THE ROLE OF BASOPHILS IN THE LATE ASTHMATIC
RESPONSE AND THE EFFECT OF ALARMIN
CYTOKINES ON BASOPHIL PRO-INFLAMMATORY
ACTIVITY
Brittany M.A. Salter
McMaster University, brittmsalter@gmail.com
BASOPHILS
AND
ALARMIN
CYTOKINES
THE ROLE OF BASOPHILS IN THE LATE ASTHMATIC RESPONSE
AND THE EFFECT OF ALARMIN CYTOKINES
ON BASOPHIL PRO-INFLAMMATORY ACTIVITY
By
BRITTANY M.A. SALTER, B.S.c.
A Thesis
Submitted to the School of Graduate Studies
In Partial Fulfillment of the Requirements
For the Degree
Doctor of Philosophy
McMaster University © Brittany M.A. Salter, June 2015
i
DOCTOR OF PHILOSOPHY (2015)
McMaster University
(Medical Sciences)
Hamilton, Ontario
TITLE:
The role of basophils in the late asthmatic response and the
effect of alarmin cytokines on basophil pro-inflammatory
activity
AUTHOR:
Brittany M.A. Salter, BSc. (University of Guelph)
SUPERVISOR:
Dr. Gail M. Gauvreau
SUPERVISORY
COMMITTEE:
Dr. Paul O’Byrne
Dr. Kieran Killian
Dr. Gail Gauvreau
Dr. Roma Sehmi
NUMBER OF PAGES: xxi, 218
ii
ABSTRACT
There is emerging evidence that the alarmin cytokines, thymic stromal lymphopoietin
(TSLP), interleukin (IL)-25 (IL-25), and IL-33 are critical mediators of T helper cell
type-2 (Th2)-driven inflammation in allergic airway disease. Although there is a myriad
of data pertaining to the interaction between these alarmins and immune cells, there is
still very little known on how alarmin cytokines can influence the activity of basophils in
allergic asthma. The overall objectives of this thesis were to examine the involvement of
basophils in the late asthmatic response (LAR) of allergic asthma and to determine
whether their pro-inflammatory activity is influenced by alarmin cytokines. Initially, we
investigated the change in activity and migration of basophils following allergen
inhalation in mild allergic asthmatics. We showed that a subset of activated basophils
migrate from the bone marrow into peripheral blood and then to the airways during an
airway allergen challenge (Chapters 2-5). We found expression of surface activation
markers and intracellular Type 2 cytokines was increased after an allergen challenge for
up to 24 hours (Chapter 3). Next, we examined the change in expression on basophils of
receptors for TSLP, IL-25, and IL-33 following an allergen challenge (Chapters 3 and 4).
We reported that basophil surface receptor expression for alarmin cytokines was
markedly increased post-allergen challenge within the peripheral blood and airway
samples. In addition, we investigated the effect of these alarmin cytokines on basophil
activity in vitro, demonstrating that they can directly stimulate basophil activation and
Type 2 cytokine intracellular expression, as well as prime eotaxin-induced migration
(Chapters 3 and 4). Finally, to explore the pro-inflammatory response of basophils to the
airway microenvironment, we examined the effect of airway samples collected post-
iii
allergen on basophils in vitro (Chapter 4). We showed that basophil activation and Type
2 cytokine intracellular expression were up-regulated following incubation with 7 hour
post-allergen airway samples, and could be inhibited following treatment with a
monoclonal antibody (mAb) to the common β-chain (βc) for IL-3/IL-5/granulocytemacrophage colony-stimulating factor (GM-CSF). The novel work presented herein
augments the available evidence that basophils play a role in the LAR of allergic asthma
that may be in part mediated by interaction with alarmin cytokines. These observations
support the notion that the basophil/alarmin cytokine axis is a potentially important
therapeutic target for allergic asthma. Continued investigation is required to determine if
blockade of this axis can inhibit or attenuate allergic airway inflammation and
bronchoconstriction.
iv
ACKNOWLEDGEMENTS
To be completely honest, never in a million years, would I see myself completing
a PhD. Just thinking about it now seems so surreal. I had such a concrete plan when I
started out doing my Bachelors degree… I would get good grades, ace the MCAT, and
hopefully get scooped up by a Canadian medical school. But what I never planned for
was the spark ignited after I took my very first immunology course. The next thing I
didn’t anticipate was my love for research to creep up after I did an undergrad thesis.
Now- almost 5 years later following the completion of my Bachelors, I am sitting here
writing this PhD thesis, with the underlying excitement that I will be attending medical
school in Australia come February 2016. Yes… another 4 years of school… plus
residency. But what I have realized is that I can’t have one or the other… research versus
medicine… it’s just too hard of a decision to make. So why not combine both and make a
stellar clinician scientist career out of it? Sure, the path I am taking is not the most direct
way of getting to my final career goal, but the journey has been incredible and I wouldn’t
change a thing.
Throughout my PhD I have had an incredible support system, which I would like
to now take the time to thank from the bottom of my heart. First and foremost, I would
like to thank Dr. Gail Gauvreau and Dr. Roma Sehmi. You have both been wonderful
role models and mentors for me. I can only hope that one day I will be half the
researcher’s you are. The valuable research and clinical experience I have learned from
you has surpassed any original expectation that I had. I especially thank you both for
trying to slow me down and prevent me from self-combusting with experiment ideas.
v
Lastly, I sincerely believe that your support and toughness has helped to shape me into
the scientist that I am today.
Next, I would like to thank my committee members Dr. Kieran Killian and Dr.
Paul O’Byrne for their time and commitment to my thesis. I really appreciate the positive
feedback you have expressed over my research. To Dr. Kieran Killian- in the beginning I
was very terrified of you but over time I saw through your tough exterior and I now think
of you as a big teddy bear- one that provides a wealth of constant statistical and clinical
knowledge. To Dr. Paul O’Byrne- I cannot begin to express the gratitude I have for your
teaching and guidance. You have helped me determine the path I would like to take in the
future (as a clinician scientist) and I am eternally grateful. I will always have the utmost
respect and admiration for you.
To the students and staff of the Cardiorespiratory Lab, what would I have done
without you? You guys provided a steady stream of blood that I could steal- and for that I
am truly thankful but also sorry to be such an annoying recruiter. I simply wouldn’t have
had the same PhD experience if I didn’t get to be around all of the wonderful people in
this lab. Steve and Adrian, you are two of the best up and coming researchers that I know
and without your support I wouldn’t have completed my PhD. Steve- I probably owe you
over a million dollars by now in consulting fees. You guys have also been a huge source
of entertainment and laughter in my life. I always look forward to coming into the lab to
giggle at the expense of Strin’s feelings (sorry for never taking your side Strin!),
Takashi’s crazy Japanese facial expressions, Damian’s “Euro” nature, or Tina’s weird
Chinese butterfly pants (which she should never have worn in public- sorry Tina!). Most
of all, I would like to thank JP- you have simply been the best lab mate anyone could
vi
ever ask for! Not only did you always go out with me for sushi when I had the craving,
but you would reply to my frantic texts (when the flow cytometer broke or experiments
failed) and always find a way to keep me from going insane. Last but not least, Graeme,
you were the best minion ever- thanks for coming in at weird “Bwatts’ hours” and
helping finish up experiments that I needed to get done.
Next, I would like to thank my boyfriend, friends, and family. A special shout out
goes out to my mommie, sister, and aunt for sacrificing their blood in the name of
science. It just so happened that they had lots of basophils- and that is a big deal when
they are such a rare population of cells! Needless to say, I took their blood a lot. To my
amazing close friends who have always been here for me (Marija, Laurie, Alysha, Sam,
Nirooya, Kara, and Justin)- thanks for always giving me encouragement and a shoulder to
cry on if I got too stressed. Most of all- thanks for sticking with me, when I have gone
into complete “hermit mode” for months at a time. Laurie and Sam- if it wasn’t for our
crazy fun road trips (NY, NJ, and NH) to visit Alysha, I would probably have gone
insane. Above all, thanks to my wonderful boyfriend, Nate. Thank you for being so
supportive, patient, and loving. You always calmed me down when I was stressed about
experiments with either a huge hug or some dark chocolate (I think I like that the best). I
am so happy that we went on all of our amazing trips and raised a puppy together (Ellieour “child” is the cutest dog in the world I swear!), which helped break up the monotony
of PhD life full of writing and experimenting. I love that you even got involved in my
research by becoming a lab rat! It was great to have you there all day with me as you
were put through allergen challenges on no sleep from night shifts.
vii
This thesis, without a doubt, is dedicated to my mommie. You have supported and
guided me every step of the way. Even in the midst of anxiously waiting for me to be
done my degree and go to medical school- you stayed patient and loved me
unconditionally.
Once again, I would like to thank everyone from the bottom of my heart! Your
support has truly been priceless.
<3 Bwatts
viii
TABLE OF CONTENTS
Abstract ………………………………………………………………………….........
iii
Acknowledgements ……………………………………………………………...........
v
Table of Contents ……………………………………………………………..............
ix
List of Tables ……………………………………………………………...................
xii
List of Figures ……………………………………………………………..................... xiii
List of Abbreviations ……………………………………………………..................... xv
Declaration of Academic Achievement …………………………………..................... xx
Chapter 1: Introduction
1.1 Asthma ……………………………………………………………......................... 1
1.1.1 Allergic Asthma and Th2 Polarization
1.1.2 The Role of Th2 Cytokines in Allergic Asthma
1.2 Early and Late Asthmatic Responses …………………………............................. 4
1.3 Basophils in Allergic Disease …………………………........................................... 7
1.3.1 Basophil Background
1.3.1.1 Basophil Growth and Development
1.3.1.2 Basophil Migration
1.3.1.3 Basophil Activation
1.3.1.4 Basophil Mediators
1.3.1.5 Tools for Basophil Research
1.3.1.6 Role of Basophils in Allergic Asthma
1.3.1.7 Basophils and Allergen Challenges
1.3.1.8 Activation-Linked Markers on Basophils
1.4 Airway Epithelium ……….……………………………......................................... 23
1.4.1 Airway Epithelium and Allergic Asthma
1.5 Thymic Stromal Lymphopoietin ………………………........................................ 24
1.5.1 TSLP
1.5.2 Human Expression of TSLP
1.5.3 Human Expression of TSLPR
1.5.4 Cellular Targets of TSLP
ix
1.5.4.1 Dendritic Cells
1.5.4.2 T cells and B cells
1.5.4.3 HPCs
1.5.4.4 Mast Cells and Eosinophils
1.5.4.5 Basophils
1.5.5 TSLP, Mouse Models, and Human Clinical Trials
1.6 IL-25 …………………………………………..…………........................................ 38
1.6.1 IL-25 and IL-25R Expression
1.6.2 Cellular Targets of IL-25
1.6.3 IL-25 and Mouse Models
1.7 IL-33 ………………………...................................................................................... 43
1.7.1 IL-33 and IL-33 Receptor
1.7.2 IL-33 and IL-33 Receptor in Allergic Disease
1.7.3 Cellular Targets of IL-33
1.7.3.1 T cells
1.7.3.2 Dendritic Cells
1.7.3.3 Hematopoietic Progenitor Cells
1.7.3.4 Mast Cells, Eosinophils, and Basophils
1.7.4 Lessons from Animal Models
1.8 Summary ………………………............................................................................... 51
1.9 Hypothesis and Aims …………............................................................................... 52
Chapter 2: Expression of activation markers in circulating basophils and the
relationship to the allergen-induced bronchoconstriction in subjects with mild
allergic asthma ………….......................................................................................................... 54
Chapter 3: Thymic Stromal Lymphopoietin Activation of Basophils in Allergic
Asthma is IL-3 Dependent............................................................................................. 67
Chapter 4: IL-25 and IL-33 Induce Th2 Inflammation in Basophils from Subjects
with Allergic Asthma …................................................................................................. 90
Chapter 5: Discussion
x
5.1 Basophils: Important Effector Cells in Allergic Asthma ………....................... 135
5.1.1 Basophil Activation in Allergic Asthma The Role of Th2 Cytokines in Allergic
Asthma
5.1.2 Basophil Migration in Allergic Asthma
5.1.3 Basophil Th2 Cytokine Expression in Allergic Asthma
5.2
Epithelial Cells Secrete a Triad of Alarmin Cytokines that Stimulate Basophils
…………………………................................................................................................. 144
5.2.1 Basophil Expression of Receptors for Alarmin Cytokines in Allergic Asthma
5.2.2 The Influence of Alarmin Cytokines on Basophil Pro-Inflammatory Activity
5.2.3 The Influence of the Airway Microenvironment on Basophil Effector Activity
5.3
Therapeutic Implications: Basophil/Alarmin Cytokine Axis as a Target in
Allergic Asthma …………………………....................................................... 154
5.4
Limitations …………………………................................................................ 158
5.5
Summary …………………………................................................................... 161
5.6
Future Directions: Blockade of Basophil/Alarmin Cytokine Axis in Allergic
Asthma ……….................................................................................................. 166
Chapter 6: References ……………………................................................................. 170
Appendix I: Copyright Permissions to Reprint Published Materials ..................... 204
Appendix II: Inhibition of allergen-induced basophil activation by ASM-024, a
nicotinic receptor ligand Copyright Permissions to Reprint Published Materials
……………................................................................................................................................ 209
LIST OF TABLES
xi
CHAPTER 1
Table 1. Role of Type 2 Cytokines in Allergic Asthma
Table 2. Effect of alarmin cytokines on immune cell functions
CHAPTER 2
Table 1: Subject Characteristics
CHAPTER 3
Table 1: Subject Characteristics
CHAPTER 4
Table 1: Allergen inhalation induces airway changes in mild allergic asthmatics
LIST OF FIGURES
xii
CHAPTER 1
Figure 1. Immune pathways resulting in early and late asthmatic responses
Figure 2. Differentiation of immune cells from HPCs
Figure 3. Migration of Basophils
CHAPTER 2
Figure 1. Effects of allergen inhalation on the level of basophil activation marker
expression
Figure 2. Correlation between CD203c basophil expression and disease severity
Figure E1. Basophil Gating Strategy
Figure E2. Allergen inhalation induces airway changes in mild allergic asthmatics
Figure E3. Effects of allergen inhalation on percent basophil expression of activation
markers
CHAPTER 3
Figure 1. Allergen inhalation induces airway changes in mild allergic asthmatics
Figure 2. Effects of allergen inhalation on basophil function
Figure 3. The effects of 1 h stimulation with TSLP on basophil activation
Figure 4. The effects of 18 h of incubation with TSLP on basophil function
Figure 5. Blocking TSLPR, IL-3Rα, and the common β-chain neutralizes the effects of
TSLP on basophils
Figure 6. TSLP priming increases eotaxin-induced basophil shape change
Figure 7. TSLP-basophil axis in the airways during allergic stimulation
Figure E1. Post-Isolation Basophil Purity
Figure E2. Dose response effects of 1 h stimulation with TSLP on basophil activation
Figure E3. Dose response effects with overnight incubation with TSLP on basophil
function
Figure E4. TSLP does not directly induce basophil shape change
xiii
Figure E5. Basophil gating strategy
Figure E6. Correlation between CD203c basophil expression and disease severity
Figure E7. Correlation between TSLPR basophil expression and disease severity
Figure E8. Correlation between intracellular IL-13 and IL-4 basophil expression and
disease severity
Figure E9. TSLP up-regulates basophil intracellular Th2-cytokine expression
CHAPTER 4
Figure 1. Allergen inhalation increases the expression of alarmin cytokines and their
receptors in basophils of subjects with mild allergic asthma
Figure 2. IL-25 and IL-33 up-regulate expression of their own receptors on basophils
Figure 3. The effect of IL-25 and IL-33 on expression of activation markers in basophils
Figure 4. Priming effects of IL-25 and IL-33 on eotaxin-induced basophil shape change
Figure 5. The expression of ST2 on basophils correlates positively with the magnitude of
the late asthmatic response
Figure 6. The effect of sputum-derived mediators on markers of basophil activation
Figure 7. Blocking the common β chain receptor neutralizes the effects of sputumderived mediators on basophils
Figure E1. Basophil gating strategy
Figure E2. Basophil activation marker dose response to IL-33
Figure E3. Basophil activation marker dose response to IL-25
CHAPTER 5
Figure 1. The Basophil/Alarmin Axis
LIST OF ABBREVIATIONS AND SYMBOLS
xiv
AD
Atopic dermatitis
AHR
Airway hyperresponsiveness
APC
Antigen presenting cell
AR
Allergic Rhinitis
ASM
Airway smooth muscle
BALF
Bronchoalveolar lavage fluid
BAT
Basophil activation test
βc
IL-3/IL-5/GM-CSF common β-chain
CCL
C-C motif ligand
CCR
C-C motif receptor
CD
Cluster of differentiation
CD40L
Cluster of differentiation 40 ligand
C/EBPα
CCAAT-enhancer binding proteins
c-kit
SCF receptor
COPD
Chronic obstructive pulmonary disease
CRTH2
Chemoattractant receptor homologous
molecule expressed on Th2 cells
CysLTs
Cysteinyl leukotrienes
CysLTR1
Cysteinyl leukotriene receptor 1
CysLTR2
Cysteinyl leukotriene receptor 2
CXCL
C-X-C motif ligand
CXCR
C-X-C motif receptor
DC
Dendritic cell
Der p 1
Dermatophagoides pteronyssinus-1
xv
mDC
myeloid dendritic cell
DR
Dual responder
DT
Diptheria toxin
DTR
Diptheria toxin receptor
EAR
Early asthmatic response
ECP
Eosinophil cationic protein
EDN
Eosinophil-derived neurotoxin
EPX
Eosinophil peroxidase
ERK
Extracellular signal-regulation kinase
FcεR1
IgE receptor
Fel d 1
Felis domesticus 1
FEV1
Forced expired volume in 1 second
FOXP3+ T-regs
T-regs positive for Forkhead box P3
GATA-2
GATA binding protein 2
GM-CSF
Granulocyte-macrophage colony-stimulating
factor
GMP
Granulocyte-macrophage progenitor
HBEC
Human bronchial epithelial cell
HDM
House dust mite
HPC
Hematopoietic progenitor cell
ICAM-1
Intracellular adhesion molecule-1
IER
Isolated early responder
Ig
Immunoglobulin
xvi
IFNγ
Type II Interferon
IL
Interleukin
ILC2
Innate lymphoid cell type 2
IL-1R AcP
IL-1 receptor accessory protein
IL-3Rα
IL-3 receptor α
IL-5Rα
IL-5 receptor α
IL-7Rα
Interleukin-7 receptor-α
IL-17R
Interleukin-17 receptor
IL-25R
Interleukin-25 receptor
IRAK
Interleukin-1 receptor-associated kinase
JAK
Janus kinase
JNK
C-jun N-terminal kinase
LAMP
Lysosome-associated membrane
glycoprotein
LAR
Late asthmatic response
LPS
Lipopolysaccharide
mAb
Monoclonal antibody
MAPK
Mitogen-activated protein kinase
MBP
Major basic protein
MCP
Monocyte chemotactic protein
MDC
Macrophage-derived chemokine
mDC
Myeloid dendritic cell
MHC II
Major histocompatibility complex type II
MIP
Macrophage inflammatory protein
xvii
mMCP-8
Mast cell protease 8
mRNA
Messenger ribonucleic acid
MyD88
Myeloid differentiation primary response
gene 88
NFκB
Nuclear factor kappa beta
NGF
Nerve growth factor
NKT
Natural killer T cell
OVA
Ovalbumin
OX40L
OX40 Ligand
PBS
Phosphate buffered saline
PG
Prostaglandin
PolyI:C
Polyinosinic:polycytidylic acid
PKC
Protein kinase C
PI3K
Phosphoinositide 3-kinase
PRR
Pattern recognition receptor
RANTES
Regulated on activation, normal T cell
expressed and secreted
SCF
Stem cell factor
c-Kit
Stem cell factor receptor
ST2
IL-1 receptor family member with
transmembrane
sST2
Soluble ST2
STAT
Signal transducers and activators of
transcription
xviii
TARC
Thymus and activation regulation
chemokine
TCR
T cell receptor
TGF-β
Transforming growth factor-β
Th1
T helper 1
Th2
T helper 2
Th0
Naive CD4+ T helper
TLR
Toll-like receptor
TNF
Tumor necrosis factor
T-reg
Regulatory T cell
TSLP
Thymic stromal lymphopoietin
TSLPR
Thymic stromal lymphopoietin receptor
VCAM-1
Vascular cell adhesion protein-1
xix
DECLARATION OF ACADEMIC ACHIEVEMENT
The research documented herein is presented as a “sandwich doctoral thesis”. The three
articles presented in Chapters 2-4 are three independent, but thematically related bodies
of work that, as of May 2015, have been or are being peer-reviewed prior to publication.
