced MCM, compared to ozone-
exposed rats treated with control serum. In contrast, these antiserum-treated, ozone-
exposed animals still had a similar magnitude of epithelial cell proliferation, compared to
that observed in control serum-treated, ozone-exposed animals killed the same
postexposure time. In addition, ozone exposure induced a similar increase in rMuc-SAC
mRN A in both control serum- and antiserum-treated rats.

The possible involvement of neutrophilic inflammation in the pathogenesis of ozone-
induced MCM has been investigated in recent studies in our laboratory by attenuating or
augmenting the inflammatory response in the nasal airways of ozone-exposed rats. In
one of these studies, rats repeatedly exposed to ozone (0.5 ppm, 8 h/day for 3 or 5 days)

and concurrently treated with a topical steroid, fluticasone propionate (50 ug/rat, 2

106

times/day by intranasal instillation), had significantly less neutrophilic inflammation and
markedly attenuated MCM in the NTE than did rats exposed to ozone but intranasally
instilled with saline only (Hotchkiss et al., 1998). In another recent study, rats were
exposed to ozone (0.5 ppm, 8 h/day for 3 days) and then intranasally instilled with a
potent proinflammatory agent, bacterial endotoxin (100 ug/day for 2-consecutive days),
prior to the appearance of MCM. Ozone-exposed rats instilled with endotoxin had
markedly enhanced MCM in the NTE, compared to ozone-exposed, saline-instilled rats
(Fanucchi et al., 1998). Results of these studies, like the results of the present study,
suggest that neutrophilic inflammation may play a crucial role in the pathogenesis of
MCM in the NTE of ozone-exposed rats.

A causative relationship between neutrophil accumulation and abnormal increases in
mucous cells has also been addressed by previous studies in distal airways of rodents.
Instillation of supernatant from either lysed, purified neutrophils or activated neutrophils
into the trachea of hamsters (Snider et al., 1985) or rats (Lundgren et al., 1988) results in
an increase (50 - 300% above controls) of the number of mucous goblet cells in the
tracheobronchial epithelium. The MCM or mucous cell hyperplasia induced by the
neutrophil-conditioned supernatant was inhibited by an anti-inflammatory glucocorticoid,
dexamethasone (Lundgren et al., 1988). Anti-inflammatory drugs including
dexamethasone and indomethacin have also been shown to inhibit the MCM or mucous
cell hyperplasia induced by cigarette smoke which induces neutrophilic inflammation in
pulmonary airways of rats (Jones and Reid, 1978; Rogers and Jeffery, 1986). Even

though these previous studies suggest that inflammation is an important factor in the

107

development of MCM or mucous cell hyperplasia in rodent airways, little is known about
the underlying mechanisms by which neutrophils contribute to in the abnormal
proliferation or differentiation of airway mucous cells.

Neutrophils are a primary source of inflammatory mediators. Proteases derived fi'om
neutrophils (i.e., cathepsin G, elastase) are well known mucous secretagogues in airway
epithelial cells (Breuer et al. , 1993; Sommerhoff et al. , 1990). Intra-airway instillation of
neutrophil elastase induces MCM in hamster airways (Breuer et al., 1985, 1993; Jamil et
al., 1997). Elastase inhibitors (e.g., chloromethyl ketone, eglin C) as well as
dexamethasone have been shown to prevent the MCM induced by neutrophil elastase
(Snider et al., 1985; Lundgren et al., 1988). Janoff et al. (1979, 1983) have suggested
that cigarette smoke-induced abnormal increases of mucous cells in rat pulmonary
airways may be due to an inactivation of endogenous anti-proteases (e.g., al-antitrypsin)
resulting in enhanced proteolytic activity in pulmonary airways. These findings suggest
that neutrophil-derived proteases may play an important role in the pathogenesis of
airway mucous overproduction and chronic obstructive airway disorders.

In addition to the proteases, neutrophils release several inflammatory cytokines
including tumor necrosis factor-alpha (TNF-a) and interleukin-6 (IL-6) that can stimulate
other airway resident cells to release various inflammatory mediators. Recent
investigations have focused on the role of inflammatory cytokines in abnormal airway
mucous responses. In vitro studies have demonstrated that interleukin-10 (IL-10), TNF-
or, and IL-6 can cause mucin hypersecretion and/or mucin gene upregulation in airway

epithelial cells (Levine et al., 1994, 1995; Jarry et al., 1996). Transgenic mice

108

overexpressing IL-4 or IL-5 have increased mucous cells (Rankin et al. , 1996; Jain-Vera,
et al., 1997; Lee et al., 1997) as well as mucin hypersecretion (Temann et al., 1997) in
tracheobronchial and pulmonary airways. Inflammatory cytokines have been found in the
bronchoalveolar or nasal lavage fluid and airway tissues of humans exposed to ozone
(Calderon Garciduenas et al., 1995; Koren et al., 1990; Devlin, et al., 1991). Certain
ozone-inducible cytokines such as IL-6 have been suggested as possible mediators of
cellular reparative responses in ozone-injured rat pulmonary airways by attenuating initial
injury and inflammation (McKinney et al., 1998). Future studies are needed to determine
the role of inflammatory cytokines and other soluble mediators derived from neutrophils
in the pathogenesis of ozone-induced MCM in the NTE of rats.

In the present study, we also examined the role of neutrophils in ozone-induced
proliferative responses in the NTE. Our findings suggest that neutrophils do not play a
role in the ozone-induced epithelial proliferation, although the onset of epithelial
hyperplasia seems to be delayed in antiserum-treated rats exposed to ozone (i. e., at 2 h
postexposure, no significant increase of the NTE cell number was observed in these
animals, compared to control serum-treated/air-exposed controls). Similar observations
were made in our recent studies in which the magnitude of ozone-induced epithelial
proliferation was not affected by the severity of concurrent inflammation in the NTE
(Hotchkiss et al., 1998; Fanucchi et al., 1998). Several previous studies have
demonstrated that ozone-induced epithelial injury in pulmonary airways of rats is mainly
mediated, not by neutrophils, but by direct ozone toxicity (Hyde et al., 1992; Pine eta1.,

1992b; Schuller Levis et al., 1994). It may be possible that ozone-induced pre-

109

metaplastic epithelial responses, cell necrosis and subsequent compensatory proliferation,
are events that are mediated by neutrophil-independent mechanisms in the NTE.

In addition, the present study was designed to determine the involvement of
neutrophilic inflammation in ozone-induced increase in mucin (rMuc-SAC) mRN A
levels, a potential early molecular indicator of subsequent MCM in the NTE. The results
of the present study do not support our hypothesis that neutrophils contribute to the
ozone-induced elevation of mucin mRNA expression in the NTE. Several investigators
have demonstrated that irritant-induced MCM in airway epithelium with accompanying
increases in mucin mRN A expression is modulated at transcriptional and/or post-
transcriptional levels (Jany et al., 1991; Borchers and Leikauf, 1997). In addition, some
inflammatory mediators have induced mucin gene upregulation as well as mucous
overproduction in airway epithelial cells as described above (V oynow et a1. , 1997; Breuer
et al., 1985; Temann et al., 1997). Our present results, however, suggested that the
upregulation of mucin mRNA, in the absence of concurrent neutrophilic inflammation, is
not sufficient for the full phenotypic development of ozone-induced MCM. It seems
likely that the most critical events in the pathogenesis of ozone-induced MCM is the
translation of mucin mRNA or post-translational processing of the apomucin core protein
(e.g., glycosylation, transport through rough endoplasmic reticulum and Golgi, storage
into secretory granules) in the NTE, which may be neutrophil-dependent. In addition to
the activation of biosynthesis of this characteristic functional molecule (i. e., mucin) in the
transformed mucous cells, neutrophils could play roles in the inhibition of mucin

secretion from the mucous granules or in the architectural differentiation of mucous cells.

110

Though it is clear that ozone exposure resulted in a further elevation of rMuc-SAC
mRN A (5 8%) than air exposure in antiserum-treated rats, antiserum treatment induced a
2-fold increase in rMuc-SAC mRNA levels in air-exposed control rats, compared to the
basal expression of rMuc-SAC in control serum-treated/air-exposed rats. We do not
know the reason, however, several possibilities are postulated. There would be mild
bacterial infection in the respiratory airways of these animals due to depletion of the
body’s primary defense system, which may lead to induction of certain cellular genes. It
may be also possible that small numbers of neutrophils which normally present in nasal
tissues could play a role in maintaining the homeostasis of epithelium. Otherwise,
phagocytosis of the antiserum-bound circulating neutrophils may release soluble
substances which finally signal for the induction of mucin genes. We observed an
unexpected increases of the epithelial DNA synthesis in the NTE of antiserum-treated,
air-exposed rats at 2 h postexposure, and it seems likely due to the similar reasons.

