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Z
Qﬂdy . LIBRARY
Muchigan State
University

This is to certify that the
thesis entitled

Persistent Mucus Accumulation in RAD-Affected Horses —
A Consequence of Delayed Mucous Cell Death

presented by
Lisa Renee Bartner

has been accepted towards fulﬁllment
of the requirements for the

Master of Science degree in The Department of Physiology

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Date

 

 

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2/05 p:/C|RC/DaieDue.indd-p.1

PERSISTENT MUCUS ACCUMULATION IN RAD-AFFECT ED HORSES —
A CONSEQUENCE OF DELAYED MUCOUS CELL DEATH?

By

Lisa Renee Bartner

A THESIS

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

MASTER OF SCIENCE
Department of Physiology

2005

ABSTRACT

PERSISTENT MUCUS ACCUMULATION IN RAD-AFFECT ED HORSES —
A CONSEQUENCE OF DELAYED MUCOUS CELL DEATH?

By

Lisa Renee Bartner

This study examined the role of delayed apoptosis of mucous cells (MC) as indicated by
Bel-2, an anti-apoptotic protein, as a contributing factor to mucus accumulation in horses
affected with recurrent airway obstruction (RAG). Measurements of disease severity were
collected from 6 RAG-affected and 6 control horses exposed to two different management
systems (one to induce inﬂammation [5 days] and one to partially resolve it [7 days]) prior to
euthanasia. Morphmetric analyses (Bcl-Z-positivity and stored mucosubstance [Vs]) were
performed on 8 bronchi. RAD-affected horses had more airway obstruction and luminal mucus
than control horses under both management systems. At the time of euthanasia, RAD-affected
horses had more inﬂammation and more Bcl-Z-positive MC than control animals but no
difference in MC number or Vs, and in RAD-affected animals, Vs decreased as BALF
neutrophil numbers increased. Therefore, a conclusive role for Bel-2 in prolonging MC life
cannot be determined, despite Bcl-2 antibody immunoreactivity. USDA/CSREES 2002-35204—

1259)

To Bubbe, Mom, and Dad for their enduring pride, love, and support.

iii

ACKNOWLEDGMENTS

I would like to thank my family and friends for their motivational support. My
mentor, teacher, and friend Dr. N. Edward Robinson for training me in the matters of
scientiﬁc research, philosophy, and for sharing with me some of his life experiences and
British humor—also for providing occasionally needed inspirational kickscin-the-pants. I
thank Dr. Yohannes Tesfaigzi for his direction and collaboration and my committee
members, Dr. Art Weber and Dr. Bruce Uhal, for contributing their expertise and ideas. To
Heather DeFeijter-Rupp and Dr. Annerose Berndt for years of laboratory support,
friendship, and patience, mostly for tolerating me. I would also like to thank Victoria
Hoelzer-Maddox, Cathy Berney, Sue Eberhart, Kieth Willard, and Dr. Cornelius Cornelisse
for technical support. Finally, I would like to thank the horses that made this thesis possible:

Scoot, Sioux, Prince, Merle, Ozark, Hank, Loretta, Tammy, Scout, Eddie, Cash, and Louis.

iv

TABLE OF CONTENTS

 

 

 

 

 

 

 

LIST OF TABLES ................................................................................................................................ VIII
LIST OF FIGURES ................................................................................................................................. Ix
LIST OF FIGURES ................................................................................................................................. Ix
LIST OF ABBREVIATIONS ................................................................................................................ XIV
INTRODUCTION .................................................................................................................................... 1
CHAPTER 1 LITERATURE REVIEW ..................................................................................................... 2
MUCOUS APPARATUS IN AIRWAYS .................................................................................................... 2
STRUCTURES ............................................................................................................................... 2
Luminal Mucosa .............................................................................................................. 3

Ciliated cell: ......................................................................................................... 3

Clara Cell: ........................................................................................................... 4

Stray: Cell: .......................................................................................................... 4

Neuromdom'rze Cell: ............................................... - -- ........................ 5

Baml Cell: ........................................................................................................... 5

Bria/J Cell: ........................................................................................................... 5

Semmucou: Cell: .................................................................................................. 6

Submucoml Gland: .............................................................................................. 6

Mucou: Cell: ........................................................................................................ 7

Lamina Propria ................................................................................................... 8

Submucosa ....................................................................................................................... 8

Adventia ............................................................................................................................ 8

AIRWAY MUCUS LINING AND THE MUCOCILIARY SYSTEM ................................................. 9
Mucus Constituents ...................................................................................................... 13

Mud»: ................. . - ..................................... - 13

Otlm'Pmtez'n: ................................................................. - - 14

Cawo/g'drate: ............................................................. 14

bpid: ............. ................................. .. _- - 14

DNA andActin Payment ................................................................................. 15

Purpose of Mucus ......................................................................................................... 15

 

 

Mucus Synthesis and Secretion ................................................................................... 16
Mucous Cell Metaplasia/ Goblet Cell Hyperplasia ................................................... 20
Airway Repair and Regeneration ................................................................................. 22
PHYSIOLOGICAL CELL DEATH: PROGRAMMED SUICIDE OR NECROSIS .................................... 23
NECROSIS .................................................................................................................................. 25
APOPTOSIS ................................................................................................................................ 25
Caenor/Jabditz: elegant-A Genetic Model of Cell Death ............................................... 26
Cell Death in Higher Mammals .................................................................................. 28
Caspases .......................................................................................................................... 29
The Mitochondria-Associated (Intrinsic) Pathway and Bel-2 ................................. 32
TI» BCL-Z Fanny .......................................................................... 33
Regulation of Cell Death Mae/JinegI ...................................... 37

OM Maintenance, Pore Forming Abiliy, and Mitochondrial
ng‘nnction ......................................................................................................... 40
The Death Receptor (Extrinsic) Pathway .................................................................. 43
RECURRENT AIRWAY OBSTRUCTION IN HORSES ......................................................................... 45
ETIOLOGY ................................................................................................................................ 4S
CLINICAL SIGNS ....................................................................................................................... 47
LESIONS .................................................................................................................................... 47
PATHOPHYSIOLOGY AND PATHOGENESIS .......................................................................... 50
Inﬂammation ................................................................................................................. 50
MEASUREMENTS OF DISEASE SEVERITY .............................................................................. 52
Maxrrnal' Changes in Pleural Pressure (APme) ........................................................ 52
Clinical Score ................................................................................................................. 53
Mucus Score ................................................................................................................... 53
Bronchoalveolar Lavage Fluid (BALF) ...................................................................... 53
MUCUS ACCUMULATION AND MUCOUS CELL METAPLASIA IN RAO .............................. 54
Apoptosis of MC ........................................................................................................... 56
SUMMARY.........: ...................................................................... .................................... 57

 

CHAPTER 2 PERSISTENT MUCUS ACCUMULATION—A CONSEQUENCE OF DELAYED MUCOUS
CELL DEATH IN RAD-AFFECTED HORSES ...................... - ..................................... 58

 

vi

ABSTRACT ............................................................................................................................................ 58

MATERIAL AND METHODS ............................................................................................................... 59
ANIMALS ................................................................................................................................... 59
EXPERIMENTAL DESIGN ........................................................................................................ 60
MEASUREMENT OF APPLMAX ................................................................................................... 61
TRACHEAL MUCUS SCORE ...................................................................................................... 62
BRONCHOALVEOLAR LAVAGE .............................................................................................. 62
POST-MORTEM PROCESSING ................................................................................................. 62
IMMUNOHISTOCHEMISTRY ..................................................................................................... 64
MORPHOMETRIC QUANTIFICATION OF MC AND BCL—2 EXPRESSION ........................... 65
QUAN'I‘IFICATION OF INTRAEPITHELIAL STORED MUCOSUBSTANCE ............................. 66
STATISTICAL ANALYSIS ........................................................................................................... 66

RESULTS ............................................................................................................................................... 67
INDICES OF DISEASE SEVERITY ............................................................................................ 67

Maximum Changes in Pleural Pressure (APme) ..................................................... 67
Tracheal Mucus Score ......................................... 67
Cytology of Bronchoalveolar Lavage Fluid (BALE) ................................................ 67
GOBLET CELL NUMBERS ........................................................................................................ 68
STORED MUCOSUBSTANCE IN AIRWAY MUCOUS CELLS ................................................... 69
BCL-2 STAINING AIRWAY MUCOUS CELLS .......................................................................... 69

DISCUSSION ........................................................................................................................................ 75

APPENDIX ........................................................................................................................................... 79

BIBLIOGRAPHY ................................................................................................................................... 86

vii

LIST OF TABLES

Table 1. Categorization of BCL-2 Family Members. Members can be categorized into
two groups based on function and three groups based on structure .................................... 36

Table 2. BALF total and percent neutrophils, macrophages, lymphocytes, eosinophils,
mast cells and total cell count on days 5 and 12 in RAG-affected and control
horses .............................................................................................................................................. 68

viii

LIST OF FIGURES

Figure 1. Mucous Granule Movement and Exocytosis. Binding of a mucin secretagogue
to its receptor ultimately activates both PKC and PKG; dual activation is required
for a robust secretory response. The ligand-receptor complex activates
phospholipase C (PLC), which results in the production of inositol triphophste
(1P3) and diacylglycerol (DAG), an activator of PKC. Increases in IP3 stimulates
calcium-release from the intracellular stores (i.e. mucin polymer matrix and
intracellular free-calcium). Activated PKC phosphorylates membrane-bound
myristoylated alanine-rich C kinase substrate (MARCKS), leading to its subsequent
translocation into the cytoplasm. The membrane association of MARCKS is
phosphorylation dependent; in a phosphorylated state MARCKS is cytoplasmic
while in a dephosphorylated state MARCKS is membrane-bound. At the same
time, activation of PKG, via the nitric oxide-cyclic GMP pathway, activates
cytoplasmic protein phosphatase-2A (PPZA), which dephosphorylates MARCKS.
After dephosphorylation by PP2A, MARCKS recovers its membrane-binding
abilities and becomes stabilized on membranes of cytoplasmic mucin granules.
Simultaneously, MARCKS interacts with actin and myosin, acting as a tether,
linking the granules to the intracellular contractile apparatus mediating granule
movement to the apical membrane and consequently exocytotic release. (NOTE.
Images in this thesis are presented in color). ............................................................................ 18

Figure 2. Mucin Granule-Cytosol Calcium Exchange Dynamics and Exocytosis. (1) IP3
binds to IP3-R channel and (2) there is release of resting, free calcium from the
granule lumen into cytoplasm and calcium in cytosol increases. (3) Localized
icreases in cytosol [calcium] trigger opening of a calcium~activated potassium
channel and potassium inﬂux that maintains electroneutrality and (4) mobilizes
lumenal matrix-bound calcium via Ca+2/K+ ion exchange (also increasing the
calcium diffusion gradient for release). (5) Calcium increases in the granule lumen
(6) promote calcium efﬂux, which increases cytosolic [calcium] and decreases
luminal [calcium]. (7) Large increases in calcium concentration in cytosol cause
inactivation of IP3~R while (8) the calcium-activated potassium channel remains
open, luminal [calcium] rises and cytosolic [calcium] decreases (via diffusion or
buffering). (9) The calcium-activated potassium channel is inactivated and the 1P,-
R is reactivated for new cycle (Nguyen, Chin et al. 1998). (NOTE: Images in this
thesis are presented in color). ..................................................................................................... 20

Figure 3. The Mitochondrial Regulator: “Bioenergetic Catastrophe” Or Subtle
Disappearance? Stage 1(solid arrows): a multitude of stimuli set off a signal
transduction pathway to the mitochondrial membrane, increasing its permeability.
This mitochondrial membrane perrneabilization (MMP) releases sequestered
apoptogenic factors (including endonucleases) into the cytosol and abolishes the
electrochemical gradient (ECG) in the membrane, leading to mitochondrial
dysfunction and the second stage (dotted arrows). Organellar breakdown leads to
loss of plasma membrane integrity (necrosis) and/ or activation of proteases and
endonucleases (apoptosis). These consequences, indicated by dotted arrows,

include: (a) collapse of mitochondrial inner transmembrane potential (A‘I’m), (b)
calcium efﬂux, (c) increased reactive oxygen species (ROS), indicative of

ix

uncoupling of electron transport chain. Additionally, caspase-activating proteins
are released from the intermitochondrial space. (NOTE: Images in this thesis are
presented in color). ....................................................................................................................... 24

Figure 4. Apoptosis pathways in C. elegan: and Mammals. Homologies occur between
the nematode, C. elegant, and mammals in proteins and in mechanism. Ced-4/Apaf—
1 binding is required to activate Ced-3/Caspase-9 for apoptosis to occur. Ced-
9 / Bcl-2 interacts with Ced-4/Apaf—1 and sequesters it away from Ced-3/Caspase-9,
thus inhibiting apoptosis. Egl-l /Bik binds to and inhibits Ced-4/Apaf-1.
(modiﬁed from Adams and Cory 1998). ................................................................................... 28

Figure 5. The Bel-2 Family. (modiﬁed from Adams and Cory 1998; Reed 2000). .................... 35

Figure 6. A ﬂow diagram of possible mechanisms leading to the progression and
development of disease in RAO-susceptible horses. Accumulation of neutrophils
and their products is an early feature of the disease, happening in three to ﬁve

hours after a challenge. Activated neutrophils generate IL-1B and INF-0t, which
activate NF-KB, which in turn induces their transcription initiating a positive
feedback loop (not shown). TNF-a also increases EGFR expression causing
MCM, mucus secretion, and mucin synthesis. Neutrophil products likely increase

mucus viscoelasticity and mucin (MU CSAC) expression (via TNF~0L~induced
EGFR expression and ROS-induced transactivation), which subsequently decrease
the clearance of and increases the production of mucus, respectively. Concurrently,
neutrophil elastase, a well-known MC secretagogue, induces mucus secretion by
interacting with MC (via adhesion molecule ICAM-l). Together, possibly with
decreased clearance (due to decreased mucociliary transport and increased
viscoeleasticity), accumulated mucus in the airway, and if it occurs, increased
numbers of MC (due to longer survival time, proliferation of existing MC, and/ or
differentiation of progenitor cells into MC), contributes to the visible increased
mucus score. Increased luminal mucus and airway remodeling potentiate airway
obstruction due to bronchospasm. In addition to neutrophil products,
‘bronchspastic’ neurotransmitters (ACh), inﬂammatory mediators, and nonspeciﬁc
irritants (dust) stimulate hypersensitive airway smooth muscle to contract. Because
of remodeling and increased thickness of the airway walls, this exaggerated
contraction (AHR) greatly reduces luminal diameter (bronchospasm) around mucus
plugs to cause airway obstruction that is measurable as the maximal change in
pleural pressure (APme) during tidal volume at rest. Arrow thickness denotes
known, relative contributions. Dotted line represents possible pathways. Solid

boxes are mediating factors; dotted boxes are consequential effects. Components
highlighted in yellow are the focus of this thesis. ......................................... - 49

 

Figure 7. Effect of atropine (0.02 mg/ kg IV) on the maximal change in pleural pressure
(APplmu) during tidal breathing in six stabled horses with a history of RAO.
Atropine decreased APme in all horses ................................................. 59

 

Figure 8. Expen'mental Design. One horse of each pair was brought into the stable, fed
research hay and bedded on straw for 5 days. At the end of the 5-day

environmental challenge, samples for APplmu, BALF, and mucus score were
collected. The management was changed to pellets and shavings for 7 days to
induce a partial remission of disease. At the end of the twelfth day, samples for

APplmx, BALF, and mucus score were collected once more and the horse was
euthaniszed. Post-mortem samples included 8 bronchi and 8 tissue sections. .................. 61

Figure 9. Regions Of horse lung from which sections were taken. Sequence of sampling
was randomized for each horse. Black lines specify location of clamps for the
isolation of parenchymal sections. ............................................................................................. 64

Figure 10. Negative control (left) and positive control (right) for Bcl-2 in a RAO-
affected horse. (NOTE: Images in this thesis are presented in color). ............................... 65

Figure 11. (A) Maximum changes in pleural pressure in RAO-affected and control horses

on days 5 and 12. Values are means i SEM of RAO (n26) and control (n=6)
horses after a 5-day hay/ straw challenge (day 5) and after 7 days on pellets/ shavings
(day 12). There was a signiﬁcant difference between diseased and control horses on

both days, but no effect of time in either group. (B) Individual values of 11%me
the six RAO-affected horses at days 5 and 12. There was considerable variability in

APpl“ml at day 5; horses with the greatest values decreased most by day 12. (C)
Subjective tracheal mucus score. The scoring system is as follows: 0=no visible
mucus, 1=single or a few mucus globules; 2=many mucus globules that are
beginning to coalesce, 3=many globules and a continuous mucous stream; 4=thick
continuous stream and many coalescent mucus globules around the wall of the
trachea; and 5=mucus stream ﬁlls about one quarter of the tracheal lumen. (D)
Tracheal mucus score in RAO-affected and control horses on days 5 and 12.

Values are means i SEM of RAO (n=6) and control (n=6) horses after a 5-day
hay/ straw challenge (day 5) and after 7 days on pellets / shavings (day 12). There
was a signiﬁcant difference between diseased and control horses on both days, but
no effect of time in either group. (E) Individual values of mucus score in the six
RAO-affected horses at days 5 and 12. There was no consistent change in mucus
score between the two days. (F) Total neutrophils per micoliter BALF of control

and RAO-affected horses on days 5 and 12. Values are means :1: SEM of RAO
(n= 6) and control (11: 6) horses after a S-day hay/ straw challenge (day 5) and after 7
days on pellets/ shavings (day 12). On day 12, the total neutrophils of control
horses was signiﬁcantly less than the day 5 value and signiﬁcantly different from the
RAO-affected horses. RAO-affected horses did not change from day S to day 12.
(G) Individual values of neutrophils per micoliter BALF in the six RAO-affected
horses at days 5 and 12. There were no consistent changes in neutrophils between
the two days 70

 

Figure 12. (A) Alcian Blue/ Positive Acid-Schiff (AB—PAS) stained epithelium of a
representative control (left) and RAO-affected horse (right). (B) Number of
mucous cells (MC) per millimeter of basal lamina (BL). and (C) mean numbers of
MC. No signiﬁcant regional or disease effects were detected. Values are means i
SEM of RAO (n=6) and control (n=6) of MC in each region of lung. (D) Stored
intraepithelial mucosubstance (V s) in large cartilaginous airways of control and

xi

RAO-affected horses. There were no signiﬁcant differences between groups. (B)
Relationship between the total neutrophils per microliter BALF and Vs in control
and RAO-affected horses. In RAO-affected horses, there was a negative

correlation between the numbers of neutrophils and the Vs. Black diamonds (0)
represent control horses; grey squares (I) represent RAO—affected horses. Values
are means 1' SEM. (NOTE: Images in this thesis are presented in color). ........................ 72

Figure 13. (A) Bel-2 staining cells in the epithelium of a representative control (left) and
RAO-affected horse (right). Bcl-2 staining in the RAO-affected horse was
unmistakably more abundant than that seen in the control horse. (B) Percent Bcl-
2-positive mucous cells (MC) present in each region of lung. RAO-affected horses
had signiﬁcantly greater numbers and percentages of Bcl-2-positive MC compared

to controls for each region of lung sampled. Values are means i SEM of RAO
(n=6) and control (n=6) horses in each region of lung. (C) Percent Bcl-2-positive
mucous cells (MC) in relation to total neutrophils per microliter BALF in RAO—
affected and control horses. Horses having more than 10 neutrophils per microliter
of BALF tended to have Bel-2 staining in more than half of their MC while those
horses with less inﬂammation had less expression. The one RAO-affected horse
with similar numbers of neutrophils as controls had a similar percentage of Bcl-2-

positive MC as the controls. Black diamonds (6) represent control horses; grey
squares (I) represent RAO-affected horses. (NOTE: Images in this thesis are
presented in color). ....................................................................................................................... 73

Figure 14. Bcl-Z/AB staining in three airway generations. (A-C) A second-generation
airway, a 4-6 mm diameter conducting airway, and a non- conducting airway of a
control horse, (D- F) a second-generation airway, a 4-6 mm diameter conducting
airway, and a non-conducting airway of a RAO-affected horse, (G, H) a 4-6 mm
diameter conducting and a non- -conducting airway from “horse 5. ” (NOTE:
Images in this thesis are presented in color). ............................................................................ 74

Figure 15. Mitochondria are ampliﬁers and mediators of apoptosis, rather than initiators.
(1) Cytochrome c leaks out of the intermembrane space into the cytosol where it
binds to and increases nucleotide-binding afﬁnity of Apaf—l by 10-fold. ATP (or
dATP) binding to Apaf—1/ cytochrome c induces a shapa change and
Oligomerization into a multimeric wheel-like structure to which caspase-9 binds to.
This functional complex is called an apoptosome. The apoptosome cleaves pro—
caspase-3 and releases active caspase-3, which will cleave pro-caspase—7 and release
caspase-7 in the caspase cascade, leading to nuclear fragmentation and apoptosis.
(2) Smac/Diablo is released when the functional barrier is disrupted. In the cytosol,
Smac/Diablo’s N-terminal binds to the BIR domain of IAPs (e.g. XIAP) and
removes the inhibitory effects of XIAP on caspase-3 and -9. (2’) In the presence of
high IAPS, Smac/Diablo cannot bind enough IAP and the inhibitory affects
remain. The Fas-mediated pathway can still initiate apoptosis, however, because of
caspase-8 cleaving and activating cystolic Bid, a pro-apoptotic Bcl-2 family member,
causing its translocation and insertion into the outer mitochondrial membrane
(OMM). Insertion of truncated Bid (tBid) leads to OMM permeabilization.