Although I, Brittany Salter, was the major contributor for the work presented in this
thesis, it required a collaborative effort from several individuals. As such, my
contributions, along with those who assisted with the content of each article, are
highlighted below.
Chapter 2:
Salter BM, Nusca GM, Tworek D, Oliveria JP, Smith SG, Watson RM,
Scime T, Obminski C, Sehmi R, Gauvreau GM. Expression of activation
markers in circulating basophils and the relationship to allergen-induced
bronchoconstriction in subjects with mild allergic asthma.
Submitted to J Clin Allergy and Immunol May 2015, In Revision June
2015
I was responsible for the experimental design of the study, as well as statistical
analysis and interpretation of the data. Rick Watson and Tara Scime recruited subjects for
the diluent/allergen inhalation challenge aspect of this study. They also conducted skin
prick and methacholine spirometry testing as a pre-screening component. Catie Obminski
also assisted in the diluent/allergen inhalation challenge process. I performed all of the
experiments with assistance provided by a research assistant (Graeme Nusca). Dr.
Damian Tworek provided valuable help with subject recruitment, sample collection, data
entry, media preparation, and back-up assistance for experiments. Dr. Steven Smith and
John Paul Oliveria provided technical support for immunostaining and flow cytometry.
With assistance from Drs. Gail Gauvreau and Roma Sehmi, I wrote the manuscript for
publication in the form of a letter to the editor. Dr. Gail Gauvreau further helped with
xx
replying to comments from the reviewers and revising the paper to achieve acceptance
and publication.
Chapter 3:
Salter BM, Oliveria JP, Nusca GM, Smith SG, Watson RM, Comeau M,
Sehmi R, Gauvreau GM. Thymic Stromal Lymphopoietin Activation of
Basophils in Allergic Asthma is IL-3 Dependent. Submitted to J Clin
Allergy and Immunol December 2014. Decision: Accepted and In Press
March 2015
This study started at the beginning of my PhD, upon transferring from the Masters
program at McMaster University. I was responsible for the experimental design of the
study, as well as statistical analysis and interpretation of the data. Rick Watson recruited
subjects for the diluent/allergen inhalation challenge aspect of this study. He also
conducted skin prick and methacholine spirometry testing for the study. I performed all
of the experiments with assistance provided by a research assistant (Graeme Nusca). John
Paul Olivera and Dr. Steven Smith provided valuable technical assistance with
immunostaining and flow cytometry. With assistance from Drs. Gail Gauvreau and Roma
Sehmi, I wrote and prepared the manuscript for publication. M Comeau assisted in the
editing of the manuscript. Dr. Gail Gauvreau further helped with replying to comments
from the reviewers and revising the paper to achieve acceptance and publication.
Chapter 4:
Salter BM, Oliveria JP, Nusca GM, Smith SG, Tworek D, Mitchell PD,
Watson RM, Sehmi R, Gauvreau GM. IL-25 and IL-33 Promote Basophil
Pro-Inflammatory Function in Allergic Asthma. Submitted to Allergy Aug
2015.
I was responsible for the experimental design of the study, as well as statistical
analysis and interpretation of the data. Rick Watson recruited subjects, pre-screened them
with skin prick and spirometry testing, and conducted the diluent/allergen inhalation
challenge aspect of this study. I performed all of the experiments with assistance
xxi
provided by a research assistant (Graeme Nusca). Dr. Damian Tworek provided valuable
help with subject recruitment, sample collection, data entry, and back-up assistance for
experiments. Drs. Steven Smith and Patrick Mitchelle, and John Paul Oliveria provided
technical support for immunostaining and flow cytometry. With assistance from Drs. Gail
Gauvreau and Roma Sehmi, I wrote and prepared the manuscript for publication. Dr. Gail
Gauvreau further helped with replying to comments from the reviewers and revising the
paper to achieve acceptance and publication.
xxii
CHAPTER 1:
INTRODUCTION
1.1 Asthma
Asthma
is
a
chronic
respiratory
disorder,
characterized
by
episodes
of
bronchoconstriction, airway inflammation, tissue remodeling, and airway hyperresponsiveness
(AHR), and is associated with shortness of breath, chest tightness, wheezing, and cough.
Asthmatic symptoms are induced by the exposure to various external stimuli including chemical
agents, environmental pollutants, and aeroallergens.
Asthma is common, disabling people over their entire life span and causing early death in
many. The World Health Organization views asthma as a serious public health threat, affecting
an estimated 235 million people worldwide (3%), with mortality rates reaching over 250, 000
annually [1]. By year 2025, the number of people suffering from asthma will grow by more than
100 million [1]. In Canada, the overall economic health care cost associated with asthma is
estimated at $12 billion [2]. Inhaled corticosteroids are the gold standard of asthma therapy, but
are limited due to side effects when high doses are used, and corticosteroid resistance in some
patients. The unresponsiveness to steroids, in addition to the increasing frequency of asthma
emphasizes the urgency for new therapeutic approaches.
1.1.1 Allergic Asthma and Th2 Polarization
Asthma can be classified into two main types: allergic and non-allergic asthma, which are
distinguished by the presence or absence of Immunoglobulin-E (IgE) antibodies to defined
common environmental allergens. Allergic asthma is the most commonly occurring type of
asthma, affecting roughly 60% of adult asthmatic patients, and almost all children with asthma
[3].
1
Allergic asthma is fundamentally based on disruption in the balance between T helper
cell type-1 (Th1) and Th2, with a skewing towards Type 2 inflammation, characterized by
airway eosinophilia, leukocytosis, and tissue remodeling. Exogenous aeroallergens enter into the
airway where they are taken up and processed by resident antigen presenting cells (APCs) such
as dendritic cells (DCs), B cells, and macrophages. APCs capture the foreign antigen through
phagocytosis or receptor mediated endocytosis, and presents a small peptide fragment of the
antigen to naive cluster of differentiation (CD) 4+ T-Helper (Th0) cells by forming a complex
between the major histocompatibility complex type II receptor (MHC II) on the APC and the T
cell receptor (TCR) on the Th0 cell [4].
Upon activation, Th0 cells polarize into either Th1 or Th2 cells [4]. These two types of T
cells have distinct features. Th1 cells mediate phagocytosis and production of anti-inflammatory
cytokines type II interferon (IFNγ) and IL-12, which inhibit Th2 cell growth [5]. In contrast, Th2
cells initiate IgE-dependent immune responses by releasing pro-inflammatory mediators into the
airways and inhibit formation of Th1 cells via release of IL-10 [6]. The differentiation of Th0
cells is dependent on cytokines present within the surrounding environment. Th2 differentiation
is promoted by Type 2 cytokines (IL-4, IL-5, IL-13, and IL-9) that are secreted by APCs. In
particular, Th2 cell growth and survival is maintained by elevated levels of IL-4 and decreased
levels of IL-12. The activation and prolonged survival of Th2 cells initiates a cascade of events
that define the inflammatory response of allergic disease.
1.1.2 The Role of Type 2 Cytokines in Allergic Asthma
Type 2 cytokines including IL-4, IL-13, IL-5, IL-9, IL-3, and GM-CSF orchestrate cellto-cell interactions that are crucial to allergic airway inflammation. The messenger ribonucleic
acid (mRNA) expression for these cytokines is increased in bronchial samples from asthmatic
2
individuals, compared to healthy subjects [7-10], and become further up-regulated following
exposure to allergen [11-14]. IL-4 and IL-13 are potent inducers of IgE antibody production
from B cells, amplifying IgE-dependent activation of immune cells [15-20]. Moreover, these
cytokines up-regulate the pro-inflammatory activity of Th2 cells, mast cells, eosinophils, and
neutrophils, as well as induce goblet cell hyperplasia, adhesion molecule expression, chemokine
release, and AHR [21]. In addition, IL-4 plays an important role in promoting Th0 cell
polarization into activated Th2 cells [22; 23], whereas IL-13 has been designated as a prime
effector cytokine that alone can drive allergic inflammation [24]. IL-9 plays a role in allergic
asthma by inducing AHR [25] and mucus hyper-secretion from goblet cells [26], as well as
enhancing mast cell and eosinophil survival and expansion [27-29]. IL-3 and GM-CSF, in
addition to IL-5, are potent promoters of eosinophil differentiation, survival, migration, and
activation [30-32]. IL-3 is also a critical molecular switch for differentiation, survival, and
functional effects of mast cells and basophils [33; 34], whereas GM-CSF is a strong activator of
neutrophil and macrophage pro-inflammatory function [35]. Table 1 below provides a summary
of Type 2 cytokines involved in allergic asthma.
3
Table 1. Type 2 cytokines in allergic asthma
Actions
Cytokine
Major cellular source
Major cellular targets
IL-3
Th2 cells, Basophils
HPCs, Basophils,
Eosinophils
Eosinophil and basophil
differentiation, maturation,
and activation [30-34]
IL-5
Th2 cells, Eosinophils,
Mast cells
HPCs, Basophils,
Eosinophils, Th2 cells
Eosinophil and basophil
differentiation, maturation, activation,
and survival,
AHR, activation of Th2 cells [30-32]
IL-4
Th2 cells, Basophils,
Mast cells
Th2 cells, B cells,
Basophils,
IgE isotype switching, Eosinophil and
basophil activation, mucus
hypersecretion, induction of Th2
cytokines, Mast cell development [15-23]
IL-13
Th2 cells, B cells,
Mast cells
Th2 cells, Epithelium,
Smooth muscle
Eosinophil activation and survival,
Mast cell
development, B-cell switch to IgE
production, mucus hypersecretion,
AHR [15-21, 24]
IL-9
Th2 cells, Eosinophils,
Mast cells, Neutrophils
Th2 cells, Mast cells,
Eosinophils, Epithelium,
Smooth muscle
Mast cell and eosinophil development,
mucus hypersecretion, AHR [27-29]
GM-CSF
Th2 cells, macrophages,
Mast cells
HPCs, Macrophages,
Eosinophils, Th2 cells,
Basophils
Granulocyte differentiation,
maturation, and activation [30-32, 35]
In addition to the classical Type 2 cytokines, there is emerging evidence to suggest that
alarmin cytokines such as TSLP, IL-25, and IL-33 are important mediators of Type 2
inflammation. The targeting of alarmin cytokines could provide a novel therapeutic avenue for
the treatment of allergic asthma.
1.2 Early and Late Asthmatic Responses
The allergen inhalation model was developed to efficiently explore the mechanisms
behind allergic asthma. This model allows for carefully controlled, safe, and effective
measurements of lung function in a clinical setting. During an allergen challenge the collection
of biological samples (induced sputum, blood, bronchial biopsies, and bronchoalveolar lavage
fluid (BALF)) is helpful in determining structural and inflammatory changes within the airways
4
specifically consequential of allergen inhalation. Structural and cellular changes during asthmatic
exacerbation have been described as mucus plugging, epithelial sloughing, airway edema,
leukocyte infiltration, and mediator production [36].
Models of allergen challenge to the skin, conjunctiva, nose, and airways have evolved
over the past 100 years. The allergen challenge model was first described by Charles Blackley
[37]. Subjects are given aerosolized allergen by inhalation via a nebulizer for a specific amount
of time. The dose given is calculated to induce modest bronchoconstriction without excessive
risk to the subject. This is based on the minimum dose required to induce a skin response to
prick testing further modified by the response of the airway to methacholine. Throughout the
challenge, changes in forced expired volume in 1 second (FEV1) are recorded. Subjects continue
to inhale the allergen until a drop in their FEV1 of 20% is reached. After this point the FEV1
readings are monitored throughout the day in order to disclose their physiological response [38].
The isolated early asthmatic response (EAR) involves an immediate response (20% drop
in FEV1) following allergen exposure that begins within 4 to 10 minutes, is maximal by 10 to 30
minutes, and resolves within 1 to 3 hours [38; 39]. This phase is characterized by rapid onset of
bronchoconstriction, vasodilation, AHR, and mucus hyper-secretion [38], which is promoted by
resident mast cells and basophils within the airways that release histamine, tryptase,
prostaglandins (PG), and cysteinyl leukotrienes (CysLTs), as well as an array of proinflammatory cytokines (IL-4, IL-15, IL-13, GM-CSF, and tumor necrosis factor (TNF)) [40].
Individuals that only develop an EAR following allergen exposure as designated as isolated early
responders (IER).
Approximately, 50% of adults and 70% of children develop a second bronchoconstriction
(15% drop in FEV1) approximately 3 hours later, which peaks between 6 to 12 hours, and
5
resolves spontaneously by 24 hours [32, 34, 36]. The LAR is driven by the uptake of allergens by
DCs, which in turn promotes Th0 polarization into activated Th2 cells, followed by the release of
Type 2 cytokines (GM-CSF, IL-3, IL-4, IL-5, IL-9, and IL-13), which further stimulates the
production of IgE, as well as airway infiltration, maturation, and activation of mast cells and
granulocytes [40]. Upon arrival of these cells, more pro-inflammatory mediators will be released,
which can lead to unprovoked constriction of the airways, characteristic of the LAR [40].
Eosinophilic airway inflammation is a distinct feature of the LAR, during which airway
infiltrated eosinophils release a range of pro-inflammatory and cytotoxic mediators including
leukotrienes, major basic protein (MBP), eosinophil cationic protein (ECP), and eosinophil
peroxidase (EPX), resulting in AHR, airway inflammation, smooth muscle constriction, mucus
hypersecretion, and airway remodeling [38-45]. The sustained occurrence of these actions can
lead to chronic tissue damage in the airways [44]. Individuals that only develop both an EAR and
DR following allergen exposure as designated as dual responders (DR). Figure 1 provides a
summary of the allergen-induced EAR and LAR.
6
Histamine,*CysLTS,**
PGDs,*etc*
*
Figure 1. Immune pathways resulting in early and late asthmatic responses. Following
allergen inhalation, the antigen is presented to Th0 cells via interactions between the TCR on
Th0 cells and MHC II on DCs, resulting in polarization and activation of Th2 cells. IL-4 released
from activated Th2 cells results in B cell isotype switching to produce IgE, which then crosslinks with high-affinity IgE receptors (FcεR1) on mast cells (MC) and basophils (BOS). This
cross-linking allows subsequent binding of the inhaled antigen, which then induces release of
histamine and lipid mediators from MCs and BOS, resulting in the EAR. The LAR is mediated
by Type 2 cytokines and chemokines released from activated Th2 cells, followed by MC release
of pro-inflammatory mediators, as well as recruitment and activation of BOS and eosinophils
(EOS) (adapted from [40]).
1.3 Basophils in Allergic Disease
In 1879 Paul Ehrlich discovered basophils, based on their unique microscopic appearance
upon staining with basic dye [46]. Due to their similarity to mast cells, basophils have been
regarded as redundant “circulating mast cells” and remain one of the least studied and
understood granulocytes [47]. Similar to mast cells, basophils possess high affinity FcεR1 that
7
cross-link upon engagement with allergen-bound IgE, resulting in degranulation of histamine and
CysLTs, as well as the release of pro-inflammatory mediators [48; 49]. Unfortunately, there has
been a lack of basophil-specific markers available to differentiate them from other
morphologically similar cells. This has made it difficult to develop any satisfactory purification
protocols to obtain an isolated basophil population that lacks contamination. Furthermore, given
the scarcity of these cells, collection of adequate numbers for in vitro experiments has been
difficult. There has also been a lack of true basophil knockout animal models to help clarify the
role of these cells in allergic disease due to the inability to identify a basophil-specific growth
factor. These aforementioned issues have lead to the stagnation of basophil research. However,
within the last 6 years, basophil purification techniques have advanced, allowing new insights
and a better appreciation for the role of basophils in allergic disease and inflammation [48].