Although neutrophil depletion did markedly attenuate the subsequent development of
ozone-induced MCM in the NTE of rats, it did not completely eliminate it. This
observation suggests that although neutrophilic inflammation plays a major role in

development of MCM, neutrophil-independent mechanisms may also contribute to the

ozone-induced MCM in rat NTE.

111

CHAPTER 4

Effects of Pre—Existing Rhinitis on Ozone-Induced Mucous Cell Metaplasia
in Rat Nasal Airways

This study was supported by NIHHL59391.

Manuscripts submitted to Toxicology and Applied Pharmacology.

112

Abstract

People with airway diseases may be more susceptible to the adverse effects of air
pollutants than healthy subjects. Ozone causes rhinitis and nasal epithelial alterations.
The toxicity of ozone on nasal airways with pre—existing rhinitis has not been
investigated. The present study was designed to determine the effect of endotoxin-
induced rhinitis on ozone-induced epithelial alterations, especially mucous cell
metaplasia (MCM), in the nasal transitional epithelium (NTE) of rats. Six h prior to daily
inhalation exposure, male F344/N rats were intranasally instilled with saline or endotoxin
(100 ug/day). Rats were killed 2 h or 4 days after 3-day (8 h/day) exposure to ozone (0.5
ppm) or filtered air (0 ppm). The maxilloturbinate from one nasal passage was processed
for morphometric analyses of the numbers of neutrophils and epithelial cells, and the
amount of intraepithelial mucosubstances (IM) in the NTE._ The maxilloturbinate from
the other nasal passage was processed for a mucin-specific (rMuc-SAC) mRNA analysis.
At 2 h postexposure, endotoxin/ozone-exposed rats had 48 and 3 times more neutrophils
in the NTE than did saline/air- and saline/ozone-exposed rats, respectively. Ozone-
exposed rats had 35% more NTE cells and 2-fold more mucin mRNA than did saline/air-
exposed rats, independent of endotoxin exposure. At 4 days postexposure,
endotoxin/ozone-exposed rats had 5 and 2 times more IM and mucous cells, respectively,
than did saline/air- and saline/ozone-exposed rats. Though endotoxin/air-exposed rats
killed at 2 h postexposure had more neutrophils (40 fold), epithelial cells (27%) and

mucin mRNA (2 fold) in the NTE than did saline/air-exposed rats, no MCM was present

113

in those rats killed at 4 days postexposure. The results of the present study indicated that

pre-existing rhinitis augments ozone-induced MCM.

Introduction

Ozone is the major oxidant gas in photochemical smog. Inhalation of ozone induces
morphologic and biochemical changes in the respiratory mucosa of humans as well as
laboratory animals. In F344/N rats, exposure to acute (i. e., days) or chronic (i. e., weeks
or months) ozone (0.5 - 1.0 ppm) induces rhinitis and marked mucous cell metaplasia
(MCM) in the nasal transitional epithelium (NTE) lining the lateral meatus of the
proximal nasal airways of rats (Harkema et al., 1997, 1989; Hotchkiss et al., 1991). How
ozone exposure induces MCM is unknown. However, we have recently observed that the
ozone-induced MCM is, at least in part, dependent on neutrophilic inflammation (Cho et
al. , 1998), and is markedly attenuated by an anti-inflammatory steroid, fluticasone
propionate (Hotchkiss et al., 1998). In addition, neutrophilic inflammation accompanies
an increase of mucin-specific gene (rMuc-SAC mRNA) expression prior to the onset of
the MCM in the NTE (Cho et al., 1997).

Both cellular inflammation and overproduction/hypersecretion of mucus are
important factors in the pathogenesis of airway diseases such as asthma, chronic
bronchitis, and allergic rhinitis (Robbins et al., 1984a,b; Aikawa et al., 1992). Since

ozone exposure also induces airway inflammation and mucous hypersecretion, it is

114

possible that patients suffering from chronic airway diseases may be more susceptible to
the toxic effects of ozone.

Gram-negative bacterial infection (e.g., Haemophilus influenzae) is frequently
observed in patients with chronic airway diseases (Hamacher et al., 1995; Taytard et al.,
1995; Imundo et al.,1995). Endotoxins are lipopolysaccharide-protein molecules, located
in the outer cell walls of gram-negative bacteria, which are responsible for the bacteria-
induced inflammatory responses (Brigham et al., 1986; Reyes et al., 1980). Endotoxins
are potent chemotaxinogens for neutrophils (Issekutz et al., 1982; Hewett and Roth,
1993). In rats, a single intranasal instillation of endotoxin induces a transient, but
conspicuous neutrophilic rhinitis (Harkema et al. , 1988). Though endotoxin induces
DNA synthesis and epithelial proliferation in the NTE of rats (Harkema and Hotchkiss,
1993), it does not cause the MCM that is a principal feature of ozone-induced alterations
in the NTE (Harkema and Hotchkiss, 1991).

The present study was designed to examine the influence of pre-existing, endotoxin-
induced neutrophilic rhinitis on acute ozone-induced alterations in the NTE of rats. For
this purpose, animals were treated intranasally with endotoxin prior to daily inhalation
exposure to ozone. We determined the effects of endotoxin-induced rhinitis on the
severity of ozone-induced MCM as well as epithelial hyperplasia and mucin mRNA
upregulation in the NTE. The results of this study confirmed our hypothesis that pre-

existing airway inflammation exacerbates the nasal epithelial lesions induced by ozone.

115

Materials and Methods

Animals and exposure. Sixty-four male F344/N rats (Harlan Sprague-Dawley,
Indianapolis, IN), 10 - 12 weeks of age, were randomly assigned into one of 8
experimental groups (n = 8/ group). Rats were housed two per cage in polycarbonate
shoebox-type cages with Cell-Sorb Plus bedding (A&W Products, Inc., Cincinnati, OH)
and filter caps. Water and food (Tek Lab 1640; Harlan Sprague Dawley, Indianapolis,
IN) were available ad libitum. Rats were maintained on a 12-h light/dark cycle beginning
at 6:00 am. under controlled temperature (16 - 25°C) and humidity (40 - 70%). Rats
were conditioned in whole-body exposure chambers (HC-1000, Lab Products, Maywood,
NJ) supplied with filtered air for 1 day prior to the start of the inhalation exposure. The
rats were individually housed in rack-mounted stainless-steel wire cages with free access
to food and water. The chamber temperature and relative humidity in the chamber as
well as room light setting were maintained as described above.

Six h prior to daily inhalation exposure, rats were briefly anesthetized with 4%
halothane in oxygen, after which 50 ul of bacterial endotoxin (lipopolysaccharide from
Pseudomonas aeruginosa Serotype 10; Sigma Chemical Co., St. Louis, MO) in pyrogen-
free saline (1 mg endotoxin/ml saline) were instilled into each nasal passage of 32 rats.
The other 32 rats were treated with only pyrogen-free saline (vehicle control). We have
previously reported that six h after intranasal instillation of endotoxin, rats have a marked

neutrophilic rhinitis (Harkema et al. , 1988).

116

Rats instilled with saline or endotoxin were exposed to either filtered air (0 ppm, air
control) or 0.5 ppm ozone in the whole-body exposure chambers, 8 h/day (10 pm - 6 am),
for 3 days. Ozone was generated with an OREC Model OBVI-O ozonizer (Ozone
Research and Equipment Corp., Phoenix, AZ) as explained in detail in Chapter 2. The
chamber ozone concentrations (mean 1 standard deviation) during the 3-day-exposures to
0.5 ppm-ozone were 0.501 _t 0.011. The chamber ozone concentrations during the 3-

day-exposures to filtered air were maintained less than 0.05 ppm.