xii

Caspase-8 is insensitive to XIAP (Riedl, Renatus et al. 2001). (3) AIF is released
and induces nuclear condensation, independent of any caspase activation. ........................ 82

xiii

A‘l’m

APplm,
1 S-HETE
AB
AHR
AIF
ANT
Apaf—I
ATP
Bad
Bak
BALF
Bax
Bel-2
Bcl-X
Bel-XL
Bcl-Xs
BH
Bid
Bik
BIR
Bok
CAMP
CARD
Caspases
CF
cGMP
DAG
DD
DED
Diablo
DISC
ECG
EGFR
eNAN C
FADD
F asL
GCH
IAP
ICE
IFN-y
IKK
IL

LIST OF ABBREVIATIONS

Mitochondrial Inner Transmembrane
Potential

Maximum Change in Pleural Pressure
1S-Hydroxyeicosatetraenoic acid
Alcian Blue

Airway Hyperresponsiveness
Apoptosis Inducing Factor

Adenine Nucleotide Translocator
Apoptotic Protease Activating Factor-1
Adenosine Triphosphate
Bcl-2-Antagonist of Cell Death
Bcl-2-Antagonist/ Killer
Bronchoalveolar Lavage
Bcl-Z-Associated X Protein

B Cell Lymphoma-2

Bcl-2-Related Protein

Bcl-2-Related Protein, Long Isoform
Bcl-2-Related Protein, Short Isoform
Bcl-2 Homology Domain

BH3 Interacting Domain Death Agonist

Bcl-Z-Interacting Killer
Baculovirus IAP Repeat Domains
Bcl-2-Related Ovarian Killer
Cyclic Adenosine Monophosphate

Caspase-Activating Recruitment Domain

Cysteine Aspartate-Speciﬁc Proteases
Cystic Fibrosis

Cyclic Guanosine 5’-Monophosphate
Diacylglycerol

Death Domain

Death Effector Domain

Direct IAP Binding Protein with Low pI

Death-Inducing Signaling Complex
Electrochemical Gradient
Epidermal Growth Factor Receptor
Excitatory NAN C

Fas-Associated Death Domain (MORTI)

Fas Ligand

Goblet Cell Hyperplasia

Inhibitors of Apoptosis Protein(s)
IL-l B-Converting Enzyme (Caspase-l)
Interferon-gamma

IKE-Kinase

Interleukin

iNAN C
11’;

IP3-R

LPS

LTB4
MARCKS

MC

MCM
MMC
MMP

MUC
NANC
N F -KB
OMM
OVA
PAS
PBS
PKA
PKC
PKG
PLC
PP2A
PT
PTP
PTPC
RAO
RIP
ROS
sGC

Smac

TNF
TNFR
TRADD
TRAF
TRAIL

VDAC
Vs
XIAP

Inhibitory NAN C

Inositol Triphosphate

1P3 Receptor
Lipopolysaccharide
Leukotriene B4

Myristoylated Alanine-Rich C Kinase
Substrate

Mucous Cell(s)

Mucous Cell Metaplasia
Metaplastic Mucous Cell(s)
Mitochondrial Membrane
Permeabilization

Mucin Gene Abbreviation
Nonadrenergic-Noncholinergic

Nuclear Factor-Kappa B

Outer Mitochondrial Membrane
Ovalbumin

Periodic Acid-Schiff

Phosphate Buffered Saline

Protein Kinase A

Protein Kinase C

Protein Kinase G

Phospholipase C

Protein Phosphatase-2A
Permeability Transition

PT Pore

Permeability Transition Pore Complex
Recurrent Airway Obstruction
Receptor Interacting Protein
Reactive Oxygen Species

Soluble Guanylyl Cyclase

Second Mitochondrial Activator of
Caspases

Tumor Necrosis Family

TNF Receptor(s)

TNF-R1 -Associated Death Domain
TNFR-Associated Factor(s)

TNF Related Apoptosis-Inducing
Factor

Voltage Dependent Anion Channel
Volume Density
X-Linked-Inhibitor-Of-Apoptosis
Protein

INTRODUCTION

Recurrent airway obstruction (RAO) is an equine respiratory syndrome characterized by
reversible airway obstruction that is due to bronchospasm, accumulation of mucoid secretions
often containing neutrophils, and remodeling of the airway wall. Acute exacerbations of RAO
are induced by exposure to organic dust usually from eating hay. When horses susceptible to
this disease are in remission at pasture, there is a signiﬁcantly greater amount of tracheal mucus
relative to that present in control horses, as indicated by an assigned subjective mucus score.
Once these animals are brought into a dusty environment, their tracheal mucus score increases
signiﬁcantly while the control horses’ mucus scores remain unchanged.

Increased mucus in RAO horses may be due increased mucus production and secretion
or decreased clearance or both. Although increased viscoelasticity and decreased clearance occur
in challenged RAO horses compared to normal horses, the difference does not fully explain the
existence of increased amounts of respiratory secretion (Gerber, King et al. 2000). Therefore,
increased secretion of mucus must play a role in the high levels in RAO horses.

The maintenance of signiﬁcantly greater quantities of mucus in RAO horses in remission
leads me to ask the question: “does delayed apoptosis of mucous cells contribute to the
observed mucus accumulation in RAO-affected horses?” In other words, do mucous cells have
a longer life due to the abnormal prevalence of the anti-apoptotic protein, Bel-2? I hypothesized
that delayed apoptosis, as indicated by an increased presence of Bcl-2-positive mucous cells,
contributes, at least in part, to an increase in mucus-secreting cells and subsequent airway mucus

accumulation.

CHAPTER 1
LITERATURE REVIEW

MUCOUS APPARATUS IN AIRWAYS
STRUCTURES

The lower respiratory system can be divided into the conducting airways and the gas
exchange area. The conducting airways, which serve to connect the environmental air to the
alveoli, include the trachea, bronchi, and bronchioles. Gas exchange occurs in the exchange area
that is comprised of the respiratory bronchioles, alveolar ducts and alveoli. The airways of the
tracheobronchial tree are classiﬁed based on: (1) the morphology of the surrounding
components and (2) the diversity of the cellular makeup. The distinct regional characteristics of
airway morphology and cellular composition allows for separation of functions. The
tracheobronchial tree conducts oxygen into alveoli and removes carbon dioxide from the blood
while effectively keeping large particles, pollutants, and pathogens from entering the exchange
area.

Traveling from proximal airways to distal airways, the epithelial cells change to allow for
particle trapping and removal in the large central airways while prohibiting foreign molecules
from entering the small, distal airways where gas exchange occurs. For example, in the trachea
and bronchi, the widespread surface mucous cells (MC) provide the mucus lining of the
bronchial airways that traps particles. Mucous cells are found only in larger bronchioles and
decrease in numbers until they are absent in smaller bronchioles. Likewise, ciliated cells—
playing the role of mucous transport—follow similar patterns as MC where they are abundant in
large airways and taper off in numbers in smaller airways. Cartilage is present in large
conducting airways to maintain airway patency but absent in small bronchioles. There is a

positive correlation between the size of an animal and its epithelial height, and a negative

correlation between animal size and number of airway generations (Mariassy 1992). On average,
the number of generations is around 23 in humans (Mariassy 1992).

The conducting airways have three layers (1) epithelium, basal lamina, and lamina
propria, collectively making up the luminal mucosa, (2) the submucosa, composed of connective
tissue, smooth muscle, and cartilage, and (3) the adventitia. With the exception of smooth
muscle, ﬁbroblasts, cartilage, and neural components, the airway structures are of endodermal
origin (Leff and Schumacker 1993). The mucosa is the innermost, well-vascularized continuous
lining (Mariassy 1992) that serves to condition the air and to protect and cleanse the lung’s
surface. It rests on the basement membrane composed mostly of type IV collagen and laminin,
and is separated from the underlying submucosa by a smooth muscle layer, the muscularis

mucosa

Luming Mucosa

Ciliated cells
In the cartilaginous airways, trachea and bronchi, there is a pseudostratiﬁed, ciliated

columnar epithelium; in non—cartilaginous bronchioles, the epithelium is ciliated columnar
epithelium. In a study done in dogs by Seraﬁni, Wanner et a1 (1979), ciliated cells comprised
22% of the tracheal epithelium and 25% of main bronchus, a range of 20% to 50% have been
documented in trachea of other species (Wolff 1992). Ciliated cells decrease to around 5 % of
total cells in smaller bronchi until the epithelium consists of mainly a single layer of cuboidal
ciliated cells (Seraﬁni, Wanner et al. 1976).

The distinctive appearance of ciliated cells is due to their apical cilia and microvilli. Each

of the cilia (50 to 300) is about 6 pm in length and has a diameter of 0.3 um; each of the

micovilli (100 to 150) is 2 to 3 um long with a 0.1 pm diameter (Alexander, Ritchie et al. 1975;

Mariassy 1992). Sliding microtubules in a 9 + 2 formation with dynein arms as a motor provide

the cilia with a cyclic back—and-forth movement. Three to seven short “claws” (25 to 35 nm
long) on the tips of cilia aid in movement (mechanically or chemically) of the mucus
glycoproteins by catching and attaching to the underside of the gel layer during the effective
stroke (Wanner, Salathe et al. 1996;]efcoat 2002). At the base is “the necklace” region which
houses the calcium-binding protein, centrin (Wanner, Salathe et a1. 1996). This “mucociliary

escalator” is the chief defensive mechanism of the lung.

Clara Cells
Distinct non—ciliated, secretory bronchiolar cells, also called Clara cells, serve a role of

detoxiﬁcation in the lung. They may also be progenitors of the ciliated cells, play a role in
epithelial regeneration after injury, and contribute to the secretions of small airways, however
these secretions are not thought to contain mucoid substances (Finkbeiner and Widdicombe
1992; Leff and Schumacker 1993; Wanner, Salathe et al. 1996). Horse Clara cells hold no
glycogen (Mariassy 1992). In large animals, Clara cells are restricted to the non-cartilaginous
bronchioles, but in small mammals, they are found in both cartilaginous and non-cartilaginous
airways. These cells contain membrane-bound secretory granules, extensive smooth
endoplasmic reticulum at their apex, and rough endoplasmic reticulum in the basolateral

perinuclear regions (Mariassy 1992).

emu elIs
Serous cells are mainly present in the surface epithelium of pathogen-free rodents, in

animals lacking submucosal glands, and in the human fetus (Basbaum, Jany et aL 1990). In
humans, they are conﬁned, for the most part, to the submuiscosal glands after birth. Serous
cells are identiﬁed by their small (600 nM) electron-dense homogenous secretory granules
present on the apical portion of the cell from which they secrete neutral glycoproteins

(Finkbeiner and Widdicombe 1992; Mariassy 1992; Newman, Robichaud et al. 1996; Wanner,

Salathe et al. 1996). They may also secrete lipids, lysozymes, lactoferin, and mucus proteinase
inhibitors (Wanner, Salathe et al. 1996). Their cytoplasm also contains rough endoplasmic

reticulum, a large vesicular nucleus and lacks smooth endoplasmic reticulum (Mariassy 1992).

Neuroendocrine Cells

Neuroendocrine cells, basal cells, brush cells, and seromucous cells occupy a relatively
small component of the cellular makeup of the epithelium. Little is known about
neuroendocrine cells besides their suggested function which is release of seemingly large
quantities of gastrin related peptide (GRP), dopamine, and serotonin, which may play a role in
reactive airway disease and pulmonary hypertension (Mariassy 1992; Plopper and Hyde 1992;

Leff and Schumacker 1993).

Basal Cells
Basal cells are small (5 to 15 pm) and contact the basal lamina. Proposed functional

roles for basal cells are maintenance of bronchial airways, possibly by tethering columnar

epithelium to the airway wall and serving as multipotent progenitor cells (Hong, Reynolds et al.

2004).

Brush Cells
In the trachea, there are focal areas 1 mm in diameter of non-ciliated cells with either

microvilli or brush-like projections on the center of the cell apex (Alexander, Ritchie et a]. 1975).
They may also serve as sensory receptors associated with the trigeminal nerve endings (Textbook
of Veterinary Histology 5'” Ed, Editors H. Dieter Dellmann and JO Ann Eurell; Williams and
Wilkins Baltimore, 1998; Authors: Donald R Adams and H. Dieter Dellmann; p 149-151).

These brush cells are rare and always less than 1% of the cell population (Mariassy 1992).

Seromucous Cells
Seromucous cells are those cells that were ﬁrst identiﬁed as MC but overlap with the

serous cell morphology. They have an electron-lucent rim with an electron-dense core, with

discrete homogenous “nucleated granules” as seen in serous cells (Mariassy 1992).

Submucosal Glands
Airway secretions come in part from submucosal glands, which play important roles in

hydrating the airway surfaces, facilitating mucociliary transport, and are involved in the
augmentation of the ﬂuid matrix for the macromolecules secreted by the mucosa, including
mucins, an albuminoid substance contained in mucus (Ballard and Inglis 2004). While in upper
airways of some species, the greater contributor of mucus is from submucosal glands, in the
horse few glands are present leaving the MC as the main supplier of mucus (Widdicombe and
Pecson 2002). Developmentally, glands are derived from basal cells and appear around week 10
of gestation (Leff and Schumacker 1993). These tubuloalveolar glands empty into the airway
lumen of cartilaginous airways and are usually found in tissue between the cartilage rings; they
are not present in bronchioles (Finkbeiner and Widdicombe 1992; Ballard and Inglis 2004).
Within the glands, there are ciliated cells, resembling surface epithelium, at the oriﬁce of the
primary duct and “nonspeciﬁed” cells in the collecting ducts (Leff and Schumacker 1993; Ballard
and Inglis 2004). The serous cells at the most distal end of the tubule and MC slightly proximal
carry out the exocn'ne function. It is suggested that, in some mammals, this arrangement enables
the ﬂuid from the serous cells to ﬂush out the mucins secreted from the MC and that, therefore,
these cells have predominant mediator functions over the MC (Ballard and Inglis 2004).
Submucosal glands in equine airways are very unevenly distributed, generally occurring

about 100 pm apart in longitudinal rows of about 5, with an average frequency of 1.0/mm2 of

mucosal surface (Widdicombe and Pecson 2002). Each gland puts out a volume of about 17 n1,

this volume is only around 15% of other species (Rubin 2002).

Mucous Cells
Airway MC, also called goblet cells, participate in the homeostasis and the

pathophysiology of the lung by rapidly secreting the contents of their granules and increasing in
number, respectively. Their classical “goblet” shape may be merely an artifact of ﬁxation. The
granules contain both neutral and acidic mucus with sialic acid and sulfate groups within mucin
glycoproteins, which determines the viscoelastic properties of the sputum (Leff and Schumacker
1993). Each granule measures about 800 nM in diameter (Wanner, Salathe et al. 1996). The
granules found in these are more numerous and have a larger diameter than those in serous cells
(W anner, Salathe et al. 1996). A morphologically distinguishing feature of MC is that the cytosol
is more electron-dense, relative to other epithelial cells, with electron-lucent secretory granules,
indicative of mucins (Finkbeiner and Widdicombe 1992; Newman, Robichaud et al. 1996).
Normal epithelium contains approximately 6,500 MC per mm2 (30,000-50,000 goblet cells per
mm’), comprising from 5% to 20% of the epithelium in sheep, monkey, and cat tracheas, and
25% of the bronchial epithelial cells in humans and larger mammals (Rogers 2003). In central
airways of humans, the ratio of ciliated columnar epithelial cells to MC is 5:1; absolute numbers
of both decrease from trachea to peripheral airways (Wanner, Salathe et al. 1996). In distal
airways, declining numbers of goblet cells with concurrent rise in Clara and simple cuboidal cells
consequently decreases the amount of surface mucus. Goblet cells are rare in healthy
bronchioles.

Airway MC are most recognizable by the presence of extensive Golgi network and
secretory granules. A three-dimensional reconstruction of a tracheal goblet cell showed that

each of the multiple cytoplasmic granules associates with a separate Gogi apparatus (Adler,

Hardwick et al. 1982). Mitochondria and rough endoplasmic reticulum resides between the
conspicuous secretory granules in the perinuclear region, along with the Golgi complex. The
large, round, dense nucleus is compressed near the basolateral membrane and composed of
heterogenous chromatin. Up to 60% of the contents of a MC are membrane-bound granules;
this percentage is inversely proportional to the space taken up by organelles (Mariassy 1992).
However, ﬁxation and tissue embedding do not preserve the carbohydrate component of the
mucous granules leaving the cell appearing to be “empty” (Mariassy 1992).

Lamina Propria

The lamina propria is a 50 to 100 pm thick layer of connective tissue that attaches to the

basement membrane. Within the lamina propria is an extensive capillary network.

Submucosa

Beneath the lamina propria is the submucosa made up of smooth muscle and connective
tissue. Within the trachea and bronchi, the connective tissue contains glands, vessels, and nerves
while the irregular spirals of smooth muscle partially surrounds the airway along with cartilage.
In smaller airways, the thin layer of smooth muscle completely encircles the airway and the
airway contains no cartilage or glands (Mariassy 1992). Mucosal and submucosal efferent
innervation is provided by parasympathetic nerves to paratracheal ganglia before synapsing in
airway smooth muscle, vasculature, submucosal glands, and epithelium; sympathetic neurons
synapse in cervical and thoracic ganglia (F inkbeiner and Widdicombe 1992). The sympathetic

and predominantly cholinergic parasympathetic ﬁbers intermingle in the respiratory tract

Adrienne
Beneath the submucosa lies loose connective tissue, the adventia. This is part of the

bronchovascular adventitia that surrounds airways and adjacent blood vessels.

AIRWAY MUCUS LINING AND THE MUCOCILIARY SYSTEM

Each day, approximately 2ml of ﬂuid per kilogram of body weight is secreted in cats and
rabbit—double in humans—for an estimated total resting secretion of 24 ml per day (Finkbeiner
and Widdicombe 1992); (Wanner, Salathe et al. 1996). During chronic exposure to irritants,
increased MC numbers and their increased mucin secretions can easily compromise the
mucociliary system resulting in mucus trapping and obstruction of the airways, particularly if
there is bronchospasm of the airway. This is especially the case in small non-cartilaginous
airways where MC are present but coughing cannot adequately eliminate secretions. Therefore,
removal of mucus relies on the mucociliary clearance system. Thus, in this instance, mucus
ceases to have a protective function for the epithelium and instead takes on a pathophysiological
role.

The main contribution of the respiratory epithelium to protection of the internal milieu
from the external atmosphere is attributable to its ability to secrete a thin lining of ﬂuid (average
5 pm in rats and 8 pm in humans) onto the surface of the epithelium (Hatch 1992). In 1934,
Lucas and Douglas ﬁrst proposed the notion that mucus contains two layers (Lucas and Douglas
1934; Wolff 1992; Wanner, Salathe et al. 1996). In some animals, including the horse, there is a
mucous (gel) layer and a periciliary (sol) layer collectively forming the mucous or ﬂuid lining.
There may also be a thin layer of surfactant separating the two layers (W anner, Salathe et al.
1996; Rubin 2002).

Having two layers of mucus makes conceptual sense: the effective stroke of the ciliary
beat takes place in a mucus-free solution, the periciliary ﬂuid, which has a depth approximately
equal to the cilia length. This would permit the mechanical coupling of the cilia claws with the
mucus. Simpliﬁed, it would seem that too thick of a periciliary ﬂuid would prohibit cilia

interactions with the mucus. likewise, too thin of a sol layer would bring the mucus layer over

the cilia tips and inhibit ciliary movement through both effective and recovery strokes (Wanner,
Salathe et al. 1996).

For cilia to move, mucus must be present (Eliezer, Sade et al. 1970; Wanner, Salathe et
al. 1996). However, just because mucus is present, does not mean that cilia will move. Spungin
and Silberberg (1984) experimentally demonstrated that tactile stimulation, like that provided by
entrapment of a particle, would restart ciliary beat that was formerly in a resting or unstimulated
state, if the epithelium is not deprived of mucus. The ﬂuid moved by other cilia will inﬂuence
the beat frequency of the next individual cilia. This phenomenon synchronizes the strokes of
neighboring cilia, thereby minimizing the resistance of the mucus (Wolff 1992).

Clearance of mucus has been described as being “biphasic,” having a fast “effective”
stroke and slow “recovery” stroke in the return direction (Wanner, Salathe et al. 1996). During
its recovery Stroke, the ciliurn swings almost 180 degrees backwards closer to the cell surface,
then fully extends until perpendicular to the cell surface throughout the effective stroke at a
maximum velocitly of 1 mm/ sec (Wanner, Salathe et al. 1996). The ciliurn rests shortly at the
end of the cycle, before resuming the next recovery stroke. The recovery stroke is two to three
times slower and does not drive the mucus; this provides propulsion in the direction of the
effective stroke. Furthermore, the roots of the cilia are organizes such that the effective strokes
are all in the same directions (Wolff 1992; Wanner, Salathe et a]. 1996). Mucus is not likely to be
uniformly moved in a “blanket” (Wolff 1992; Wanner, Salathe et al. 1996). Rather it is more
likely to move in “plaques” that were produced in response to the particles ﬁrst impact on the
epithelium (Spungin and Silberberg 1984).

Ciliary beat frequency plays a role in mucus clearance. In the central airways, the ciliary
beat frequencies ranges between 10 and 15 Hz in many mammals (W anner, Salathe et al. 1996).

Ciliary beat frequency of upper airway epithelium is modulated through at least two distinct

10

pathways: (1) the beta 2—adrenergic receptor produces ciliary stimulation by a pathway involving
increased intracellular cAMP levels, and (2) the muscarinic receptor increases ciliary beat
frequency by a mechanism involving production of prostaglandins, nitric oxide, and cGMP
(Yang, Schlosser et al. 1996). Additionally, pressure (Calvet, Verra et al. 1999), PKA and PKG
(Wyatt, Spurzem et al. 1998), and histamine (Schuil, van Gelder et al. 1994) have been shown to
regulate ciliary beat frequency.

The highest mucus velocity is in the upper airways, and in each increasing generation, the
velocity decreases. In the trachea, mucus travels at a velocity of 4 to 20 millimeters per minute
(VViHoughby, Ecker et al. 1991; Wanner, Salathe et al. 1996). Mucus velocity decreases in
peripheral airways because there are fewer, shorter cilia, with a slower beat frequency.
Additionally, MC numbers decrease with increasing generation until absent in the terminal

bronchioles.