1.3.1 Basophil Background
1.3.1.1 Basophil Growth and Development
Basophils develop from granulocyte-macrophage progenitor (GMP) cells, and more
specifically arise from lineage-negative CD34+FcεR1hic-Kit- hematopoietic progenitor cells
(HPCs), which can be found in cord blood, peripheral blood, and bone marrow [50]. Their
further lineage commitment is strictly regulated by the timed expression of the transcription
factors (CCAAT-enhancer-binding proteins type alpha) C/EBPα and (GATA binding protein
type 2) GATA-2 [50; 51]. For example, Arinbou et al. demonstrated that expression of C/EBPα
is necessary for late basophil development but not mast cells [51]. Basophils have further been
described to evolve from CD43+/IL-3 receptor-α subunit (IL-3Rα)+/IL-5 receptor-α subunit (IL5Rα)+ eosinophil/basophil progenitors [52]. The development of basophils is further summarized
in Figure 2. Stem cell factor (SCF) and IL-6 act early on in the survival and self-renewal of
8
HPCs, while other surrounding growth factors such as IL-3, GM-CSF, IL-5, transforming growth
factor-β (TGF-β), and nerve growth factor (NGF), are responsible for terminal differentiation
into basophils [52-61]. Out of all these mediators, it is thought that the primary growth and
differentiation factor for basophils is IL-3 [57; 62]. Once mature, basophils can be distinguished
phenotypically from mast cells by their differential surface expression of the IL-3Rα chain and
SCF receptor (c-Kit).
Although it is clear that IL-3 is a key regulator of basophil development and their
functional effects, some studies have shown that when IL-3 is removed, basophil development is
still evident, which suggests that IL-3 is a component of a larger network of redundant cytokines
that mediate basophil development [63-65]. These findings add to the frustration associated with
a lack of basophil knockout animal models given that a basophil-specific growth factor has not
been identified to date. For example, knockout of GATA-2 would effect the development of mast
cells and eosinophils in addition to basophils and C/EBPα-deficient mice would lack basophils,
eosinophils, and neutrophils [50; 51]. Recently emerging studies have identified epithelialderived TSLP as a regulator of basophil development [66; 67]. Siracusa et al. demonstrated that
TSLP promotes murine bone-marrow progenitor cell differentiation into basophils, comparable
to IL-3 [66]. Furthermore, they showed that TSLP can induce basophil differentiation
independently of IL-3 and that TSLP-elicited basophils display a distinct phenotype of basophils
as opposed to IL-3-elicited basophils. For example, TSLP-elicited basophils produced more IL-4
and IL-13 following subsequent stimulation with IL-3 or IL-33. Hui et al. demonstrated in
humans that peripheral CD34+ HPCs pre-incubated with IL-3 and TNF have enhanced sensitivity
to TSLP-mediated basophil lineage commitment, which is further increased in asthmatics
compared to normal controls [68]. Mast cell-activated bone marrow mesenchymal stromal cells
9
produce TSLP, which can enhance the differentiation of CD34+ HPCs into eosinophil/basophil
colonies [69]. Collectively, these studies suggest that alarmin cytokines play an important role in
promoting basophil differentiation. There is a need to further examine whether the alarmin
cytokines IL-33 and IL-25 can induce human basophil differentiation. Such studies will help
broaden our understanding of the growth and development of basophils in allergic asthma and in
turn identify novel therapeutic avenues to treat this disease.
HSC
MPP
CMP
IL-3*, IL-5, GM-CSF,
TGF-β, NGF
Macrophage
GMP
TSLP
IL-33?
IL-25?
Basophil
Mast Cell
Eosinophil
Neutrophil
Figure 2. Differentiation of immune cells from HPCs.
Hemopoietic stem cells (HSCs) give rise to all cell types within the bone marrow and
peripheral blood. Multipotent progenitors (MPP) differentiate into the common myeloid
progenitor (CMP), GMP. The presence of surrounding growth factors determines the terminal
differentiation of GMPs into a mature peripheral blood cell type. For example, IL-3, GM-CSF,
IL-5, TGF-β, and NGF, are responsible for terminal differentiation into basophils. *IL-3 is the
most potent promoter of basophil differentiation, however recent evidence suggests that TSLP
may also be an important growth factor for basophils [66-69]. Whether IL-25 and IL-33 can
promote basophil differentiation still remains unclear.
10
1.3.1.2 Basophil Migration
Basophil are granulocytes that are round, 12-15 µm in diameter, containing electrondense cytoplasmic granules that stain metachromatically with selected basic dyes. Basophils are
enriched with surface receptors that initiate internal signaling cascades to control cellular
functions, including migration.
Chemokines are released from resident airway cells that cause basophils to migrate from
the bone marrow and periphery into airway tissues during the allergen-induced EAR and LAR of
allergic asthma [70]. This multistep process involves the adhesion of basophils to the microvascular endothelium, trans-endothelial migration, and locomotion through airway tissues. The
first step in recruitment is initiating contact between peripheral blood basophils and the vascular
endothelium, a process known as margination. Upon contact, the basophils will roll along and
eventually adhere to the endothelial surface, which is mediated by selectins (L-selectin; Pselectin) and integrins such as, vascular cell adhesion protein-1 (VCAM-1) and intracellular
adhesion molecule-1 (ICAM-1) [71; 72]. This is followed by transmigration of basophils through
the endothelium, induced by endogenous cytokines IL-1β, IL-3, and numerous chemokines [73].
Upon entrance into the airways, basophils undergo migration to specific tissue sites via
chemotaxis, which is dependent on the binding of basophil surface receptors to surrounding
chemoattractants. Basophils express a large number of chemotactic receptors including C-C
motif receptor (CCR) 1, CCR2, CCR3, C-X-C motif receptor (CXCR) 1, CXCR3, and CXCR4
[74; 75]. Of these receptors, CCR3 is the most prominently expressed on basophils [73; 75].
Basophil migration can be mediated by several chemo-attractants such as IL-8, eotaxin,
regulated on activation, normal T cell expressed and secreted (RANTES), monocyte chemotactic
protein (MCP)-3, MCP-1, and macrophage inflammatory protein (MIP)-1α [75-79]. The
11
chemokine eotaxin, produced by activated epithelial cells, is up-regulated within the airways and
peripheral blood, and serves as the most potent promoter of eosinophil and basophil influx into
airway tissue [75, 80-82]. Basophils bind to eotaxin via CCR3 [75]. The binding of eotaxin and
basophil surface CCR3 is important to the infiltration of these cells to the airways during the
EAR and LAR [70; 75]. Recently, in vitro studies have shown IL-33 to prime basophils towards
migration to eotaxin [83]. It is therefore plausible that not only do alarmin cytokines influence
basophil differentiation and growth, but also play a role in implementing airway infiltration.
However, more studies are needed to determine whether other alarmin cytokines including TSLP
and IL-25 have an effect on basophil migration. One component of this thesis is to examine the
direct and indirect effects of TSLP, IL-25, and IL-33 on basophil migration in vitro.
1.3.1.3 Basophil Activation and Mediators
Upon the arrival of basophils to the airway, these cells can be stimulated through two
modes of activation, which are the IgE-dependent and IgE-independent pathways. IgE-dependent
activation involves the docking of allergens to IgE via high-affinity FcεRI expressed on the
surface of basophils, whereas IgE-independent activation of basophils entails the use of nonantigen-derived inputs such as complement products (C5a and C3a), purines, eosinophil toxins,
and cytokines (IL-3, IL-4, IL-9, IL-5, and IL-13).
Both IgE-dependent and -independent
mechanisms are enhanced via priming, exerted through autocrine and paracrine mechanisms. For
example, IL-3 produced by neighboring immune cells or basophils themselves, prime basophils
for increased degranulation of histamine and secretion of IL-4, IL-13, and CysLTs [84-87]. IL-5
and GM-CSF can also prime basophil pro-inflammatory function following IgE-dependent or
IgE-independent activation [88; 89]. Both IgE-dependent and IgE-independent modes of
basophil activation contribute to the effector role of these cells in allergic asthma. Recent reports
12
have shown additional IgE-independent mechanisms of basophil activation, with are mediated by
the alarmin cytokines TSLP, IL-25, and IL-33. More extensive research needs to be done to
elucidate these newly found IgE-independent mechanisms.
Following activation via inhaled allergen, basophils degranulate to release histamine and
secrete lipid mediators (CysLTs and PD2), as well as cytokines (IL-3, IL-4, IL-6, IL-9, and IL13,) [90-96]. Basophils, along with mast cells, are the major sources of the pre-formed granule
product histamine. Histamine acts to induce airway smooth muscle cell (ASM) contraction,
increase vascular permeability, microvascular leakage, and mucus secretion. The effects of
CysLTs such as broncho-constriction, airway obstruction, and airway eosinophilia, are mediated
through binding to Cysteinyl leukotriene receptor 1 (CysLT1) and Cysteinyl leukotriene receptor
2 (CysLT2), which are found on lung tissue, macrophages, monocytes, eoisnophils, basophils,
mast cells, dendritic cells, and lymphocytes [97].
Recent studies have shown basophils to be sources of IL-4 and IL-13 in several Type 2
inflammatory diseases, including parasitic infections and allergic inflammation [98-107]. For
example, Deovaussoux et al. demonstrated that following in vitro allergen stimulation with Felis
domesticus 1 (Fel d 1), basophil production of IL-4 and IL-13 accounted for a significant
frequency of cells producing these cytokines, next to T cells [103]. Nouri-Aria et al. found both
the number of basophils and IL-4+ mRNA cells to increase within bronchial mucosa 24 hours
post-segmental allergen challenge in asthmatics. When they colocalized the IL-4 to determine
what cells produced this cytokine, basophils were responsible for producing 72% of IL-4 found
24 hours post-allergen challenge [105]. A real-time quantitative polymerase chain reaction-based
assay in whole blood also identified basophils as a prominent source of IL-4 and IL-13 after in
vitro stimulation with Fel d 1 allergen extract [104]. Schroeder and coworkers demonstrated that
13
following a segmental allergen challenge, 20 hour BALF samples cultured with basophils
induced significant IL-4 protein production 4 to 5 hours after incubation, whereas IL-4 was not
detected in BALF cell fracitions cultured with lymphocytes and eosinophils [107]. Other studies
have revealed that the propensity of basophils to store IL-4 is increased in atopic donors and
similar obervsations have been made with respect to spontaneous IL-13 production in circulating
basophils from AR patients following intranasal allergen provocation [108; 109]. The
aforementioned studies suggest that basophil production of IL-4 and IL-13 may play an
important role in initiating and maintaining Type 2 inflammation. However, more in vivo models
are required to monitor the change in basophil production of IL-4 and IL-13 following an
allergen challenge and if their contribution of these cytokines is important to initiating and
mediating allergic inflammation.
Interestingly, murine and human studies have also shown basophils to be significant
sources of IL-25 and TSLP [106; 110]. For example Sokol and colleagues found basophils to be
significant sources of IL-4, IL-13, and TSLP within the lymph nodes following a challenge with
papain extract in a murine model [106]. Wang et al. demonstrated that basophils express mRNA
for IL-25 following IgE stimulation, with could be up-regulated in synergy with IL-3 [110].
These findings suggest that basophils assist the epithelium in promoting Type 2 inflammation by
producing alarmin cytokines. Furthermore, given that basophils respond to these cytokines, it is
possible that the effector role of basophils can be additionally mediated through an IL-25 or
TSLP autocrine positive feedback loop.
Collectively, these findings suggest that the presence of basophils within the airways
following allergen exposure and their release of pro-inflammatory cytokines helps to generate
and maintain the Type 2 response during allergic asthma exacerbations [111-114]. A second
14
objective of this thesis aims to assess whether alarmin cytokines TSLP, IL-25, and IL-33 can
promote basophil pro-inflammatory activity.
1.3.1.4 Tools for Basophil Research
The study of basophils has been hampered due to their rarity and lack of tools for their
detection and functional analyses. As the number of roles ascribed to basophils in vitro has risen,
there is a growing demand for animal models that lack basophils. During the past 5 years, there
have been numerous groundbreaking tools that have aided in the development of new basophil
research methods.
Monoclonal Antibodies:
Basophils are quickly recruited to sites of inflammation at peripheral tissues in a variety
of allergic diseases. To assist with the detection of cellular infiltration into peripheral tissues a
panel of mAbs has been established that are specific to basophils. These mAbs include BSP-1,
2D7, BB-1, and J175-7D4 [115-123]. BSP-1 is an IgM class mAb that recognizes an epitope
expressed on the surface of human basophils [118]. Unfortunately, BSP-1 is not suitable for
immunohistochemical staining given that it has a low sensitivity [118]. 2D7 is an IgG1κ mAb
that reacts with ligand localized to the secretory granules of human basophils [119].
Immunological activation of basophils leads to the loss of attenuation of the staining intensity,
indicating release of the 2D7 antigen [119]. This mAb has been used for immunohistochemical
staining of basophils in both the skin and airways during the LAR after an allergen challenge
[120; 121]. BB-1 is an IgG2a mAb that recognizes a 124-kD antigen localized to basophil
secretory granuales with a comparatively small expression of the suface [122; 123].
J175-7D4 is a mAb generated against the recombinant pro-form of human eosinophil granule
major basic protein 1, which stains only basophils [117]. Ugajin et al. have also recently
15
established TUG 8, a mAb specific to mouse mast cell protease 8 (mcpt-8), which is useful for
immunohistochemical identification of mouse basophils [124]. Mcpt-8 is a granzyme-like serine
protease that is selectively expressed by basophils and stored in their secretory granules. Despite
the benefits of using these basophil-specific mAbs to monitor cellular movement in and out of
tissues, additional models are required to look at basophil function using an in vivo model.
Basophil Depletion:
There has been the discovery of natural mutant mice deficient in mast cells such as
WBB6F1-kitW/W-v and C57BL/6-kit W-sh/W-sh mice, which has allowed us to gain understanding in
mast cell function in vivo [125]. On the other hand, murine models that are deficient only in
basophils have long been lacking until recently. The development of basophil-depleting mAbs
has helped to overcome this obstacle, including the in vivo administration of Ba103, a mAb
specific to CD200R3, or MAR-1, a mAb specific to FcεR1 [106, 126, 127]. These mAbs have
been shown to preferentially deplete circulating basophils, which has allowed researchers to
begin to test the functions of basophils in vivo [106, 126-128]. For example, Mukai et al.
demonstrated that following the transfer of FcεR1-expressing basophils into FcεR1-deficient
mice there was a restoration of the development of the delayed-onset chronic allergic
inflammation, whereas mast cells were essential for the immediate and late-phase responses
[129]. Noti and coworkers used mAb depletion of basophils using CD200R3 to test whether
basophils contributed to the maintenance of eosinophil esophagitis-like disease [130]. They
demonstrated that mice with eosinophil eosophatitis-like disease that had CD200R3-mediated
basophil depletion resulted in decreased esophageal eosinophilia and a reduction in the total cell
numbers infiltrated [130]. This was similar to the results in TSLPR-deficient mice, suggesting
that the basophil-TSLP axis is important to pathogenesis of eosinophilic esophagitis.
16
Unfortunately, there has been concern raised over the validity of the mAb depleted
basophil model due to the unwanted side effects on mast cells, such as cell activation and their
partial depletion. For example, intravenous injection of Ba103 or MAR-1 can stimulate mast
cells in vivo and induce systemic anaphylaxis, and repeated intraperitoneal administration of
MAR-1 leads to reduction of mast cells in the peritoneal cavity [126, 127, 131]. Due to the
effects on mast cells, this method of antibody-mediated basophil depletion cannot provide clear
evidence for the role of basophils in allergic inflammation and thus the results of these studies
must be taken with caution.
In order to overcome the limitations related to mAb-mediated basophil depletion, Wada
et al. generated Mcpt8DTR mice where only basophils express the human diphtheria toxin
receptor (DTR), and therefore administration of diphtheria toxin (DT) will induce selective and
transient ablation of basophils, without affecting other cell lineages like mast cells [132]. The
mouse model was specifically created by inserting the DTR-encoding gene into the 3’
untranslated region of the Mcpt8 gene in a bacterial artificial chromosome. Wada et al. used the
Mcpt8DTR mouse model to test the role of basophils in acquired resistance to tick infection. They
demonstrated that following treatment with DT there was abolishment of the secondary
infection, however transfer of basophil-enriched CD49b+ splenocytes isolated from sensitized
mice was sufficient to confer resistance in naive mice. In the context of allergic inflammation,
Reber et al. demonstrated that Mcpt8DTR mice given DT or treatment of wild-type mice with
basophil-depleting antibody Ba103 had significant reduction in peanut-induced hypothermia
[133]. These results suggest that basophils play a critical nonredundant role in acquired
immunity. Similarly, the DTR gene has also been introduced under the control of mouse
CD203c, resulting in selective depletion following administration of DT [134]. In this basophil-
17
depletion model, the DT administration attenuated a drop in the body temperature of IgGmediated systemic anaphylaxis in a dose-dependent manner and significantly abolished the
development of ear swelling in IgE-mediated chronic allergic inflammation. Lastly, a transgenic
mouse model called Mcpt8Cre has been made, which is constitutively deficient in basophils.
Studies using these mice have demonstrated that basophils function in protective immunity
against helminths and help to orchestrate chronic allergic inflammation, whereas primary Th2
cell responses commence efficiently in the absence of basophils [131]. Collectively, the
generation of basophil-specific depletion models has allowed us to gain a more comprehensive
understanding for the in vivo role of basophils in allergic inflammation that is distinct from mast
cells. In the future, these basophil-specific depletion models should be used to study allergic
inflammation in the context of asthma to understand the role of basophils from an in vivo level in
both the EAR and LAR.
1.3.1.5 Role of Basophils in Human Allergic Asthma
Over the last several years, we have gained a more comprehensive understanding of the
role of basophils in human allergic asthma. Kimura et al. were one of the first researchers to
demonstrate that asthmatics had elevated peripheral basophil numbers compared to healthy
controls [135]. Immediately before an asthmatic attack, peripheral basophil numbers increase
significantly and then decline during the exacerbation. These findings suggest that basophils
migrate to the airways when asthmatic symptoms arise. This was further supported by Kimura et
al. and Braunstahl et al., who found basophil numbers to decrease within the peripheral blood
but significantly increase within the sputum, bronchial, and nasal mucosa during an asthmatic
exacerbation, followed by a return to baseline levels at the conclusion of symptoms [136; 137].
Additional studies have reported that the total number of airway basophils correlates
18
significantly with asthma severity. For example, Kimura et al. showed that patients with severe
asthma had greater airway frequency of basophils compared to that of mild asthmatics [136].
Furthermore, post-mortem lung samples from severe asthmatics were found to have significant
numbers of basophils within the airway lumen, bronchial epithelium, and submucosa [138].