Necropsy and tissue preparation for morphometric analyses and RNA isolation. Rats
were killed 2 h or 4 days afier the end of 3—day inhalation exposure. Rats were deeply
anesthetized using 4% halothane in oxygen and killed by exsanguination via the
abdominal aorta. Immediately after death, the head of each rat was removed from the
carcass. After the eyes, lower jaw, skin and musculature were removed from head, the
nasal airways were opened by splitting the nose in a sagittal plane adjacent to the midline.
The maxilloturbinate from one nasal passage (Figure 2-1A in page 49) was excised by
microdissection, and immediately homogenized in Tri-Reagent (Molecular Research
Center, Cincinnati, OH). The homogenate was snap frozen in liquid nitrogen and stored
at -80°C until further processed for isolation of total RNA and analysis of mucin mRNA.
The opposite nasal passage was fixed in a large volume of ice-cold 2%
glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for at least 2 h. After fixation, a
portion of the maxilloturbinate in the nasal passage was microdissected from the

proximal nasal airway. The dissected portion of the maxilloturbinate extended from the

117

level of the upper incisor tooth to the distal end of the turbinate (Figure 2-1A in page 49).
The dissected tissue was decalcified with 10% EDTA in 0.2 M phosphate buffer for 7
days at 4°C with gentle shaking. The decalcified maxilloturbinates were post-fixed in 1%
phosphate-buffered osmium tetraoxide (OsO4), dehydrated in increasing ethanol
concentration, and rinsed with propylene oxide. The tissues were then infiltrated with
epoxy resin (Poly/Bed 812-Araldite) sequentially (50% resin in propylene oxide,
overnight; 100% resin for 8 h) at room temperature, and epoxy resin was polymerized at
60°C for 2 days. Semi-thin (1 pm in thickness) tissue sections were cut from the anterior
face (i.e., level of the incisor tooth) of each tissue block for light microscopic analyses.
One tissue section from each block was histochemically stained with 1% toluidine
blue for morphological identification of epithelial cells and infiltrated neutrophils in the
NTE. Another tissue section from each block was stained with Alcian Blue (pH
2.5)/Periodic Acid-Schifi‘s sequence (AB/PAS) to identify acidic and neutral

mucosubstances in the surface epithelium.

Morphometry of neutrophilic inflammation and epithelial cell numeric density. The
NTE lining the maxilloturbinate of each animal were analyzed using image analysis and
standard morphometric techniques (Hotchkiss and Harkema, 1992; Hotchkiss et al. ,
1991). Neutrophilic inflammation (intraepithelial neutrophils/mm basal lamina) and
epithelial cell numeric density (epithelial nuclei/mm of basal lamina) were determined
using a Power Macintosh 7100/66 computer and the public domain image analysis

software (NIH Image; written by Wayne Rasband at the US. National Institutes of Health

118

i” . mmJ

 

 

and available on the Internet at http://rsb.info.nih.gov/nih-irnage/) as described in detail in

Chapter 2.

Morphometry of stored intraepithelial mucosubstances and mucous cells. To
estimate the amount of the intraepithelial mucosubstances in NTE lining the
maxilloturbinates, the volume density (Vs) of AB/PAS-stained mucosubstances was
quantified using the computerized image analysis system and standard morphometric
techniques as described in Chapter 2. The percentage of mucous cells (epithelial cells
containing AB/PAS-stained mucosubstances) in the NTE lining the maxilloturbinates
was also morphometrically determined by dividing the number of mucous cell nuclei by

the total number of epithelial cell nuclei and multiplying by 100.

Analysis for mucin mRNA in maxilloturbinates. Total cellular RNA was isolated from
the maxilloturbinate homogenate according to the method of Chomczynski et al. (1987).
The RNA was then analyzed by reverse transcriptase polymerase chain reaction (RT-
PCR) with rat MUC-SAC-specific primers to determine the steady-state levels of rMuc-
SAC mRNA in maxilloturbinates. Cyclophilin mRNA was used as an internal standard
in this semi-quantitative RT-PCR analysis. The abundance of mucin mRNA was

determined by densitometric analysis. All of these techniques are described in detail in

Chapter 2.

119

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Statistical Analyses. All data were expressed as the mean group value :1: the standard
error of the mean (SEM). The natural logarithms of the data were used for statistical
analyses to make the variances approximately equal for all groups. The data were first
analyzed using three-way analysis of variance (ANOVA) to determine the potential
effects of inhalation exposure (air to ozone), intranasal instillation (saline to endotoxin),
and time after inhalation exposure (2 h to 4 days). Student-Neuman-Keuls Method, an all
pairwise multiple comparison procedure, was followed to identify differences in the
group means. Statistical analyses were performed using a commercial statistical analysis

package (SigmaStat; Jandel Scientific Software, San Rafael, CA). The level of statistical

significance was set at p g 0.05.

120

Results

Histopathology of nasal mucosa. No exposure-related lesions were observed in the
nasal mucosa of maxilloturbinates lined by NTE in saline/air-exposed rats (control) at
either postexposure time. Two h after 3 days of inhalation exposure to either air or
ozone, rats intranasally instilled with endotoxin had a similar degree of severe
neutrophilic influx (i.e., neutrophilic rhinitis) principally observed in the dorsal and
medial aspect of the maxilloturbinates (Figure 4-1). The neutrophilic rhinitis was
characterized by endothelial margination of neutrophils in the large capacitance vessels of
the lamina propria and an influx of neutrophils in the adjacent interstitial tissues, which
extended into the NTE. Saline/ozone-exposed rats had only mild-to-moderate
neutrophilic inflammation at the same postexposure time. Epithelial hyperplasia was the
principal morphologic alteration concurrent with the neutrophilic inflammation in the
NTE of rats exposed to either endotoxin or ozone alone, or in combination. The
hyperplastic NTE was approximately 3 - 4 cells in thickness, compared to 1 - 2 cells in
the NTE of the saline/air-exposed control rats. There were no recognizable differences in
the severity of the epithelial hyperplasia among the rats exposed to either endotoxin or
ozone alone, or in combination 2 h after the end of the inhalation exposure.

There was a marked MCM, characterized by the appearance of AB/PAS-stained
mucous cells in the NTE of rats killed 4 days after 3 days of ozone exposure, regardless
of the type of nasal instillation (i. e., saline or endotoxin) (Figure 4-2). However, the
magnitude of the metaplastic response was greater in rats exposed to both endotoxin and

ozone. In the rats containing MCM, the majority of the mucous cells were present in the

121

dorsal and medial aspects of the NTE lining the maxilloturbinates. Epithelial hyperplasia
persisted with the MCM in those rats, but the magnitude was less severe than that
observed in the similarly exposed rats killed 2 h after inhalation exposure. Endotoxin/air—
exposed rats had only occasional isolated mucous cells in the NTE at 4 days after the
inhalation exposure. In addition, epithelial hyperplasia was not observed in these rats
killed at this time point. Neutrophils were rarely present in the turbinate mucosa of rats

killed 4 days afier inhalation exposure.

Neutrophilic inflammation. Exposure to either ozone or endotoxin alone, or to both
agents, resulted in a transient influx of neutrophils in the NTE lining the
maxilloturbinates (Figure 4-3). Two h afler 3 days of inhalation exposure, there were 40-
, 8- and 48-times more neutrophils in the NTE of endotoxin/air-exposed, saline/ozone-
exposed and endotoxin/ozonc-exposed rats, respectively, compared to those in saline/air-
exposed controls. Rats exposed to both endotoxin and ozone had significantly more (3-
times) neutrophils in the NTE than did saline/ozone-exposed rats. At 4 days after 3 days
of inhalation exposure, there were no significant differences in the number of

intraepithelial neutrophils in rats of all exposure groups.

Epithelial cell numeric density. Rats exposed to either ozone or endotoxin, or both, had
increased numbers of NTE cells lining the maxilloturbinates, compared to saline/air-
exposed controls (Figure 4-4). At 2 h after the end of the inhalation exposure, epithelial
cell numeric densities in endotoxin/air-exposed, saline/ozone-exposed, and

endotoxin/ozone-exposed rats were 28 - 37% greater than those in saline/air-exposed

122

controls. No significant differences were observed in the number of NTE cells of rats in
these three exposure groups. At 4 days after the inhalation exposure, only rats exposed to
ozone had more epithelial cells in the NTE (12 - 17%) than did saline/air—exposed
controls, independent of endotoxin instillation. However, these saline/ozone-exposed and
endotoxin/ozone-exposed rats killed 4 days after inhalation exposure had significantly
fewer epithelial cells in the NTE (14% and 9%, respectively), compared to the similarly-

exposed rats killed 2 h after inhalation exposure.

Stored intraepithelial mucosubstances. At 4 days after the end of the inhalation
exposure, saline/ozone-exposed and endotoxin/ozone-exposed rats had significantly more
intraepithelial mucosubstances (6- and 12- times, respectively), compared to air/saline-
exposed controls (Figure 4-5). The amounts of intraepithelial mucosubstances in the
NTE of endotoxin/ozone-exposed rats was 2—fold greater than those of saline/ozone-
exposed rats. There was no significant difference in the volume densities of
intraepithelial mucosubstances between endotoxin/air-exposed and the saline/air-exposed
rats. Only scant amounts of AB/PAS-positive mucosubstances were present in the NTE

of rats killed 2 h after the inhalation exposure.