The composition of the mucus predicts its effectiveness in the entrapment and removal
of particulates during invasive challenges. The airway secretions have both viscous and elastic
properties to trap airborne particles and transport them up to the pharynx to be swallowed; the
effectiveness of the interactions between cilia and secretions is determined by viscoelasticity of
mucus. The thin ﬂuid mucus lining in the nasal passage, trachea, and conducting airways is an
aqueous solution containing high molecular weight mucous glycoproteins called mucins, and a
suspension of proteins, lipids, and glycoconjugate components.

Mucus is elastic because it deforms with an applied force and that it can store energy, but
because permanent deformation does not occur, mucus is not perfectly elastic (Wolff 1992). In
the same respect, the viscous properties of mucus are that it ﬂows when force is applied to it.

Likewise, viscosity fails as the mucous layer gets thinner with an increased applied force (Wolff

ll

1992; Wanner, Salathe et al. 1996). Taken together, the transport of mucus is indirectly
proportional to its viscosity and directly proportional to its elasticity (Wanner, Salathe et a1.
1996)

Mucus viscoelasticity is the main controlling factor of mucus transportability by cilia,
rather than its biochemical characteristics. With each applied force to the mucus, viscosity
decreases giving it a relaxation time up to 30 seconds long, enough time to trap and retain
foreign particles (Wanner, Salathe et al. 1996). However, because the effective stroke of cilia is
much faster than the recovery stroke, the cilia encounter mucus in a more solid state having a
higher viscosity than if it were having a shearing force applied to it. Experiments done in 1977-
1978 showed that the fastest transport rate was mucus with high elasticity and low viscoSity
(Shih, Litt et al. 1977). In chronic bronchitis, alternately, viscosity is the controlling factor, as
elasticity does not change (Wolff 1992). Thus, decreased clearance (mucokinesis) is due to
decreased mucociliary transport. In addition to rheological properties, the surface tension,
thread—forming ability, and thickness of the mucus play a role in its movement (Wanner, Salathe
et al. 1996; Wolff 1992).

Other factors that play a role in clearance include environmental factors, temperature
and humidity, mechanical stimulation (cg. particle deposition), pharmacological agents,
infectious diseases, and chronic inﬂammation, which leads to MC hyperplasia and metaplasia.
Particle deposition may promote gel layer formation (Art 2002). While beta-agonists, xanthine,
and cholinergic agents increase ciliary activity, anesthetics cause it to decrease. Xylazine and
detomidine hydrochloride given to normal horses cause tracheal clearance rates to decrease
signiﬁcantly, ranging from 18 to 54% (Willoughby, Ecker et aL 1991). The effects of mucolytics
and expectorants have only been tested in humans (Art 2002). Bacterial diseases and purulent

secretions impair mucociliary functions. Respiratory viral infections target the epithelial cells of

12

the lung producing desquamation, microvascular dilatation, edema, and an inﬂammatory cell
inﬁltrate (Hogg 2000). Interactions between infectious agents modify the infections of a single
agent, where viral infections pave the way for bacterial agents. Combined viral and bacterial
infections, acting in synergy, aggravates clinical diseases by impairing the ciliary function and

promoting mucus secretion (Degre 1986).

Mucus Constituents

Mucus is about 95% water and the remaining 5% is proteins, lipids, and glycoconjugate
components combined. The main role of water is regulating the depth of the sol layer bathing
the cilia. The important water sources are air condensation during expiration and osmotic
secretion from the plasma whereas removal is through reabsorption, concentration and

evaporation (Hatch 1992).

Mucins

High molecular weight glycoconjugates, or mucins, are rapidly secreted and, despite
accounting for less than 2% of the wet weight, serve as a MC marker. The colocalization of the
special stains alcian blue/ periodic acid-Schiff (AB / PAS) indicates speciﬁc mucin components,
speciﬁcally, AB stains the acidic radicals and PAS stains the neutral glycoproteins.

Mucins are 70% to 80% carbohydrates, 20% protein, and 1% to 2% sulfate with attached
oligosaccharide side chains (Rubin 2002). Mucins are extremely polyanionic at a neutral pH due
to sulfation of oligosaccharide and terminal sialic acid (Wanner, Salathe et al. 1996). The core of
the mucin, called an apoprotein, is a single poylpetide chain (Wolff 1992) with extensive
tandemly repeated amino acid sequences, largely threonine and serine. Mucins link together via
disulﬁde bonds in an end-to-end fashion to form molecules 0.5 to 6 um in length (Wanner,

Salathe et al. 1996).

13

Other Proteins
Proteins in the sputum account for only 0.1% to 0.5% (Wolff 1992) of the total volume

and, for the most part, are secreted directly from the plasma through tight junctions, or to a
lesser extent, co-packaged with other secretions (Hatch 1992). Reabsorption and elimination of
proteins is achieved through a type of endocytosis called transcytosis, which may be selective
(Hatch 1992). In dogs, mucus protein concentrations are increased during pharmacological
stimulation and this increased Viscosity of the gel layer (Hatch 1992). The most common

proteins are albumins, immunoglobulin A, lysozyme, and lactoferrin.

Carbobzdrates
Carbohydrates comprise 0.2% to 3 % of the wet weight of mucus (Hatch 1992; Rogers

2003) and are the major contributors to the viscous and elastic properties (Hatch 1992;]efcoat
2002; Rogers 2003). Speciﬁcally, the high molecular weight glycoconjugates modify the
viscoelastic properties by forming a polymer matrix with strong afﬁnity to water and positively
charged ions and proteins. There are three enzymes required for biosynthesis and elimination is
through ciliary action and lysozomal activity in the mucous gel. De nooo synthesis of
carbohydrates occurs in MC and submucosal glands, the latter contributes the larger percentage
of airway secretion (Hatch 1992).
Liv—ids

Lipids represent 0.3% to 0.5% in the sol phase (Wolff 1992). They occur in saliva and
synovial ﬂuid and therefore are normal in the mucus. However, lipids collected in
bronchoalveolar lavage ﬂuid poorly serve as an indicator of mucus quantity as most lipids are
from the surfactant. However, damaged epithelium releases products such as phospholipids into

the airway ﬂuid that could be an indicator of bacterial infection Widdicombe 1995).

14

DNA and Actin Polymers
DNA and actin are also present in insigniﬁcant amounts normally, but become

Signiﬁcant during inﬂammation (Wanner, Salathe et al. 1996) as they increase mucus
viscoelasticity (Gerber, King et al. 2000). These constituents mostly arise from cellular debris

such as bacteria, luminal leukocytes, and epithelial cells.

Human periciliary ﬂuid is slightly hypoosmotic and acidic, contains les sodium and
chloride, and more potassium than plasma with the salt and pH levels determining hydration of
the mucus (Wanner, Salathe et al. 1996). In obstructive airway diseases, or bronchitis, and
particularly during inﬂammatory and infectious episodes, mucus dehydration is associated with
an increase in secreted molecules and with marked augmentation of DNA content. This in turn

increases viscoelasticity of the surface mucus (Puchelle, de Bentzmann et al. 1995).

ngose of Mucus

Three main functions of mucus are: (1) clearance of inhaled particulates, (2), providing a
chemical screen and biological barrier and (3) protection of the lung from dehydration. The
mucociliary activity is a result of the sol and gel layers.

The proteins and lipids in the mucus—acting independently or in combination—
inactivate bacterial, viral, and fungal pathogens. Chemically reactive airborne pollutants undergo
reactions with antioxidants in mucus. Phagocytosis by macrophages in conjunction with the
mucociliary escalator and coughing are most important mechanisms for clearance of pathogens
and particulates.

Prevention of dehydration relies. on the maintenance of ionic composition of the mucus
layer, via the active transport of ions and water in the ciliated cells, and regulation of the rate of

mucin secretion in the goblet cells (Hatch 1992). Ciliated epithelial cells appear to be involved in

15

the secretion and absorption of ions and water into and out of the airway surface ﬂuid (Hatch

1992).

Mucus Synthesis and Secretion
There are 19 mucin genes in humans (MUCl through MUC19), at least eight of which

are expressed in the respiratory tract. Of these, MUCZ, MUCSAC (major MC mucin), and
MUCSB (major submucosal gland mucin, also in goblet cells) are the main gel-forming mucins
secreted in both normal and diseased airways of rats and humans Oefcoat 2002; Gerber,
Robinson et al. 2003; Rogers 2003). Equine homologues have been identiﬁed for MUCSAC and
MUCZ (eqMUCSAC and eqMUC2, respectively); while eqMUCSAC expression is high,
eqMUCZ has not been detected in equine lungs (Gerber, Robinson et al. 2003). No MUCSB
homologue has been found in the horse, possibly due to fewer numbers of submucosal glands
relative to human lung.

Mucin molecules are assembled within the extensive Golgi apparatus of the goblet cells.
After the core mucin apoprotein is assembled, glycosylation and scaffold formation occur—
hydroxy ions binding to threonine and serine—to allow binding of positively charged, acidic
oligosaccharide side chains (Wolff 1992;]efcoat 2002). The mucous glycoproteins inﬂuence the
viscoelasticity of the mucus layer (Majima, Harada et al. 1999).

Once synthesized, the mucins are tightly packaged into granules. Mucin molecules are
polyanionic so that upon secretion, there is rapid and massive expansion. However, in order to
ﬁrst be tightly packaged into granules, this biochemical restriction must be overcome. These
repulsive forces are overcome by interlacing a positively charged ion amongst the negative mucin
polymer matrix (Nguyen, Chin et al. 1998; Rogers 2003). This arrangement gives the mucin

“ion-exchange properties” (Nguyen, Chin et al. 1998) to allow for rapid exocytosis of the granule

l6

contents into the airway lumen; exocytosis of one mucin granule takes less than 100 milliseconds
(Rogers 2003) and it will expand up to 600-fold in the airway lumen (Rogers 1994).

Since mucus is required for ciliary beat, it is possible that ciliated cells can speciﬁcally or
nonspeciﬁcally signal mucus secretion. While the former is unlikely (Wanner, Salathe et a1.
1996), airway epithelial cells are capable of producing many substances shown to modulate
mucus secretion in a nonspeciﬁc manner. Extensive studies have investigated these substances,
including, nitric oxide (Runer and Lindberg 1999;]ain, Rubinstein et al. 1993), prostaglandins
(Gayner and McCaffrey 1998), and leukotriene C4 (Schuil, van Gelder et al. 1994).

Newman, Robichaud et al. (1996) categorized the stage or characteristics of exocytosis
into three types that have been used in subsequent studies (Rogers 2003). Simple exocytosis is
“constitutive secretion” in which a single granule fuses with the plasma membrane and is
secreted. Compound exocytosis is due to fusion of membranes of multiple granules
intracellularly and with the plasma membrane soon after. Apocrine-like secretion is the loss of

the central apical granules and some components of the cytoplasm.

The secretory process relies on synergistic events of concurrent activation of both
protein kinase C (PKC) and cyclic GMP—dependent protein kinase G (PKG) and translocation
of intracellular granule to the internal surface of the apical membrane Oefcoat 2002; Rogers
2003). The key regulator mediating this dynamic process is myristoylated alanine-rich C kinase
substrate (MARCKS) (Li, Martin et al. 2001). Binding of a mucin secretagogue to its receptor

initiates the sequence of events illustrated in Figure 1 (Li, Martin et al. 2001).

17

 

Figure l. Mucous Granule Movement and Exocytosis. Binding of a mucin secretagogue to its receptor
ultimately activates both PKC and PKG; dual activation is required for a robust secretory response. The
ligand-receptor complex activates phospholipase C (PLC), which results in the production of inositol
triphophste (1P3) and diacylglycerol (DAG), an activator of PKC. Increases in IP; stimulates caldum-release
from the intracellular stores (Le. mucin polymer matrix and intracellular free—calcium). Activated PKC
phosphorylates membrane—bound myristoylated alanine-rich C kinase substrate (MARCKS), leading to its
subsequent translocation into the cytoplasm. The membrane association of hL-XRCKS is phosphorylation
dependent; in a phosphorylated state MARCKS is cytoplasmic while in a dephosphorylated state MARCKS is
membrane—bound. At the same time, activation of PKG, via the nitric oxide-cyclic GMP pathway, activates
cytoplasmic protein phosphatase-2A (PPZA), which dephosphorylates MARCKS. After dephosphorylation by
PP2A, MARCKS recovers its membrane—binding abilities and becomes stabilized on membranes of
cytoplasmic mucin granules. Simultaneously, MARCKS interacts with actin and myosin, acting as a tether,
linking the granules to the intracellular contractile apparatus mediating granule movement to the apical
membrane and consequently exocytotic release. (NOTE: Images in this thesis are presented in color).

After docking of the granule to periphery of the cell, it must go through a priming
process before opening and releasing its mucins onto the airway epithelium (Figure 2)
(Rogers 2003). After the initiation of exocytosis, there is an efﬂux of calcium from the

mucin matrix via a CaIZ/K‘ exchanger on granule membrane (Rogers 2003). As mentioned

above, intragranular calcium, sequestered in the mucin polymer matrix serves as one calcium

18

extracellular sodium. There may also be a free cystolic calcium pool (Nguyen, Chin et al. 1998).
Nguyen, Chin et al. 1998 devised a model to illustrate the complex mechanisms involved in the
storage and release of intracellular calcium from these two probable stores (Nguyen, Chin et al.
1998). The steps are described in Figure 2. The calcium efﬂux creates an “electrostatic
repulsion”, due to the high number of anions, that rapidly expands the mucin polymer matrix
(Rogers 2003). Further enhancement for the expulsion of mucins is the simultaneous uptake of
water and “hydration” of the mucus. This process is likened to a Jack-in-the-box requiring no

more than 100 milliseconds to complete, and mucus expands up to 600-fold due to hydration

(Wanner, Salathe et al. 1996; Rogers 2003).

19

 

         

f. Mucin Polymer,“
' m‘ ‘ ‘ ‘v

   

 

Figure 2. Mucin Granule—Cytosol Calcium Exchange Dynamics and Exocytosis. (1) IP; binds to IPs-R
channel and (2) there is release of resting, free calcium from the granule lumen into cytoplasm and calcium in
cytosol increases. (3) Localized icreases in cytosol [calcium] trigger opening of a calcium-activated potassium
channel and potassium inﬂux that maintains electroneutrality and (4) mobilizes lumenal matrix-bound calcium
via Ca*2/K* ion exchange (also increasing the calcium diffusion gradient for release). (5) Calcium increases in
the granule lumen (6) promote calcium efflux, which increases cytosolic [calcium] and decreases luminal
[calcium]. (7) Large increases in calcium concentration in cytosol cause inactivation of IPa-R while (8) the
calcium-activated potassium channel remains open, luminal [calcium] rises and cytosolic [calcium] decreases
(via diffusion or buffering). (9) The calcium-activated potassium channel is inactivated and the IP3-R is
reactivated for new cycle (Nguyen, Chin et al. 1998). (NOTE: Images in this thesis are presented in color).

Mu 11 Me lasi let Cell H ia
After either acute or chronic exposure to an environmental challenge, there is airway
remodeling, characterized by thickening of the mucosa, increased bronchial vascularity, and
hypertrophy of the smooth muscle (Rogers 2002). The plasticity of the epithelium allows for
an increase in the number of epithelial cells and/ or replacement of large patches of
mucociliary epithelium by metaplastic MC given its ability to proliferate, differentiate, and

change the proportions of speciﬁc cell (Basbaum and Jany 1990).

20

Increases in MC numbers may be due to de novo synthesis of goblet cells or
transformation form existing cells (metaplasia) resulting in more MC (hyperplasia). New goblet
cells may arise from the differentiation of non-granulated progenitor cells, and less from goblet
cell division. Since no increase in mitotic rates has been observed in secretory cells, the increase
in mucous cell numbers is attributed to hyperplasia and/ or metaplasia (Rogers 2003). The term
“hyperplasia” is usually reserved for large airways where MC normally occur and “metaplasia” is
speciﬁc to the origin of MC in small airways less than 2 mm in diameter where MC are absent in
healthy lungs. This is not to say that metaplasia cannot occur in large airways, but it is difﬁcult
to identify with conﬁdence. Mucous cells increase not only in number, but also in size as seen
from increased alcian blue-staining (Basbaum and Jany 1990). The electron density of secretory
granules in the goblet cell cytoplasm also decreases. These changes in histochemical staining and
electron density appear to be general responses of the airway epithelium to injury (Basbaum and
Jany 1990).

The progenitor cells—serous, basal, and Clara cells—have the capacity to divide
followed by differentiation into “mature” ciliated or secretory cells. Some investigators
hypothesize that Clara and serous cells differentiate into goblet cells. This is because non-
ciliated, electron lucent mucus granule-containing cells display many similarities as Clara cells
(Hayashi, Ishii et a]. 2004). While others claim that basal cells are progenitors for MC after
injury, which in turn are progenitors for ciliated cells. When MC are absent, as in the peripheral
airways, Clara cells serve as progenitor cells for ciliated cells (Wanner, Salathe et al. 1996).
However, it is unclear if these ‘new’ cells maintain a Clara cell disposition or if they become

genuine MC.

21

Airway Repair and Regeneration

Under normal conditions, mucosal cell turnover is very slow resulting less than 1% of all
epithelial cells turning over in a 24 hour period (Wanner, Salathe et al. 1996). Within 12 hours
after minor injury, this process speeds up considerably so that in approximately 2 weeks a highly
differentiated epithelium is restored (W anner, Salathe et al. 1996).

When 90 percent of epithelium is removed from chicken trachea, mucociliary function is
restored in 14 days and complete epithelial restoration in 30 days (Battista, Denine et al. 1972).
Half of the mucociliary activity can be restored by regeneration of only 10 percent of ciliated
cells, suggesting a functional reserve for the mucociliary apparatus (Wanner, Salathe et al. 1996).

Tesfaigzi (2003) categorized the airway epithelial recovery process into ﬁve steps: (1)
epithelial cells migrate to the damaged area within minutes; (2) epithelial cells begin the processes
of proliferation and differentiation to replace injured epithelial cells and to establish squamous or
MC metaplasia, respectively; (3) apoptosis of some cells occurs in order to allow restoration of
the epithelium by the newly proliferated epithelial cells; (4) once reduction of cell numbers
occurs, to that of an unexposed epithelium, normal proportions of cell types are restored; (5)
epithelial cells can develop a memory for chronic exposure. This last point is important because
it permits a quick response to minimize further injury (T esfaigzi 2003).

There appears to be a Th1 cytokine proﬁle involved in the resolution of mucous cell
metaplasia (MCM) with IFN-‘y crucial for induction of MC apoptosis (O-Quan Shi, Fischer et al.
2002). IFN-‘y induces Fas-mediated apoptosis in airway epithelial cells (Wen, Madani et al.
1997), probably by increasing the expression of Bax, a pro-apoptotic member of the Bcl-2 family
of proteins (T esfaigzi, Fischer et al. 2002). It is unclear if 1) only newly formed cells express
Bax, or 2) if pre-existing cells can also undergo apoptosis during the resolution of MCM

(T esfaigzi, Fischer et al. 2002).

22

PHYSIOLOGICAL CELL DEATH: PROGRAMMED SUICIDE OR NECROSIS

Life is deﬁned by death. Death at a cellular level plays many roles having widespread
biological signiﬁcance in defense, differentiation and development, proliferation/ homeostasis,
and aging. Cell death occurs through two courses of action, one physiological (apoptosis) and
the other accidental (necrosis), each having distinguishing features. The relative rates of these
processes determines whether the cell undergoes apoptosis or necrosis as the primary means of
removal (Kroemer, Dallaporta et al. 1998). According to Kroemer et al (1998), both methods go
through a two-step process involving the mitochondria (Figure 3).

The morphological characteristics of apoptosis are caused by activation of cysteine
proteases, termed caspases, which leads to chromatin condensation, nuclear fragmentation
(pyknosis), cytoplasmic blebbing, cell shrinkage, and breakup into small membrane-bound
vesicles called apoptotic bodies (Reed 2000). Release of these apoptotic bodies gave this form
of programmed cell death its name; “apoptosis” is from the Greek, meaning “to fall away from”
(Reed 2000). The apoptotic bodies express phosphotidylserine making them recognizable for
phagocytosis. Because they are phagocytized and there is no release of cellular contents, there is

no inﬂammation.

There is evidence for an alternative nonapoptotic programmed cell death pathway
(‘paraptosis) that does not fulﬁll morphological criteria of apoptosis; however, since it requires

gene expression, it is not necrosis either (Sperandio, de Belle et al. 2000).

23

Stimulus ‘- " ,,, ,
Xx ‘ Apopluucnic ‘
t

Fw‘utois‘

I ctllux [

“'II....._..¢“

 

    

      
    
  

Plasma Membrane
Disruption

Necrosis

Caspase
Activation

Endonuclease
Activity

T-cell activation Apoptotic Body Formation
Inﬂammation Degradation

Figure 3. The Mitochondrial Regulator: “Bioenergetic Catastrophe” Or Subtle Disappearance? Stage 1(solid
arrows): a multitude of stimuli set off a signal transduction pathway to the mitochondrial membrane, increasing
its permeability. This mitochondrial membrane perrneabilization (BIMP) releases sequestered apoptogenic
factors (including endonucleases) into the cytosol and abolishes the electrochemical gradient (ECG) in the
membrane, leading to mitochondrial dysfunction and the second stage (dotted arrows). Organellar breakdown
leads to loss of plasma membrane integrity (necrosis) and/or activation of proteases and endonucleases
(apoptosis). These consequences, indicated by dotted arrows, include: (a) collapse of mitochondrial inner
transmembrane potential (A‘l’m), (b) calcium efﬂux, (c) increased reactive oxygen species (ROS), indicative of
uncoupling of electron transport chain. Additionally, caspase—activating proteins are released from the
intemiitochondrial space. (NOTE: Images in this thesis are presented in color).