Kepley et al. demonstrated that basophils within post-mortem samples of large and small
airways, airway epithelium, submucosa, and alveolar walls were markedly higher in patients
with fatal asthma compared to healthy controls [139]. Similar results were shown by Koshino et
al. who measured basophil numbers within bronchial biopsies of normal, mild non-atopic, and
mild-atopic subjects before and after an acetylcholine bronchial challenge [140]. Basophils were
present in the airway mucosa of asthmatics, as opposed to mild non-atopic or healthy controls
and there was an inverse correlation between these airway basophil numbers and methacholine
PC20. Lastly, Lewis et al. demonstrated that an increase in peripheral basophils was associated
with impairment of FEV1, AHR, and IgE levels [141]. The aforementioned studies indicate that
the frequency of basophils within the airways is associated with disease severity, suggesting that
basophils contribute to the pathogenesis allergic asthma. However, further research is needed to
determine the exact role of basophils in allergic asthma and how this may result in increased
disease severity.
Although basophils have been traditionally regarded as cells that discharge large amounts
of histamine and CysLTs during the EAR, recent findings suggest that basophils have an
additional role in the LAR. For example, basophils accumulate significantly within the skin,
airways, and nasal mucosa during the LAR of patients with asthma and seasonal allergic rhinitis
(AR) following an allergen challenge to those respective organs [120, 121, 141-145]. Our lab
has shown that following allergen exposure, there are increased numbers of airway basophils and
19
up-regulated circulating basophil activation marker expression of CD203c for up to 24 hours
post-allergen challenge [Appendix II]. Furthermore, Ono et al. have reported basophil CD203c
expression to increase and correlate with airway limitation during asthmatic exacerbation and to
decrease during remission [146]. The emerging findings that basophil number and activation
increases during the LAR, in combination with studies showing that basophils produce IL-4 and
IL-13, provide evidence that basophils are effector cells that may help initiate and maintain the
Type 2 inflammatory response in allergic asthma. Additional research is needed to better
understand the kinetics and effector role of basophils during the LAR. Furthermore, little is
known about the basophil response to the airway microenvironment during the LAR, such as
mediators released by the epithelium and other inflammatory cells. A third objective of this thesis
is to determine the change in basophil pro-inflammatory activity during the LAR following a
whole lung allergen inhalation in mild allergic asthmatics. In addition, a fourth objective of this
thesis is to examine how the inflammatory airway microenvironment created following a whole
lung allergen challenge can influence basophil activity in vitro.
1.3.1.6 Activation-Linked Markers on Basophils
Traditional diagnostic testing for allergic individuals has been conducted through skin
prick tests and serology (ie. measuring serum IgE levels). Unfortunately these two types of
diagnostic tests correlate poorly with each other and are not as reliable or accurate as originally
theorized. More recently, in vitro tests have been developed as additional diagnostic tools. For
instance, measurement of histamine-release following allergen exposure, however this method is
time consuming and economically unfeasible.
In an effort to improve diagnostic testing, researchers have looked towards basophil
biomarker measurements via flow cytometry. There are various receptors on the surface of
20
basophils, which can be used as markers to reflect cellular activation. In recent years, the
basophil activation test (BAT) has been developed to assess human basophil activation in cases
of immediate-type allergic responses. These studies specifically monitor surface expression of
the markers CD63, CD203c, and CD69.
CD63 is a member of the lysosome-associated membrane glycoprotein (LAMP) family
[147], which has been thought to be reflective of basophil histamine containing granules [148150]. CD63 basophil expression is currently used as a biomarker of IgE-mediated allergy [151155]. CD69 has been established as an activation marker for cytokine-mediated eosinophil and
basophil activation. Yoshimura et al. demonstrated that basophil CD69 expression is strongly upregulated by IL-3 in vitro [156]. They found basophil CD69 expression to be significantly higher
in asthmatic individuals compared to normal subjects, and CD69 expression to be higher in
BALF then peripheral blood samples in asthmatics. Lastly, studies have demonstrated that
basophil CD69 expression becomes up-regulated following in vitro exposure to recombinant
allergen [156; 157].
CD203c is a member of the ectonucleotide pyrophosphatase-phosphodiesterase family
consisting of seven structurally related molecules that can hydrolyze pyrophosphate and
phosphodiester bonds in various substrates [158]. CD203c has been identified on CD34+ cells
and mast cells, but is the most highly expressed on basophils [159]. CD203c is constitutively
expressed on basophils and becomes rapidly up-regulated following IgE-dependent or IgEindependent cell activation [159-165]. This marker is stored intracellularly and becomes
transported to the cell surface after activation in a manner that is dependent on the mobilization
of intracellular Ca2+ stores, as well as protein kinase C (PKC) and Phosphoinositide 3-kinase
(PI3K) intracellular signaling, which can be induced by IgE-dependent and IgE-independent
21
mechanisms [159]. The exact function of CD203c has remained elusive until recently, where a
study by Tsai and co-workers in 2015, demonstrated that CD203c has the ability to hydrolyze
ATP and thus negatively affect basophil and mast cell function [166]. ATP is released during
inflammation and has been shown to activate immune cells via the P2X7 receptor. Tsai et al.
demonstrated that following IgE stimulation, CD203c becomes upregulated on mast cells and
basophils, and that they produce ATP, which can in turn further activate these cells [166].
CD203c knockout mice had greater numbers of basophils and mast cells within peripheral blood,
decreased ability to clear ATP, and were highly sensitive to allergen airway challenges. From
these findings it can be concluded that CD203c can assist in negatively regulating acute and
chronic allergic inflammation by suppressing ATP-dependent activation of immune cells.
Multiple studies have turned to monitoring basophil surface expression levels on CD203c
to assist in the diagnosis of allergy to hymenoptera venom, grass pollens, and cat dander [159165]. In addition, CD203c measurement has been used as a biomarker of asthma status. For
example, Ono et al compared constitutive or anti-IgE stimulated basophil expression of CD63,
CD69, and CD203c from patients with stable or exacerbated asthma and healthy controls [146].
They reported that spontaneous CD203c basophil expression was significantly higher in patients
with asthma exacerbation than stable asthma (following treatment with inhaled corticosteroids)
or healthy patients, whereas no differences were found in CD63 or CD69 expression. In vitro
anti-IgE-induced basophils had significantly higher expression of CD63 and CD20c in patients
with asthma exacerbation compared to healthy controls. Moreover, in vitro stimulation with the
allergen Dermatophagoides pteronyssinus-1 (Der p 1) or IL-3 up-regulated CD203c expression
in a dose-dependent manner, with clinically improved asthma patients experiencing less of an
increase versus patients with asthma exacerbation. Furthermore, patients with clinical
22
improvement had a correlation between spontaneous CD203c basophil expression and %
predicted FEV1. Ono et al. established the difference of CD203c basophil expression at baseline
across varying severities of asthma in comparison to healthy controls, which continued to be
notable after in vitro stimulation with anti-IgE. Further investigation is needed to determine the
change in basophil activation following allergen inhalation and whether the BAT can be used to
monitor allergic asthma status. A fifth objective of this thesis is to investigate the change in
surface basophil activation marker expression following allergen inhalation in both isolated IER
and DR asthmatics, and determine whether basophil surface activation markers can be used to
assess allergen-induced bronchoconstriction.
1.4 Airway Epithelium
1.4.1 Airway Epithelium and Allergic Asthma
The regulation of immune homeostasis within the airways has long focused on leukocytes
as the key orchestrators of the immune systems; however various genetic, structural, and
functional studies suggest that the airway epithelium is required for normal homeostasis. Airway
epithelial cells form a continuous, highly regulated physical barrier that lines the airway lumen,
creating an interface between the host and environment, which acts as the first line of defense
against inhaled microorganisms and allergic antigens. Epithelial cells express pattern recognition
receptors (PRRs) and toll-like receptors (TLRs), which detect environmental stimuli, including
viral and allergic antigens, and subsequent activation of PRRs and TLRs leads to epithelial cell
release of soluble cytokines, chemokines, growth factors, and anti-microbial peptides, which can
alert and activate the immune system to any potential threats. Among the cytokines released
from the epithelium are TSLP, IL-33, and IL-25, which are produced upstream of Type 2
cytokines in response to allergens within the airways. TSLP, IL-33, and IL-25 have been in the
23
past referred to as epithelial-derived cytokines, however given that these cytokines can be
produced by other immune cells we have chosen to term them as alarmins, which by definition
are mediators that function by signaling cell and tissue damage in the airways.
Recent evidence indicates that damage to or dysregulated activation of the airway
epithelium brought on by exposure to allergens may have a central role in driving Type 2
responses and subsequent chronic airway inflammation. For example, compared to healthy
controls, airway epithelial cells within asthmatics release significantly more pro-inflammatory
mediators such as IL-13, TSLP, IL-25, IL-33, thymus and activation regulation chemokine
(TARC), RANTES, and MCP-3 [167]. The airway epithelium is conveniently situated to
potentiate the adaptive immune response, and further act as an interface between innate and
adaptive immune regulation. Thus, the airway epithelium is no longer considered a simple
immunological barrier, but a key player in initiating and amplifying airway inflammation. The
below sections will highlight the important roles of alarmin cytokines TSLP, IL-25, and IL-33 in
the pathogenesis of allergic asthma.
1.5 Thymic Stromal Lymphopoietin
1.5.1 TSLP
TSLP is an IL-7-like cytokine that is part of the IL-2 cytokine family, characterized as a
140-amino acid 4-helix bundle [168]. TSLP was originally discovered by Friend et al. in murine
thymic stromal cell line supernatants, which supported B-cell development in the absence of IL-7
[169; 170]. The human gene for TSLP is localized on chromosome 5q22.1, adjacent to the atopic
cytokine gene cluster, 5q31, encoding the cytokines IL-4, IL-5, IL-9, and IL-13 [171]. Recent
genetic studies have shown that polymorphisms of the TSLP gene are associated with chronic
obstructive pulmonary disorder (COPD), allergic asthma, AHR, as well as increased serum IgE
24
levels and eosinophilia [172-178].
1.5.2 Human Expression of TSLP
Initially TSLP was thought to be exclusively produced by thymic and epithelial-derived
linings of the thymus, lungs, gut, and skin, with the highest level of expression in lung and skinderived epithelial cells [179-181]. However, recent studies report that TSLP is also secreted by
structural and immune cells such as, fibroblasts, ASM cells, endothelial cells, mast cells,
macrophages/monocytes, granulocytes, and DCs [182-190]. Despite having multiple sources of
TSLP it is unknown what cellular source of this cytokine is the most crucial to mediating allergic
asthma.
TSLP mRNA and protein expression is regulated by a variety of factors, including
exogenous stimuli (allergic antigens, trauma, mechanical injury, bacterial and viral infection, and
TLR ligation) and host-derived pro-inflammatory cytokines (IL-1β, TNF, TGF-β, IL-4, and IL13) [183, 186, 191-194]. The aforementioned stimuli can promote TSLP expression through
several intracellular signaling pathways. For example, studies have shown that pro-inflammatory
cytokine-induced TSLP expression is mediated through extracellular signal-regulation kinase
(ERK) 1/2, Janus kinase (JAK)/Signal transducers and activators of transcription (STAT) 6, and
p38 Mitogen-activated protein kinase (MAPK) signaling, as well as the up-regulation of nuclear
factor kappa beta (NFκB) in human bronchial epithelial cells (HBECs) and ASM cells [193-195].
Rhinovirus and respiratory syncytial virus promote TSLP expression in HBECs through STAT6
signaling and the activation of transcription factor NFkB [183, 191, 195]. Together, these studies
demonstrate that TSLP expression can be regulated through a wide-range of intracellular
signaling mechanisms, which themselves could pose as novel targets to regulate TSLP
production within the airways.
25
Several important studies that have measured the expression of TSLP mRNA within
asthmatic airways and highlight the importance of this cytokine in inflammation. Ying et al.
were the first to find evidence showing a link between TSLP mRNA and human asthma [187].
They compared the level of TSLP mRNA expression in the airways of asthmatics versus healthy
controls. Through in situ hybridization and immunohistochemistry they determined that the level
of TSLP mRNA expression in bronchial epithelial cells and macrophages, as well as the submucosa endothelial cells and neutrophils was significantly greater in asthmatic subjects
compared to healthy controls. Furthermore, they observed an inverse correlation between FEV1
and TSLP mRNA expression. They additionally confirmed that TSLP mRNA was expressed by
epithelial cells, endothelial cells, macrophages, neutrophils, and mast cells. Another study by
Ying et al. expanded on these findings, through monitoring the expression of TSLP mRNA
across a range of airway disease severities [185]. They found increased TSLP mRNA expression
within the BALF of severe asthmatics and COPD compared to healthy subjects. Other research
groups have shown that the number of TSLP mRNA+ mast cells is significantly elevated in
asthmatics versus healthy controls and notably, 90% of the bronchial mucosa TSLP + cells were
identified as mast cells [185; 196]. TSLP has further been measured at the protein level in
airways. For example, the level of TSLP protein within BALF and airway epithelium is
significantly higher in asthmatic patients compared to healthy controls [197]. TSLP protein has
also been reported to be significantly elevated in the airway epithelium and lamina propria of
asthmatic patients and even more so in severe asthmatics, which further correlated with the
severity of airflow obstruction [197]. Collectively, the aforementioned studies provide evidence
that TSLP mRNA and protein expression within the airways is associated with increased allergic
inflammation and decline in lung function.
26
1.5.3 Human Expression of the TSLP Receptor
The effects of TSLP are exerted through a high-affinity TSLP receptor complex,
consisting of the TSLP-binding chain (TSLPR) and interleukin-7 receptor-α (IL-7Rα) subunit
[198; 199]. TSLP can bind to the TSLPR with low affinity, but when combined with the IL-7Rα
subunit, the affinity is greatly enhanced. The IL-7Rα/TSLPR complex has been shown to exert
its effect through JAK1/JAK2-dependent STAT-1, STAT-3, and STAT-5 signaling [171, 198,
200-206]. Various studies have confirmed both mRNA and surface expression of TSLPR on
several structural and immune cells including DCs, CD4+ and CD8+ T cells, regulatory T cells
(T-regs), B cells, mast cells, natural killer (NK) T cells, monocytes, CD34+ progenitor cells,
eosinophils and basophils, as well as ASM cells [66, 167, 198, 205, 207-217]. Furthermore,
mRNA expression of the IL-7Rα has been confirmed in monocytes, CD4+ and CD8+ T cells, NK
cells, B cells, mast cells, basophils, and eosinophils [205]. This multi-cellular expression of the
receptor complex for TSLP on both structural and immune cells suggests that this cytokine is
involved in widespread activity within the airways.
Unfortunately, there has been a lack of studies that have looked at baseline or allergeninduced levels of TSLPR or IL-7Rα expression in asthmatics versus healthy controls. Smith et
al. observed that 24 hours following an allergen challenge there were significantly higher
absolute numbers of HPCs with the sputum expressing TSLPR+ and IL-7Rα+ [215]. Corrigan and
colleagues observed up-regulation of TSLPR and IL-7Rα expression on DCs for up to 48 hours
following a cutaneous allergen challenge in atopic subjects [216]. Interestingly, a recent study
has shown that basophil TSLPR expression is increased in atopic asthmatics compared to nonatopic controls, and is further up-regulated following IgE receptor ligation to a greater extent
then TSLPR on DCs [217]. The magnitude of this increase was found to correlate with total
27
serum IgE levels. Collectively, these findings suggest that immune cells may become more
sensitive to TSLP following allergic stimulation, which may in turn contribute to Type 2
inflammation observed during allergen-induced exacerbation. The functional consequence of
allergen-induced changes in immune cell TSLPR expression within the airways needs to be
investigated further.
1.5.4 Cellular Targets of TSLP
1.5.4.1 Dendritic Cells
The most well established epithelial mechanism for the development of Type 2
inflammation is the interaction of DCs and TSLP. This has many implications due to DCs being
considered the primary APC in most immune responses, and thus the key initiator and director of
inflammation.
DCs have been traditionally thought to be activated by CD40 ligand (CD40L) and TLR
ligands such as lipopolysaccharide (LPS) and Polyinosinic:polycytidylic acid (polyI:C). Studies
have observed that similar to the above stimuli, TSLP can bind to the TSLPR on DCs and exert
various pro-inflammatory effects. TSLP can induce human myeloid DC (mDC) maturation
through the up-regulation of expression of MHC II along with co-stimulatory molecules CD40,
CD86, CD80, CD83, and CD54 [167]. In contrast to TLR ligands, TSLP does not stimulate
production of pro-inflammatory TNF, IL-1β, and IL-6 cytokines or Th1-polarizing cytokines IL12 and IFNs [167, 218, 219]. On the other hand, TSLP can stimulate the production of multiple
chemokines from mDCs, such as IL-8 and eotaxin-2, that act as chemo-attractants for neutrophils
and eosinophils [167, 200, 218, 219]. TSLP can also induce production of TARC and
macrophage-derived chemokine (MDC), as well as CCL1, which attract Th2-polarized cells
[167, 189, 200, 218, 219].
28
Most notably, TSLP can up-regulate the Th2-polarizing molecule, OX40 ligand (OX40L)
on mDCS. Up-regulation of OX40L, in turn, drives Th0 cells to polarization into activated Th2
cells, which leads to production of pro-inflammatory cytokines TNF, IL-4, IL-5, and IL-13, but
not Type 1-cytokine IL-10 [167, 188, 199, 218, 219]. Interestingly, the presence of the Type 1cytokine IL-12 prevents the ability of TSLP and OX40L to induce DC promotion of Th2
differentiation. TSLP is the only mediator that can activate mDCs without the induction of IL-12
and other Th1-polarizing cytokines [167; 219]. It is unclear as to the molecular mechanisms
through which TSLP can stimulate Th2-polarizing signals, as opposed to Th1-polarizing signals.
However, the activation of DCs by TSLP subsequently provides a permissive condition for Th2
development by up-regulating Th2-polarization, without inducing Th1-polarizing signals, thus
contributing to Type 2-driven allergic disease.
In addition, TSLP-activated DCs can potently expand and promote the function of the
CRTH2 (chemoattractant receptor homologous molecule expressed on Th2 cells) on CD4+ Th2
memory cells, without altering their central memory phenotype and Th2 commitment [220].
Furthermore, TSLP has the capacity to activate and expand CD8+ T cells, as well as induce them
to differentiate into pro-inflammatory IL-5 and IL-13 producing cytolytic T cells [221]. In turn,
these cells can induce eosinophilia and increase IgE production in atopic dermatitis (AD) [222].