Number of mucous cells. The percentage of mucous cells was significantly increased
(5- and 10-times) in the NTE of saline/ozone-exposed or endotoxin/ozone-exposed rats,
respectively, compared to that of saline/air-exposed controls (Figure 4-6). Rats exposed
to both endotoxin and ozone had 2-fold more mucous cells in the NTE than did the

saline/ozone-exposed rats. There was no significant difference in the number of mucous

123

cells between endotoxin/air-exposed and saline/air-exposed rats. Few mucous cells were

present in the NTE of all the rats killed 2 h after the end of the inhalation exposure.

Mucin (rMuc-5A C) mRNA expression. Exposure to either ozone or endotoxin alone,
or in combination, resulted in marked increases of rMuc-SAC mRNA expression in the
maxilloturbinates 2 h after the inhalation exposure (Figures 4-7 and 4-8). There were
approximately 2-fold increases in rMuc-SAC mRNA levels in endotoxin/air-exposed,
saline/ozone-exposed, and endotoxin/ozone-exposed rats, compared to those in the
saline/air-exposed controls. No significant differences in mucin mRNA levels of
maxilloturbinates were present among those rats exposed to ozone and/or endotoxin. The
rMuc-SAC mRNA levels remained elevated in the maxilloturbinates of endotoxin/air-
exposed rats killed 4 days after the inhalation exposure. However, mucin mRNA levels
in maxilloturbinates of ozone-exposed rats (regardless of the type of nasal instillation)

were not significantly different from those of saline/air-exposed controls at 4 days after

the inhalation exposure.

124

 

 

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Discussion

The results presented in this study demonstrate that preexisting nasal inflammation
(i.e., neutrophilic rhinitis caused by endotoxin) can augment ozone-induced MCM in the
NTE of rats. There were 2-fold greater amounts of mucosubstances and numbers of
mucous cells in the NTE of rats intranasally instilled with endotoxin and exposed to
ozone for 3 days, compared to those in rats instilled with saline and exposed to ozone.

A few previous studies have suggested that pre-existing airway inflammation
enhances the pulmonary toxicity of inhaled pollutants (e.g., nitrogen dioxide, sulfur
dioxide, or automotive emission) in laboratory animals (Gilmour, 1995; Menzel, 1994;
Hubbard et al., 1994; Mauderly et al., 1989). The present study is the only animal study
to our knowledge that reports the effects of pre-existing airway inflammation on ozone-
induced nasal toxicity.

Several investigators have demonstrated that people with chronic respiratory diseases
have increased adverse respiratory effects from ozone exposure. Asthmatics exposed to
acute ozone (0.2 - 0.4 ppm, 2 - 6 h) have greater increases in airway obstruction and
inflammation, and bronchial hyperresponsiveness as well as further decrements in lung
function, compared to non-asthmatics (Basha et al., 1994; Kreit et al., 1989; Koenig,
1995; Scannell et al., 1996). Patients with allergic rhinitis also have enhanced nasal
inflammation and bronchial allergen responsiveness after exposure to ozone (0.25 - 0.5
ppm, 3 - 4 h), compared to ozone-exposed healthy subjects (Bascom et al., 1990; Jorres et

‘11-. 1996). In addition, epidemiologic studies have suggested that chronic ozone

135

exposure in many urban areas may account for the increased incidence of asthma-related
summertime hospital admissions (Cody et al., 1992; Dockery et al. , 1989).

Specific cellular and molecular mechanisms underlying ozone-induced MCM are
unknown. However, the presence of copious neutrophils in the NTE before the histologic
appearance of MCM have led us to hypothesize that neutrophilic inflammation plays a
role in the development of ozone-induced MCM. This hypothesis was supported by the
results from our most recent study in which intranasal treatment with an anti-
inflammatory steroid, fluticasone propionate (100 pig/day) resulted in a marked
attenuation of acute (0.5 ppm, 8 h/day for 3 days) ozone-induced neutrophilic
inflammation and MCM in the NTE of rats (Hotchkiss et al., 1998). In addition, we have
also reported that depletion of circulating neutrophils significantly attenuated the MCM
in the NTE of rats exposed to ozone (0.5 ppm, 8 h/day for 3 days) (Cho et al., 1998). In
the present study, we observed that endotoxin alone did not induce MCM in the NTE, but
it markedly magnified the ozone-induced MCM. This suggests that the potentiating
effects of endotoxin exposure on ozone-induced MCM may be mediated, not by direct
effects of endotoxin on airway epithelium, but by endotoxin-induced neutrophilic
inflammation.

In another recent study, we have addressed the effects of endotoxin exposure
following ozone exposure on nasal airway lesions in rat NTE (Fanuch et al., 1998). In
that study, rats were consecutively exposed to ozone for 3 days (0.5 ppm, 8 h/day), and
then intranasally instilled with endotoxin (100 pig/day) for 2 days. The endotoxin-

induced neutrophilic rhinitis markedly magnified the MCM induced by the previous

136

ozone exposure in the NTE. From our present and previous studies, it is postulated that
neutrophilic inflammation plays an important role in the pathogenesis of MCM, and thus,
neutrophilic rhinitis present in the NTE, either prior to or simultaneously with the onset
of MCM, can amplify the mucous metaplastic responses to ozone.

The role of inflammatory mediators derived from neutrophils in the pathogenesis of
MCM or secretory cell hyperplasia has been investigated in rodent tracheobronchial
airways by others. Instillation of supematants from either lysed purified neutrophils or
activated neutrophils into the trachea of hamsters (Snider et al., 1985) or rats (Lundgren
et al., 1988) results in an increase (50 - 300% above controls) in the number of mucous
goblet cells in the tracheobronchial epithelium. MCM induced by the neutrophil-
conditioned supernatant was inhibited by an anti-inflammatory steroid, dexamethasone
(Lundgren et al., 1988). Intra-airway instillation of neutrophil elastase induces MCM in
hamster airways (Breuer et al., 1993, 1985), and elastase inhibitors (e.g., chloromethyl
ketone) as well as dexamethasone have been shown to prevent the MCM induced by
neutrophil elastase (Breuer et al. , 1985; Lundgren et al. , 1988).

In the present study, we also determined the effect of endotoxin-induced rhinitis on
ozone-induced epithelial proliferation. Pre-existing rhinitis did not alter the severity of
ozone-induced epithelial cell hyperplasia in the NTE. This result was consistent with our
recent findings that attenuation of neutrophilic inflammation in the NTE by depletion of
circulating neutrophils (Cho et al., 1998) or by intranasal instillation of an anti-
inflammatory steroid (fluticasone propionate) (Hotchkiss et al., 1998) did not alter the

severity of the ozone-induced NTE cell proliferation. The results suggest that ozone

137

directly induces epithelial proliferation, independent of the preceding or concurrent
inflammatory response.

The present study was also designed to determine the effects of preexisting
neutrophilic rhinitis on ozone-induced mucin mRN A upregulation in maxilloturbinates.
Rats exposed to ozone had a similar magnitude of rMuc-SAC mRNA expression in the
nasal tissues, regardless of endotoxin instillation, at 2 h after the end of last exposure.
The elevated mRNA levels did not persist with MCM at 4 days after the end of last
exposure. We also observed that rats repeatedly exposed to endotoxin had an increased
mucin mRN A expression in the nasal tissues without significant histologic evidence of
MCM, as seen in another study (Fanucchi et al., 1998). The results of our study indicate
that pre-existing neutrophilic inflammation does not magnify the ozone-induced mucin
mRNA upregulation. In addition, the elevated rMuc-SAC mRNA levels in the nasal
tissues may not accurately predict or reflect the magnitude of the production of mucous
glycoprotein in the NTE.

In conclusion, pre-existing rhinitis augmented the ozone-induced MCM in the NTE.
Endotoxin exposure, alone, did not induce MCM in the NTE. This augmentation may be
mediated by the intraepithelial influx of neutrophils induced by the inflammatory agent,
endotoxin. The results from this airway study suggest that people with rhinitis may be
more susceptible to some, but not all, of the epithelial lesions caused by exposure to high

ambient concentrations of ozone.