24

NECROSIS

Death by necrosis is un-programmed and occurs in acute, non-physiological injury
(Hetts 1998). Cells passively (i.e. energy is not required) take up extracellular ﬂuid, swell, and
undergo lysis releasing their cytoplasmic and nuclear contents (Hetts 1998). This release results
in an inﬂammatory response involving maturation of dendritic cells into antigen presenting cells
to stimulate CD4+ T helper cells and CD8+ cytotoxic T lympthocytes (Schultz and Harrington
2003). Although important in acute injury and certain inﬂammatory responses, this is not the

normal mechanism for cell death (Hetts 1998).

APOPTOSIS

Apoptosis is a highly organized process that has been evolutionarily conserved from
worms to mammals. In contrast to necrotic cell death, apoptosis is a non-inﬂammatory, energy-
dependent form of genetically programmed cell death (Hetts 1998). It occurs under normal,
physiological circumstances where cells commit suicide ‘for the good of the organism,’ although
apoptosis also can play a primary or secondary role in pathological states.

Defects in programmed cell death can lead to disease, either with cell accumulations, as
in cancer or restenosis, or with an excess loss of cells, seen in a stroke, heart failure, AIDS, or
neurodegeneration (Reed 2000). Speciﬁc stimuli induce apoptosis, necrosis occurs instead

because there is failure by the cells to maintain homeostasis following damage (Schultz and

Harrington 2003).

The apoptotic process is divided into four phases. In the ﬁrst phase, the cell receives a
stimulus and “decides” whether or not to commit. This stimulus provokes an apoptotic

response which ultimately leads to activation of the effectors: cysteine-dependent aspartate-

25

speciﬁc proteases called capsases (V aux and Strasser 1996). The signal can be external such as
ligation of surface receptors, lack of survival signals, contradictory cell cycle signals, and
developmental death signals; the signal may also be internal from drugs, toxins, or radiation that
may cause DNA damage due to a defect in repair mechanisms, or cytotoxic drug treatments

(V aux and Strasser 1996).

After stimulus detection, signal transduction pathways reach death effector machinery to
begin the second phase. Many components of the apoptotic machinery are ready and waiting
for activation and, for this reason, tight regulation and multiple checkpoints ensure appropriate
execution. At this point, in the “execution phase”, activation of proteases and positive and
negative regulators occurs (V aux and Strasser 1996). Apoptosis is carried out by caspases; these
proteases are ready in the cytosol as precursors called pro-caspases. A death signal leads to
proteolytic cleavage of pro-caspases to release the active caspase, which then cleaves the next
pro-caspase to release its active caspase in a caspase cascade. Completion of this pathway
depends on the ratios of positive and negative regulators.

Finally, in the postmortem phase, morphological characteristics appear (chromatin
condensation, DNA degradation) and apoptotic bodies are formed. Changes in the makeup of
cell surface proteins and lipids occur, speciﬁcally, externalization of phosphatidylserine, to ﬂag
the apoptotic bodies for phagocystosis is the earliest detectable morphological event (Liu,

Brouha et al. 2004). After phagocytosis, lysosomes degrade the packaged bodies.

Caenorbabditis clams-A genetic Model of Cell Death
It was not until the late 19803 to early 19905 that cell death was hypothesized to be a

normal fate during development. This idea developed through study of C. elegant and ﬁnding

that not only is apoptosis a normal process, but it is a very precise one at that. During this

26

nematode’s development, speciﬁc genes are activated to kill exactly 131 cells and leave 959 cells
(Hetts 1998). These and subsequent studies have provided the basics for apoptosis in higher
multicellular animals, as the homology is widespread, in the mechanisms and the molecules.
Genes discovered in C. elegans are extremely conserved from worm to humans (Figure 4). The
four sequential steps of apoptosis—commitment, signal transduction, execution, and
phagocytosis and degradation—have been described using C. elegam.

The protein products of the ced-3 gene implement the execution of apoptosis. These
are cysteine aspartyl proteases (caspases) that are activated after cleavage behind a conserved
aspartate and go on to cleave their substrates, including polyadenosine diphosphate ribose
polymerase, components of nuclear membrane, and endonucleases (responsible for cleaving the
apoptotic cell’s DNA) (Reed 2000). Mutations of red-3 gene block all developmentally
programmed cell deaths (Yuan, Shaham et al. 1993); the animals age normally with normal life-
spans and no apoptosis (V aux and Strasser 1996). This would suggest that different processes
than those for programmed cell death regulate aging. Perhaps during evolution of multicellular
organisms, aging evolved q?” the two lineages (germ and somatic) formed (V aux and Strasser
1996). Likewise, defense and development mechanisms may have evolved earlier in single-cell
organisms.

There are two cad (cell death abnormal) genes involved in carrying out apoptosis and one
inhibiting it. Upstream to CED-3 is the cad—4 gene. Ced4 receives the death commitment signal,
binds to and cleaves pro-CED-3 and releases active CED-3 (Hetts 1998). The antagonist for
apoptosis, CED-9, is a multifunctional protein. Cad-9 can inhibit both cad-3 and cad-4; CED-9 is
localized to the mitochondrial membranes and by binding to CED-4, it prevents the activation

of pro-CED-3 by anchoring CED-4 away (Hetts 1998).

27

  

 

Apoptosis

   

Mammals '4. ‘7 7 7 1.3 " Apoptosis

 

Figure 4. Apoptosis pathways in C. elegant and Mammals. Homologies occur between the nematode, C. elegant, and
mammals in proteins and in mechanism. Ced-4/Apaf-1 binding is required to activate Ced-3/Caspase-9 for
apoptosis to occur. Ced-9/Bcl-2 interacts with Ced-4/Apaf-1 and sequesters it away from Ced-3/Caspase-9, thus
inhibiting apoptosis. Egl-l /Bik binds to and inhibits Ced-4/Apaf-1. (modiﬁed from Adams and Cory 1998).

Cell Death in HQ her Mammals
Studies of cell death in higher mammals showed that commitment to death occurs

through some ubiquitous pathways but many times, the pathways are speciﬁc for cell type,
stimulus, or even time (Hetts 1998). It is far more beneﬁcial for an organism to have many
apoptotic pathways than a single one; if only one pathway was present, all the cells would die
from a given signal. Fortunately, with the array of triggers that are present in the environment,
only a speciﬁc subset of the cells undergo programmed cell death while other are spared (Hetts
1998). These mechanisms for survival (or death) allow for independent regulation of pathways
that converge at a common endpoint, namely the caspase cascade and morphological
characteristics of apoptosis (V aux and Strasser 1996).

Several differences between the worm and mammals are present. In the worm, Ced-4
binds directly to Ced—3, but in mammals, Apaf-l (Apoptotic Protease Activating Factor—1)
cannot directly bind to caspase-9. It requires a co-factor to mediate Apaf-l-caspase—9

interactions, identiﬁed as cytochrome c. The second variation concerns Bcl-2 and its

28

involvement as a death suppressor. Although in the worm model Ced—9 binds to Ced-4, Bel-2
has not been shown to bind directly to Apaf-l (Moriishi, Huang et al. 1999). Instead, its main
function may be through indirect constraints via prevention of mitochondrial release of
cytochrome c (Kluck, Bossy-Wetzel et al. 1997; Yang, Liu et al. 1997). This is still up for debate
as Bel-2 is seen to retain its pro-survival activity even with cytochrome c in the cytosol
(Brustugun, Fladmark et al. 1998).

Even with such great similarities, many pathways exist for commitment to death and
many components of “the executioner” exist (Hetts 1998). Although all cells have the capacity
to undergo cellular suicide, not all are susceptible (Chao and Korsmeyer 1998). A cell’s

apoptotic threshold depends on the ratio of positive and negative regulators.

There are two principal pathways for the initiation of apoptosis. One is the ‘intrinsic’
pathway controlled by the mitochondria and the Bel-2 family of proteins to regulate activation of
caspase-9. The second pathway is the ‘extrinsic’ pathway mediated by death receptors belonging ’
to the tumor necrosis factor (TN F) family and CD95 (also known as Fas or APO—1) and the
resultant activation of caspase-8. Although initiated by separate signals, one at the plasma
membrane (extrinsic) and the other at an organelle (intrinsic) level, crosstalk occurs and the two
pathways converge for a common outcome: activation of downstream proteases termed

caspases.

Caspases

Caspases are cysteine aspartyl-speciﬁc proteases that are responsible for the
morphological characteristics associated with apoptosis; they are the conserved downstream

machinery that carries out the execution phase of programmed cell death. Caspase—l or ICE

(IL-IB—Converting Enzyme), was the ﬁrst homologue of Ced-3 to be identiﬁed in mammals

29

(Miura, Zhu et al. 1993). Regardless of pathway of activation or trigger, the same endpoint is
reached at a caspase cascade. Caspases are present in most, if not all, nucleated cells (Hetts
1998)

Caspases are synthesized in precursor form of zymogens, called pro-caspases, containing
a large (p10) N-terminal prodomain and a small (p20) catalytic subunit, sometimes separated by a
linker peptide (Reed 2000). While both domains are common among caspases, the N-terminal
prodomain differs greatly (Sprick and Walczak 2004).

There at least 14 caspases in mammals and these form two funWentally different
subgroups based on amino acid similarities of the prodomain (Reed 2000). The ﬁrst group is the
upstream, or ‘initiator’, caspases, characterized by a long N-terminal prodomain, which provide
protein interaction modules to allow for recruitment of and interaction with pro-caspases at an
activating protein complex. Caspases included in this group are caspases-1, -2, -4, -5, -8, -9, -10,
~11, and —12 (caspases-11 and -12 are in mice only, caspases-4 and -5 in humans only). The N-
terminal prodomain of caspases-8 and --10 has a death effector domain (DED) and the other
caspases listed have N-terminal prodomains with a caspase-activating recruitment domain
(CARD) to enable activation of the second group, the downstream ‘effector’ caspases (Sprick
and Walczak 2004). Caspases-1, -4, -5, -11, and -12 are involved in processes other than
apoptosis and will not be discussed in further detail. Caspase-2 is activated during cytotoxic
stress and is required for the permeabilization of mitochondria and the release of apoptosis-
inducing molecules (Lassus, Opitz-Araya et al. 2002). Pro-caspases-S and -10 are recruited and
bind to the adapter protein FADD/MORTI via their DED regions to complete the formation
0f the scaffold, called a DISC (Death Inducing Signaling Complex), to which additional pro-
caspases can bind to and autocatalytically cleave and activate caspases (Boldin, Goncharov et al.

1 996; Muzio, Chinnaiyan et al. 1996). Therefore, caspase-8 is regarded as the ‘apical caspase’ in

30

this pathway. Caspase-9 is the apical caspase in the intrinsic pathway and its recruitment to a
complex analogous to the DISC forms the apoptosome. Unlike the DISC—activated caspase—8,
caspse-9 likely requires the apoptosome to carry out its functions.

The second group of effector caspases, also called executioner caspases, is responsible
for the majority of the cellular destruction. These caspases, caspases—3, -6, and -7 have a short
N-terminal prodomain having an unknown function (Reed 2000). These caspases depend on

the upstream, initiator caspases for proteolytic processing and activation.

Downstream pro-caspases require post-translational activation by an activated initiator
caspase. A model for initiator caspase activation has been proposed for each pathway. In the
extrinsic pathway, the mechanism for activation has been proposed in the ‘induced proximity
model’ where the DISC serves as a scaffold to which pro-caspases are recruited, cleaved, and the
active caspases then cleave other inactive zymogens to release active enzymes in a proteolytic
cascade, that has been likened to the complement cascade or blood clotting (Hetts 1998). This
‘trant-processing’ model assumes a very important fact: zymogens are not completely inactive;
they have weak yet measurable proteolytic activity that is at around 1 percent of fully active
enzymes for procaspase-8 (Denault and Salvesen 2002).

A somewhat similar activation pathway to the induced proximity model is described for
the intrinsic pathway. After the release of cytochrome c, a caspase-activating protein, from the
mitochondria, it binds to ATP or dATP, with Apaf-l and pro-caspase-9 to form the so-called
apoptosome. Procaspase-9 is a monomer and its dimerization leads to its release of caspase-9 in
much the same manner as caspase-8 in the extrinsic pathway (Denault and Salvesen 2002).

Caspase-9 goes on to activate the effector pro-caspases-3, -6, and -7 to amplify the death signal.

31

Activation of executioner caspases occurs in a two-step mechanism. First, proteolysis
separates the large and small subunits; this is normally carried out by an active initiator caspase
(i.e. DISC-activated caspase-8 or apoptosome-activated caspase-9) (Sprick and Walczak 2004).
Even after this initial cleavage step, the enzyme is still inhibited by its own prodomain. In the

second, autocatalytic step, the prodomain is removed and the active effector caspase is ready.

The Mitochondria-Qsogiated (Intrinsic) Pathway and Bel-2

A role for mitochondria in apoptosis was ﬁrst illustrated in Xenoput oocyte extracts
where apoptosis required a membrane fraction that was enriched with mitochondria (N ewmeyer,
Farschon et al. 1994). Induction of apoptosis by the mitochondria-dependent pathway can be in
response to many stimuli including growth-factor withdrawal, irradiation, my overexpression,
hypoxia, UV radiation, chemotherapeutic agents, oxidants, and calcium overload. A lack of
exogenous signals causes activation of an endogenous default death program, the intrinsic
pathway. Proteins associated with the outer mitochondrial membrane (OMM) regulate
cytochrome c leakage and its integrity. Release of cytochrome c induces formation of a complex
analogous to the DISC of the TNF pathway, the apoptosome, and activation of the most
upstream protease, caspase-9 (Li, Nijhawan et al. 1997).

In a similar mechanism to the close proximity model involving the DISC complex, the
apoptosome aides in the cleavage of pro-caspase-9. Activated caspase-9 cleaves and activates
caspase-3, the ﬁrst caspase common to both the intrinsic and extrinsic pathways. However,
caspase-9 is not released into the cytosol after cleavage (Reed 2000). While Apaf—l requires a
cofactor (i.e. cytochrome c) to bind to pro-caspase-9 (Hengartner 1998), it was shown that pro-
caspase-9 is not required for the active complex (Acehan, Jiang et al. 2002). Furthermore, the

apoptosome may serve to amplify caspase activity instead of to initiate it because Bel-2 is shown

32

to regulate caspase activation independently of the apoptosome (Marsden, O'Connor et al.

2002).

The BCL-Z Family
Bel-2, isolated from a B-cell lymphoma, was ﬁrst described in 1984 as an oncogene, on

the human chromosome t (14:18), that prolongs cell survival (Pegoraro, Palumbo et al. 1984;
Vaux, Cory et al. 1988). Most of these members are associated with the membranes of
mitochondria, endoplasmic reticulum, and nucleus, anchored by a stretch of hydrophobic
residues near the carboxyl terminal (Schendel, Xie et al. 1997; Reed 2000).

Bel-2 gene expression may result in an inhibition of cell division cycle progression from
the G1 to S phases resulting in a 30°/o-60°/o increase in the length of G1 phase (Mitzel, Burtrum
et al. 1996), although conﬂicting evidence was seen in rats (T esfaigzi, Harris et al. 2004). With
conﬁdence, it can be said that Bel-2 proteins either positively or negatively control cell death
commitment, independent of caspases, and they regulate the release of cytochrome c (Reed
2000), although the functional mechanisms are unclear. Several theories have been proposed:
(1) Bel-2 binds directly to Apaf-l, sequestering it away from caspases and/ or (2) Bel-2 prevents
cytochrome c, as well as other apoptogenic proteins, from escaping the conﬁnes of the
mitochondria, or (3) both events occur (Hetts 1998).

In the ﬁrst theory, borrowed from C. elegant, Bcl-2 directly controls caspase activation.
However, Moriishi, Huang et al (1999) demonstrated that Apaf—l does not bind to Bcl-2
proteins. The second scenario gives Bel-2 family members a more central role in which they
guard mitochondrial activation. Many groups have found Bel-2 to protect mitochondrial
integrity and control cytochrome c release (Kluck, Bossy-Wetzel et al. 1997; Shimizu, Natita et
al. 1999). Likewise, as mentioned above, others argue against this involvement for Bel-2

(Marsden, O'Connor et a1. 2002). There is also some discrepancy whether Bel-2 and the close

33

homolog Bcl-xL may also be able to inhibit F as-mediated apoptosis. While some groups claim
F as-induced pathways are separate from Bel-2 regulated pathways (Huang, Hahne et al. 1999;
Robinson, Jeffcoat et al. 2002) others disagree (Armstrong, Aja et al. 1996).

Bcl-2 acts both upstream and downstream from cytochrome c; cytochrome c activates
caspases, and activated caspases, in turn, stimulate cytochrome c release. In one experiment,
Bcl-2 failed to block Bax-induced cytochrome c release, regardless of co-localizing with Bax, and
the cells survived with cytochrome c in their cytoplasm showing Bel-2 working downstream of
cytochrome c (Rosse, Olivier et al. 1998). Another group found that Bcl-2 blocked apoptosis
induced by injected cytochrome c (Zhivotovsky, Orrenius et al. 1998). Alternatively, Bel-2 and
Bcl-xL can prevent cytochrome c release from the mitochondria (Kluck, Bossy—Wetzel et al.
1997; Finucane, Bossy—Wetzel et al. 1999); they set a threshold in the amount of cytochrome c
required to activate caspases (Cosulich, Savory et al. 1999). The ability of Bcl-2 to act upstream
and downstream of cytochrome c allows it to suppress caspase activation at several distinct steps

(Cosulich, Savory et al. 1999).

34

Anti-Apoptotic
Bcl-Z Subfamilv

 

 

 

 

(Ltcrminus a] (12 1:14— ii (17 N-terminus
. , . Bel-2
(‘1 .. I I 5'qu Loop I BH3] I BHZ] [TMD ] 1 Rd”
'3‘” Mel-1
Bel-w

Pro—Apoptotic
Bax Subfamilv

 

, ‘ . Bax
(.lass l r ] BH3 ] BHZ 'TLID Balk

Bok/Mtd

(Ilassll m Loop BH3 [TMD J | Bcl-xs

 

BI 13-( )nlv Subfamilv
Bik
[BH3 I [TMDJ | 1::
Elk
Bad
Bid

 

 

Class lll

Figure 5. The Bel—2 Family. (modiﬁed from Adams and Cory 1998; Reed 2000).

The Bcl—2 protein family is a large group of more than 20 members categorized by
the presence of at least one of four (BHl-BH4) conserved motifs called Bcl-2 homology
(BH) domains (Figure 5). There are two functional classes of proteins, those that promote
survival and those that promote cell death, and three structural subgroups, the anti-apoptotic
Bc1-2-like proteins (the Bcl-2 subfamily), the pro-apoptotic Bcl—Z-like proteins (the Bax
subfamily) and the pro-apoptotic BH3—only proteins (Table 1). After sub—dividing them in
this manner, two facts become evident: (1) the anti—apoptotic proteins contain at least three
BH domains—3H1, BH2, and BH3—and (2) the pro-apoptotic protein must have, at

minimum, the BH3 domain (Figure 5) (Kelekar and Thompson 1998).

35

 

 

 

 

 

Anti-Apoptotic Pro-Apoptotic
Bel-2 Subfamily Bax Subfamily BH3-Only Proteins
Bel-2 Bax Bad
Bel-XL Bak Bid
Bel-w Bok/Mtd Bik
Mel-1 Diva BimL
Bﬂ-l/ A1 Bel-x5 Blk
NR-13 Hrk
BHRFI Bmf
LMWS-HL Noxa
ORF16 Spike
KS-Bcl-Z Puma
BIB-19K BNIP3
CED-9 BimL
EGL-l

 

 

Table 1. Categorization of BCL-2 Family Members. Members can be categorized into two groups based on
function and three groups based on structure.

Further division of these groups, based on their homology with Bel-2, places them into
two anti—apoptotic and three pro-apoptotic classes (Figure 5). The pro-survival proteins making
up class I (Bel-2, BCl-XL, and Mel—1) contain a loop region and are (at-helical pore-like proteins
(Reed 2000). Other members of the Bel-2 subfamily lack the ‘loop’ domain, putting them into
class II. Although, they contain all four BH domains, their functions are different. For instance,
the class I Mel-1 behaves like Bcl-2 in a cytoprotective manner, while the function of the class II
protein, A1, remains unknown (V aux and Strasser 1996).

Of the pro-death proteins, class I contains members of closest relation to Bel-2 (Bax,
Bak, and Mtd/Bok), having BH1, BH2, and BH3 domains and transmembrane domains but
lacking the loop regions. These proteins may interact with and antagonize the anti-apoptotic
members (via the BH3 domain) or act independently of them. Both classes II and III pro-death
proteins must interact with survival proteins to elicit their apoptotic promoting function.

Proteins in Class III are members containing only the central short (9 to 16 residue) BH3

36

domain, and thus called ‘BH3-only proteins’, act as sentinels over some organelles and processes
(Adams and Cory 1998; Sprick and Walczak 2004). Although normally inactive, stimulation
causes BH3-only proteins to become activators for pro-apoptotic members, therefore, in order
for the BH3-only proteins to act on the mitochondria, Bax and Bak must be present (Sprick and
Walczak 2004). However, they are unable to dimerize with pro-apoptotic proteins. BH3-only
proteins contain amphipathic a—helix that ﬁts into hydrophobic “crevices” of anti—apoptotic
proteins like Bcl-2 and Bcl-xL (Reed 2000).

Alternative splicing of the Bcl-x results in two distinct bcl-x mRNAs, protein products
from which are the longer, anti-apoptotic Bcl—xL and the shorter, pro-apoptotic Bcl-xS proteins
(Boise, Gonzalez—Garcia et al. 1993). Bcl-xs, can be placed in its own class II having only BH3
and BH4 and the transmembrane domain, however it has been placed in the Bel-2 subfamily
because it is shares the BH4 domain (T sujimoto 1998). Bel-x5 has also been categorized as part

of the Bax subfamily (Tsujimoto 1998).

Reﬂaa’on of Cell Death Machinery
Regulatory mechanisms among Bel—2 members vary. A recurring theme in apoptosis is

the important role protein-protein interactions play and as expected, Bel-2 proteins are no
exception. These interactions are involved in dimerization, sequestration through proteolytic
cleavage via caspases, and phoshorylation by kinases.