TSLP has also been shown to promote DC-induced differentiation of regulatory T cells (T-regs)
and development of T-regs positive for Forkhead box P3 (FOXP3+ T-regs) [214, 220, 223, 224].
Lastly, DCs themselves can produce TSLP, upon TLR stimulation, which suggests that TSLP
can act in an autocrine manner to further drive DC-initiated Type 2 inflammation [188].
The above findings suggest that TSLP can program human DCs to initiate Type 2
responses by (1) up-regulating Th2-polarizing signals (OX40L); (2) limiting production of Th1-
29
polarizing signals (IL-12); (3) recruiting neutrophils, eosinophils, and Th2 cells via production of
chemokines; (4) inducing differentiation of naive CD4+ and CD8+ T cells, and T-regs; and, (5)
promoting expansion of memory Th2 cells.
1.5.4.2 T cells and B cells
As mentioned earlier, the effects of TSLP on T cells are mainly mediated through a
mDC-dependent mechanism. However, various murine and human studies demonstrate that
TSLP can directly activate CD4+ T cells, independent of mDCs. CD4+ T cells do not
constitutively express TSLPR and therefore maintain insensitivity to TSLP under baseline
conditions. However, following cellular activation, the expression of TSLPR becomes upregulated on their cell surface. For example, it has been shown in a murine model that following
stimulation via the TCR, TSLP can preferentially expand Th0 cells and differentiation into Th2
cells [225; 226]. Furthermore, TSLP can induce IL-4 gene transcription, and this subsequent
production of IL-4 can then up-regulate TSLPR expression of Th0 cells, thus creating a positive
feedback loop [225; 226]. Furthermore, activated T-regs have been reported to express TSLPR
and respond directly to TSLP [223]. For example, TSLP-treated T-regs demonstrate impaired IL10 production and diminished suppressive activity [223; 227]. Lastly, recent studies have shown
a direct effect of TSLP on NKT cells. Nagata et al. observed that TSLP has a pro-inflammatory
effect on murine NKT cells via stimulation of IL-13 production but not IFN-γ or IL-4 [228].
TSLP can directly support B cell lymphopoiesis. Scheeren et al. demonstrated that in
vitro culturing of progenitor cells with TSLP led to differentiation and proliferation of pro and
pre-B cells from fetal progenitor cells, as well as an increase in the absolute number of mature B
cells [229; 230]. Additional studies have shown TSLP can promote the development of IgM+
immature B cells in fetal liver and bone marrow cultures through STAT-5 phosphorylation [230;
30
231]. The role of TSLP in normal B cell development or during an allergic inflammatory
response has not been described. Collectively, the above findings suggest that TSLP has a
primary role in regulating the adaptive immune response.
1.5.4.3 Hematopoietic Progenitor Cells
CD34+ HPCs have been thought to contribute to allergic inflammation through their
differentiation into mature effector cells such as eosinophils, basophils, and mast cells, which in
turn promote Type 2 inflammatory responses within sites of inflammation. However, recent
studies demonstrate that progenitor cells can themselves function as effector cells through their
production of Type 2 cytokines, and that expression of these cytokines can be up-regulated
following antigenic stimulation. Given that HPCs are recruited to the airways during an
asthmatic exacerbation [48], interaction with the alarmin cytokines may further potentiate their
differentiation and effector activity.
Allakhverdi et al. investigated HPC expression of TSLPR, and they found that human
cord CD34+ cells express the TSLPR and IL-7α chain subunits [208]. They determined that
overnight stimulation of human CD34+ cells with TSLP caused a dose-dependent release in Type
2 cytokines (IL-5, IL-13, and GM-CSF) and chemokines (CCL22, TARC, IL-8, and CCL1).
Moreover, these CD34+ cells could be activated in a TSLP-dependent manner by supernatant
acquired from polyI:C-stimulated airway epithelial cells and nasal explants from patients with
non-allergic and AR [208]. To test the effects of TSLP on CD34+ cell differentiation, Hui et al.
cultured human cord blood CD34+ cells overnight with TSLP and found a significant increase in
IL-5Rα expression [68]. 14-day cultures of human cord blood CD34+ cells with TSLP led to a
significant increase in colony-forming units grown with IL-5 and GM-CSF [68]. Lastly, Smith et
al. determined the in vitro effect of TSLP on human peripheral HPCs activity [215]. Pre-
31
exposure to TSLP primed migration of HPCs to CXCL12, and incubation with TSLP induced
HPC production of IL-5, IL-13, but not IL-4, which was blocked by a mAb to TSLPR.
Collectively, these findings suggest a key role for TSLP in driving HPC differentiation, lung
homing, and effector function in allergic asthma.
1.5.4.4 Mast Cells and Eosinophils
Mast cells initiate allergic inflammation following cross-linking of high-affinity FcεR1
with IgE, which leads to cell activation and subsequent release of mediators such as histamine,
leukotrienes, cytokines, and chemokines. A study by Allakhverdi et al. observed that TSLP
synergistically reacts with IL-1β and TNF to stimulate mast cell production of Type 2 cytokines
(IL-5, IL-13, IL-6, and GM-CSF) and chemokines (IL-8 and CCL1) [69]. Their study also
demonstrated that supernatants collected from airway epithelial cell cultures with measurable
levels of TSLP, in combination with IL-1β/TNF, were sufficient to induce IL-13 and IL-5
production from mast cells. TSLP was also shown to dose-dependently induce the release of IL5, IL-13, IL-6, IL-10, GM-CSF, and IL-8 from mast cells for up to 24 hours following
stimulation, whereas it suppressed the release of TGF-β. Interestingly, mast cells within the
airways of asthmatic subjects have been shown to express TSLP mRNA and protein [183, 185,
187]. Okayama et al. further demonstrated that human mast cells are able to produce significant
amounts of TSLP following cross-linking with IgE and priming with IL-4, suggesting a possible
mast cell/TSLP positive feedback loop [196]. Mast cells have also been shown to regulate
epithelial TSLP expression in allergic disease [232-234]. In a mouse model of AR, nasal
epithelial TSLP expression was up-regulated in ovalbumin (OVA)-sensitized and nasally
challenged mice, and abolished in mast cell-deficient and FcεR1-deficient mice compared with
32
wild-type controls [234]. Treatment with a neutralizing mAb to TSLP inhibited the OVAinduced development of AR.
With respect to eosinophils, these cells have been shown to express both chains of the
TSLPR complex (TSLPR and IL-7Rα) [205, 210, 235]. In vitro stimulation of purified
eosinophils with TSLP has been shown to induce the following: degranulation, delay apoptosis,
up-regulate release of eosinophil-derived neurotoxin (EDN), IL-5, IL-8, CXCL1, and MCP-1,
increase expression of adhesion molecules CD19 and ICAM-1, and down-regulate expression of
L-selectin [210; 235]. As a whole, these findings suggest that TSLP can promote mast cell and
eosinophil activation at sites of Type 2 cytokine-associated inflammation.
1.5.4.5 Basophils
Emerging evidence demonstrates that TSLP can affect basophil activity. Initial
experiments with HPCs have shown that TSLP can promote murine and human differentiation
into basophils [66]. Further studies have expanded on these findings to show that human HPCs
pre-incubated with IL-3 and TNF have enhanced sensitivity to TSLP-mediated basophil lineage
commitment, and mast cell-activated bone marrow mesenchymal stromal cells produce TSLP,
which can promote the differentiation of CD34+ progenitors into eosinophil/basophil colonies
[68]. Hui et al. demonstrated that CD34+ cell expression of TSLPR could be up-regulated
following stimulation with IL-3 and that IL-3Rα could be increased following stimulation with
TSLP. Studies investigating the effect of TSLP on murine basophil activity have shown that
TSLP, in the presence or absence of IL-3, can elicit various basophil functional markers such as
increased CD69, L-selectin, CD11b, IL-3Rα, and ST2 surface marker expression [66]. Siracusa
et al. was the first to identify TSLPR on peripheral human basophils and demonstrate that this
expression could be further increased following overnight incubation with IL-3 [66]. Agrawal et
33
al. reported that basophil TSLPR expression in atopic asthmatics is up-regulated following
stimulation with Der p 1 or anti-IgE [217]. Noti and coworkers found a correlation between
elevated airway TSLP expression and exaggerated basophil responses in patients with
eosinophilic esophagitis [130]. They further showed that in a mouse model of eosinophilic
esophagitis TSLP neutralization and/or basophil depletion ameliorated disease-like symptoms,
suggesting that the interaction between TSLP and basophils may be an important component of
airway inflammation [130]. Lastly, it is noteworthy that basophils are also a significant source of
TSLP, supporting the possibility of a positive feedback loop [106]. Collectively, these findings
suggest an important relationship between TSLP and basophils. Although TSLP has been
identified as an important initiator of Type 2 inflammatory responses, the basophil/TSLP axis in
human allergic asthma has not been well characterized and the aforementioned findings warrants
further investigation. A sixth aim of this thesis is to further elucidate the relationship between
basophils and TSLP in human allergic asthma.
1.5.5 TSLP, Mouse Models, and Human Clinical Trials
Animal models act as an invaluable tool to investigate the pathogenesis of allergic
disease. Findings from asthmatic animal models have provided extensive information pertaining
to the contribution of TSLP to allergic airway responses and the cellular mechanisms underlying
TSLP-induced inflammation.
Through the examination of TSLP transgenic mice (increased expression of TSLP within
the lungs), Zhou et al. determined that these animals had robust airway infiltration of eosinophils
and CD4+T cells compared with wild-type controls [219]. Moreover, these transgenic mice had
increased numbers of multinucleated macrophages, activated bone marrow DCs, elevated serum
IgE levels, as well as enhanced airway remodeling as evidenced by goblet cell hyperplasia and
34
subepithelial fibrosis. The airways of these transgenic mice were also more responsive to
methacholine than wild-type controls, thus indicating increased AHR.
Bronchoprovocation challenges with OVA in murine models have also been used to
determine the impact of TSLP on allergic asthma. Following OVA challenges, TSLP transgenic
mice have greater eosinophil infiltration and enhanced AHR compared with phosphate-buffered
saline (PBS)-challenged transgenic mice or OVA challenged wild-type mice. For example, AlShami et al. assessed the importance of TSLP in mounting Type 2 inflammation, by using an
OVA challenge model to compare TSLPR knockout and wild-type mice airway responses [236].
Following the OVA challenge, compared to the wild-type mice, the TSLPR knockout mice
demonstrated a decline in AHR and a Type 1 response, characterized by down-regulated
production of Type 2 cytokines and decreased airway infiltration of eosinophils and neutrophils.
In contrast, TSLPR knockout mice had a strong Type 1 airway response, with high levels of IL12, IFN-γ, and IgG2a.
Multiple murine studies have expanded on the above findings to confirm that blockade of
TSLP signaling can down-regulate antigen-induced airway responses [236]. These experiments
involved pre-treating mice with either a TSLPR mAb or istoype control, followed by
sensitization with OVA or PBS, with airway inflammatory assessments following airway
challenges. Pre-treatment with the isotype control, followed by sensitization with OVA was
found to significantly elevate TSLP expression within BALF samples, which correlated with
BALF eosinophil numbers and IL-5 protein levels [237]. In contrast, treatment with TSLPR
mAb prior to sensitization with OVA yielded the following changes: decreased total cell
numbers, eosinophils, and lymphocytes in BALF; reduced IL-4 and IL-5 levels; elevated IFN-γ;
and, inhibited expression of co-stimulatory molecules CD40, CD80, CD86, and MHC II on
35
pulmonary DCs [237].
In addition to studying the role of TSLP in the pathogenesis of asthma using an OVA
challenge model, other researchers have employed the chromic house dust mite (HDM)-induced
mouse model. Chen et al. sensitized mice to HDM, followed by intranasal treatment with TSLP
mAb or an isotype control 60 minutes prior to challenging the mice with HDM [238]. TSLP
mRNA and protein expression within the lung tissue were increased for up to 24 hours following
HDM exposure as compared with PBS control group. Treatment with TSLP mAb prior to HDM
exposure led to significantly higher TSLP levels within the lungs, however there was significant
attenuation of methacholine-induced AHR, and down-regulated production of airway Type 2
cytokines IL-3 and IL-13, with an increase in IFN-γ production. Lastly, Chen et al. observed that
expression levels of OX40L, CD80, and CD86 on airway CD11c+ cells significantly increased
following HDM exposure, but this was attenuated following treatment with TSLP blockade
compared with the isotype control [238].
Another avenue to investigate TSLP-induced pathogenesis in asthma was undertaken by
Seshsayee et al., where they determined the effect of the OX40L blockade on TSLP-driven
allergic airway inflammation [239]. Mice were OVA-sensitized and treated with OX40L mAb or
an isotype control, followed by a challenge with TSLP and OVA. The airway challenges with
TSLP and OVA induced significant lymphocyte and eosinophil airway infiltration, increased
CD4+ effector and memory cells numbers, as well as up-regulated IgE, IL-4, IL-5, and IL-13
protein levels in the BALF. However, treatment with OX40L mAb was able to markedly reduce
the above TSLP/OVA-induced effects.
In addition to mouse models, monkeys have also been employed to study immune
regulation and effector functions in asthma due to their genetic and physiological similarity to
36
humans. To further elucidate the importance of TSLP in the allergic asthmatic response, Cheng
et al. assessed TSLPR blockade in a primate model of allergic asthma [240]. Sensitized
cynomologus monkeys were dosed weekly with a TSLPR mAb or isotype control for 6 weeks,
and challenged with Ascaris suum allergen extracts at baseline and again at 2 and 4 weeks postdosing. Lung function and airway inflammation were assessed at 8 and 24 hours post-allergen
challenge. Following 6 weeks of treatment, the monkeys receiving TSLPR mAb had
significantly reduced allergen-induced BALF eosinophils, IL-13 levels, and airway resistance
compared with those receiving the isotype control treatment.
Collectively, the above studies demonstrate a distinct role of TSLP in allergen-driven
asthma and support modulation of TSLP levels within the airway as a therapeutic avenue to treat
this disease. Thus, these studies have provided evidence to support initiating human clinical trials
to determine the therapeutic potential of TSLP neutralization to treat allergic asthma. Gauvreau
et al. has been the first group to examine the effect of a TSLP mAb to treat human allergic
asthma [241]. They demonstrated that treatment with TSLP mAb (AMG 157) in subjects with
mild allergic asthma significantly attenuated allergen-induced EAR and LAR in parallel with a
reduction in blood and sputum eosinophils. Given that allergen-induced EAR and LAR are
mediated by effector cells (eosinophils, basophils, and mast cells), the above findings suggest
that TSLP may activate these cells, thereby further amplifying their role in driving Type 2
inflammation. Lastly, these findings suggest that the neutralization of TSLP may be used a novel
treatment for asthma by reducing consequential airway inflammation and AHR. To fully
understand the impact of TSLP on allergic asthma, further research is required to determine the
effect of TSLP neutralization on the effector function of immune cells involved in potentiating
the Type 2 response of allergic asthma.
37
1.6 IL-25
1.6.1 IL-25 and IL-25R Expression
IL-25/IL-17E is a member of the structurally related IL-17 cytokine family. Homologybased cloning has resulted in the discovery of six IL-17 family members: IL-17A to IL-17F. The
genes encoding murine and human IL-25 are both located on chromosome 14 [242]. Studies
have shown murine IL-25 to form a homo-dimer and contain a total of 10 cysteine residues,
allowing it form a cysteine knot-like structure. The IL-25 cysteine residues are conserved in
human IL-25, which share 80% homology with murine IL-25.
IL-25 mRNA expression has been reported in epithelial-rich tissues including the colon,
kidney, lung, prostate, testis, and trachea [243]. Murine studies have found IL-25 to be upregulated in sensitized lung epithelium following Aspergillus fumigatus infection, or after OVAsensitization [244-246]. In addition to epithelial cells being a key source of IL-25, there have
been other identified cells which can produce this cytokine including basophils, eosinophils, Th2
cells, and mast cells, as well as in vitro and in vivo in the gut during chronic colitis [110, 247.
248]. IL-25 has been shown to be up-regulated in allergic disease [110] and polymorphisms in
the gene coding for IL-17RB have been associated with asthma [249]. For example, analysis of
human lung tissue has revealed markedly increased IL-25 expression in asthmatics and the skin
of AD subjects, compared to healthy controls [110]. These findings suggest an important role for
IL-25 in the induction of Type 2 allergic responses.
The cytokine IL-17 signals through a heterodimeric receptor complex, consisting of IL17 receptor (IL-17R) A and IL-17RC [250; 251], whereas IL-25 signals through IL-17RB
(Interleukin-25 receptor (IL-25R)) [251; 252]. However, recent studies suggest that IL-25
signaling also requires heterodimerization of IL-17RA and IL-17RB [251; 252]. A number of
38
structural and immune cell types have been described as expressing IL-25R including ILC2s,
APCs, ASM cells, asthmatic lung tissue, NKT cells, CD4+ Th2 cells, eosinophils, mast cells, and
basophils [253-257]. The exact cellular target of IL-25 and the effects the cytokine exerts on
these cells are not well understood.
The kinetics and cellular origins of IL-25 and IL-25R expression in allergic asthma
remains unclear. However, a recent study by Corrigan et al. has shown that for up to 24 hours
following an allergen bronchoprovocation there was increased expression of IL-25 and IL-25R in
asthmatic bronchial mucosa and dermis of sensitized atopic subjects, which correlated with the
magnitude of late-phase allergen-induced clinical responses [258]. They observed endothelial
cells, epithelial cells, macrophages, and mast cells to express IL-25 within the airways.
Interestingly, Cheng et al. found a specific subset of asthmatics to have high-IL-25 expression
[259]. Compared to the low-IL-25 subset, they had greater AHR, airway eosinophilia, serum IgE
levels, subepithelial thickening, and Type 2 cytokine gene expression. Their plasma IL-25 levels
correlated with epithelial IL-25 expression and airway eosinophilia. Furthermore, the IL-25-high
subset was associated with improvement in FEV1 and AHR in response to inhaled corticosteroid
treatment.