138

CHAPTER 5

Ozone-Inducible Cytokines and Their Role in Mucin Gene Expression
in Rat Nasal Tissues

I39

Introduction

In our previous studies, we demonstrated that acute ozone exposure induced mucin
mRNA upregulation in nasal tissues within a few hours after the start of exposure. It
preceded the onset of mucous cell metaplasia (MCM) by several days and persisted with
MCM. Ozone-induced MCM appears to be a multi-stage process. Ozone alone induces
premetaplastic events including epithelial cell injury and proliferation and induction of
mucin gene expression prior to the development of MCM. However, full phenotypic
expression of MCM is dependent on the presence of inflammatory cells (i. e., neutrophils).
Although the results from our studies are not enough to elucidate the mechanisms of
ozone-induced MCM or the dependency of MCM on the activation of mucin genes, and
mucin mRNA levels in nasal tissues do not always predict the histologic appearance of
mucin glycoproteins in the NTE, the increases in the steady-state levels of mucin mRN A
precede the onset of the ozone-induced MCM.

Little is known about regulatory mechanisms of mucin gene expression in healthy
and diseased airway epithelium. A few recent studies have reported that cytokines
including TNF-a (Levine et al., 1995), IL-6 (Levine et al., 1994) and IL-4 (Temann et
al., 1997; Rankin et al., 1996), or neutrophil elastases (Voynow et al., 1997) induce
mucin mRNA upregulation in pulmonary epithelium or in cultured airway tissues or cells.
Some of these factors are also known to induce MCM and/or mucin hypersecretion in
rodent airways or in cultured airway cells (Jamil et al., 1997; Rankin et al., 1996; Levine

et al., 1994, 1995). Elevated TNF-a or IL-6 levels have been correlated clinically with

140

airway obstructive disorders such as asthma which is characterizzd by mucous
hypersecretion and overproduction (Marini et al. , 1992; Broide et al. , 1992)

Inhalation exposure of humans to ozone (0.1 - 0.4 ppm, 1 - 24 h) induces the release
of various cytokines (e.g., TNF-a, IL-1, IL-6, IL—8) into respiratory airways (Devlin et
al., 1991, 1996; Koren et al., 1989; Pendino et al., 1994; Rusznak et al., 1996). Among
them, TNF-a and IL-6 are multifunctional proinflammatory cytokines, which are thought
to be markers of inflammation, tissue injury and repair in ozone-exposed airways
(Leikauf et al., 1995; Pendino et al. , 1994, 1995). Both TNF-a and IL-6 are expressed by
airway epithelial cells afier ozone exposure in the absence of inflammatory cells (Devlin
et al., 1994; Beck et al., 1994). However, the specific roles these cytokines play in
ozone-induced airway epithelial alterations have not been investigated. In addition, very
little is known about the time course of expression of these mediators in laboratory
animals following exposure to ozone.

The present studies were designed to test the hypotheses that (1) acute ozone
exposure causes TNF-a and IL-6 expression in nasal airways of rats, and that (2) these
soluble mediators can induce mucin gene expression in rat nasal tissues. We first
examined the time-dependent changes in the steady-state levels of TNF-a and IL-6
mRNAs in nasal airways during and after in vivo ozone exposure. Next, we examined the
potential role of soluble forms of these cytokines on mucin (rMuc-SAC) mRNA
expression in normal nasal tissues in vitro. Microdissected maxilloturbinates maintained

in culture at an air-liquid interface were exposed to these cytokines to investigate the

141

direct effects of TNF-a or IL-6 on the expression of rMuc-SAC mRNA. A quantitative

RT-PCR assay was used to evaluate the expression of rMuc-SAC mRN A in nasal tissues.

STUDY 1: TIME-DEPENDENT CHANGES OF TNF-a AND IL-6 mRNA LEVELS
IN NASAL AIRWAYS AFTER SINGLE AND REPEATED OZONE
EXPOSURE

Materials and Methods

Animal, exposure, sacrifice and RT-PCR analysis. RNA samples from the study
described in Chapter 2 (n = 8/group) were also used for this study. In brief, total RNA
was isolated from microdissected maxilloturbinates of rats exposed to 0 ppm ozone
(filtered air) for 7 days, or rats exposed to 0.5 ppm ozone for 1 day or 2 days (8 h/day)
and killed 2 h after the end of exposure, or rats exposed for 3 days and killed 2 h or 1, 2,
and 4 days afier the end of exposure.

The abundance of mRN A for each cytokine was determined by semi-quantitative
RT-PCR and densitometric analysis as described in Chapter 2 using cyclophilin mRNA
as an internal standard. The PCR primers specific for rat TNF-a and IL-6 cDNA
sequences were synthesized and purified by the Macromolecular Structure Facility at
Michigan State University. The sequences of rat TNF-a forward and reverse primers for
PCR amplification were 5’-ATG AGC ACA GAA AGC ATG ATC-3’ and 5’-TAC AGG

CTT GTC ACT CGA ATT-3’, respectively, and the predicted size of TNF-a cDNA

142

products was 276 bp (Nanji etal., 1995). The sequences of rat lL-6 forward and reverse
primers were S’-CTT CCC TAC TTC ACA AGT C-3’ and 5’-CTC CAT TAG GAG
AGC ATT G-3’, respectively, and the predicted size of IL-6 cDNA products was 476 bp
(Farges et al. , 1995). PCR amplification of TNF-a cDNA started with 3 min incubation

at 94°C followed by 33-cycles of a three-step temperature cycle; denaturation at 94°C for
15 sec, annealing at 55°C for 30 sec and extension at 72°C for 30 sec. PCR amplification
of lL-6 cDNA started with 3 min incubation at 95°C followed by denaturation at 95°C for
30 sec, annealing at 56°C for l min and extension at 72°C for l min for 30 cycles. Each
amplification reaction was finished with a final extension step at 72°C for 10 min.
Cyclophilin mRNA was separately reverse transcribed and PCR-amplified following the
three-step cycle (23 cycles, 95°C for 30 sec - 56°C for 1 min - 72°C for 1 min) using
cyclophilin forward and reverse primers described in Chapter 2. The densitometric
volume of the TNF-a and IL-6 cDNA bands on an agarose gel was divided by that of the

cyclophilin cDNA band achieved from the same RNA sample.

Statistical analyses. Data were expressed as the mean group value i the standard error
of the mean (SEM). Data presenting the changes of TNF-a mRNA and IL-6 mRNA
abundance were natural log- and square root-transformed, respectively, to make variances
homogeneous. The data were analyzed for potential effects of exposure duration on
cytokine mRNA expression using one-way ANOVA. Significant differences among the

experimental groups were determined by use of Dunnett’s Method (p _<_ 0.05).

143

Results

Ozone-induced TNF-a mRNA expression. The mean steady-state level of TNF-a
mRNA was increased by 70%, compared to the air-exposed controls, in rats killed 2 h
afier l and 2 days of ozone exposure (Figure 5-1). Two h following the third day of
exposure, steady-state levels of TNF-a message were 110% greater than controls. The
elevated levels of TNF-a mRNA significantly decreased in ozone-exposed rats killed 1,

2, and 4 days after 3 days of exposure.

Ozone-induced IL-6 mRNA expression. Ozone exposure induced an increase in IL-6
mRNA expression in nasal tissues (Figure 5-2). A significant elevation of IL-6 mRNA
level was observed in rats killed 2 h afler 1 day of ozone exposure (9-fold increase,
compared to air-exposed controls). However, there was no difference in the nasal IL-6
mRNA levels between the control rats and the rats exposed to ozone for 2 or 3 days,

regardless of the sacrificed time.

144

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Discussion

In the present study, we demonstrated a distinctive induction of mRNAs for two pro-
inflammatory cytokines, TNF-ot and IL-6, in rat nasal airways after single and/or repeated
exposure to ozone. There was a tendency of increases in nasal TNF-a mRNA levels in
rats killed 2 h after exposure to ozone for 1 and 2 days (70% more than controls),
although significant increases were observed only in rats killed 2 h after 3 days of
exposure (110% more than controls). The level of IL-6 mRNA was elevated only in rats
killed 2 h after 8 h (i.e.,l day) of ozone exposure. Numerous studies have correlated
increased cytokine mRN A levels with increased production of this cytokine (Elias et al. ,
1990). Therefore, our results suggest that acute ozone induces a rapid local production of
TNF-a and IL-6 in nasal tissues.