Pro- and anti-apoptotic Bel-2 family members can homodimerize and/ or heterodimerize
and one may titrate the other’s function. In this manner, the ratio of inhibitors to activators can
establish a cell’s susceptibility to death taggers. For instance, heterodimerization of Bax and
Bel-2 allow one to regulate the other’s function. Although heterodimerization is not required for

survival function, it is for apoptotic function.

37

Through X-ray crystallography and NMR methods, the structures of Bcl-2 proteins have
provided insight into the architectural basis for dimerization (Chittenden, Flemington et al. 1995;
Muchmore, Sattler et al. 1996). The three-dimensional structure of Bcl-xL showed that there are

two central hydrophobic a—helices, a5 and a6, surrounded by ﬁve amphipathic helices, a1, a2,
a3, 0L4 and a7, arranged so that the BH1, BH2, and BH3 domains are closely juxtaposed,
forming a hydrophobic pocket on the surface. This pocket seems to serve a receptor-like
function by interacting with the hydrophobic face of the BH3-containing a2 helix of the Bcl-2
protein it needs to bind (Reed 2000). In a monomeric state, the hydrophobic side chains of the
amphipathic a2 helix are pointed inward, making these residues unavailable for protein
interaction (Sattler, Liang et al. 1997). Thus, structural changes, such as rotation of the a2 helix,
may be required before BH3 can take part in dimerization. Such a rotation is a strong possibility

based on the prediction that ihe (12 helix is ﬂanked by highly ﬂexible loops on either end.

There seems to be some degree of selectivity and hierarchy of dimerization. Using yeast-
two hybrid systems, Bax homodimers were shown to induce cell death, heterodimerization of
Bcl-2 and Bax inhibit death by sequestration of Bax function, and Bel-x3 binding to Bcl-2 does
not allow Bcl-2 to neutralize Bax and cell death occurs (Sato, Hanada et al. 1994). Bcl-2/Bcl-2,
Bel-2 / Bax, Bax/ Bax, and Bax/Bcl-xL interactions have been proven in mammalian cells (Sedlak,
Oltvai et al 1995).

Mutagenic studies have identiﬁed the BH1, BH2, and BH3 regions to be critical in
dimerization and induction of apoptosis. Mutations in BH1 and BHZ that disrupt the repressive
functions of Bcl-2 or Bcl-xL also disrupt their abilities to dimerize with Bax or Bak (Yin, Oltvai
et al. 1994; Chittenden, Flemington et a1. 1995). Thus, BH1 and BH2 domains are essential for

dimaization with pro.apoptotic proteins and the survival function of Bel-2 and BCl-XL.

38

Although Bax and Bak contain BH1 and BH2, the BH3 domain appears to contribute to their
apoptotic function and interactions with pro-survival proteins (Kelekar and Thompson 1998).
Bel-xS lacks both BH1 and BH2 regions and does not interact with pro-apoptotic members.

Cytosolic members may lack the C-terminal transmembrane domain, as in the BH3-only
proteins (Bid, Bim, and Bad), or have a regulatory C-terminal domain (Bax). While Bax is free in
the cytosol, its C-terminal transmembrane domain is masked (N echushtan, Smith et al. 1999)
and upon receipt of apoptotic trigger, possibly pH (Khaled, Kim et al. 1999), it undergoes a
conformational change, exposing the C- and N-terminals for insertion into the OMM and
regulation of cell death machinery.

Proteolytic cleavage by caspases is required for activation of some BH3-only proteins.
This is evident in caspase-8—induced translocation of truncated Bid to the mitochondrial
membrane. Removal of the N—terminal 52 amino acids exposes a hydrophobic core and the
BH3 dimerizing domain, both of which are required for insertion into the OMM (Schendel,
Azirnov et al. 1999) and mitochondrial leakage. Thus, tBid links the death receptor pathway to
the mitochondrial pathway. Other proteins, such as Bim and Bad, are sequestered in the
cytoplasm. Bim associates with the dynein light-chain of microtubules; once freed, Bim
dimerizes with anti-apoptotic Bel-2 members via their BH3 domains (Reed 2000).

The phosphorylation state of some members determines their colocalization. In a
phosphorylated state, Bad is inactive and is bound to the cytosolic protein 14—3-3, which
interacts with signaling enzymes (N agata 1997). Dephosphorylation of Bad activates it and
enables dimerization with Bel-2 or Bel-xL to promote apoptosis. Kinases actively regulating Bcl-
2 members include Akt, PKA, Raf1, Rskl, Pakl (Reed 2000).

Transcriptional regulation for members varies. Bel-2 and Bax are under Stat3 and p53

control (Miyashita, Krajewski et al. 1994) while, Bcl-xL expression partly depends on p53, Stat5,

39

and NF-KB activation (Merchant, Loney et al. 1996; Tsukahara, Kannagi et a1. 1999; Bureau,

Vanderplasschen et al. 2002).

OMM Maintenance, Pore Forming Abilim and Mitochondrial Dysﬁmction

Members of the Bcl-2 family of proteins maintain mitochondrial homeostasis and OMM

 

integrity either directly (e.g. ion-channel forming activity) or indirectly—via OMM proteins like
those of the Voltage Dependent Anion Channel (VDAC)——to regulate leakage of cytochrome c
and other apoptogenic factors from the mitochondria (Donovan and Cotter 2004). Loss of this
results in a permeability transition (PT), loss of membrane potential and swelling, allowing the
rapid release of cytochrome c.

Crystal structures of Bcl-xL and Bcl—2 demonstrate similarities to pore forming bacterial
toxins, such as colicins A1 and E1 and diphtheﬁa toxin (Muchmore, Sattler et al. 1996), in the
BHl- and BH2-ﬂanked helices (15 and (1.6, which are speculated to participate in pore formation
(Kelekar and Thompson 1998). This indicates survival proteins, as well as Bax and tBid may
form ionic pores that allow electrochemical homeostasis in organelles, primarily the
mitochondria (Hetts 1998) (Antonsson, Conti et al. 1997; Luo, Budihardjo et al. 1998).
Homologous and non-homologous protein-protein interactions are likely to regulate pore
formation. For example, interaction with the co—chaperone, survival protein BAG-1 enhances
Bel-2 pore formation, while interactions with Bel-XS and Bad interferes with it (Schendel, Xie et
al. 1997). In any case, it is unclear if these channels directly control cytochrome c release.

Bax must oligomerize in order to form pores (Antonsson, Montessuit et al. 2000), doing
so in a membrane potential— and pH-dependent manner (Antonsson, Conti et al. 1997). Not
. only do pores formed by Bax abolish Bel-2 channel formation, they can directly induce
cytochrome c release, instead of indirectly by inhibiting regulators of caspases ([urgensmeier, Xie

et al. 1998), and allow the transport of ions or proteins across the membrane, opposite to Bel—2

40

(Schendel, Xie et al. 1997). At a neutral pH, Bax channels are mildly cation selective having a
permeability ratio of sodium to chloride of 2:1 (Antonsson, Conti et al. 1997) but overall, Bax
pores display a much broader range of pH than Bel—2 channels (Schlesinger, Gross et al. 1997).
The ability of Bel—2 family members to interact with PT pore (PTP) proteins (e.g.
VDAC) on the OMM allows the formation of much larger pores than would otherwise have
been allowed with Bel-2 alone (Reed 1997; Hengartner 2000). Bid, the BH3-only protein that
links the death receptors to the membrane pathway, can trigger cytochrome c release in puriﬁed
mitochondria after cleavage by caspase-8 by closing the VDAC, resulting in decreased
metabolite exchange between mitochondria and the cytosol resulting in mitochondrial
dysfunction, without swelling and PT (Luo, Budihardjo et al. 1998; Rostovtseva, Antonsson et
al. 2004). Although Bax has been shown to induce cytochrome c release, Bid is far more potent
in releasing 100% of mitochondrial cytochrome c at a 500-fold lower concentration than Bax,
which causes only 20% of the total cytochrome c to be released (Luo, Budihardjo et aL 1998).
Bid may also oligomerize with a pro-apoptotic member (Bax) to trigger cytochrome c release

and / or inactivate an anti-apoptotic member (Luo, Budihardjo et al. 1998).

Completion of programmed cell death, regardless of stimulus and pathway, relies on
disruption of the OMM and the rapid release of various caspase-activating factors into the
cytosol. Most apoptosis-inducing conditions involve induction of mitochondrial PT, a critical
early event (Zamzami, Susin et al. 1996) due to the opening of a large conductance channel

termed the PT pore (FTP), and the disruption of the mitochondrial inner transmembrane

potential (A‘l’m). During PT, there is a sudden increase in the A‘l’m and this allows molecules up
to 1.5 kD to enter the intermembrane space. Maintenance of A‘I’m relies on impermeability of

the inner mitochondrial membrane; thus, PT leads to disruption of A‘l’m. Collapse of A‘l’m is

41

accompanied by immediate shutdown of biological homeostasis in the mitochondria (i.e.
uncoupling of oxidative phosphorylation and the generation of ROS) (Attardi and Schatz 1988;
Zamzami, Marchetti et al. 1996). The increased ROS oxidize lipids, proteins, and nucleic acids
to cause further disruption of A‘l’m (Marchetti, Decaudin et al. 1997). These changes occur
before apoptotic nuclear events (Zamzami, Marchetti et al. 1996) and in enucleated cells
([acobson, Burne et al. 1994; Schulze—Osthoff, Walczak et al. 1994), indicating this mitochondrial
dysfunction is independent of the nucleus.

After discussing disruption of the inner mitochondrial membrane, the next question to
ask is how these larger molecules breach the OMM. The most prominent scenario involves the
opening of the FTP (Green and Reed 1998). The PTP, also called the megachannel, normally
appears to regulate matrix calcium, pH, A‘I’m, and volume, functioning as a calcium-, voltage,
pH-, and redox-gated channels with several levels of conductance ([acotot, Costantini et al.
1999). At high levels of conductance, the mitochondrion undergoes irreversible A‘l’m dissipation
and matrix swelling. Opening of this nonselective channel causes volume dysregulation in the
mitochondria caused by equilibrium of ions between the matrix and inner membrane space,
dissipation of H+ gradient across the membranes, and uncoupling of the electron transport chain
and eventually, expansion of the matrix due to it hyperosmolarity (Green and Reed 1998).
Because the inner membrane is convoluted and folded into cristae, its large surface area can
handle the expansion. Unfortunately, the same is not true for the OMM and it will rupture as a
result, releasing caspase-activating proteins into the cytosol. Bel-2 family members regulate PTP
opening and closing. Bel-2 can prevent PT while Bax induces PT and apoptosis (Zamzami,
Susin et al. 1996). Opening of the PT? seems to be required for Bax-, p53-, TNF-, and

glucocorticoid-induced death ([urgensmeier, Xie et al. 1998).

42

The effects of cytochrome c likely depend on the cell type (Green and Reed 1998).
Dying cells that have a pre-existing excess of cytochrome c are able to maintain electron
transport, oxygen consumption, and ATP production with docked cytochrome c, even after the
released cytochrome c activates caspases. These cells experience apoptotic demise. On the
other hand, in cells with an excess of endogenous caspase inhibitors, despite cytochrome c

release there is failure to induce caspase-independent apoptosis. The mitochondria experience
loss of electron transport, PT, ‘I’m collapse and inner membrane swelling, and mitochondrial

rupture tending towards necrosis.

The Death Receptor (Extrinsic) Pathway
Once extracellular binding of a membrane-bound or soluble ligand to death receptors

occurs, the signals are transmitted via a death domain on the cytoplamsic tail that activates the
proteases homologous to CED-3, capsases (14 have been described to be involved in the cell
death pathway) (Schultz and Harrington 2003). Caspase-8 is considered the apical caspase for
the extrinsic pathway.

In the death receptor pathway, apoptotic signals are transmitted via the TNF family, with
at least 16 members, including TNFR1, TNFRZ, Fas, NGFR, CD40, CD27, CD30, DR3, TNF-
related apoptosis-inducing ligand (TRAIL), and DR6 death receptors (Schultz and Harrington
2003); (Nagata 1997). Most is known about the Fas (CD95 or Apo 1) receptor and TNF
receptor-1 (TNFR-l) (p55 or CD120a); these receptors share cytoplasmic region similarities,
along with TNFRZ (Nagata 1997).

As we have seen with caspase activation and Bel-2 proteins, protein to protein
interactions play a prominent role in conducting a death signal from its origin to its destination
in most, if not all, apoptotic pathways. The intermolecular domains required for these

interactions are described in Appendix 1.

43

The Fas receptor is central in signaling apoptosis and activating the transcription factor
nuclear factor kappa B (N F-KB). This transcription factor protects cells from apoptosis by
inducing the expression for pro-survival factors, such as the inhibitor of apoptosis protein (IAP)
family (Schultz and Harrington 2003). The F as receptor ligand, FasL, is inactive in its
membrane—bound form (Nagata 1997). After proteolysis by metalloprotienases it is released
from activated CD4+ and CD8+ T cells in its functional, soluble form (N agata 1997). Binding of
F asL to Fas induces trimerization of the Fas receptor (Nagata 1997). Activated F as recruits
FADD /MORT1 via interactions between death domains (DD), which activate caspase-8
(FLICE/ MACH) through its DED. Similarly to F asL, TNF is normally in an inactive,
membrane-associate form; upon proteolysis by metalloproteinases, it is converted into its
functional, soluble form and binds to TNFR1 or TNFRZ (Nagata 1997). TNFR trimerize and
the DD of TRADD, an adapter protein, interacts with the receptor’s DD. This complex attracts
additional proteins, FADD /MORT1 and RIP.

FADD /MORT1 —induced apoptosis can be counteracted by RIP-induced transcription
of NF -KB. TNFR1-mediated IKK (IKB kinase) activation requires both RIP and TRAF2
proteins, possibly because RIP stabilizes the interaction between TRAF2 and IKK and mediates
the activation of IKK (Devin, Cook et al. 2000). Transcription of NF-tcB can also occur

through the TNFRZ receptor in some cells, but it is more likely to use the major signaling

receptor, TNFR1 (N agata 1997). Thus, since both receptors can trigger the two major functions
of TNF—activation of NF-KB and induction of apoptosis—some overlap does exist (Hsu,

Huang et al. 1996). NF -KB has been shown to inhibit TNF-mediated apoptosis by inducing

transcription of BCl-XL, A1, cIAP, etc. (Wang, Cusack et al. 1999). Alternatively, overexpression

44

of RIP protein was shown to induce both NF-KB and apoptosis (Hsu, Huang et al. 1996). This

is suggested to be possible because, in low RIP conditions, TRAF2 binds and induced NF-KB
transcription and survival due to uncoupling of the death pathway. Once death triggers are
received, an increase in RIP associated with TRADD bridges TNF R to FADD/MORT1 and the
apoptotic machinery (Pimentel-Muinos and Seed 1999). Thus, the dual functions of TRADD

may cause TNFR to “nullify” their own apoptosis-inducing activity.

RECURRENT AIRWAY OBSTRUCTION IN HORSES
Recurrent airway obstruction (RAO) is a chronic, recurring inﬂammatory lung disease,

most commonly found in horses stabled during the winter in environmental conditions prone to
having dust and allergens present in the air (Gerber 1973; Cook 1976). N eutrophilic inﬁltration
into the airways, mucus accumulation within the airways, bronchospasm, and airway
hyperreactivity (AHR) are hallmark features of this disease (Obel and Schmiterlow 1948;
Schatzmann, Buergi et al. 1973; Nicholl 1978; Robinson, Derksen et al. 1996). Described below
are: the etiology, clinical signs, lesions, pathophysiology and pathogenesis, treatments, and

indices of severity for RAO in the equine lung.

ETIOLOGY

Many factors present in the horse’s environment elicit an airway response and disease
exacerbation. These pro-inﬂammatory agents include thermophilic molds and actinomycetes
Wergillwﬁlm'gatut, Tbemoaetinomycet vulganlt, and Faertia mtivz'rgula) mold spores, endotoxins,
proteinases, forage mites, and particles of feed grains, feces, dander, and pollen that become
aerosolized around the horse’s breathing zone while it is feeding (McPherson, Lawson et al.

1979a; Derksen, Robinson et al. 1988; McGorum, Dixon et al. 1993a). When RAO-susceptible

45

horses are exposed to such agents, they develop inﬂammation, AHR, and obstruction, whereas
horses not affected with RAO do not have the same severity of inﬂammation and do not
develop obstruction or AHR. Changing the management of RAO-susceptible horses to either
keeping them on pasture or feeding them pellets or grass silage and bedding them on shavings
greatly reduces the dust exposure (Woods, Robinson et al. 1993; Vandenput, Votion et al. 1998)
and severity of disease exacerbation (Thomson and McPherson 1984). When RAO-affected
animals are allowed to be at pasture for 1 or 2 weeks, their BALF leukocyte count is no different
from that of control horses and airway obstruction is minimal (Derksen, Scott et al. 1985). The
time taken for horses to become asymptomatic correlates with age, duration of illness, and
severity of disease as measured by the non-elastic work of breathing (Thomson and McPherson
1984). There may be genetic predisposition for horses to develop RAO, although the
inheritance pattern in unclear (Marti, Gerber et al. 1991). Breed, gender, body weight, and
season are not factors in disease susceptibility (McPherson, Lawson et al. 1979).

Exposure to the dusty conditions described above causes acute inﬂammation, with
neutrophil accumulation in the airway lumina. Many believe repeated exposure and
inﬂammation causes MC proliferation and that this aids in protecting the lung through increased
mucus secretion, presumably to trap and transport inhaled particles. Normally useful, this
becomes a problem in combination with the edematous airways resulting in thickened walls,
decreased airway diameter, and mucus plugs.

One consequence of RAO to horses is poor gas exchange, indicated by low PaO2
(hypoxemia) (Robinson, Derksen et al. 1996). Interestingly, this is not accompanied by
hypoventilation, or increased PaCOz, despite the horse having to work harder to breathe air
through obstructed airways (bronchospasm) to maintain tidal volume. The drop in PaOz, due to

ventilation/ perfusion mismatching, causes an increase in respiratory rate. The most common

46

and effective treatments for horses affected with RAO are environmental management to reduce
dust exposure, bronchodilators (Murphy, McPherson et al. 1980; Robinson, Derksen et al. 1993),

and corticosteroids (LaPointe, Lavoie et al. 1993).

CLINICAL SIGNS

RAO-affected horses display all or some of the following clinical signs during acute
exacerbations of disease: 1) difﬁcult breathing characterized by expiratory abdominal effort
(heaving), 2) increased breathing sounds with wheezing, and 3) increased bronchial secretions
(Gillespie and Tyler 1969). In more severe instances, these ‘abnormal ﬁndings’ worsen and
horses develop chronic cough, double expiratory effort, and hypertrophy of the external
abdominal oblique muscle (“heaves line”) (Breeze 1979). Copious amounts of mucus are visible
in the trachea within 24 hours of exposure and are maintained in RAO—affected horses, even at

pasture (Gerber, Straub et al. 2001).

LESIONS

Originally, RAO was thought to be an emphysema-like disease because the lungs do not
collapse when the chest is opened (Lowell 1964; Thurlbeck and Lowell 1964; Tyler, Gillespie et
al. 1971). However, unlike emphysema where failure of collapse is due to the loss of elastic
tissue in the alveolar septa, failure of collapse in RAO is due to the trapping of gas behind
mucus plugs and the consequential hyperinﬂation of alveoli (Robinson, Derksen et al. 1996).

The primary lesion of RAO is inﬂammation in small diameter (<2 mm) airways with
widespread epithelial hyperplasia and metaplasia (Breeze 1979). Inﬂammation, largely consisting
of luminal neutrophils, is a prominent feature of RAO. Peribronchial inﬁltration of

lymphocytes, plasma cells, mast cells and few eosinophils have been documented in addition to

47

the intraluminal neutrophils (Breeze 1979; Fairbairn, Page et al. 1993; Robinson, Derksen et al.
1996). Mast cells may be found in the intercellular clefts (Kaup, Drommer et al. 1990a).
Mucous cell metaplasia (MCM) has been described in bronchioles of affected horses,
where MC are normally absent, and the changes increase with disease severity (Kaup, Drommer
et al. 1990b). This becomes more problematic farther out the tracheobronchial tree as the
diameter of airways decreases, contributing to the stasis of mucus. Thus, the small diameter
together with increased mucus viscoelasticity (Gerber, Lindberg et al. 2004) and the resultant
decreased tracheal transport rates (Coombs and Webbon 1987; Turgut and Sasse 1989) expedite
the formation of plugs composed of mucus and neutrophils (Breeze 1979) in more peripheral
airways. Descriptive pathology studies report some ﬁbrosis in small diameter airways, necrosis
of type I cells, their replacement with type II cells, and acinar overinﬂation in alveoli (Kaup,
Drommer et al. 1990b). In larger airways, focal loss of ciliated cells followed by replacement
with undifferentiated cells leading to development of a hyperplastic epithelium is described
(Kaup, Drommer et al. 1990a). This was also seen in smaller airways (Winder, Gruenig et al.
1989). While many of these changes correlate with severity of disease, some horses Show ciliary
malformations that appear to be independent of disease severity (Kaup, Drommer et al. 1990a).
In larger airways, remodeling occurs (i.e. hyperplasia and/ or hypertrophy of mucosa,
submucosa, and smooth muscle) that coincides with neutrophilic inﬁltration (Robinson,
Derksen et al. 1996) so that only a small amount of smooth muscle contraction is required to
cause in a big change in airway diameter. This exaggerated narrowing of the airways or,
nonspeciﬁc AHR, contributes to airway obstruction; airway remodeling is most likely due to the

inﬂammatory response (Figure 6).