In addition, studies have shown increased IL-17RB surface expression on human effector
cells in allergic models. For example, Wang et al. observed both basophil mRNA and surface
expression of IL-17RB, which increased following overnight stimulation with IL-3 and a 1-week
allergen challenge, compared to the diluent, in seasonal AR patients, whereas no change was
seen in healthy controls [260]. This study suggests that both IgE-dependent and IgE-independent
mechanisms can increase the sensitivity of basophils to IL-25, which may be contributing to their
role in allergic asthma. Our own lab has demonstrated that IL-17RB expression on mDCs
39
significantly increases within the peripheral blood and sputum 24 hours following a whole lung
allergen challenge in mild allergic asthmatics (data not published). Tang et al. examined plasma
levels of IL-25 and IL-17RB expression on eosinophils in allergic asthmatics, atopic nonasthmatics, and normal controls [257]. They observed significantly higher eosinophil surface
expression of IL-17RB and levels of plasma IL-25 in allergic asthmatics, compared to atopic
non-asthmatics, and healthy controls. Furthermore, they found a negative correlation between
plasma IL-25 levels and % predicted FEV1. This study indicates that increased IL-25 expression
and sensitivity of eosinophils to IL-25 in asthmatics may be in part contributing to the
pathogenesis of this disease. To better understand the interaction between effector cells and
alarmin cytokines during asthmatic exacerbations, future studies are required to determine the
change in IL-17RB surface expression on effector cells following allergen exposure.
1.6.2 Cellular Targets of IL-25
The effect of IL-25 on immune cells has been studied with respect to eosinophils,
basophils, DCS, Th2 memory T cells, and NKTs. Wong et al. was the first research group to
demonstrate that stimulation of human eosinophils with IL-25 induced activation of signaling
molecules c-jun N-terminal kinase (JNK), p38, MAPK, and NF-κB, suggesting an intracellular
signaling pathway which may be activated following IL-25R ligation [253]. In addition, they
observed IL-25 significantly up-regulated eosinophil mRNA and protein expression of
chemokines (MCP-1, MIP-1α, and IL-8) and IL-6. Other reports have shown IL-25 to
significantly up-regulate eosinophil surface expression of adhesion molecule ICAM-1, but
suppressed ICAM-3 and L-selectin in a dose-dependent manner, which was mediated through
NFκB, p38 MAPK, and JNK signaling pathways [261]. With respect to basophils, Wang et al.
determined that stimulation of basophils with IL-3 up-regulated surface expression of IL-17RB,
40
and that activation with IL-25 prolonged basophil survival, and increased IgE-mediated
degranulation [260]. Basophils and eosinophils have also been shown to express mRNA for IL25 following IgE cross-linking, which was further increased in allergic subjects, suggesting that
these cells have an IL-25 autocrine feedback loop to regulate their effector function [110]. TSLPactivated DCs have been found to strengthen allergy-inducing properties of Th2 memory cells by
up-regulating their gene expression of IL-17RB, suggesting a possible role for IL-25 in the
regulation of Th2 memory cells [110]. This was confirmed by Wang et al. who observed that IL25 enhanced TSLP-activated DCs induction of Th2 memory cell proliferation and Type 2
cytokine production (IL-4, IL-5, and IL-13) [110]. A study with NKTs has shown that a
population of CD4+IL-25R+NKT cells was necessary for the induction of AHR and that
depletion of these cells by targeting IL-25R with an anti-IL-25R mAb prevented the
development of AHR, suggesting a role for the IL-25R+ population in promoting Type 2
inflammation [262]. Lastly, our own lab has reported IL-25 to prime human eosinophils and
eosinophil progenitors to eotaxin and SDF-1α, respectively (data not published). The above
findings indicate that IL-25 can enhance the pro-inflammatory function of immune cells, thereby
further developing and maintaining the Type 2 response in allergic disease. More studies are
needed to determine the effects that IL-25 may have on the effector cells of allergic asthma. A
seventh aim of this thesis is to further examine the relationship between basophils and IL-25 in
human allergic asthma.
1.6.3 IL-25 and Mouse Models
Various studies in mouse models have provided evidence to support the notion that IL-25
is an important player in Type 2 responses. Evidence suggesting the significance of IL-25 in
allergic asthma were first reported by Tamachi et al. who found that following antigen inhalation
41
in mice, IL-25 mRNA expression markedly increased within the lungs, and neutralization of IL25 by soluble IL-25R decreased antigen-induced eosinophil and CD4+ T cell infiltration within
the airways [263]. These findings were expanded by Yao et al. who carried out intranasal
challenges with IL-25 in a murine model, which in turn induced delayed, but predominantly
eosinophilic and lymphocytic infiltration into the airway lumen, as well as increased production
of Type 2 cytokines and chemokines, mucus hypersecretion, goblet cell hyperplasia, and AHR,
but no increase in IgE or IgG1 levels [264]. Similar reports have been made in transgenic mouse
models, where overexpression of IL-25 resulted in significantly enhanced Type 2 cytokine
production, airway infiltration of eosinophils, macrophages, and CD4+ T cells, as well as mucus
hypersecretion, and goblet cell hyperplasia compared to wild type mice [265; 266]. Lastly,
neutralization experiments have not only helped to confirm the above reports but have also
shown promising results for a new therapeutic avenue for allergic asthma. Treatment with antiIL-25 mAb prior to allergen inhalation challenges has led to significant reduction in IL-5 and IL13 secretion within the airways, eosinophil infiltration, goblet cell hyperplasia, and serum IgE
secretion, as well as prevents AHR [266]. This was further confirmed in a mouse model
involving pre-treatment with anti-IL-25 mAb and subsequent exposure to HDM, which resulted
in reduction of pulmonary eosinophilia and Type 2 cytokine levels (IL-5 and IL-13), and AHR
[267]. In addition, Rickel et al. has shown that blockade of IL-17RA and IL-17RB completely
abolished IL-25-induced pulmonary inflammation and AHR in wild-type mice [268].
Collectively, these studies demonstrate that IL-25 is an important mediator of airway
inflammation and AHR, which in turn may be a novel therapeutic target for asthma. These
murine studies underscore the need to conduct studies in humans to determine whether blockade
IL-25/IL-25R signaling can inhibit allergen-induced asthmatic exacerbations.
42
1.7 IL-33
1.7.1 IL-33 and IL-33 Receptor
IL-33 was originally identified as a nuclear protein expressed in lymph node-associated
endothelial cells [269]. In 2005 the cytokine was identified as the ligand for an orphan receptor,
ST2, which was discovered 12 years prior [270]. IL-33 is now known as a nuclear cytokine that
is a member of the IL-1 cytokine family.
IL-33 mRNA expression has been identified in multiple tissue-cell types such as
endothelial cells, bronchial smooth muscle, and mucosal epithelial cells [270; 271]. The
epithelial cells of the skin, gut, and lung are the most prominent IL-33-expressing areas of the
body. Studies have also shown IL-33 production by immune cells, including DCs, macrophages,
and mast cells [270; 271]. Immunohistochemical studies have shown IL-33 protein expression is
found within the nucleus, suggesting that localization is an important property of the molecule.
IL-33 has been proposed as a dual-function alarmin-like cytokine that can be released from the
nucleus as a signal of tissue damage to local immune cells [270; 272]. IL-33 is secreted by
damaged cells in response to exogenous triggers such as viral infection, smoke inhalation, or
airborne allergens. For example, epithelial cells that are commonly exposed to the environment
have increased IL-33 expression following activation with allergens [273].
IL-33 exerts its cytokine activity through the IL-33 receptor complex (IL-33R) consisting
of IL-33-bound IL-1 receptor like-1 (ST2) and a ubiquitously expressed, shared co-receptor
called IL-1 receptor accessory protein (IL-1RAcP). ST2 and IL-1RAcP recruit the adaptor
molecules myeloid differentiation primary response gene 88 (MyD88) and interleukin-1
receptor-associated kinase (IRAK) to induce activation of the transcription factor NF-κB and
MAP kinases (p38 and JNK) [273-277]. ST2 is expressed on a wide range of immune cells
including group 2 innate lymphoid cells (innate lymphoid cell type 2 (ILC2s), natural helper
43
cells, nuocytes, innate helper 2 cells), HPCs, DCs, macrophages, mast cells, basophils,
eosinophils, Th2 cells (but not Th1 cells), and NKT cells [278-284]. Over time, the body has
evolved mechanisms to regulate the cytokine activity of IL-33, where IL-33 can be sequestered
and neutralized by a soluble form of ST2. Soluble ST2 (sST2) essentially acts as a decoy
receptor to bind and inhibit IL-33 activity.
1.7.2 IL-33 and IL-33 Receptor in Allergic Disease
Genetic links and polymorphisms in genes encoding for IL-33 and ST2 have been
associated with allergic disease. For example, polymorphisms in IL-33 (chromosome 9q) and IL1RL1 (chromosome 2q) are associated with AR, AD, and asthma [285-290]. There is also
accumulating non-genetic evidence that IL-33, ST2, and sST2 expression is associated with
allergic disease. Perfontaine et al. measured airway mRNA IL-33 expression in mild and severe
asthmatics, and control subjects [291]. They found higher levels of IL-33 mRNA expression in
epithelial and ASM cells of bronchial biopsies in asthmatics compared to controls, with severe
asthmatics having the highest level of expression. Similarly, Hamzaoui et al. measured the level
of sST2 expression and IL-33 in asthmatic children across varying degrees of disease activity
[292]. They found sST2 and IL-33 protein levels to be significantly higher within induced
sputum and serum of asthmatic children compared to healthy controls. Other studies have shown
serum protein IL-33 levels in patients with asthma to be markedly increased versus non-allergic
controls, and that asthmatic patient serum IL-33 was negatively correlated with % predicted
FEV1, and positively correlated to asthma severity [293; 294]. Oshikawa and colleagues further
demonstrated that sST2 protein levels in the serum of patients with asthma are increased during
an acute exacerbation compared to normal controls [295]. Additional research groups have
shown that IL-33 and ST2 expression is elevated within the serum, sputum, lung tissue, and
44
BALF of patients with asthma, anaphylactic shock, and AR, compared to healthy controls [296303]. Lastly, Smith et al. examined the change in CD34+ HPC ST2 expression following allergen
inhalation in asthmatic subjects and investigated the effect of IL-33 on HPC migration [215].
They found significant increases in the absolute numbers of HPC cells within the sputum 24
hours post-allergen inhalation that expressed ST2. Collectively, these findings indicate that
expression of sST2, ST2, and IL-33 is significantly greater in asthmatics compared to healthy
controls, and is associated with disease severity, which provides indirect evidence for asthmaassociated activation of the IL-33-axis, which may further contribute to the pathogenesis of this
disease.
1.7.3 Cellular Targets of IL-33
1.7.3.1 T cells
Studies have shown that Th2 cells express ST2 and that they can be affected by IL-33.
Human Th2-polarized cells from allergic donors respond to IL-33 in the presence of TCRdependent antigen stimulation, by producing elevated levels of IL-5 and IL-13 [270].
Interestingly, IL-33 can also induce Type 2 cytokine release from Th2 cells in the absence of
TCR stimulation, suggesting that it can act as a direct signal for Th2 cell pro-inflammatory
function [270]. Furthermore, IL-33 can promote the release of IFN-γ from NKT cells in humans
[270]. The induction of both Type 2 and Type 1 cytokines reflects the mixed Type 1/Type 2
cytokine profile that is found in some human asthmatics. Lastly, IL-33 has been shown to
selectively attract mouse and human Th2 cells, indicating IL-33 has a dual function where it can
both recruit and activate Th2 cells [278]. On the other hand, the ability of IL-33 to activate Th0
cells and regulate Th2 cell polarization is not well understood. There is evidence to suggest IL33 can affect T cell polarization via mouse models demonstrating that through a DC-dependent
45
mechanism, IL-33 can induce IL-5 and IL-13 production from Th0 cells, independent of IL-4
[304; 305]. Collectively, the above studies demonstrate that IL-33 can promote Th2 cell
activation both directly and indirectly, thereby initiating and prolonging the Type 2 inflammatory
response of allergic asthma.
1.7.3.2 Dendritic Cells
Recent published work has presented DCs as master regulators of the IL-33-axis. ST2 is
expressed on DCs at both the mRNA and protein level. Besnard et al. showed that IL-33 could
drive bone marrow-derived DC maturation in vitro via up-regulation of expression of the costimulatory molecules CD40, CD80, and OX40L, but had no effect on MHC II or CD86
expression [306]. Conversely, Rank and colleagues demonstrated that IL-33 increased expression
of MHC II and CD86 on DCs, but not that of CD40 or CD80 [304]. The above studies also found
IL-33 to directly induce DC production of IL-6, TNF, IL-1β, and TARC [304; 306]. These
findings suggest that IL-33 plays a role in promoting DC-induced Th2 cell polarization.
Interestingly, Su et al. reported that DCs produce IL-33 via TLR/NFκB signaling following
microbial stimulation, which suggests that Type 2 inflammatory responses might be amplified
via DC-produced IL-33 through a potential autocrine feedback loop [307]. In summary, various
studies have shown DCs to express ST2 and respond to IL-33 with increased proliferation,
maturation, and promotion of Type 2 inflammation.
1.7.3.3 Hematopoietic Progenitor Cells
There is a considerable body of evidence demonstrating that IL-33 directly influences the
function of HPCs. Freshly isolated circulating human CD34+ progenitor cells respond to IL-33
directly by rapidly releasing high levels of Type 2 cytokines (IL-5, IL-13, GM-CSF, and IL-6)
and chemokines (IL-8, CCL1, and TARC) [69]. With respect to mast cell development, human
46
CD34+ mast cell progenitors express ST2 and 6-8 weeks of stimulation with IL-33 can markedly
accelerate their maturation into tryptase-containing mast cells, which can be further enhanced by
TSLP [208]. Lastly, Smith et al. reported that pre-exposure to IL-33 primed migration of HPCs
to the chemoattractant SDF-1 and incubation with IL-33 stimulated production of IL-5 and IL13, but not IL-4 by HPCs, which was blocked by mAb to ST2 [215]. The above findings suggest
that IL-33 may orchestrate increased differentiation, airway infiltration, and activation of HPCs
in allergic asthma.
1.7.3.4 Mast Cells, Eosinophils, and Basophils
Studies have shown human mast cells stably express ST2 and respond directly to
stimulation with IL-33 by producing pro-inflammatory cytokines and chemokines. The effects of
IL-33 can be further enhanced when in combination with TSLP or IgE cross-linking. IL-33 by
itself cannot induce mast cell release of lipid mediators, such as PG2 and CysLTs, or histamine
via degranulation, suggesting that it cannot substitute IgE-dependent signaling [308]. However,
IL-33 can prime the degranulation of mast cells in response to free IgE [308]. IL-33 has also
been shown to prolong human mast cell survival, adhesion to fibronectin, and directly stimulate
the release of cytokines IL-6, IL-13, and TNF [308-311], therefore providing a signal to not only
activate mast cells but also maintain localization their localization within the airways.
With respect to eosinophils, IL-33 has been shown to augment a variety of their cellular
functions. IL-33 can enhance eosinophil survival, CD11b expression, and adhesion to
extracellular matrix proteins [312; 313]. IL-33 can also stimulate production of eosinophil
superoxide and IL-8 [312]. Lastly, IL-33 can induce human eosinophil shape change (surrogate
marker for chemotaxis) and dose-dependently enhance their chemokinetic activity [313]. These
47
findings collectively demonstrate that IL-33 induces mobilization and activation of eosinophils,
which may in turn contribute to airway eosinophilia during asthmatic exacerbations.
There have been several studies, which have highlighted the effect of IL-33 on human
basophils. Pekaric-Petkovic and colleagues provided evidence of ST2 and sST2 mRNA
expression in peripheral basophils, which became up-regulated following stimulation with IL-3
[314]. sST2 became up-regulated as early as 2 hours, whereas ST2 increase was evident by 6
hours post-IL-3 stimulation. IL-33 induced significant release of IL-13 and IL-4 from basophils
that was enhanced in synergy with IL-3 and/or anti-IgE. IL-33-induced stimulation of basophils
could be inhibited following treatment with sST2. Suzukawa and coworkers demonstrated that
basophil express mRNA for ST2, which became up-regulated following stimulation with IL-33
[83]. Expression of IL-13 and IL-4 mRNA in basophils was increased following stimulation with
IL-33. Basophil CD11b expression significantly increased after 30 minutes and 18 hours of
culturing with IL-33. Degranulation and migration towards eotaxin was also enhanced by IL-33,
which was blocked following neutralization of ST2. Smithgall et al. found freshly isolated
human basophils to express high levels of ST2 and respond to IL-33 by producing several proinflammatory cytokines including IL-4, IL-5, IL-6, IL-8, IL-13, and GM-CSF [284]. Blom and
colleagues additionally showed that basophils express the ST2 receptor, which could be upregulated following stimulation with IL-3, and IL-33 can induce IL-9 basophil production [315].
Lastly, Rivellese et al. found IL-33 to directly induce basophil production of IL-4 and histamine
degranulation, independently of IgE-mediated activation, but no change in CD63 expression was
evident [316]. These findings demonstrate that IL-33 can contribute to basophil migration,
adhesion, and Type 2 cytokine production, which may further promote the role of these cells in
allergic disease. The aforementioned studies show that IL-33 acts as a signal to sustain the
48
accumulation, maturation, and pro-inflammatory function of effector cells (mast cells,
eosinophils, and basophils) under pathological conditions. An eighth aim of this thesis is to
further investigate the relationship between basophils and IL-33 in a model of human allergic
asthma.
1.7.4 Lessons from Animal Models
Several animal model studies have underlined the important functional role of the IL33/ST2 axis in allergic asthma. Studies with OVA and HDM challenged mice have yielded
significantly higher levels of IL-33 mRNA and protein within lung tissue compared to PBSchallenged mice [317; 318]. Furthermore, administration of IL-33 to mice has been shown to
promote AHR, goblet cell hyperplasia, eosinophilia, and accumulation of Type 2 cytokines (IL4, IL-5, and IL-13) [319]. Zhiguang et al. have demonstrated that over-expression of IL-33 in
transgenic mouse models leads to spontaneous massive airway inflammation, characterized by
the infiltration of eosinophils, hyperplasia goblet cells, as well as pro-inflammatory cytokine
(IL-5, IL-8, and IL-13) and IgE accumulation within BALF [320]. Similar studies in models of
AD have shown that IL-33 transgenic mice had markedly increased eosinophil, mast cell, and IL5-producing ILC2 infiltration within the skin, as well as elevated circulating histamine and IgE
levels [321]. In contrast, Oboki and colleagues demonstrated that OVA sensitized IL-33deficient mice have attenuated eosinophil and lymphocyte recruitment to the lung, as well as
decreased AHR and inflammation [322]. Moreover, Louten et al. reported that endogenous
administration of IL-33 induces airway inflammation in OVA-induced IL-33-deficient mice
[323]. The above studies have been expanded on through treatment of transgenic mice with sST2
complementary DNA [324]. Following OVA challenges, these mice had significant reduction in
eosinophil infiltration, as well as IL-4, IL-5, and IL-13 within the BALF, compared to wild-type
49
mice [324]. Similarly, intranasal administration using adenovirus-mediated delivery of sST2
prior to OVA challenging the mice, yields significantly reduced serum IgE levels, eosinophil
infiltration, as well as IL-4, IL-5, and IL-13 within the BALF [325].