In the previous study (Chapter 2), we observed an ozone-induced transient
neutrophilic influx in the NTE during the first 3 days of ozone exposure, which was
resolved afier the end of exposure (See Figure 2-5 in page 65). TNF-a and IL-6 mRNA
levels increased at the same time of neutrophilic influx in the nasal tissues. Our
observations suggest that TNF-a and IL-6 may be involved in the recruitment of
neutrophils to the site of ozone-induced injury, or that these cytokines may be produced
by infiltrating neutrophils. Increased levels of TNF-a and IL-6 mRNA were also
concurrent with the initial increases of rMuc-SAC mRNA levels in the ozone-exposed
nasal tissues as we observed in Chapter 2 (See Figure 2-11 in page 71). To determine

the direct effects of these ozone-inducible cytokines on mucin gene expression in nasal

147

tissues, we conducted an in vitro exposure of explants of microdissected
maxilloturbinates to soluble TNF-oc or IL-6 in the absence of neutrophils or other soluble
substances (See Study 2 in page 149).

Previous studies suggested that TNF-a and IL-6 are involved in airway repair or
adaptive responses. TNF-a has been linked to the initiation of silica-induced tissue
reparative adaptation, such as fibrosis in rat lungs (Piguet et al., 1989, 1990). It has also
been reported that administration of TNF-a can attenuate ozone-induced epithelial cell
injury in rat NTE (Hotchkiss and Harkema, 1992). In the present study, we observed that
TNF-a was elevated during the first 3 days of ozone exposure, which may be the most
critical time for the molecular and cellular changes to develop MCM in the NTE.
Therefore, TNF-a may be an important factor in ozone-induced MCM. Though the
overexpression of IL-6 message was more brief than that of TNF-a after ozone exposure
in our study, IL-6 was persistently expressed in pulmonary airways of rats exposed to
chronic ozone (0.5 ppm, 56 days) (Bree et al., 1996). In addition, results from a recent
study indicated that inhibition of IL-6 by pretreatment with antibody exacerbated the
acute ozone-induced neutrophilic inflammation and tissue injury in rat lungs (McKinney
et al., 1998). These previous findings suggest a possible role of IL-6, as an anti-

inflammatory cytokine, in airway repair and adaptation after ozone exposure.

I48

STUDY 2: EFFECTS OF SOLUBLE TNF-a AND IL-6 ON RAT MUC-SAC
mRNA EXPRESSION

Materials and Methods

Animal, microdissection of maxilloturbinate and culture. Twelve male F3 44/N rats
(Harlan Sprague-Dawley, Indianapolis, IN), 10 - 12 weeks of age, were used to determine
effect of TNF.a on rMuc-SAC mRNA expression. Eighteen rats were used to determine
the effect of IL-6 on rMuc-SAC mRNA expression. Rats were sacrificed and
maxilloturbinates from nasal passages were removed immediately by microdissection as
described in Chapter 2. Each microdissected maxilloturbinate was randomly assigned to
one of 6 experimental groups in each study (n = 4/ group for TNF-a study, 11 = 6/ group
for IL-6 study). The maxilloturbinates were placed on 0.4 pm-pore-size Transwell
inserts (Costar, Pleasanton, CA) in 12-well plates (1 maxilloturbinate/well). The
maxilloturbinates were placed in the center of the mesh insert with medial aspects of the
turbinates facing the air phase (Figure 5-3). Medium was added to both the top (100 pl)
and bottom (400 pl) compartments of the Transwell. The culture medium (supplemented
Ham’s F12), a modification of that used by Wu and coworkers (Wu et al., 1985),
contained 1 uM hydrocortisone, 5 pg/ml transferrin, 5 ug/ml insulin, 25 ng/ml EGF,
bovine pituitary extract, 1 pM retinol, 100 U/ml penicillin, and 0.1 mg/ml streptomycin.

The tissues were cultured (37°C, 5% C02) overnight.

149

 

 

 

 

 

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150

 

In vitro exposure. Recombinant human TNF-a (rhTNF-a; R&D System, Minneapolis,
MN) or recombinant human IL-6 (rhIL-6; Gibco BRL, Gaithersburg, MD) was dissolved
in sterile phosphate-buffered saline containing 0.1 % bovine serum albumin (Sigma
Chemical Co., St.Louis, MD) as a carrier. Maxilloturbinates in culture were exposed to
either TNF-a or IL-6 by replacing the medium in the upper (80 p1) and lower (400 pl)
chambers of the Transwell with fresh supplemented F12 containing 0 (control), 2, or 20
ng/ml of the respective cytokine. Maxilloturbinates cultured with TNF-a were removed
and analyzed afier 4 and 24 h of exposure. Maxilloturbinates cultured with IL-6 were
removed and analyzed after 8 and 24 h of exposure. The concentration and the exposure
duration were chosen based on previously published studies in which exogenous TNF-a
or IL-6 was shown to effect various cellular and molecular changes including activation
of cellular genes in vitro without cytotoxicity (Levine et al., 1995, 1994; Roncero et al.,
1995; Ravid et al., 1995; Rokita et al., 1989; Putowski and Kotarski, 1995). Lactate
dehydrogenase (LDH) level in the culture medium was measured as an indicator of tissue
cytotoxicity caused by each cytokine. At the end of culture, the activity of LDH in the
medium was determined spectrophotometrically from the disappearance of NADH using
pyruvate as substrate. Total RNA was isolated from each tissue, and analyzed for rMuc-

5AC mRN A expression using quantitative RT-PCR assay.

Preparation of internal standard for rMuc-SAC RT-PCR. We followed the simple and

rapid general PCR-based method reported by Heuvel et. al. (Vanden Heuvel et al. , 1994)

for generating rcRNA templates for use as internal standards in quantitative RT-PCR.

151

The basis for this method is amplification of genomic DNA using primers containing the
T7 promoter, target (i.e., rMUC-SAC), and spacer RNA (i.e., human glutathion S-
transferase; GST) primer sequences and polydeoxythymidylic acid (poly(dT),8) (Figure
5-4). Genomic DNA was used to generate PCR products of an appropriate size to be
resolved from the target RNA-PCR products. The T7 promoter and poly(dT)18 are
needed to produce a rcRNA with a polyadenylated tail following in vitro transcription of
the PCR products. Using this procedure, the rcRNA molecules which contained rMuc-
SAC mRN A forward and reverse primer sequences were able to be synthesized, and a
RT-PCR product was produced and easily resolved from that generated from the rMuc-
SAC mRNA. Construction of the rcRNA internal standards was accomplished using the
following procedure. The forward rcRNA primer contained the T7 promoter, rMUC-
SAC mRN A forward primer, and GST mRNA forward primer. The reverse rcRNA
primer contained the GST mRN A reverse primer, rMuc-SAC mRN A reverse primer, and
poly(dT),8. The rcRNA primers were approximately 60 base pairs in length and
synthesized by the Southwest Scientific (Albuquerque, NM). PCR reactions were
conducted in a final volume of 50 ml containing PCR buffer, 3 mM MgC12, 0.2 mM each
of deoxynucleotide triphosphate, 30 pmol each of rcRNA forward and reverse primers,
100 ng human genomic DNA, and 1.25 units Taq DNA polymerase. The reactions were
assembled at 85°C, heated to 94°C for 3 min, and cycled 30 times through a 15-sec
denaturing step at 94°C, a 30-sec annealing step at 55°C, and a 30-sec extension step at
72°C. Following the final cycle, a 10—min extension step at 72°C was included. The

PCR products were diluted 1:100 in water, and 2 ml were reamplified using the

152

 

conditions stated above. PCR products from several second amplification reactions were
pooled and purified (Magic-Prep DNA purification system; Promega, Madison, WI). The
pooled PCR products were transcribed into RNA by the T7 promoter using the Riboprobe
Gemini II in vitro transcription system (Promega, Madison, WI). The rcRNA was
subsequently treated with RNase-free DNasc to remove the DNA template, extracted
sequentially with water-saturated phenol-chloroforrn (24:1) and chloroform-isoamyl
alcohol (24:1), and precipitated with ethanol. The rcRNA pellet was washed with 70%

ethanol, suspended in nuclease-free water, and quantitated by absorbance at 260 nm.