48

Environmental Particulates

I TNF-ct , 11.-113, Elastase, ROS, Inﬂammatory Mediators I

 

 

 

 

 

 

 

 

 

 

 

____)I- Ach . l ﬁdhesion Molecules I

 

 

 

 

 

 

 

 

 

 

 

 

|
UPG 14.3; Histamine EGFRj \ I
. - . ‘ I I |
I Airway I ﬂ 301'? I |
IHypersensitivityI Expression ﬂ Mucins l |
I i M CM (eqMUCSAC) I I
Smooth Muscle 101:?“ v (I V {I l I
Activation e ) Mucus ( Mucus I
l OCH” Secretion Production \ I
I MC 1””le wI ’
__ Remodeled I Neoplasia 1) ABC v f'
T i 0) : I
i..___,. mi Mucus (
MC V Accumulation U Clearance I
. , , Hyperplastic I A I
DIVISIon E . - MW--. mm,
pithehum 3 . I
v ﬂ Mucus I U Mucociliaty _
Bronchospasm I Score I Action I
E Alma), {—1 ﬂ Viscoehsadty ]
Obstruction (3-Fold) |
v I .
ll APplmax Altered Glycoprotein ide Chains]

Figure 6. A flow diagram of possible mechanisms leading to the progression and development of disease in
RAO-susceptible horses. Accumulation of neutrophils and their products is an early feature of the disease,
happening in three to ﬁve hours after a challenge. Activated neutrophils generate IL-lB and TNF-0t, which
activate NF-KB, which in turn induces their transcription initiating a positive feedback loop (not Shown).
TNF-a also increases EGFR expression causing MCM, mucus secretion, and mucin synthesis. Neutrophil
products likely increase mucus viscoelasticity and mucin (MUCSAC) expression (via TNF-a-induced EGFR
expression and ROS-induced transactivation), which subsequently decrease the clearance of and increases the
production of mucus, respectively. Concurrently, neutrophil elastase, a well-known MC secretagogue, induces
mucus secretion by interacting with MC (via adhesion molecule ICAM-l). Together, possibly with decreased
clearance (due to decreased mucociliary transport and increased viscoeleasticity), accumulated mucus in the
airway, and if it occurs, increased numbers of MC (due to longer survival time, proliferation of existing MC,
and/ or differentiation of progenitor cells into MC), contributes to the visible increased mucus score. Increased
luminal mucus and airway remodeling potentiate airway obstruction due to bronchospasm. In addition to
neutrophil products, ‘bronchspastic’ neurotransmitters (ACh), inﬂammatory mediators, and nonspeciﬁc
irritants (dust) stimulate hypersensitive airway smooth muscle to contract. Because of remodeling and
increased thickness of the airway walls, this exaggerated contraction (AHR) greatly reduces luminal diameter
(bronchospasm) around mucus plugs to cause airway obstruction that is measurable as the maximal change in
pleural pressure (APplnm) during tidal volume at rest. Arrow thickness denotes known, relative contributions.
Dotted line represents possible pathways. Solid boxes are mediating factors; dotted boxes are consequential
effects. Components highlighted in yellow are the focus of this thesis. (NOTE: Images in this thesis are

presented in color).

49

PATHOPHYSIOLOGY AND PATHOGENESIS

Inﬂammation

While RAO-susceptible horses are in remission, macrophages and lymphocytes are the
main white blood cells found in the bronchoalveolar lavage ﬂuid (BALF) of horse lungs
(Derksen, Scott et al. 1985; McGorum, Dixon et al. 1993). During an acute exacerbation,
neutrophils inﬁltrate the lung between three and ﬁve hours after the onset of exposure
(Fairbairn, Page et al. 1993) and are detectable in BALF at ﬁve hours (McGorum, Dixon et al.
1993). In general, percentage of neutrophils increases with severity of disease; however,
neutrophils can be present without dyspnea (Fairbairn, Page et al. 1993). Likewise, because
neutrophilic accumulation is not always observed in the above allotted time frames, neutrophils
may not be required for airway obstruction early on and may have more prominent roles later in
disease pathogenesis (Fairbairn, Page et al. 1993).

Paramount in the pathogenesis of RAO is neutrophil inﬁltration into the airway lumen.
Speciﬁc mechanisms for neutrophil chemotaxis involve macrophages and lymphocytes that
produce and secrete cytokines. IL-8, a strong neutrophﬂ chemoattractant, correlates tightly with
the percentage of neutrophils in tracheal aspirates of asthmatics (Ordonez, Shaughnessy et al.
2000) and has been found in the BALF of RAO-affected horses (Giguere, Viel et al. 2002).
However, since neutrophilic inﬂux can occur in the presence of low IL-8, other mediators must

be involved in neutrophil chemotaxis. During disease exacerbation of RAO-affected horses, IL-
10 and TNF-a are increased in the BALF (Giguere, Viel et al. 2002). The latter

proinﬂammatory cytokines increase ICAM-l expression in endothelial and epithelial cells (T osi,
Stark et al. 1992), which, in concert with elevated levels of both mRNA and protein of IL-8, may

contribute to the observed neutrophilia in diseased horses. Activated neutrophils of RAO-

affected horses generate IL-IB and TNF-0t, which activate NF-KB, which in turn induces their

50

transcription initiating a positive feedback loop (Bureau, Delhalle et al. 2000). The persistent
NF—KB expression in RAO-affected horses (Bureau, Bonizzi et al. 2000), which may partially
explain higher cytokine gene expression (Giguere, Viel et al. 2002) may explain the neutrophilic
nature of RAO inﬂammation. Another proinﬂammatory mediator, leukotriene B4 (LTB4), that is
related to neutrophil-mediated tissue damage, is increased by tumor necrosis factor and
interleukins as well as complement fragments and endotoxins (Crooks and Stockley 1998).
Production of LTB4 may be increased in RAO-affected horses and also may contribute to
neutrophil inﬁltration into the lung (Lindberg, Robinson et al. 2004). LTB4 stimulates the
activation and production of NF-KB and IL-8 cytokine (Aoki, Qiu et al. 1998) and may prolong
the life of neutrophils (Lee, Lindo et al. 1999). Hence, LTB4 promotes transcription of
inﬂammatory genes and cytokines.

There is a debate as to whether RAO has a polarized cytokine proﬁle and if it is Th1,
Th2, or some combination. In support for a predominantly Th2 response, Komai et a1 (2003)
have found this cytokine proﬁle to be responsible for airway remodeling and increased mucus
secretion, via MU CSAC induction, in mice (Komai, Tanaka et al. 2003). Increased expression of
cytokines implicated in the Th2 response, such as IL-4, IL-5, and IL-l3, suggest RAO in horses
may be an allergic condition like asthma (Lavoie, Maghni et a1. 2001 ; Bowles, Beadle et al. 2002;
Cordeau, Joubert et al. 2004). Additionally in animal models, IgE generation and its
maintenances in BALF and serum (Halliwell, McGorum et al. 1993; Komai, Tanaka et al. 2003)

relies on IL-4 and IL-13.
Alternatively, IFN-‘y, IL-8, IL-12 (Ainsworth, Grunig et al. 2003) and several forms of

IgGs have been found in serum and BALF samples (Ainsworth, Appleton et al. 2002) of MO

horses refuting a polarized Th2 cytokine proﬁle and suggestive of a Th1 type response. Finally,

51

it may be that RAO-affected horses have a Th0 response, characteristic of both Thl and Th2
cytokines (Ledru, Lecoeur et al. 1998).

Neutrophils can have a large impact on lung function and can indirectly induce airway
obstruction by releasing ROS, proteases, and inﬂammatory mediators, which cause increased
mucus secretion as well as other possible outcomes (Figure 6). The products of neutrophils are
described as causing ‘adverse changes’ in mucus rheology, inducing mucus hypersecretion,

increasing mucin gene expression, and causing glycoprotein alterations to mucin molecules.

MEASUREMENTS OF DISEASE SEVERITY

Mm’ a1 Changes in Pleural Pressure (A_P_pl,mI

Placement of an esophageal balloon distal to the heart is commonly used to measure of
maximal changes in pleural pressure (APplmx) during tidal breathing (Derksen and Robinson
1980; Robinson, Derksen et al. 1999). The determinants for APpl1m are: pulmonary resistance
(RI), tidal volume (V T), dynamic compliance (Cm), and air ﬂow rate (V).

APme = VT/Cdyn + Flow"‘R.L

In RAO-affected horses, increased APplm, is indicative of airway obstruction (Figure 6).
The airway obstruction increases RL and decreases Cd”. Respiratory rate increases but tidal

volume remains constant, thus horses must increase minute ventilation to move air through

obstructed airways at increased ﬂow rates resulting in larger differences in pleural pressure. The
increased RU decreased Cd”, and increased ﬂow rate all contribute to the increase in APplmﬂ.
Horses having a APplm, greater than 15 cm H20 are considered to have RAO (Robinson 2001).

Normal horses have APme of 5 to 10 cm H20.

52

Clinical Score

A subjective clinical score with a signiﬁcant relationship to changes in lung function, has
been validated (Robinson 2001). Upon clinical examination, two scores ranging from 1 to 4 are
assigned indicating the degree of visible abdominal effort (abdominal score) and ﬂaring of the
nostrils (nasal score). These two scores are totaled (total clinical score) and serve as a good
measure of disease severity when the total score is greater than 5 as compared to measurements
of pulmonary resistance and dynamic elastance (Costa, Seahorn et al. 2000; Robinson, Olszewski
et a1. 2000). However, scores lower than 5, implying minimal impairment of lung function, fail
to reﬂect low-grade airway obstruction detected with lung function tests (Robinson, Olszewski

et al. 2000).

Muc c re
Severe mucus accumulation is one of the most notable ﬁndings in RAO-affected horses
(Costa, Seahorn et al. 2000). Recently, a subjective scoring system—via tracheal endoscopy—
was shown to be good measure of mucus volumes and it was reproducible between blinded
observers (Gerber, Straub et al. 2004). This is a good indicator for the presence of disease, but

not necessarily the severity of the disease.

Br n h r L v e Fl ' AL
Collection of BALF is a useful investigative tool for the quantiﬁcation of inﬂammatory
cell inﬁltration into the airways and analysis of the airway lining ﬂuid. In both control and
RAO-affected horses, a single BALF sample is representative of the entire lung (McGorum,
Dixon et al. 1993). During collection, passage of an endoscope into the lung allows sampling of
peripheral airways, through which, typically of 1-3 aliquots of 100 ml of sterile physiological
saline are instilled and aspirated. From the pooled samples, total and differential white blood

cell counts may be determined to assess total leukocyte density.

53

During disease exacerbation in RAO—affected horses, neutrophils increase to signiﬁcantly
higher levels than those of controls, with an insigniﬁcant increase in eosinophils and mast cells
(Derksen, Scott et al. 1985; Winder, Grunig et al. 1991; Robinson 2001; Gerber, Lindberg et al.
2004). The number of neutrophils present in BALF aspirated from the lungs is a good indicator
to the degree of airway inﬂammation present. RAO-affected horses generally have greater than
20% neutrophils in BALF (Robinson 2001). In control horses, the primary leukocytes present
are alveolar macrophages (60%) and lymphocytes (35%), while neutrophils, eosinophils, and
mast cells are negligible (Robinson 2001).

Colleted BALF provides a more useful diagnostic aid over transtracheal washes in
evaluating RAO, even if the amount of BALF recovered is very small (Pickles, Pirie et al. 2002).
In control horses, there is less variability in leukocyte populations in BALF compared to
transtracheal washes while in RAO-affected horses, the clinical usefulness of cytological
evaluations of transtracheal aspirates may be limited (Derksen, Brown et al. 1989). Though
increased leukocytes, found by cytology performed on BALF, allows early detection of

inﬂammatory respiratory disease, is not speciﬁc for RAO (Couetil, Rosenthal et al. 2001).

Mucus ACCUMULATION AND Mucous CELL METAPLASIA IN RAO

Mucus accumulation contributes to airway obstruction (Figure 6) and leads to changes in
breathing strategy, abnormal sounds upon auscultation, and the overall appearance of clinical
signs. Persistent mucus accumulation is a consistent ﬁnding in RAO-affected horses before,
during, and after exacerbation of disease (Gerber, Lindberg et al. 2004). This may be a
consequence of changes in the rates of production, secretion and clearance.

Increased production and secretion may result from one or all of the following: the

presence of secretagogues (neutrophils and their products), induction of mucin gene expression

54

(eqMUCSAC), or increased MC numbers. Increased mucin gene expression has been repeatedly
shown in many animal models, including horses (Fischer and Voynow 2000; Takeyama,
Dabbagh et al. 2000; Gerber, Robinson et al. 2003). Neutrophils play a central role in MC
degranulation and mucin secretion. Neutrophil elastase and other factors (e.g. IL-8, IL-13, ROS,
EGF, TNF-0t) are important inducers of mucin production and secretion (Agusti, Takeyama et
al. 1998; Takeyama, Agusti et al. 1998; Nadel, Takeyama et al. 1999).

During exacerbations of RAO, there is indeed a decreased rate of mucociliary clearance
in horses with RAO compared to control horses (Turgut and Sasse 1989) accompanied with a
three—fold increase in mucus viscoelasticity, apparently a result of alterations to the glycoprotein
side chains of mucins, within 24 hours of exposure (Gerber, King et al. 2000; Jefcoat, Hotchkiss
et al. 2001). The latter causes decreased clearability and thus stasis and accumulation of mucus.
Increased mucus viscoelasticity also results from actin and DNA released from degranulated
neutrophils, which aggregate, increase the viscoelasticity, and affect both ciliary and cough
clearability of mucus (Gerber, King et al. 2000; Pietra, Guglielmini et al. 2000). Decreased
mucociliary action is also likely to play a role in decreased clearance. While in remission,
however, there is no difference in mucus rheology (i.e. viscoelasticity) between RAO-affected
and control horses but there was up-regulation of mucin genes that may increase mucin
secretion and overcome mucociliary clearance mechanism$ to contribute to the persistent
changes in mucus transport (Gerber, Lindberg et al. 2004). Thus mucus accumulation in
remission is likely due to hypersecretion i.e. mucin upregulation, and perhaps to increased
numbers of MC.

Goblet cell hyperplasia (GCH) is a common ﬁnding in many respiratory diseases.
Qualitative ﬁndings of GCH and MCM in bronchi and bronchioles, respectively, have been

reported numerous times where existing and newly formed cells can differentiate into mucus-

55

producing cells (Winder and von F ellenberg 1988; Kaup, Drommer et al. 1990b; Nyman,
Lindberg et al. 1991; Lakritz, Wisner et al. 1995; Costa, Seahorn et a1. 2000; Shi, Fischer et al.
2002). This may be due to proliferation (i.e. MCM and neoplasia), to prolonged cell life
[increased anti-apoptotic factors (Bcl—2)], or to a combination. Activation of epidermal growth
factor receptor (EGFR) can also lead to MCM and eventually GCH (Lee, Takeyama et al. 2000).
Initiation of neutrophil-induced oxidative stress by cytokines, such as IL-13, causes
phosphorylation of the EGFR (Figure 6) (Takeyama, Dabbagh et al. 2000; Shim, Dabbagh et al.
2001). Increased expression of EGFR by TNF-0t (N adel 2001) and activation of its tyrosine
kinase in airways increases the synthesis of mucin MUCSAC mRNA and protein (T akeyama,
Dabbagh et al. 1999; Takeyama, Jung et al. 2001). Morphometric techniques can be applied to
measure the amount of store intraepithelial mucins (Vs) in the airways in order to assess mucus
production (Harkema and Hotchkiss 1992). A role for Bcl-2 in MCM is suggested by the
observation that Bcl—2 expression occurs in ~20—30°/o of metaplastic MC (MMC) in epithelium
of ozone-challenged rats (T esfaigzi, Hotchkiss et al. 1998; Tesfaigzi, Fischer et al. 2000) and that

MCM does not resolve as long as Bcl-2 is expressed (Foster, Gott et al. 2003).

A t i f M

Determining mechanisms for maintenance of MCM is an ongoing topic of investigation
in both spontaneous and induced airway disease. One thought that has gained interest, as a
causative mechanism is prolonged life of MC. Challenges with ozone, endotoxin, and allergens
in rats, increase Bel-2 expression in epithelial cells in the proximal septum of the rat nose and
different regions of the lungs (Tesfaigzi, Fischer et al. 2000; Tesfaigzi 2002; Foster, Gott et al.
2003; Tesfaigzi, I-Iarn's et al. 2004). These observations suggest a role for Bel-2 in MCM,
possibly by inhibiting progression of the cell division cycle (Mazel, Burtrum et al. 1996).

However, more recently published was the observation that approximately one-half of the MMC

56

are derived from proliferating cells and the other half differentiate from pre-existing epithelial
cells (T esfaigzi, Harris et al. 2004). Moreover, since Bel-2 is expressedin MMC derived from
preexisting and proliferating cells, Bel-2 may not have a cell cycle regulatory function, at least
not one that was seen in rat epithelial cells (T esfaigzi, Harris et al. 2004). The expected role for

Bel-2 is maintenance of MCM.

SUMMARY
This literature review describes the mucous apparatus in airways, physiological cell

death and the pathology and diagnosis of RAO in horses. Increased and persistent mucus
accumulation is a common ﬁnding of RAO. The contribution of increased numbers of MC
has not been investigated. This thesis addresses GCH as a possible consequence of delayed
apoptosis. I hypothesized that delayed apoptosis, as indicated by an increased presence of
Bcl-2-positive MC, contributes, at least in part, to an increase in the number of mucus-

secreting cells and airway mucus accumulation.

57

CHAPTER 2
PERSISTENT Mucus ACCUMULATION—A CONSEQUENCE OF DELAYED
Mucous CELL DEATH IN RAO-AFFECTED HORSES

ABSTRACT
This study examined the role of delayed apoptosis of mucous cells (MC) as indicated

by Bel-2, an anti-apoptotic protein, as a contributing factor to mucus accumulation in horses
affected with recurrent airway obstruction (RAO). Measurements of disease severity were
collected from 6 RAO-affected and 6 control horses exposed to two different management
systems (one to induce inﬂammation [5 days] and one to partially resolve it [7 days]) prior to
euthanasia. Morphmetric analyses (Bcl-2-positiVity and Stored mucosubstance [Vs]) were
performed on 8 bronchi. RAO-affected horses had more airway obstruction and luminal
mucus than control horses under both management systems. At the time of euthanasia,
RAO-affected horSes had more inﬂammation and more Bcl-2-positive MC than control
animals but no difference in MC number or Vs, and in RAO-affected animals, Vs decreased
as BALF neutrophil numbers increased. Therefore, a conclusive role for Bel—2 in prolonging
MC life cannot be determined, despite Bel-2 antibody immunoreactivity. USDA/CSREES

2002352041259)

58

MATERIAL AND METHODS
ANIMALS

Six control and six RAO-affected adult horses (eight males and four females; ages 10
to 29, weights 399 to 538 kg) were used in this study. The RAO-affected animals were from
a herd of such horses maintained by the Pulmonary Laboratory at Michigan State University
and met the criteria deﬁned at the International Workshop on Equine Chronic Airway
Disease (Robinson 2001). That is, in the presence of organic dust in a stable they had airway

obstruction that was decreased by administration of atropine (0.02 mg/ Kg IV) (Figure 7).

-0- Horse 2
-I— Horse3
+ Horse 5
-0- Horse 10
-3K- Horse 11
-0- Horse 12

APplnW (cm H20)
8 S

8

 

 

Baseline Atropine

Figure 7. Effect of atropine (0.02 mg/ kg IV) on the maximal change in pleural pressure (APpla...) during tidal
breathing in six stabled horses with a history of RAO. Atropine decreased APplm, in all horses.

Horses in the control group had no known history of chronic airway disease

and developed no clinical signs of obstructive airway disease when stabled.

59

EXPERIMENTAL DESIGN

In this blinded study (Figure 8), which was approved by the Animal Use and Care
Committee of MSU, each horse and sampling time-point was assigned a random code.
Horses were studied in pairs of one RAO and one control. After being at pasture for a
minimum of two weeks (average of 8.5 weeks), one horse from the pair was stabled, fed
poor quality hay and bedded on straw for 5 days. These stabling conditions induce airway
inﬂammation in the RAO—susceptible animals. At this point, the horse’s diet and bedding
were changed to pellets and Shavings, respectively, in order to reduce the severity of airway
obstruction and inﬂammation in the RAO-affected horses (Thomson and McPherson 1984;
Vandenput, Votion et al. 1998). After 7 days on pellets and shavings (day 12), the horse was
euthanized. On days 5 and 12, the severity of airway obstruction was quantiﬁed by
measurement of the maximal change in pleural pressure dunng tidal breathing (APplmu),
bronchoalveolar lavage was performed, and a tracheal mucus accumulation score was
assigned. This protocol was repeated the following week with the other horse from the pair.
The sequence of the pairs—RAO-affected and control—was randomized.

Horses were euthanized with pentobarbital (86 mg/ kg IV). The thorax was opened;
the lungs were dissected away from other structures, extracted, and grossly examined. From
each of eight regions (Figure 9), samples of peripheral parenchyma and an adjacent
cartilaginous bronchus (2-6 mm diameter) were collected. The sequence in which the

regions were sampled was randomized a priori.

60

     

 

 

 

5 Day 7 Day
Barn Challenge Partial Remission
Day 0 Day 5 Day 12
APleax APleax 8 Bronchi
BALF BALF o Tri-Reagent
Mucus Score Mucus Score a Fixation
8 Tissue Sections
° Perfusion/
Fixation

Figure 8. Experimental Design. One horse of each pair was brought into the stable, fed research hay and

bedded on straw for 5 days. At the end of the 5-day environmental challenge, samples for APplmu, BALF, and
mucus score were collected. The management was changed to pellets and shavings for 7 days to induce a

partial remission of disease. At the end of the twelfth day, samples for APplm, BALF, and mucus score were
collected once more and the horse was euthaniszed. Post-mortem samples included 8 bronchi and 8 tissue
sections.