In 2009, Liu et al. investigated whether an anti-IL33 mAb could reduce airway
inflammation in OVA challenged mice [326]. Intraperitoneal injection of anti-IL-33 significantly
reduced serum IgE secretion, eosinophil and lymphocyte numbers, as well as IL-4, IL-5, and IL13 within the BALF compared to treatment with an isotype control antibody. This was confirmed
by Lee et al., where they observed that anti-IL33 antibody and sST2 exerted a negative
regulation on OVA-mediated allergic airway inflammation [327]. Both treatments reduced the
total cell counts and eosinophil numbers with the BALF, as well as AHR to methacholine, and
down-regulated Type 2 cytokine levels (IL-4, IL-5, IL-13) in the BALF. Similar results have
been shown in a model of AR, where anti-IL-33 mAb treatment significantly reduced the AR
symptoms, eosinophilic infiltration in the nasal cavity, as well as IL-4, IL-5, and IL-13 in the
BALF [328]. Collectively, the above data suggest that IL-33 is involved in allergic asthmatic
responses and supports the concept of ST2 as a novel therapeutic target in asthma. Although
there are numerous mouse models demonstrating the effectiveness of IL-33 blockade in allergic
asthma, there is a need for future studies in human models of allergic asthma to further
substantiate IL-33 as a therapeutic target. The table below summarizes the effects of alarmin
cytokines on immune cells.
50
Table 2. Effect of Alarmin Cytokines on Immune Cells
1.8 Summary
The inflammatory response of allergic asthma is comprised of a very broad, complex, and
intricate system, involving interaction between the alarmin cytokines and immune cells, which
still remains poorly understood. Given that emerging evidence suggests basophils to be effector
cells involved in the allergic disease, it is important to understand how their activity can be
mediated by interaction with alarmin cytokines. The key purpose of this thesis is to establish the
role of basophils in allergic asthma and determine whether interaction with alarmin cytokines
can further potentiate the activity of these cells.
51
1.9 Hypothesis and Aims
The objective of this thesis was to examine the role of basophils in the LAR and determine how
interaction with alarmin cytokines can enhance the pro-inflammatory activity of basophils. As
such, we divided the thesis into two distinct parts, the first being to investigate the role of
basophils in the LAR, and the second, to explore the effect of alarmin cytokines (TSLP, IL-33,
and IL-25) on basophil pro-inflammatory activity.
OVERALL HYPOTHESIS:
The overall hypothesis of this thesis is that alarmin cytokines promote the effector role of
basophils in the LAR of allergic asthma.
Collectively, the goal of these studies was to broaden our understanding of the relationship
between basophils and the alarmin cytokines, and how this interaction can subsequently
contribute to the LAR. This research will help determine whether the basophil/alarmin cytokine
axis poses as a novel target for the treatment of allergic asthma.
52
SPECIFIC HYPOTHESES:
1.9.1 Hypothesis: Allergen inhalation induces up-regulation of peripheral blood basophil
surface activation markers, which can be used to monitor allergen-induced
bronchoconstriction in mild allergic asthmatics.
Aim 1: Investigate the change in basophil surface activation marker expression (CD203c,
CD63, and CD69) following allergen inhalation, and determine what marker best reflects
basophil activation.
1.9.2 Hypothesis: Allergen inhalation leads to up-regulation of basophil pro-inflammatory
activity during the LAR, which in part, contributes to the Type 2 response of allergic
asthma.
Aim 1: Examine the change in basophil pro-inflammatory activity (surface activation
marker and Type 2 cytokine intracellular expression, and migration) following allergen
inhalation in mild allergic asthmatics, within multiple biological compartments (bone
marrow, periphery, and airways).
1.9.3 Hypothesis: Allergen inhalation leads to up-regulation of basophil pro-inflammatory
activity during the LAR, which is in part, promoted by interaction with alarmin cytokines
TSLP, IL-25, and IL-33.
Aim 1: Investigate the change in surface expression for TSLP, IL-25, and IL-33 receptors
on basophils following allergen inhalation in mild allergic asthmatics, within multiple
biological compartments (bone marrow, periphery, and airways).
Aim 2: Determine how TSLP, IL-25, and IL-33 can affect basophil pro-inflammatory
activity (surface activation marker expression, Type 2 cytokine intracellular expression,
and migration) in vitro.
Aim 3: Determine the effect of airway microenvironment samples on basophil proinflammatory activity in vitro, and whether blockade of Type 2 and receptors for alarmin
cytokines can inhibit this effect.
53
CHAPTER 2:
Expression of activation markers in circulating basophils and the
relationship to the allergen-induced bronchoconstriction in subjects
with mild allergic asthma
Submitted to Journal of Allergy and Clinical Immunology. May 2015.
CD203c, CD63, and CD69 are basophil surface activation markers that have been used
previously to diagnose and monitor various allergic diseases; however the relationship of this
marker expression to allergic asthma remains unclear. Traditionally, basophils have been thought
to play an important role in the development of the EAR through IgE-mediated histamine
degranulation and CysLT release, which promote bronchoconstriction, mucosal edema, and
mucus hypersecretion. However, little is known about the kinetics of basophil activation during
the EAR and LAR, and whether this changes across different asthmatic responses to inhaled
allergen. Our study investigated basophil activation marker expression at varying time-points
post-allergen inhalation in both IER and DR mild allergic asthmatics. Furthermore, we
investigated what surface marker was the most effective at detecting peripheral blood basophil
activation, and whether this marker could reflect allergen-induced bronchoconstriction. Firstly,
we observed that pre-allergen basophil activation was significantly higher in DR compared to
IER. Secondly, we found that in IER subjects there was a significant up-regulation in circulating
basophil CD203c expression during the EAR, whereas DR had increased expression during both
the EAR and LAR. Pre-allergen basophil activation had a significant correlation with the %
predicted values for FEV1. Lastly, CD203c expression at 7 hours post-allergen had a significant
correlation with maximum % fall in FEV1. This study demonstrated that CD203c expression
could be used as an additional tool to help monitor asthma status and differentiate between
asthmatic responses to inhaled allergen. Most importantly, our study showed a positive
correlation between the level of basophil activation and allergen-induced bronchoconstriction,
54
providing indirect supporting evidence that basophils may contribute to the decline in lung
function during the LAR.
55
Letter to the Editor
Expression of activation markers in circulating basophils and the relationship to allergeninduced bronchoconstriction in subjects with mild allergic asthma
Brittany M. Salter, BSc1, Graeme Nusca, BSc1, Damian Tworek, MD, PhD1, John-Paul Oliveria,
BSc1, Steve G. Smith, PhD2, Rick M. Watson, BSc1, Tara Scime, BSc1, Catie Obminski, BSc1,
Roma Sehmi, PhD2, Gail M. Gauvreau1.
1
Department of Medicine, McMaster University, Hamilton, ON, Canada
2
Firestone Institute for Respiratory Health, St Josephs, Hamilton, ON, Canada
Corresponding author:
Gail M Gauvreau, McMaster University, HSC 3U26, 1200 Main St West, Hamilton, ON Canada
Telephone: 905-525-9140 x22791
Fax: 905-528-1807
E-mail: gauvreau@mcmaster.ca
Key Words: Basophils, Allergic Asthma, CD203c, CD63, CD69, Type 2 Cytokines
Key Messages:

CD203c increases on human basophils following allergen inhalation

CD203c basophil expression correlates significantly with decline in lung function postallergen challenge
Capsule Summary: Allergen inhalation up-regulates CD203c expression on circulating
basophils in subjects with allergic asthma.
56
Expression of activation markers in circulating basophils and the relationship to allergeninduced bronchoconstriction in subjects with mild allergic asthma
To the Editor:
Asthma is a chronic respiratory disease characterized by reversible airway obstruction, airway
hyperresponsiveness, and eosinophilic airway inflammation. In sensitized individuals, basophils
play an important role in airway responses to allergen through IgE-mediated release of histamine
and cysteinyl leukotrienes, which are both potent mediators of bronchoconstriction. The number
of basophils in the airways has been reported to be significantly higher in asthmatics compared
to healthy patients,1 and this number increases during asthmatic exacerbation.2 Furthermore,
basophil infiltration of the airways increases during the late phase asthmatic response (LAR)
following allergen inhalation,3, 4 where they provide a significant source of IL-4.5 In recent years,
the basophil activation test has been developed to assess human basophil activation in cases of
immediate-type hypersensitivity allergic responses. These tests monitor surface marker
expression of CD63, CD203c, and CD69 following immediate type sensitivity reactions. Given
that the use of a basophil activation test in the context of allergic asthma has not been well
established, we examined the change in peripheral blood basophil surface activation markers
after allergen inhalation in subjects with allergic asthma.
Nineteen subjects with mild allergic asthma were enrolled in a diluent-controlled allergen
bronchoprovocation study (See Online Repository, Table 1). Subjects were required to have a
skin prick test positive to common aeroallergens, methacholine PC20≤16 mg/mL, and FEV1
≥70% of predicted. All subjects developed a fall in FEV1 ≥20% within 2 h post-allergen
inhalation. Those those with a subsequent fall in FEV1 ≥ 15% between 3-7 h post-allergen were
57
considered to be isolated early responders while those who developed a subsequent fall in FEV1
≥15% between 3-7 h post-allergen were defined as dual responders.
Baseline lung function and peripheral blood was measured on day 1. Subjects were then
randomized to inhale diluent or allergen on day 2, followed by blood sample collection at 3 h, 7
h and 24h post-challenge. This was repeated with allergen or diluent after a 2-week recovery
period. Airway challenges are described in Online Repository Methods. Basophils were defined
as CD45+/HLA-DR-/IL-3Rα+ (Online Repository Fig E1); whole blood was immuno-stained
with monoclonal antibodies to CD45-Alexa Fluor 700 (BD Biosciences), HLA-DR-APCH7 (BD
Biosciences), and IL-3Rα-PE Cy7 (Ebioscience), and markers of activation (CD203c-APC
(MACS Miltenyi), CD63-V450 (BD Biosciences), and CD69-PE (BD Biosciences)). Cells were
acquired with a LSR II flow cytometer (BD Biosciences) and analyses were performed using
Flow-Jo software (TreeStar), as described in Online Repository Methods. Statistical analysis is
described in Online Repository Methods.
Six subjects developed isolated early responses with a 30.2±12.9% maximum fall in
FEV1 0-2 h post-allergen (p<0.05 versus diluent) with no difference from diluent challenge at
LAR 3-7 h post-allergen (Online Repository Fig E2, A). Thirteen subjects developed dual
responses with a 34.2±10.9% maximum fall in FEV1 during the early response and a 25.1±14.8%
maximum fall in FEV1 during the late response, and no change following diluent inhalation
(Online Repository Fig E2, B).
Dual responders had significantly higher pre-allergen CD63 sMFI expression compared
to isolated early responders (Online Repository Fig E4). Compared to diluent challenge, there
was a significant increase in CD203c sMFI expression in basophils from isolated early
responders at 3 h post-allergen, whereas CD69 and CD63 showed no change (Fig 1A; C). A
58
similar response was observed in basophils of dual responders at 3 h, however CD203c levels
remained elevated at 7 h and 24 h post-allergen (Fig 1B; D). In addition, dual responders had a
peak in circulating CD63 sMFI basophil expression at 7 h post-allergen, compared to diluent
(Fig 1B), whereas no change was seen in CD69 expression post-allergen. The same results were
found when activation markers were expressed as percent positive basophils (Online Repository
Fig E3). When measures of basophil activation were compared to lung function, there was a
positive correlation between the maximum % fall in FEV1 during the LAR (7 h post-allergen)
and CD203c expression (P=0.0018, R=0.661) (Fig 2). Lastly, there was a significant correlation
between the predicted FEV1 (%) and pre-allergen CD203c % cell expression (P=0.0248,
R=0.265) (Online Repository Fig E5).
Ono et al used flow cytometry to compare spontaneous and stimulated expression of
CD63, CD69, and CD203c on basophils from healthy subjects and from patients with stable or
exacerbated asthma.6 Spontaneous CD203c basophil expression was significantly higher in
patients with asthma exacerbation compared to patients with stable asthma or healthy subjects,
whereas no differences were found in CD63 or CD69 expression. In vitro anti-IgE-induced
basophils had significantly higher expression of CD63 and CD203c in patients with asthma
exacerbation compared to healthy controls. Stimulation with Der p 1 or IL-3 up-regulated
CD203c expression in a dose-dependent manner with clinically improved asthma patients
experiencing less of an increase versus patients with asthma exacerbation. Lastly, patients with
clinical improvement demonstrated a significant correlation between CD203c expression on
basophil and predicted FEV1 values. Ono et al. established that there are differences in CD203c
expression on basophils at baseline across asthma severity groups and healthy controls, and this
continued to be notable after in vitro stimulation.
59
The current study aimed to expand upon the above findings by examination of the
change in the expression of activation markers on circulating basophils following allergen
inhalation, and by comparing subjects with isolated early versus dual responses to allergen.
Subjects with isolated early responses showed a significant increase in basophil CD203c
expression at 3 h post-allergen compared to diluent; a return of CD203c expression to baseline
by 7 h and 24 h post-allergen is consistent with the lack of the LAR in these subjects. Following
allergen inhalation, subjects with dual responses experienced a significant up-regulation of
CD203c from 3 h to 24 h post-allergen, which is consistent with the allergen-induced
bronchoconstriction during the EAR and LAR. These findings are also consistent with our
previous reports demonstrating that circulating basophil expression of CD203c remains elevated
for up to 24 h post-allergen inhalation in dual responders.4
To conclude, this study has demonstrated that CD203c expression on circulating
basophils post-allergen challenge can be used to differentiate between isolated early and dual
asthmatic responses to an inhaled allergen. Given that dual responders had significantly higher
basophil activation pre-allergen compared to isolated early responders, and that pre-allergen
basophil activation correlated with percent predicted FEV1, this suggests that basophil activation
tests can be used as a marker to reflect pre-allergen airflow limitation and the occurrence of the
late phase response. Furthermore, changes in basophil CD203c levels together with a positive
correlation between allergen-induced bronchoconstriction and CD203c expression suggests that
activated basophils contribute to the decline in lung function during LAR.
60
Brittany M, Salter, BSc1
Graeme Nusca, BSc1
Damian Tworek, MD, PhD1
John-Paul Oliveria, BSc1
Steve G. Smith, PhD2
Rick M. Watson, BSc1
Tara Scime, BSc1
Catie Obminski, BSc1
Roma Sehmi, PhD2
Gail M. Gauvreau, PhD1
1
Department of Medicine, McMaster University, Hamilton, ON, Canada
2
Firestone Institute for Respiratory Health, St Josephs, Hamilton, ON, Canada
61
REFERENCES
1. Kimura I, Tanizaki Y, Saito K, et al. Appearance of basophils in sputum of patients with
bronchial asthma. Clin Allergy 1975;5:95-8.
2. Maruyama N, Tamura G, Aizawa T, et al. Accumulation of basophils and their
chemotactic activity in the airways during natural airway narrowing in asthmatic
individuals. Am J Respir Crit Care Med 1994;150:1086-93.
3. Gauvreau GM, Lee JM, Watson RM, Irani AA, Schwartz LB, O’Byrne PM. Increased
numbers of both airway basophils and mast cells in sputum after allergen inhalation
challenge of atopic asthmatics. Am J Respir and Crit Care Med 2000; 161(5): 1473-1478.
4. Salter BM, Oliveria J, Nusca G, et al. Thymic Stromal Lymphopoietin Activation of
Basophils in Allergic Asthma is IL-3 Dependent. J Allergy and Clin Immunol 2015 (In
Press).
5. Nouri-Aria KT, Irani AM, Jacobson MR et al. Basophil recruitment and IL-4 production
during human allergen-induced late asthma. J Allergy Clin Immunol 2001;108(2):205211.
6. Ono E, Taniguchi M, Higashi N, et al. CD203c expression on human basophils is
associated with asthma exacerbation. J Allergy Clin Immunol 2010;125:483-9.
62
FIGURES
B. Dual Responders
1500
CD203c + (sMFI)
1500
1000
*
500
*
500
0
20000
20000
15000
10000
5000
10000
5000
0
200
Diluent
Allergen
200
CD69 + (sMFI)
CD69 + (sMFI)
*
15000
0
150
100
50
0
*
*
1000
0
CD63 + (sMFI)
CD63 + (sMFI)
CD203c + (sMFI)
A. Isolated Early Responders
Pre
3 Hr
7 Hr
Post
24 Hr
150
100
50
0
Pre
3 Hr
7 Hr
24 Hr
Post
Figure 1- Effects of allergen inhalation on the expression of basophil activation markers
63
Basophils sMFI expression of CD203c, CD63 and CD69 before, 3 h, 7 h and 24 h postallergen (black bars), compared to diluent (open bars) in (A) Isolated Early Responders (N=6)
and (B) Dual Responders (N=13), as well as CD203c APC concatenate histograms for (C)
Isolated Early Responders and (D) Dual Responders. Data are presented as meanSEM. *
Significantly different from diluent control; P<0.05.
CD203c + (%)
7 Hr Post-Allergen
100
80
60
40
P<0.05
R=0.661
20
0
0
5 10 15 20 25 30 35 40 45 50 55 60
LAR Maximum % Fall in FEV 1
Figure 2- Relationship between CD203c expression on circulating basophils and maximum
% fall in FEV1 during the late asthmatic response.
Expression of CD203c on circulating basophils from Isolated Early Responders (open
circle) (N=6) and Dual Responders (closed circle) (N=13) at 7 h post-allergen compared to
maximum % fall in FEV1 during the late response.
64
ONLINE REPOSITORY
METHODS
Inclusion Criteria
Subjects were excluded if they were current or ex-smokers with more than 10 pack-years,
developed lower respiratory infections or used inhaled or oral steroids 4 weeks prior to study.