Quantitative RT -PCR. Quantitative RT-PCR was performed as described by Gilliland
et al. (Gilliland et al., 1990). For each RNA sample, 6 equal aliquots (100 ng total RNA)
were prepared, and a dilution series (0 or 10’ - 107 molecules/tube) of the internal
standard RNA was spiked into the RNA aliquot in each tube. Reverse transcription of
RNA was performed following the same procedure described in serniquantitative RT-
PCR analysis of rMuc-SAC mRNA (Chapter 2). The PCR amplification of cDNA
products were followed by 3-min incubation at 95°C, 33—cycling of the three-step
temperature reactions (95°C 30 sec, 56°C 1 min, 72°C 1 min), and final extension step
at 72°C for 10 min using same reagents described in Chapter 2. Aliquots (15 ml) of the
PCR reaction were electrophoresed as described previously. Quantitation of the amount
of rMuc-SAC mRN A was performed using the method by Gilliland et al. (1990). Briefly,
the ratio of the densitometric volume of internal standard cDNA spot to that of rMuc-

5AC cDNA spot was plotted against the amount of internal standard RNA (number of

153

 

molecules) added to each reaction. The internal standard RNA concentration at which the
volume ratio was equal to one (i.e., where the volume of rMuc-SAC cDNA matches that
of the internal standard RNA) represented the amount of rMuc-SAC mRN A present in the
initial RNA sample. A broad range of internal standard concentration (i.e., 10' to 108
molecules/tube) was examined in order to estimate an approximate concentration of the
rMuc-SAC mRNA in each tissue sample. In a second RT-PCR, a much narrower internal
standard range (i.e., 105 to 107) was examined for accurate quantitation of mucin message

levels.

Statistical analyses. All data were expressed as the mean group value 1: the SEM. The
mucin mRNA data from individual cytokine study were analyzed by two-way AN OVA to
determine the potential effects of TNF-a or IL-6 concentration and exposure duration on
mucin mRN A expression. Student-Newman-Keuls Method was followed to determine
the significant differences in IL-6-induced group mean values (p S 0.05). Because there
was no effect of exposure duration (i.e., 4 or 24 h) on TNF-a-induced rMuc-SAC mRNA
expression, data from each concentration of TNF-a-exposed groups (i.e., 0, 2 and 20
ng/ml) were pooled. The pooled data were then analyzed by the Analysis of Contrast to
determine the significant differences in the mean rMuc-SAC mRNA levels among the
combined experimental groups (p S 0.05). The data from LDH assay of each study were
also analyzed by two-way AN OVA and following Student-Newman-Keuls Method to

determine the potential cytotoxicity of individual cytokines in nasal tissues (p s 0.05).

154

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Results

Effect of TNF-a on rMuc-SAC mRNA Expression. Figure 5-5 depicts digitized images
of representative agarose gels containing rMuc-SAC and IS cDNA bands and standard
plots used in the densitometric determination of rMuc-SAC mRNA levels in
maxilloturbinates. The volume ratio of the internal standard cDNA band to rMuc-SAC
cDNA band versus the amount of the internal standard added was a linear relationship
(correlation coefficient; 98 - 100 %). A slight increase in rMuc-SAC mRNA level (2.5 to
4.1 X 106 molecules/mg RNA) was observed in cultured tissues receiving 2 ng/ml of
TNF-a, but this was not significant, compared to the message level in control tissues
(Figure 5-6). However, exposure of maxilloturbinates to 20 ng/ml of TNF-a resulted in a
3-fold increase (2.5 to 7 X 10° molecules/mg RNA) in the rMuc-SAC mRNA expression.

No evidence of TNF-a-induced cytotoxicity was detected by LDH determination at both

concentrations (Figure 5-7).

Effect of IL-6 on rMuc-SAC mRNA Expression. The steady-state level of rMuc-SAC
mRNA in the cultured maxilloturbinates increased 2-fold (0.9 to 1.7 x 107 molecules/mg
RNA) after 24 h exposure to 2 or 20 ng/ml of IL-6 G’igure 5-8). A linear relationship
(correlation coefficient; 77 - 97 %) was observed in the ratio of the volume of the internal
standard to rMuc-SAC mRNA versus the amount of the internal standard added. No

evidence of IL-6-induced cytotoxicity was detected by LDH determination at both

concentrations (Figure 5-9).

156

(a) Control (0 nglml)

0 10‘ 5x105 10‘ 5x10“ 4107 Is Molecules

un- ‘- ......... “we" rMuc-5A0 (320 bp)
“our use “I“ new 4” IS (265 bp)

 

 

 

 

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o 2 4 6 a 10 12
Is Molecules (x 10‘)
(b) TNF-a (20 nglml)

0 10‘ 5x105 10‘ , 5x10“ 107 _ IS Molecules

 

 

 

 

—r w w w W f rMuc-5A0 (320 bp)
' 2...... an. m - -“ IS (265 bp)
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Is Molecules (x 10‘)

Figure 5-5. Digitized images of representative agarose gels and standard plots
for rMuc-SAC mRNA quantitation using rMuc-SAC-specific
internal standard (IS) rcRNA. As the amount of IS in the reaction
increases, the volume ratio increases. The number of rMuc-SAC
mRNA molecules is determined by the number of IS where the
volume ratio equals 1.

157

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Figure 5-6. Effect of TNF-a on rMuc-SAC mRNA expression

in explants of maxilloturbinates. Bars represent
the group mean i SEM. (n = 4/group).
*Significantly different from the control (p 5 0.05).

158

160 _

120 -

80—

LDH Release
(% of the Control)
3
I

 

 

2 nglml 20 nglml 2 nglml 20 nglml

 

 

4h 24h

Figure 5-7. Cytotoxicity of TNF-a to maxilloturbinate explants indicated
by % LDH release compared to time-matched, vehicle-
exposed controls. Bars represent the group mean i SEM
(n = 4/group).

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(% of the Control)

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8h 24h

Figure 5-9. Cytotoxicity of IL-6 to maxilloturbinate explants indicated
by % LDH release compared to time-matched, vehicle-
exposed controls. Bars represent the group mean i SEM
(n = 6/group).

161

Discussion

The results of the present study demonstrate that the ozone-inducible pro-
inflammatory cytokines, TNF-a and IL-6, can directly induce increased rMuc-SAC
mRNA levels in explants of microdissected maxilloturbinates. Exposure of nasal tissues
to 20 nglml of TNF-a induced a 3-fold increase in rMuc-SAC mRNA, compared to
vehicle-exposed controls. At 2 nglml of TNF-oc, there was a slight but not significant
increase in mucin message levels, compared to the controls. In contrast, either 2 or 20
nglml of IL-6 induced 2-fold increases in rMuc-SAC mRNA after 24 h exposure. Eighth
exposure to IL-6 did not alter the steady-state level of mucin mRNA at either
concentration.

Our observations were in accord with the results of recent studies conducted by
Levine et al. (1994 and 1995) in which exposure to either TNF-a (2 - 20 ng/ml) or IL-6
(20 ng/ml) induced upregulation of MUC-2 mRNA (0.5 - 24 h by TNF-a, 8 - 72 h by IL-
6) in cultured human airway epithelial cells. In those studies, TNF-a and IL-6 also
induced concurrent mucin hypersecretion in cultured cells (TNF-a; 4 - 72 h, IL-6; 8 - 72
h) or bronchial organ explants (TNF-a; l - 24 h, IL-6; 4 - 48 h) at the same
concentrations. In addition to TNF-a and IL-6, IL-4 and IL-5 have been implicated in
mucin gene upregulation, mucin hypersecretion and/or MCM in distal airways of rodents
(Rankin et al., 1996; Lee et al., 1997; McBride et al., 1994). Though IL-lb is a strong
mucin secretagogue in airway epithelial cells (Jarry et al., 1996), little is known about its

role in mucin gene expression. The results of these recent studies indicate that certain

162

cytokines (i. e., TNF-a , IL-4 and IL-6) can have multiple effects on airway mucous cells
- inducing mucin gene expression, mucous hypersecretion, or overproduction. Therefore,
it is strongly postulated that TNF-a and/or IL-6 may play roles, not only in rMuc-SAC
mRNA expression, but in mucin secretion or MCM in the nasal tissues of rats exposed to
ozone.

In the present study, in vitro tissue culture was employed to assess the direct effects
of individual cytokines on mucin gene expression. By using this culture system, we
eliminated possible effects of neutrophils or neutrophil-mediated inflammatory responses
on mucin mRNA regulation in nasal tissues. The results of the present study suggested
that local mucosal production of the cytokines, TNF-a and IL-6, may have had direct
effects on the ozone-induced mucin gene upregulation in nasal tissues. Further studies
are required to determine if these cytokines are expressed by nasal epithelial cells or other

mucosal cells in ozone-exposed nasal airway.