MEASUREMENT OF APPL“Ax

Horses were intubated with an esophageal balloon (Trojan condom, Carter-Wallace
Inc, NY) sealed over the end of a 240—cm long polyethylene catheter (3 mm internal
diameter, 4.4 mm external diamﬁer) (PE240, Becton, Dickinson & Co, Franklin Lakes, NJ)
with lateral holes drilled in the portion covered by the condom. The catheter was inserted so
that the balloon was placed caudal to the heart base but cranial to the diaphragm. This
location provides a good estimate of the pressure within the pleural cavity at that location
(Derksen and Robinson 1980). Pressure changes within the balloon were detected by means
of a pressure transducer (Model DP/45-35, Validyne, Northridge, Ca) and were recorded on
a portable physiograph (Dash model II, Astro-Med, West Warwick, RI). The equipment was
calibrated daily against a water manometer. The ﬂuctuations in esophageal pressure were

recorded for 20 consecutive breaths. The differences between the peak inspiratory and peak

expiratory pressures were calculated to derive the APpllm.

61

TRACHEAL Mucus SCORE

Horses were intravenously sedated with xylazine hydrochloride (1.1 mg/ kg) and
butorphanol tartrate (0.02 mg/ kg) approximately ﬁve minutes before the bronchoscopy was
performed with a 3—m endoscope (Olympus GIF 300, Olympus America Inc., Melville
Maryland, USA). The bronchoscope was passed via the nares into the trachea where the
amount of accumulated mucus was scored using a subjective grading system (Figure 11C)

that is reproducible between Observers (Gerber, Straub et al. 2004).

BRONCHOALVEOLAR LAVAGE

The endoscope was passed further into the trachea and into a bronchus. Once
wedged, three-100 ml aliquots Of sterile Dulbecco’s phosphate buffered saline (PBS) were
infused into the lungs and aspirated after each aliquot infusion and the samples were pooled.
Leukocyte density per microliter BALF was assessed in a 10-Iil aliquot by use Of a
hemacytometer. The percentage of each type of white blood cell was determined from
counting 200 cells on a cytocentrifuged preparation stained with Wright-Giemsa. The

percentage Of each cell type and the total cell counts were used to calculate the total number

of each cell type present per pl BALF.

POST-MORTEM PROCESSING

Parenchyma from each region Of lung (Figure 9) was securely clamped using two
curved bowel-clamps, tips touching to form two sides of a triangle, the third side being the
lung margin. One large cartilaginous bronchus (2 to 6 mm diameter) also was dissected (1

cm length) from each region adjacent to the clamped region. A small cross-section (3 mm in

length, 500 mg) of this bronchus was submerged into 5 ml of tri-reagent and stored at —80°C

62

for future use. The remainder of the bronchus was ﬁxed by immersion in 1%
paraformaldehyde/O.1% glutaraldehyde solution (pH 7.4) for 45 minutes, and was then
transferred to 30% ethanol and washed three times in a fresh ethanol bath every ﬁve
minutes. The ﬁxed bronchus was stored in the ﬁnal wash Of 30% ethanol at room
temperature.

The parenchymal sample was separated from the remaining lung tissue by incising
along the outside of the clamps, which were then removed. The lung ‘pouch’ was carefully
perfused with 1% paraformaldehyde/0.1% glutaraldehyde solution (pH 7.4); through a 19-
gauge needle, inserted into the parenchymal pouch and connected via tubing to a system that
maintained a constant perfusion pressure of 30 cm HZO. After 15 minutes, the perfusion
apparatus was disconnected from the lung tissue, and the lung sample remained an
additional 30 minutes in a bath of the ﬁxative solution before undergoing three ethanol

washes and being stored in the last wash. Blocks of tissue and bronchi were embedded in

parafﬁn and 5pm sections were cut. Two slides were prepared; one stained with Alcian-Blue
(AB)-Positive Acid Shiff (PAS) and the other irnmunohistochemically Stained for Bch and

counterstained with AB.

63

Apex
Apex

9 ‘A

v
unsure-urn“

Body v v

Q 6

Intermediate Lobe

 

 

Figure 9. Regions of horse lung from which sections were taken. Sequence of sampling was randomized for
each horse. Black lines specify location of clamps for the isolation of parenchymal sections.

IMMUNOHISTOCHEMISTRY

For immunohistochemical examination, sections of lung tissue were deparafﬁnized
and rehydrated by routine methods. Antigen retrieval was accomplished by incubation of
slides in antigen retrieval solution (Dako, Carpinteria, CA) in a steamer at 98°C
(Black&Decker) for 20 min followed by gradual cooling of slides within the retrieval solution
for 20 min at room temperature. Endogenous peroxidase was blocked for 5 min at room
temperature with 3% hydrogen peroxide. Endogenous avidin and biotin activity was
blocked by incubating the slides in an avidin and bition blocking reagents (Dako, Carpinteria,
CA) for 10 min each at room temperature. Non-speciﬁc irnmunoglobulin binding was
blocked by incubation of slides for 10 min at room temperature with a protein-blocking
agent (Dako, Carpinteria, CA) prior to application Of the primary antibody. Sections were

stained in a Dako autostainer apparatus. The slides were incubated with the primary

64

antibody against Bcl-2 (PharMingen) at a dilution of 1:200 for 30 minutes at room
temperature. A streptavidin-immunoperoxidase staining procedure (Dako, Carpinteria, CA)
was used for immunolabeling (Webster, Kiupel et al. 2004). The immunoreaction was visible
with Nova Red (Vector Laboratories). Sections were counterstained with alcian blue at a pH
of 2.5 and Gill’s 3 haematoxylin. Positive immunohistochemical controls included
hyperplastic lymph nodes and lymph nodes with malignant lymphoma from archived equine
tissues. For negative controls, the primary antibodies were replaced with TBS buffer (Figure
10).

Sections serial to those airways quantiﬁed for MC and Bel-2 were stained with Alcian
Blue (pH 2.5)/ periodic acid-Schiff (AB / PAS) reagent for stored acidic and neutral mucin

components as described previously (Harkema and Hotchkiss 1993).

 

Figure 10. Negative control (left) and positive control (right) for Bel-2 in a RAO-affected horse. (NOTE:
Images in this thesis are presented in color).
MORPHOMETRIC QUANTIFICATION OF MC AND BCL-Z EXPRESSION

Morphometric analysis of airway mucosa and Bcl—2—positivity was performed using
the Scion Image program (Scion Corporation, Frederick, MD). The regions of mucosa used
for counting mucus—containing cells met the following conditions: 1) the basal lamina was

intact, 2) the section of airway wall was transverse rather than oblique so that the apical

65

membrane of MC was aligned with the adjacent surface epithelium. The length of basal
lamina underlying the surface epithelium was calculated from a contoured line on the
digitized image Of the airway epithelium and the number Of Bcl—2—positive and negative MC
per millimeter of basal lamina were counted. Total MC numbers were also quantiﬁed from

AB / PAS stained tissue to conﬁrm numbers.

QUANTIPICATION OF INTRAEPITHELIAL STORED MUCOSUBSTANCE

Using Scion image analysis software, pixel density and threshold of intensity were
adjusted to determine the volume density (Vs) and the areas of acidic and neutral (AB / PAS-
stained) stored mucosubstance along a measured perimeter as described previously
(Harkema, Plopper et al. 1987; Harkema, Plopper et al. 1987). Data were expressed as the
mean volume density (Vs; nl/mm2 of basal lamina.) Of AB / PAS-positive mucosubstance 1'

SEM.

STATISTICAL ANALYSIS

Data were examined and if not normally distributed, log10 transforms were made. A
two way repeated measures ANOVA was used to determine the effects of 1) time and
disease on APplmx, mucus score, total and differential cell counts, and 2) the effects of
disease and lung region on the mucous cell counts and their Bcl-2 expression. When
signiﬁcant main effects were detected (p<0.05) multiple comparisons were made using the
Student-Newman-Keuls method. Because of the small number of animals, signiﬁcance Of
the interaction term was set at p<0.1. The Vs in the two groups of horses was compared by

the Student’s t-test. Statistical analyses were performed by means Of a computer software

program (SigmaStat, SPSS Science, Chicago, IL).

66

RESULTS
INDICES OF DISEASE SEVERITY

Maximum Changes in Pleural Pressure (£21m;

On day 5, RAO-affected horses had signiﬁcantly greater APplmx compared with
control horses (Figure 11A). On Day 12, the mean APplmx had decreased but continued to
be Signiﬁcantly greater than in controls. The APme Of control horses was unchanged
between days 5 and 12.

Although RAO-affected horses had signiﬁcantly greater APplmx than controls (Figure
11A), they greatly ranged in values among horses (Figure 11B). Two horses had APplm,
greater than 40 cm HZO at day 5, two had intermediate values, and two had low values. The
decrease in APplmu that occurred between day 5 and day 12 was positively correlated with its

magnitude on day 5.

Tracheal Mucus Score

The RAO-affected horses had signiﬁcantly higher mucus scores (>25) than controls

on both days 5 and 12 (Figure 11D). There was no effect of time in either group. Unlike
APme, in which the trend in RAO-affected horses was to decrease between days 5 and 12,

the change in mucus score was highly variable (Figure 11E).

1 Br n h e Fl ° AL
The only statistically signiﬁcant change in leukocyte numbers was in neutrophils

(T able 2, Figure 11F). On day 5, control and RAO-affected horses had 11.8 i: 5.1 and 216.3
1' 204.7 (mean i SEM) neutrophils/pl BALF, respectively. This difference was not

signiﬁcant because one RAO-affected animal had a low neutroth count (9 cells/pl) (Figure

67

11G). On day 12, the neutrophil count for RAO-affected animals was statistically
unchanged from Day 5 (102.7 i 48.4). In control horses, the count on Day 12 was 1.0 i
0.6, signiﬁcantly less than controls on Day 5 and RAO-affected animals on Day 12. The
mean count Of macrophages, lymphocytes, mast cells and eosinophils was 22.5 i' 6.0, 51.1 i

16.8, 2.5 i 1.3, and 0.03 i 0.03, respectively.

Table 2. BALF total and percent neutrophils, macrophages, lymphocytes, eosinophils, mast cells and total cell
count on days 5 and 12 in RAO-affected and control horses.

 

 

 

 

 

 

BALF CYTOLOGIES
Day RAO-Affected Horses (n=6) Control Horses (n=6)
. 5 44.9: 12.3b 17.7: 8.6
% Nemmph‘l‘ 12 40.3: 14.59 1.6: 1.0:
Total Netitrophils "5‘” 216.3: 204.7 11.8: 5.1
(Cons/In) 12 102.7: 48.4b 1.0: 0.6 .
% Macrophages 5 14.8: 4.3b 29.8: 4.4
12 11.4: 2.7b 37.5: 7.3
Total Macrophages 5 23.01 8.0 21.7i 3.9
(Cells/Ill) 12 19.3i 6.1 25.8i 5.8
5 34.1: 8.3 50.7: 5.6
% Lymphm’y‘“ 12 46.6: 13.3 53.3: 6.7
Total Lymphocytes 5 463i 13.5 37.1i 6.1
(Cells/ 111) 12 75.9: 32.4 45.2: 15.1
. . 5 0.2: 0.2 0.0: 0.0
% E°sm°9hd° 12 0.1: 0.1 0.2: 0.2
Total Eosinophils 5 0.0: 0.0 0.0i 0.0
(Cells/I11) 12 0.0: 0.0 0.1: 0.1
5 2.3: 1.0 1.8: 0.7
% Mm Cd" 12 1.7: 0.1 7.4: 4.5
Total Mast Cells 5 20¢ 1.0 1.5i 0.7
(Cells/ 111) 12 1.4: 0.9 4.9: 2.6
TCC 5 292.9: 211.2 72.1: 7.1
JCells/ul) 12 199.2: 52.1 77.1: 18.3

 

Values are means i SEM
' Signiﬁcantly different from day 5 within group
5 Signiﬁcantly different from control at same point
GOBLET CELL NUMBERS
AB-PAS staining was present in MC of control and RAO horses (Figure 12A).

There was no signiﬁcant disease or regional effect on MC numbers (Figure 12B and 6C) in

either AB / PAS or Bcl—2/ AB stained tissue.

68

STORED MUCOSUBSTANCE IN AIRWAY MUCOUS CELLS
There was no signiﬁcant difference between groups in Vs (Figure 12D). In the
RAO-affected horses, Vs was negatively correlated (r = -0.886, p=0.033) with the log10 of

the number of neutrophils in BALF (Figure 12E).

BCL-2 STAINING AIRWAY MUCOUS CELLS

In comparison to control horses, RAO-affected horses had noticeably more Bel-2-
positive MC in their epithelium (Figure 13A). Bcl-2 staining, seen as Nova Red counter-
stain, was so abundant it masked the AB. However, morphological characteristics and serial
sections stained with AB—PAS (Figure 12A) ensured these were MC.

In every region, the bronchi of RAO-affected horses contained signiﬁcantly more
Bcl-2~positive MC per millimeter of basal lamina (p30.011) than controls (Figure 13B).
Because there was no difference in total MC between the two groups, the Bcl—2—positive MC
constituted a signiﬁcantly (p: 0.009) greater percentage of MC in MO-affected animals.
Examination of large second-generation bronchi and bronchioles demonstrated comparable
levels of Bel-2 expression in each horse (Figure 14A-F).

Figure 13C suggests that horses with more than 10 neutrophils per microliter of
BALF (day 12) expressed Bel-2 in their MC while those with fewer neutrophils (including
one RAO-affected animal) had very little observable Bel-2 staining. There was no

correlation between Bel-2 positivity and Vs (data not shown).

69

 

 

A I Control B

40 IRAO ‘0
A A 50
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37; 10 E 20
1:. <1 10
<

0 0

Day 5 Day 12 Day 5 Day 12

   

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Clean Singular Multiple Confluent Pool Profuse

 

 

 

 

D IControl 5 0
5.0 ERAO E '
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Day 5 Day 12 Day 5 Day 1?-
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1 1‘3; -‘ "ml: 1 _
Day 5 Day 12 Day 5 Day 12

Figure 11. (A) Maximum changes in pleural pressure in RAO—affected and control horses on days 5 and 12.
Values are means i SEM of RAO (n=6) and control (n=6) horses after a 5-day hay/ straw challenge (day 5) and

70

after 7 days on pellets/ shavings (day 12). There was a signiﬁcant difference between diseased and control
horses on both days, but no effect of time in either group. (B) Individual values of APplmam in the six RAO-
affected horses at days 5 and 12. There was considerable variability in APplm at day 5; horses with the
greatest values decreased most by day 12. (C) Subjective tracheal mucus score. The scoring system is as follows:
0=no visible mucus, 1=single or a few mucus globules; 2=many mucus globules that are beginning to coalesce,
3=many globules and a continuous mucous stream; 4=thick continuous stream and many coalescent mucus
globules around the wall of the trachea; and 5=mucus stream ﬁlls about one quarter of the tracheal lumen. (D)
Tracheal mucus score in RAO-affected and control horses on days 5 and 12. Values are means i SEM of
RAO (n=6) and control (n=6) horses after a 5—day hay/ straw challenge (day 5) and after 7 days on
pellets/ shavings (day 12). There was a signiﬁcant difference between diseased and control horses on both days,
but no effect of time in either group. (E) Individual values of mucus score in the six RAO-affected horses at
days 5 and 12. There was no consistent change in mucus score between the two days. (F) Total neutrophils per

micoliter BALF of control and RAO-affected horses on days 5 and 12. Values are means i SEM of RAO
(n=6) and control (n=6) horses after a 5-day hay/ straw challenge (day 5) and after 7 days on pellets/ shavings
(day 12). On day 12, the total neutrophils of control horses was signiﬁcantly less than the day 5 value and
signiﬁcantly different from the RAO-affected horses. RAO-affected horses did not change from day 5 to day
12. (G) Individual values of neutrophils per micoliter BALF in the Six RAO-affected horses at days 5 and 12.
There were no consistent changes in neutrophils between the two days. (NOTE: Images in this thesis are
presented in color).

‘ Signiﬁcantly different from day 5 within group; b Signiﬁcantly different from control at same point

71

   

I Control

     

 

 

 

 

 

,4 g 80 IRAO
°° s
E a
E t.
\ o
E) a.

D

2
Region
D E

8 12- E
3 8
5; A s 10-
m A *- A
gum "g a 8" O I
8 g 8'} 6
= U "‘
O
E 5 5 $2 41’ 0 a
m -
> I
0 l l I I
0 1 10 100 1000
Neutrophils/ul BALF

Figure 12. (-\) Alcian Blue/ Positive Acid-Schiff (AB-PAS) stained epithelium of a representative control (left)
and RAO-affected horse (right). (B) Number of mucous cells (MC) per millimeter of basal lamina (BL). and
(C) mean numbers of MC. No signiﬁcant regional or disease effects were detected. Values are means i SEM
of RAO (n=6) and control (n=6) of MC in each region of lung. (D) Stored intraepithelial mucosubstance (V5)
in large cartilaginous airways of control and RAO-affected horses. There were no signiﬁcant differences
between groups. (E) Relationship between the total neutrophils per microliter BALF and Vs in control and
RAO—affected horses. In RAO-affected horses, there was a negative correlation between the numbers of
neutrophils and the V s. Black diamonds (0) represent control horses; grey squares (I) represent RAO-
affected horses. Values are means i SEM. (NOTE: Images in this thesis are presented in color).

72

   

 

 

' \ \
B IContlol C
100 IRAO 0
m 10 I
55’ 53 so I
g E l
’5 E 60 I
n. .2
:3 E 40
U I
E g 20
1: I: I
8 a 0 o. O
8: §
3.: '20 I l I I l
0 l 10 100 1000
Region 1 2 3 4 5 6 7 8 Neutrophils/ul

Figure 13. (A) Bcl-2 staining cells in the epithelium of a representative control (left) and RAO-affected horse
(right). Bel-2 staining in the RAO—affected horse was unmistakably more abundant than that seen in the
control horse. (B) Percent Bcl-2-positive mucous cells (MC) present in each region of lung. RAO—affected
horses had signiﬁcantly greater numbers and percentages of Bcl—Z-positive MC compared to controls for each
region of lung sampled. Values are means 1 SEM of RAO (n=6) and control (n=6) horses in each region of
lung. (C) Percent Bcl—2-positive mucous cells (MC) in relation to total neutrophils per microliter BALF in
RAO-affected and control horses. Horses having more than 10 neutrophils per microliter of BALF tended to
have Bcl-2 staining in more than half of their MC while those horses with less inflammation had less
expression. The one RAO-affected horse with similar numbers of neutrophils as controls had a similar
percentage of Bcl-Z-positive MC as the controls. Black diamonds (0) represent control horses; grey squares
(I) represent RAO—affected horses. (NOTE: Images in this thesis are presented in color).

“ Signiﬁcantly different from control in the same region.

73

        
 

$15.4. ...~« we ‘a-‘f’ .
Figure 14. Bcl-Z/AB staining in three airway generations. (A-C) A second-generation airway, a 4—6 mm
diameter conducting airway, and a non—conducting airway of a control horse, (D-F) a second—generation airway,
a 4—6 mm diameter conducting airway, and a non-conducting airway of a RAO-affected horse, (G, H) a 4—6 mm
diameter conducting and a non-conducting airway from “horse 5.” (NOTE: Images in this thesis are presented
in color).

74

DISCUSSION
Although the expression of Bel-2 by MC has been well established in rodents

challenged with LPS, ozone, and allergens, its expression in spontaneously occurring airway
disease has only been examined in cystic ﬁbrosis (CF) (Harris, Fischer et al. 2005). In the
latter, BCl—2 was expressed in MC Of CF patients but not Of controls. The present study
examined the differences in expression of MC Bel-2 between control horses and those with
RAO, an environmentally induced inﬂammatory Obstructive airway disease. Bel-2 was
expressed in more than half of bronchial MC in RAO-affected animals but expression was
trivial in the MC Of controls that had undergone the same challenge.

The design of the experiment incorporated an organic dust challenge (hay feeding)
known to induce inﬂammation and obstruction in RAO-susceptible animals followed by
reduction of dust exposure (pellet feeding). Because there is little stored mucin in some
RAO-affected horses during periods of acute inﬂammation, the week in the low—dust
environment allowed time for both synthesis and storage of mucins and for GCH. As
expected, the 5-day dust challenge resulted in airway obstruction and accumulation of
mucoid secretions in RAO-affected animals—their APplm and tracheal mucus scores were
signiﬁcantly greater than in controls. Although there were these functional differences
between the two groups of animals, there was no Signiﬁcant difference in the severity Of
inﬂammation on Day 5. Others also have reported that control animals develop
neutrophilic airway inﬂammation without changes in lung function in response to a similar
dust challenge (T remblay, Ferland et al. 1993; Gerber, Straub et al. 2004). After one week in
the lower-dust environment, inﬂammation had signiﬁcantly waned in controls but not in
RAO-affected animals so that at the time of euthanasia, control animals had signiﬁcantly

fewer neutrophils in BALF than did the RAO-affected horses.

75

At the time Of euthanasia, there were signiﬁcant differences in Bcl-2 expression
between the two groups of horses. In RAO-affected animals, Bel-2 was expressed, on
average, in over 40 percent Of MC whereas less than 1 percent of cells expressed Bel-2 in
controls. Because I was concerned that disease may not be uniform throughout the lung, I
examined eight different regions and found the Bel-2 expression to be similar among
bronchi. Although Bel-2 expression was virtually absent in all control animals, there was
some variation between individual RAO-affected horses. The animal that had very little
inﬂammation on both Days 5 and 12 had Bcl-2 levels like a control animal in both large and
small airways (Figure 14F and 6G). Figure 13C shows that Bel-2 was only expressed in
animals in which the neutrophil count exceeded 10 cells / I11 and in RAO-affected animals,
expression tended to increase with severity of neutrophilic inﬂammation.

A similar association between the magnitude of Bel-2 expression in MC and numbers
Of neutrophils in airways also has been reported in rats challenged with two doses of LPS
(Foster, Gott et al. 2003). However, neutrophils are not essential for Bel-2 expression

because, in the latter model, neutrophil depletion did not reduce Bel-2 expression. The

immune mechanisms of RAO are not well understood but TNF-0t, IL-1, IL-4, IL-5, IL-8,

IL-13 and IFN- 7 are increased during the development and/ or maintenance Of the disease

(Joubert, Silversides et al. 2001; Cordeau, Joubert et al. 2004). Furthermore there is evidence
that oxidant stress is increased during acute exacerbations of RAO (Kirschvink, Smith et al.

2002). Many of these factors have been associated with changing levels of Bel-2. In

particular, TNF-a induces cell death by reducing the Bcl—2/ Bax ratio (Luo, Xia et al. 2005)
but this effect can be inhibited by IL—1 and NF-KB-induced up-regulation of Bcl-2

expression via degradation of the IKB inhibitor (Van Antwerp, Martin et al. 1996; Bouts,

76

Bonizzi et al. 2000). The latter. pathway is of interest in the case or the horse because NF-KB
activity in airway brushings is positively correlated with the magnitude of airway Obstruction
and is maintained by persistent inﬂammation as indicated by TNF-01 expression (Bours,
Bonizzi et al. 2000). Another possibility is that increased Bel-2 expression is induced by
activation Of the 15-lipoxygenase (Tang, Chen et al. 1996) pathway because the lungs of
horses with RAO produce more IS-HETE than do controls (Gray, Derksen et al. 1992).
Although Bel-2 was expressed in a greater percentage of MC in RAO-affected horses
with neutrophilic inﬂamrmtion, this was not associated with an increase in either the number
of MC or the amount of stored mucins (V 5). One possible explanation for this observation
is that the horses were euthanatized before GCH had occurred. This is unlikely because, in
rats, MCM develops within 3 to 5 days of an allergen, LPS or ozone challenge (Tesfaigzi,
Fischer et al. 2000). If the time course of events is similar in horses, the up to 12-day period
of inﬂammation should have been sufficient to cause GCH. The negative correlation of Vs
with neutrophil numbers (r = -0.886, p-0.033) in RAO horses supports neutrophil products
being potent secretagOgues. Forthis reason, I may not have been able to identify empty MC
. and could therefore have underestimated the MC numbers in horses with heaves. Assuming
that there was adequate time for GCH and that all MC were identified, what is the role for
Bel-2? Myinitial hypothesis that Bcl-Z prolonged MC life and thereby increased MC
numbers is clearly not supported by the data. An alternative role for Bel-2 would therefore
be prevention of premature MC death due to the oxidant stress and other factors produced
during the inflamnmory response. Over expression of Bcl-2 has been shown to induce
accumulation of the active form caspase-9 in the mitochondria, rendering the cells resiStant
to the redox stress (Katoh, Tomimori et al. 2004). AS neutrophils of RAO- affected horses
produce reaCtive oxygen species (ROS), Bel-2 may protect their MC from dying, while MC

77

of control horses undergo apoptosis, despite the generation of fewer ROS (Art, Kirschvink
et al. 1999).

With regard to the MC numbers, they were similar to those reported in other species.
The absence of GCH agrees with an earlier description of inconsistent morphological
changes in the epithelium Of larger airways Of horses with (DPD, a term that includes
horses with RAO (Kaup, Drommer et al 1990).

The presence of Bel-2 in three different airway generations indicates that RAO is a
condition that affects the entire tracheobronchial tree and is not limited to the peripheral
airways although the inflamrmtion may be most obvious in that region. Further support for
the generalized nature of RAO is provided bythe Observations of up- regulation of
eqMUCSAC (Gerber, Robinson et al. 2002) at all airway levels and the absence of iNANC
function in large airways (Robinson, Derksen et al. 1996).

This study addressed the role of increased MC numbers, as a result of delayed death,
as a potential cause of accumulated amounts of mucoid secretions in the airways of RAO-
affected aninuls. Mydata indicate that MC numbers are not increased but that stored
mucins are released in response to neutrophilic inﬂammation in RAO-affected horses. The
increased expression of eqMUCﬁAC is necessaryto replenish the secreted mucins (Gerber,
Robinson et al. 2003). Finally the decreased mucus clearability further contributes to
accumulation of secretions (Coombs and Webbon 1987).

78

APPENDIX

Activation of caspases require cleavage after an aspartate residue at an active cysteine in the
middle of the conserved motif, QACRG present in all proteases, (V aux and Strasser 1996).
This cleavage results in the large (~20 kd) and small (~10 kd) subunits and their subsequent
assembly into heterotetramers (V aux and Strasser 1996). Each heterotetramer consists of
two large and two small subunits with two active sites per molecule (Reed 2000). Substrates
observed in humans are both nuclear and cytoplasmic proteins for DNA repair and
replication, RNA splicing, cytoskeletal structure, and cell division (V aux and Strasser 1996).

Once activated, executioner caspases cause the breakdown of normal cellular compartrnental
barriers and despite the intracellular disassembly, the plasma membrane is left intact (Hetts
1998). It is important to realize that caspase-mediated cell demise can be inhibited at set
points. The second step of executioner caspase activation can be inhibited by the inhibitor
of apoptosis proteins (IAPs), so there is no autocatalytic cleavage (Sprick and Walczak 2004).
XIAP, the best described homolog, interacts with caspase-3 and the initiator caspase,
caspase-9 (Salvesen and Duckett 2002).

Inhibition of caspases still leads to cytochrome c release and to non-apoptotic cell death;
therefore, another caspase-independent mechanism for commitment, via the mitochondria
must exist. Amarante-Mendes, Finucane et a1 (1998) described this as cell death without
activation of caspases, externalization of phosphatidylserine, nuclear condensation, or DNA
fragmentation. Instead, cytoplasmic and nuclear vacuolization is a morphological
characteristic (Amarante-Mendes, Finucane et al. 1998). However, caspase-8 inhibitors will
prevent Fas-induced cytochrome c release (Green and Reed 1998). Also, Bel-2 (an anti-
apopototic protein) expression inhibits this death and preserves the clonogenic potential of
the cells (Amarante-Mendes, Finucane et al. 1998), while Bax (a pro-apoptotic protein) still
induces cytochrome c release and apoptosis even with caspase inhibition (Xiang, Chao et al.
1996;}urgensmeier, Xie et al. 1998).

Knockouts targeting caspase—9 (and also caspases-3) have severe defects in the brain and
undergo perinatal mortality (Zheng, Hunot et al. 1999). Caspase-8 deﬁcient embryos die
after 12 days due to ablation of cell death induction by TNFRs, Fas/Apol, and DR3

(V arfolomeev, Schuchmann et al. 1998). These embryos have impaired heart muscle
development and congested accumulation of erythrocytes contained in larger and smaller
blood vessels.

Inhibitor ofAQothsr’s Proteins

Inhibitors of apoptosis proteins (IAPs) are a family of evolutionarily conserved intracellular
proteins that suppress apoptosis whether induced by the TNFR-dependent (extrinsic)
pathway or the mitochondrial—dependent (intrinsic) pathway. This could be because its
targets are caspases that are common to both pathways (Reed 2000). Yet, some will inhibit
initiator caspases in the mitochondria pathway, perhaps reﬂective of the later evolution of
the death receptor (TNFR) pathway. By deﬁnition, all IAP proteins contain 1 to 3 copies of
the zinc-binding fold, baculovirus inhibitor of apoptosis proteins repeat (BIR) domains, and
also may contain CARDS (Reed 2000). Members of this protein family, such as XIAP,

79

cIAPl, and cIAPZ, can inhibit initiator caspase-9, via the third BIR domain, in addition to
already activated effector caspases-3 and -7, via the ﬁrst and second BIR domains (Reed
2000).

Birth by the Media_t0_r

Mitochondria have been ﬁttingly referred to as the understudy for the executioner in
triggering apoptosis (Green and Reed 1998). Mitochondria, originally viewed as initiators of
apoptosis are now recognized as ampliﬁers of caspase activity (Borner and Monney 1999;
Lassus, Opitz-Araya et al. 2002). This is evident by the positive feedback loop seen
following cytochrome c activation; cytochrome c is essential for caspase activation (Liu, Kim
et al. 1996) while caspases cause cytochrome c release from intact mitochondria (Hengartner
1998; Lassus, Opitz-Araya et al. 2002). If this is true, this system would require
“dampeners” to prevent unwarranted death such as the anti-apoptotic Bcl-2 proteins and the
MP5 to protect OMM integrity and inhibit particular caspases, respectively.

Mitochondria kill a cell through three mechanisms: (1) disruption of electron transport chain
activity, (2) alteration of reduction-oxidation (redox) potential, and (3) release of sequestered
caspase-activating proteins.

An early feature of apoptosis at the mitochondria is disruption of the electron transport
chain and oxidative phosphorylation that results in a reduced production of the ATP
required for many processes, thus loss of ATP kills the cell. However, this occurs very late
in the apoptotic process and is not likely to be the means of induction (Green and Reed
1998).

Multiple mechanisms engage mitochondrial involvement, leading to release of cytochrome c
and other factors normally residing in the inter—mitochondrial space (Figure 15). (\Wang
2001). (Acehan, Jiang et al. 2002).

Mitochondria are major producers of superoxide, where 1% to 5% of electrons traveling

down the electron transport chain are lost and form 0; and other reactive oxygen species
(ROS) (Green and Reed 1998). For this reason, a good hypothesis would be a decrease in
the coupling efﬁciency of the respiratory chain leads to increased ROS production. But like
disruption of the electron transport chain, this may be a late event (Green and Reed 1998).
Furthermore, experiments (Jacobson and Raff 1995) have shown that ROS are not required
for apoptosis induced by stimuli that do not necessarily generate ROS, as originally thought.
ATP depletion and severe oxidative stress can push an apoptotic cell towards necrosis
(Orrenius 2004). Bel-2 can protect cells in anaerobic conditions through means that do not
require inhibition of ROS production. However, ROS can still be generated in complete
anaerobiosis; thus, a role for ROS must still be considered (Degli Esposti and McLennan
1998).

During apoptosis, the permeablized OMM nonselectively releases proteins, in addition to
cytochrome c, that is also implicated in apoptosis. These apoptogenic proteins are involved
in caspase—dependent and -independent cell deaths and aid in ensuring valid execution.
Cytochrome c, Smac/Diablo, and apoptosis inducing factor (AIF) are the main proteins and

80

all require modiﬁcations within the mitochondria to become inducers of cell death; heme
attachment to cytochrome c, ﬂavin adenine dinucleotide (FAD) aMcMent to AIF and
proteolytic processing of the N -terminal mitochondrial leader peptide for AIF and
Smac/Diablo (Reed 2000; Wang 2001). This prohibits premature apoptosis from taking
place (e.g. during biosynthesis) and functionally links apoptosis to a disturbance in the
barrier function of the mitochondria (Reed 2000). Endonuclease G (EndoG), Omi/HtrAZ
(protease), and caspases-2, -3, and —9 are also released from the mitochondria (Reed 2000;
Wang 2001; Donovan and Cotter 2004).

Smac/Diablo, a 25-kD protein, is released from inside the inner mitochondrial membrane
with cytochrome c (Du, Fang et al. 2000). The N-terminal of mature Smac/Diablo binds to
IAPs and removes their inhibitory effects on caspases (Wang 2001). There is a direct
competition between activated caspases and Smac/Diablo providing a feedback system. The
presence of high amounts of IAPs terminates the activity of any caspases activated by newly
formed apoptosomes, thus providing a safety net for transient or accidental release of
cytochrome c (Wang 2001). Smac/Diablo is a larger molecule than cytochrome c and does
not escape the conﬁnes of the mitochondria in this event (Chai, Du et al. 2000). However, if
there is persistent and / or severe damage to the OMM, a large amount of Smac/Diablo is
released to remove the inhibition of the IAPs (inhibitor of apoptosis proteins), for example
the X-linked-inhibitor-of-apoptosis protein (XIAP) (Figure 15). In addition to the
cytochrome c pathway, Smac/Diablo and IAPs regulate the death receptor pathway because
the two pathways converge at caspase-3. Since XIAP does not inhibit Caspase—8 of the
extrinsic pathway, apoptosis can still occur via cleavage of a pro-apoptotic Bcl-2 family
member, the BH3-only Bid, by caspase-8. Activated Bid (tBid) inserts into the
mitochondrial membrane and releases Smac/Diablo, cytochrome c, and AIF.

Apoptosis inducing factor (AIF), a 57-kD ﬂavoprotein that resembles bacterial
oxidoreductase, also resides in the intermembrane space of mitochondria and induces
apoptosis in a caspase-independent manner (Daugas, Nochy et al. 2000; Wang 2001).
Apoptosis induces AIF’s translocation to the nucleus, condensation of chromatin, and DNA
fragmentation (Susin, Lorenzo et al. 1999). The mechanisms are unclear how AIF brings
about these changes since it itself has no measurable DNase activity (Susin, Daugas et al.
2000).

81

F

   
 

0:“!
ATP Cas ase-9 FADD
DISC

   
    
   
 
   
   
 

   
   
 
    

Pro-caspase-B

Apo tosome

‘ ‘2 <2)
I 9
Smac ! Di ablo XIAP

Pro-Caspase-3

Caspasc-3
_Pro-Caspase-7

,Qaspase-7

   

Caspase
Cascade ‘ y

 

 

Figure 15. Mitochondria are ampliﬁers and mediators of apoptosis, rather than initiators. (1) Cytochrome c
leaks out of the intermembrane space into the cytosol where it binds to and increases nucleotide-binding
afﬁnity of Apaf—l by 10—fold. ATP (or dATP) binding to Apaf—l / cytochrome c induces a shapa change and
oligomerization into a multimeric wheel-like structure to which caspase—9 binds to. This functional complex is
called an apoptosome. The apoptosome cleaves pro-caspase—3 and releases active caspase-3, which will cleave
pro-caspase-7 and release caspase—7 in the caspase cascade, leading to nuclear fragmentation and apoptosis. (2)
Smac/Diablo is released when the functional barrier is disrupted. In the cytosol, Smac/Diablo’s N-terminal
binds to the BIR domain of L~\Ps (e.g. XIAP) and removes the inhibitory effects of XIAP on caspase-3 and -9.
(2’) In the presence of high IAPs, Smac/Diablo cannot bind enough IAP and the inhibitory affects remain.
The Fas-mediated pathway can still initiate apoptosis, however, because of caspase-8 cleaving and activating
cystolic Bid, a pro—apoptotic Bcl—2 family member, causing its translocation and insertion into the outer
mitochondrial membrane (Oth). Insertion of truncated Bid (tBid) leads to OMM permeabilization. Caspase—
8 is insensitive to XL-\P (Riedl, Renatus et al. 2001). (3) AIF is released and induces nuclear condensation,
independent of any caspase activation.

82

EXTRINSIC PATHWAY PROTEINS
Dea th Domains

Many proteins involved in death pathways have so-called death domains (DD).
Oligomerization of one protein’s DD with another’s facilitates execution of apoptosis by
bringing required molecules into close proximity with a receptor or protease, for example.
This leads to initiator caspase activation followed by activation of downstream effector
caspases.

Death Effector Domains

Death effector domains (DED) are found in the initiator caspases, caspase-8 and caspase-10,
in humans (Reed 2000). Cleavage of these pro-caspases requires interaction of the two pro-
caspase DEDs with the DED on the adapter protein, Fas-associated death domain (FADD),
also called MORTl. This adapter protein is absolutely crucial in caspase activation because
it contains both a DD and DED (Zhang, Cado et al. 1998), linking it to the receptor (via DD)
to the pro—caspases (via DED). Additional DED-containing modulators are described in the
review by Reed (2000).

Casgase-Associated Recruianent Domains

Caspase-associated recruitment domains (CARD) are found on the N-terminals of pro-
caspases for caspase-1, -2, —4, -5, and -9 in humans and caspase-1, -2, -9, -11, and -12 in mice
(Reed 2000). They are similar in structure as the DD and DED. Ablation of CARD-
carrying caspases has signiﬁcant effects on disease, including, reduced tissue loss after stroke,
increased resistance to endotoxin-induced sepsis, slowing of Huntington’s disease
progression (Bergeron, Perez et al. 1998; Ona, Li et a1. 1999). Importantly, Apaf—l contains
a CARD that mediates interaction with the CARD on pro-caspase-9 to initiate caspases by
the induced proximity model.

Ligand binding to the receptor induces a shape change on the intracellular portion of the
receptor to expose the DD and facilitate Oligomerization with the DD of an adapter protein,
and possibly binds additional downstream adapter proteins. Pro-caspase—8 binding to the
adapter protein FADD /MORT1 via their DEDs completes the formation of the DISC, to
which additional pro-caspases can bind and autocatalytically cleave and activate caspases
(Boldin, Goncharov et al. 1996, Muzio, 1996 #100). Therefore, FADD /MORT1 indirectly
links pro-caspase-8 to the cystolic domains of TNF family of death receptors [c.g. Fas
(Apol /CD95)].

Numerous experiments have been performed to characterize the functions, interactions, and
regulations of members of the Bcl-2 family. Gene disruptions experiments have shown that
Bel-2 is widely expressed during embryogenesis and becomes more restricted postnatally

(Chao and Korsmeyer 1998). Newborn bcl-Z"- or bcl-xL-l— animals have extensive cell death
and usually die within a few weeks. Mice bcl-f/- knockouts are born viable where bcl-x #-

83

animals die at around embryonic day 13 (Chao and Korsmeyer 1998). The [rd-2 -/— mice turn
gray at 5 to 6 weeks reﬂecting decreased melanocyte survival and have polycystic kidney
disease-like changes in the renal tubules (Nakayama, N egishi et al. 1994). These mice
appeared to have a normal nervous system unlike Bcl-x ablated mice. Mice lacking Bcl—x
have substantial death of hematopoietic cells and postrnitotic immature neurons in brain,
spinal cord, and dorsal root ganglia (Motoyama, Wang et al. 1995).

There is evidence to support a two-class model for BH3 domains: 1) Bid-like domains that
activate, and 2) Bad-like domains that sensitize (Letai, Bassik et al. 2002). Bid and Bim with

a OL-helical BH3 domain are capable of activating and mediating Bax and Bak
Oligomerization, while Bad and Bik preferentially interact with the pockets of the anti-
apoptotic members like Bel-2 and Bcl-xL, which displaces Bid-like BH3 domains. Most of
the BH3—only proteins act as trans-dominant inhibitors of anti-apoptotic members and are
unable to homodimerize (Reed 2000). Of those listed in Table 1, only Bik and Blk are
similar to each other and all others are unrelated to any known protein (Adams and Cory
1998).

The function of either Bax or Bak is required to initiate most forms of apoptosis.
Independently, Bax inactivation has only slight effects on apoptosis with modest phenotypic
abnormalities and Bak. inactivation shows no effects (Lindsten, Ross et al. 2000) and in both
models underwent Bim- and Bad-induced apoptosis (Zong, Lindsten et al. 2001). However,
Bax‘l-Bak-I' double-knockouts showed dramatic impairments of apoptosis (preservation of
interdigital webs, an irnperforated vaginal canal, and accumulations of excess cells within the
central nervous and hematopoietic systems) and die perinatally (Lindsten, Ross et al. 2000).

Bax-/-rrrice have apparent normal external development, however, they exhibit hyperplasia
of thymocytes and B cells and the gonads, in both ovaries and testis, as well as hypoplasia
accompanied by multinucleated giant cells and dysplastic cells (Knudson, Tung et al. 1995).
Overexpression of Bax accelerated cytokine deprivation-induced cell death in a IL-3-
dependent cell line (Oltvai, Millirnan et al. 1993). Constitutively active forms of Bim and
Bad fail to induce apoptosis in the mice without Bax or Bak (Zong, Lindsten et al. 2001).

In a recombinant Bel-2 protein experiment, Schendel, Xie et a1 (1997) found that Bel-2
forms distinct ion-conducting, cation-selective channels. Such pore forming abilities are
dependent on a low pH and acidic lipid membranes to permit proper orientation. Acidic pH
has not only been shown to induce pore formation, but it stabilizes hetero- or

homodimaized BCl-XL to Bel-2, Bax, and Bid (Xie, Schendel et al. 1998). The perpendicular

insertion of an estimated minimum of four dimerized or oligomerized hydrophobic a-
helices into the lipid bilayer provide the make up of the channel’s core. This channel is in a
closed state in vitro at neutral pH; however pH-dependent mechanisms for channel formation
are unlikely because low in viva pH does not induce channel formation but only promotes
association of pore-forming fragments with the mitochondrial membrane (Schendel, Xie et
al. 1997).

84

Both Bel-2 and Bax insert into experimental lipid membranes in a pH-dependent fashion
with maximum activity at pH of 4.0 (Schlesinger, Gross et al. 1997). Bax channels have a
minimal conductance of 22pS with at least three subconductance levels (Schlesinger, Gross
et al. 1997) that decrease with an increase in pH (0.731 nS at pH 4.0 to 0.329 nS pH 7.0) and
mild chloride anion selectivity in a predominantly open state. In contrast, Bel-2 or Bel-xL
channels are slightly potassium cation selective (Schendel, Xie et al. 1997) and have a
prominent conductance of 80pS (Chao and Korsmeyer 1998), although 20pS has been
reported in lipid membrane experiments (Schendel, Xie et al. 1997). Despite these moderate
differences in ion selectivity of the Bel-2 and Bax channels, other characteristics such as
conductance, voltage dependence, and rectification are exhibited making either protein a
candidate for exerting regulatory or functional components on the PT pore and cytochrome
c release (Schlesinger, Gross et al. 1997).

PT is always followed by A‘i’m, but A‘I’m is not always caused by PT, and cytochrome c may

still be released without A‘I’m (Bernardi, Scorrano et al. 1999; Kroemer and Reed 2000). This
may be possible due to the rapid opening and closing of the channel, at a low conductance

state, to allow for a repeated, respiratory-driven re-establishment of A‘I’m. This explains why
OMM disruption and cytochrome c release precede A‘I’m collapse (Green and Reed 1998).

Evidence exists, however, for PT to induce apoptotic A‘I’m dissipation (Zamzami, Marchetti
et al. 1996).

85

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