163

CHAPTER 6

 

Summary and Conclusions

164

Epithelial alterations in the nasal airways of rats exposed to acute or chronic ozone
have been well characterized in the previous studies from our laboratory. Acute exposure
of rats to high ambient concentrations of ozone (3 - 7 days, 2 0.5 ppm) induces MCM and
epithelial hyperplasia in the NTE lining the lateral meatus of proximal nasal airways in
rats (Harkema et al., 1989, 1994; Hotchkiss et al., 1989). A transient neutrophilic
inflammation precedes the development of epithelial hyperplasia and MCM (Harkema et
al., 1989; Hotchkiss et al., 1989, 1997). These previous studies, however, were not
designed to investigate how the epithelial alterations (i. e., neutrophilic inflammation,
epithelial hyperplasia, MCM) relate to each other. In addition, the effects of ozone
exposure on airway mucin-specific gene expression and its relationship to the ozone-
induced MCM had not been previously investigated. To further investigate the
pathogenesis of ozone-induced MCM, we designed the present research to test the
guiding hypothesis that neutrophilic inflammation plays a key role in the development of
MCM in the NTE.

To understand how and when ozone induces nasal cell injury and the reparative and
adaptive changes (i.e., epithelial proliferation and MCM) that occur in the NTE of ozone-
exposed rats, we first determined the time-dependent relationships of neutrophilic
inflammation with epithelial responses including airway mucin-specific gene (rMuc-
SAC) expression and epithelial proliferation (i. e., NTE cell BrdU-labeling index and
numeric density) which occurred early after the start of exposure and during the
development of MCM. We also investigated the temporal relationships of ozone-induced
mucin mRNA upregulation with epithelial proliferation and MCM. The results from our

studies demonstrated that acute ozone exposure (0.5 ppm, 8 h) induced an increase in the

16S

 

steady-state levels of rMuc-SAC mRNA in nasal tissues within hours after the start of
exposure. This ozone-induced upregulation of the airway mucin gene preceded the
phenotypic expression of MCM by three days. The upregulation of this mucin gene was
initiated concurrently with neutrophilic inflammation in the NTE, but persisted with
epithelial hyperplasia and MCM even afier the initial neutrophilic inflammation was
resolved two days later. Although temporal correlations of those molecular and cellular
responses in the present study did not prove causality, these findings suggested that the
early ozone-induced increases in mucin mRNA are associated with the neutrophilic
inflammation and epithelial cell proliferation. In addition, ozone-induced MCM may be
dependent on these important pre-metaplastic responses (i.e., mucin mRN A upregulation,
neutrophilic inflammation and epithelial proliferation).

Based on these initial findings, we further investigated the role of infiltrating
neutrophils in the ozone-induced epithelial alterations (i.e., hyperplasia and MCM) and
mucin gene upregulation in the NTE. For this purpose, we depleted rats of their
circulating neutrophils using an anti-rat neutrophil antiserum prior to the repeated, acute,
ozone exposures. The results from this study indicated that ozone-induced MCM is, at
least in part, neutrophil-dependent (i. e., ~70 % decrease of ozone-induced MCM in
antiserum-treated animals, compared to control serum-treated animals). In contrast, we
found that the induced epithelial proliferation (i.e., epithelial DNA synthesis and
hyperplasia) and the increase in mucin mRN A levels were independent of the ozone-
induced neutrophilic influx in the NTE. Therefore, it was concluded that ozone alone is

sufficient to induce mucin gene upregulation and epithelial proliferation in the NTE

166

before the onset of MCM. However, for full phenotypic expression of MCM, neutrophil-
mediated molecular and cellular events are needed.

Another study was then designed to support the neutrophil-dependency of the ozone-
induced MCM. We hypothesized that pre-existing neutrophilic inflammation in the NTE
(i.e., rhinitis) augments the ozone-induced MCM. For this purpose, neutrophilic rhinitis
was induced by exposing rats to a strong pro-inflammagen, endotoxin (100 gig/day,
intranasal instillation), 6 h prior to daily ozone exposure (0.5 ppm, 8 h/day for 3 days).
Pretreatment of endotoxin enhanced the severity of the ozone-induced MCM by 2 fold,
while it did not affect the severity of either NTE cell proliferation or mucin mRN A
upregulation induced by ozone. Although endotoxin alone induced neutrophilic
inflammation and mucin mRNA upregulation, it did not induce MCM in the NTE. The
results of this study suggested that the augmentation of ozone-induced MCM could be
mediated by endotoxin-induced inflammatory responses. However, we cannot rule out
the direct contribution of endotoxin to the pathogenesis of MCM in the NTE of ozone-
exposed nasal airways.

Our in viva findings suggested that mucin mRNA levels in nasal tissues do not
always match with or predict the histologic appearance of the mucin glycoproteins.
However, significant increases of the mucin mRN A levels were consistently detected
prior to the onset of the ozone-induced MCM in the NTE. A few previous studies have
also indicated that other airway irritants (e. g., sulfiir dioxide, acrolein) also induce
upregulation of mucin mRN A concurrently with or prior to the development of MCM in

distal airway epithelium of rats (Jany et al., 1991; Borchers and Leikauf, 1997).

167

However, underlying mechanisms of the elevated expression of airway mucin gene have
not been fully investigated. Recently, several cytokines (e.g., TNF-oc, IL-4, IL-5, IL-6)
have been reported to play important roles in mucin mRNA upregulation in association
with elevated mucous production in or secretion from airway epithelial cells (Levine et
al., 1994 and 1995; Rankin et al., 1996; Temann et al., 1997; Lee et al., 1997). Among
them, TNF-a and IL-6 are known to be produced by airway epithelial cells after ozone
exposure in the absence of inflammatory cells (Devlin et al., 1994; Beck et al., 1994). In
our rat model of ozone exposure, we observed that expression of mRNAs for these two
pro-inflammatory cytokines, TNF-a and IL-6, was markedly increased in nasal tissues as
early as 10 h after the start of ozone exposure. Induction of these cytokine genes
coincided with increases in mucin mRNA levels in nasal tissues. Therefore, we
conducted in vitro studies to investigate the putative role of those ozone-inducible
cytokines (i.e., TNF—a, IL-6) in mucin gene expression in nasal tissues. When soluble
forms of either TNF-a or IL-6 (2 or 20 nglml, 4 - 24 h) were applied to the explants of
microdissected maxilloturbinates, the steady-state level of rMuc-SAC mRNA was
elevated by 2 - 3 fold. These results suggested that local mucosal production of TNF-a
and IL-6 may directly contribute to the ozone-induced mucin mRN A upregulation in
nasal tissues.

Major findings of our studies are summarized in a diagram in Figure 6-1. Ozone
directly induces early overexpression of mucin mRNA and epithelial cell proliferation in
the absence of neutrophilic inflammation. These neutrophil-independent epithelial events

contribute to some extent to the development of the ozone-induced MCM. However,

168

neutrophilic inflammation plays a key role in the full phenotypic development of MCM,
probably by accelerating mucin apoprotein translation or mucin glycosylation (e. g.,
increases in N-acetylgalactosaminetransferase activity or its expression, mucin transport
through RER and Golgi), or by stabilizing stored mucin in granules or inhibiting mucin
exocytosis.

How these inflammatory cells mediate this epithelial transformation is yet to be
determined. Future studies must be designed to determine if soluble substances derived
from neutrophils (e. g., proteases, cytokines, peptide growth factors) are responsible for
the metaplastic transformation of NTE from a nonsecretory epithelium to a secretory
epithelium containing numerous mucous cells. In addition, further research is needed to
understand what specific epithelial cells (e.g., basal, cuboidal) are involved in these
metaplastic processes. What are the progenitor cells for the newly appeared mucous
cells ? Answers to these questions will not only add to our understanding of how ozone
causes MCM in the NTE of rats, but will also contribute to a greater understanding of the
cellular and molecular mechanisms involved in this basic metaplastic process which is a
key pathologic feature in most chronic airway diseases (e.g., asthma, chronic bronchitis,

allergic rhinitis, and cystic fibrosis).

169

 

l

Nasal Transitional Epithelium

/ \
TNF-a ? Mucin Gene

 

Chemokines
. . IL-6 7 ~ s Transcription
Eplthellal . 7 (1?¢Post-mnscfipfion
DNA Synthes's N; u:m;3=.‘;:.'ic _ _ Mucin mRNA
Inffigmima :ion 7‘" pf; Translation
. . I I '
EP'thfha' Dell I l \ \ Mucin Apoprotein
Proliferation I ‘\ \ \\
,L I \ \ \ \ \ Glycosylation
I \ \\ T ‘ - ..) Transport of
Epithelial 7 I \ \ ? (4.) mucin through.
Hyperplasia (7') : \\ \\ v RER and 3019!
V \ \\ Mucin Glycoprotein
\\ \ \
\\ 7 7:? ¢ Storage
? \\ lVlucin in Granules
‘ '~ - 9 Secretion
7 J H

 

 

l

Mucous Cell Metaplasia

Figure 6—1. Summarized diagram of results

170

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171

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