Anti-histamines, caffeine, and non-steroidal anti-inflammatory agents were prohibited 48 h
before study visits.
Methacholine Challenge
The methacholine inhalation challenge was carried out via tidal breathing from a Wright
nebulizer, as previously described.E1 Doubling concentrations of methacholine chloride
(Methapharm) were inhaled orally from a Hans Rudolph valve every 2 min. The FEV1 was
measured following inhalation at 30 s and 90 s or until it stopped falling. The percent fall was
calculated from the post-diluent FEV1 value. The test was terminated when a fall in FEV1 of at
least 20% of the lowest post-saline value occurred. The methacholine PC20 was then calculated
using linear interpolation.
Allergen and Diluent Challenge
Allergen challenges were conducted by administering doubling inhaled concentrations of
allergen extract, as previously described,E2 while diluent challenges were conducted using 3
inhalations of 0.9% saline for 2 min each. The early asthmatic response (EAR) was the largest
percent fall in FEV1 between 0 and 2 h, and the LAR was the largest percent fall in FEV1
between 3 and 7 h post-challenge.
Flow cytometry staining and analysis
Whole blood samples were immunostained with isotype or specific monoclonal antibodies to
CD45-Alexa Fluor 700 (BD Biosciences), HLA-DR-APCH7 (BD Biosciences), and IL-3Rα-PE
65
Cy7 (Ebioscience), CD203c-APC (MACS Miltenyi), CD63-V450 (BD Biosciences), and CD69PE (BD Biosciences) for 30 min at 4ºC. Following incubation the samples were washed with
FACs buffer at 300 g for 10 min at 4ºC then re-suspended in 2 mL of FACS buffer and stored at
4ºC until flow cytometry could be conducted. Cells were acquired with a LSR II flow cytometer
(BD Biosciences) and analyses were performed using Flow-Jo software (TreeStar). Basophils
were identified through a commonly used basophil gating strategy as the CD45+/HLA-DR-/IL3Rα+ population (Online Repository Fig E1), as previously described.4 A first gate was made in
the SSC vs. FSC dot plot around the monocyte/lymphocyte populations, which are known to
contain basophils (Online Repository Fig E1, A). A second gate was placed around the CD45+
population in the SSC vs. CD45 AF700 dot plot (Online Repository Fig E1, B), followed by
gating around the HLA-DR- population in the SSC vs. HLA-DR APCH7 (Online Repository Fig
E21 C), and a final gate around the IL-3Rα+ population in the SSC vs. IL-3Rα PE Cy7 plot
(Online Repository Fig E1, D). The isotype control for the markers of interest was set to 2%,
which was compared to the specific markers to detect the percentage of cells expressing the
marker (Online Repository Fig E1, E; F). To maintain consistency across samples, at least 300,
000 CD45+ events were acquired, with collection of the same cell number for each time-point
pre/post-diluent/allergen challenge per subject.
Statistical Analysis
Statistical analysis was conducted using Graph Pad Prism. All data are presented as mean
± SEM. The flow cytometry results are expressed as mean percent positive cells or specific Mean
Fluorescent Intensity (sMFI). Statistical analysis for the inhalation challenges was performed
using a 2-way ANOVA and post-hoc Bonferroni test. Correlation analyses were conducted using
a Pearson correlation test. Statistical significance was set to P<0.05.
66
REFERENCES
E1. Cockcroft DW. Measure of airway responsiveness to inhaled histamine or methacholine;
method of continuous aerosol generation and tidal breathing inhalation. In: Hargreave FE,
Woolcock AJ, eds. Airway responsiveness: measurement and interpretation. Mississauga: Astra
Pharmaceuticals Canada Ltd, 1985: 22 - 28.
E2. O'Byrne PM, Dolovich J, Hargreave FE. Late asthmatic responses. Am Rev Respir Dis 1987;
136 (3):740-51.
67
ONLINE REPOSITORY TABLE
Sex
Age
(Years)
Predicted
FEV1
(%)
Baseline
Methacholine
PC20
(mg/ml)*
Post-Diluent
Methacholine
PC20
(mg/ml)*
Post-Allergen
Methacholine
PC20
(mg/ml)*
Allergen
Extract
Inhaled
Dilution
of Inhaled
Extract
Early
Asthmatic
Response (%)
Late
Asthmatic
Response (%)
Subject
Classification
M
F
24
21
99
83
2.3
13
2.6
10
6.7
3.0
1:128
1:16
22
32
3
9
IER
IER
M
F
M
F
F
F
M
36
22
25
19
21
47
29
88
93
86
97
91
84
104
5.7
1.4
0.43
1.2
5.3
0.41
0.39
9.8
1.2
0.74
0.51
3.3
0.46
0.36
12
0.80
0.68
0.34
7.8
0.16
0.38
1:512
1:64
1:1024
1:8
1:64
1:256
1:8
64
43
29
21
31
22
49
19
8
0
2
25
43
14
DR
IER
IER
IER
DR
DR
IER
M
M
M
F
63
28
23
43
88
85
83
91
5.9
3.6
0.48
4.2
6.2
2.8
1.6
3.2
11
1.8
0.29
1.9
1:128
1:64
1:128
1:32
34
22
35
22
16
22
19
15
DR
DR
DR
DR
F
F
18
21
102
106
2.4
8.6
8.6
17
9.4
0.50
1:128
1:16
26
36
15
23
DR
DR
M
M
F
F
Mean
±
SEM
50
53
27
21
31±3
96
96
93
83
92±2
19
1.1
6.2
5.6
2.6
6.9
0.61
9.7
3.02
2.6
5.4
0.6
14
1.38
1.6
GRASS
RAGWE
ED
GRASS
TREES
HORSE
HDM
HDM
CAT
RAGWE
ED
CAT
HDM
HDM
RAGWE
ED
HDM
RAGWE
ED
CAT
HDM
HDM
HDM
1:64
1:32
1:4
1:1024
37
27
41
23
32.4±3.1
19
16
17
56
18±3.7
DR
DR
DR
DR
68
Table E1: Subject Characteristics
DR, Dual Responder; F, female; HDM, house dust mite; IER, Isolated Early Responder; M, male. * Geometric mean.
69
Ph.D. Thesis- B.M. Salter
McMaster University- Medical Sciences
ONLINE REPOSITORY FIGURES
Figure E1- Basophil Gating Strategy
Representative whole blood dot plots showing basophil gating strategy used for
flow cytometry analysis. (A) SSC vs. FSC; (B) SSC vs. CD45 Alexa Fluor; (C) SSC vs.
HLA-DR PerCP Cy5.5; (D) SSC vs. IL-3Rα PE Cy7; (E) Dot plot demonstrating isotype
vs. specific CD203c APC expression; and (F) Histogram demonstrating isotype vs.
specific CD203c APC expression on basophils.
62
Ph.D. Thesis- B.M. Salter
McMaster University- Medical Sciences
Isolated Early Responders
0
*
*
*
*
FEV1
(% Change)
*
-20
*
*
*
Diluent
Allergen
-40
0
1
2
3
4
5
6
7
*
*
6
7
Time
Dual Responders
FEV1
(% Change)
0
*
-10
*
*
*
*
*
-20
**
*
-30
-40
0
1
2
3
4
5
Time
Figure E2- Allergen inhalation induces airway changes in mild allergic asthmatics.
Percent change in FEV1 in (A) Isolated Early Responders (N=6) and (B) Dual
Responders (N=13). Data presented as meanSEM. * Significantly different from diluent
control; p<0.05.
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Ph.D. Thesis- B.M. Salter
McMaster University- Medical Sciences
B. Dual Responders
A. Isolated Early Responders
100
CD203c + (%)
80
*
60
40
20
80
100
80
80
CD63 + (%)
100
20
60
40
20
0
0
100
100
80
80
60
40
20
0
Pre
3 Hr
7 Hr
Post
*
20
0
40
*
40
0
60
*
60
CD69 + (%)
CD69 + (%)
CD63 + (%)
CD203c + (%)
100
60
40
20
0
24 Hr
Diluent
Allergen
Pre
3 Hr
7 Hr
24 Hr
Post
Figure E3- Effects of allergen inhalation on percentage of basophils expressing
activation markers
Percentage of basophils expressing CD203c, CD63 and CD69 before, 3 h, 7 h and
24 h post-allergen (black bars), compared to diluent (open bars) in (A) isolated early
responders (N=6) and (B) dual responders (N=13). Data are presented as meanSEM. *
Significantly different from diluent control; p<0.05.
64
Ph.D. Thesis- B.M. Salter
McMaster University- Medical Sciences
Baseline Pre-Allergen
A.
CD203c + (sMFI)
2000
1500
1000
500
0
IER
DR
CD63 + (sMFI)
B.
*
20000
15000
10000
5000
0
CD203c + (%)
C.
IER
DR
IER
DR
100
80
60
40
20
0
CD63 + (%)
D.
100
80
60
40
20
0
IER
DR
Figure E4- Pre-Allergen Basophil Expression of Activation Markers of Isolated
Early versus Dual Responders
Basophil (A) CD203c sMFI, (B) CD63 sMFI, (C) CD230c % cell expression, and
(D) CD63 % cell expression pre-allergen of IER (N=6) (open circle) compared to DR
(N=13) (closed circle). Data are presented as meanSEM. * Baseline of IER and DR are
statistically significant if p<0.05.
65
Ph.D. Thesis- B.M. Salter
McMaster University- Medical Sciences
CD203c + (sMFI)
A.
1500
P=0.18
R=0.106
1000
500
0
70
80
90
100
120
Predicted FEV1 (%)
B.
25000
CD63 + (sMFI)
110
P=0.31
R=0.06
20000
15000
10000
5000
0
70
80
90
100
110
120
Predicted FEV1 (%)
CD203c + (%)
C. 100
P=0.026
R=0.265
80
60
40
20
0
70
80
90 100 110
Predicted FEV 1 (%)
120
Figure E5- Relationship between pre-allergen CD203c and CD63 expression on
circulating basophils and Predicted FEV1 (%).
Circulating basophil (A) CD203c sMFI, (B) CD63 sMFI, and (C) CD63 % cell
expression from Isolated Early Responders (open circle) (N=6) and Dual Responders
(closed circle) (N=13) at pre-allergen compared to Predicted FEV1 (%). Data are
presented as meanSEM.
66
Ph.D. Thesis- B.M. Salter
CHAPTER 3:
McMaster University- Medical Sciences
Thymic stromal lymphopoietin activation of basophils in
allergic asthma is IL-3 dependent
Accepted to Journal of Allergy and Clinical Immunology. March 2015.
Basophil numbers within the periphery and airways are significantly higher in
asthmatics compared to healthy patients [135-141], and further increase during asthma
exacerbation [120, 142]. Studies have also shown increased infiltration of basophils
during the LAR following allergen inhalation into the airways [120], where they act as a
significant source of IL-4 and IL-13 [103-109]. There is emerging evidence that
basophils both produce and respond to TSLP [66, 106, 130, 217]. The relationship
between human basophils and TSLP during the LAR needs to be further examined. Our
study attempts to better understand the role of basophils in the LAR and determine
whether interaction with TSLP can further potentiate their pro-inflammatory activity. We
investigated this by measuring the change in basophil activity following allergen
inhalation in multiple compartments, and further examined the change in basophil
TSLPR surface expression to understand how allergen inhalation can influence the
sensitivity of basophils for TSLP. Lastly, we determined the in vitro effect of TSLP on
basophil activity. We observed that allergen inhalation significantly increased airway
basophil numbers, and this influx of basophils may have been primed by TSLP. We have
also shown that basophils exhibit up-regulated surface TSLPR and activation marker
expression, as well as Type 2-cytokine intracellular expression during the LAR, which is
consistent with their in vitro response to TSLP stimulation. We conclude that initiation
and potentiation of basophil pro-inflammatory activity during the LAR post-allergen
inhalation in human allergic asthma is partially due to the interaction with TSLP.
67
Ph.D. Thesis- B.M. Salter
McMaster University- Medical Sciences
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McMaster University- Medical Sciences
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McMaster University- Medical Sciences
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McMaster University- Medical Sciences
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McMaster University- Medical Sciences
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McMaster University- Medical Sciences
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McMaster University- Medical Sciences
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McMaster University- Medical Sciences
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McMaster University- Medical Sciences
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McMaster University- Medical Sciences
ONLINE REPOSITORY MATERIALS AND METHODS
Subject Exclusion Criteria
Subjects were excluded if they were current or ex-smokers with more than 10
pack-years, developed lower respiratory infections or used inhaled or oral steroids 4
weeks prior to study. Anti-histamines, caffeine, and non-steroidal anti-inflammatory
agents were prohibited 48 hours before study visits.
Methacholine Challenge
The methacholine inhalation challenge was carried out by tidal breathing from a
Wright nebulizer.E1 Doubling concentrations of methacholine chloride (Methapharm,
ON) were inhaled orally from a Hans Rudolph valve at 2 min intervals. The FEV1 was
measured following inhalation at 30 s and 90 s or until it stopped falling. The percent fall
was calculated from the post-diluent FEV1 value. The test was terminated when a fall in
FEV1 of at least 20% of the lowest post-saline value occurred. The methacholine PC20
was then calculated using linear interpolation.
Allergen and Diluent Challenge
Allergen challenges were conducted by administering doubling inhaled
concentrations of allergen extract, as previously describedE2 and diluent challenges were
conducted using 3 inhalations of 0.9% saline for 2 min each. The early asthmatic
response was the largest percent fall in FEV1 between 0 and 2 h, and the late asthmatic
response was the largest percent fall in FEV1 between 3 and 7 h post-challenge.
1-h Dose-Response Basophil Cultures
Purified basophils were cultured for 1 h at 37ºC with increasing concentrations of
TSLP (1, 10, 100 ng/mL) (R&D Systems). Following incubation, the cell pellet was
collected, then washed with PBS and centrifuged at 300 g for 10 min at 4ºC. The cell
77
Ph.D. Thesis- B.M. Salter
McMaster University- Medical Sciences
pellet was then re-suspended in FACS buffer and immunostained to assess CD203c
expression by flow cytometry. In addition, the supernatant was collected from culture to
measure basophil histamine release via a commercially available ELISA (IBL-America).
Basophils heated for 10 min at 90ºC provided a measure of total histamine release.
18-h Basophil Cultures
Purified basophils were incubated for 18 h at 37ºC in RPMI-C with the golgiblocking agent, monensin (Biolegend) with or without increasing concentrations of TSLP
(0.1, 1, 10, 100 ng/mL). Basophils were immunostained to measure extracellular
receptors CD203c, CCR3, and TSLPR, and intracellular cytokine expression of IL-3, IL4, IL-5, IL-13, and GM-CSF by flow cytometry.
Flow cytometry staining and analysis
Cells for flow cytometry experiments were immunostained with isotype controls
or antibodies to extracellular receptors CD45, HLA-DR, CCR3 (BD Biosciences),
TSLPR, IL-3Rα (Ebioscience), CD203c (MACS Miltenyi) for 30 min at 4ºC. To measure
intracellular cytokine expression, cells were washed, fixed and permeabilized, then
stained with isotype controls or antibodies to IL-4, IL-13 (Ebioscience), GM-CSF (BD
Biosciences), IL-3, IL-5 (R&D Systems). Cells were washed and acquired with a LSR II
flow cytometer (BD Biosciences). Analyses were performed using Flow-Jo software
(TreeStar). Basophils were defined as the CD45+/HLA-DR-/IL-3Rα+ population
(Supplementary Figure 2). For whole staining of blood and bone marrow, at least 300,
000 CD45+ events were acquired, with the collection of the same cell number for each
time-point pre/post-diluent/allergen challenge per subject. Similarly, at least 1, 000, 000
events were acquired for sputum samples, with the same cell number collected for each
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time-point pre/post-diluent/allergen challenge per subject. Lastly, for the in vitro culture
studies, at least 10, 000 CD45+ events were acquired per culture condition.
Statistical Analysis
All data in the manuscript are presented as mean ± SEM. In this study the EAR
and LAR were assessed as the maximum allergen-induced fall in FEV1 %, based on
analysis of the minimum FEV1 % remaining over 0-3 h and 3 to 7 h, respectively, postchallenge.
Methacholine PC20 values were log-transformed before analyses, and expressed as
the geometric mean and range. The Methacholine PC20 between Day 1/Day 3 of both
diluent and allergen inhalation challenges were compared.
Flow cytometry results are expressed as mean percent positive cells. In cases
where basophils constitutively express the marker of interest the results are expressed as
specific Mean Fluorescent Intensity (sMFI). Histamine is expressed as percent of total
histamine release. The cytokines measured by ELISA are shown as concentration
(pg/mL).
Statistical analysis for the inhalation challenges was performed using 2-way
ANOVA and post-hoc Bonferroni test. The in vitro experiments were analyzed using 1way ANOVA with a post-hoc Tukey test. Significance was accepted at P<0.05.
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ONLINE REPOSITORY TABLE
Sex
Age (y)
% Predicted Methacholine
FEV1
PC20, mg/mL
Ag Inhaled
Final [Ag]
Inhaled
M
61
96.00
0.66
CAT
1:32
F
24
80.00
1.46
RGW
1:16.15
M
52
95.00
0.11
HDM
1:68
F
40
104.00
0.18
CAT
1:120
M
25
86.00
1.44
RGW
1:83
F
24
93.00
0.45
HDM
1:84
M
26
93.00
0.33
HDM
1:32
M
20
94.00
3.71
GRS
1:4
F
34
70.00
0.26
CAT
1:512
M
26
96.00
0.64
CAT
1:83
Mean
90.7±9.6
Geometric
Mean= 0.55
Range= 0.11 to
3.71
No.
Mean
M/F 6:4 33.2±13.6
Table I: Subject Characteristics
Ag, Allergen; EAR, early asthmatic response; F, female; HDM, house dust mite; LAR, late
asthmatic response; M, male; RGW, ragweed; GRS, grass. Data presented as mean ±
SEM for Age and FEV1 and geometric mean with range for methacholine PC20.
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ONLINE REPOSITORY FIGURE LEGNEDS AND FIGURES
Figure E1. Basophil purity post-isolation. (A-D) Representative dot plots showing flow
cytometric analysis of basophil purity through gating around CD45+/CD123+(IL3Rα)/HLA-DR- population. The final purity after gating around the basophil population
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is 98. 8%. (E) Representative toluidine blue-stained cells at 40x magnification (0.65mm
diameter field).
Figure E2. Basophil gating strategy. Representative whole blood dot plots showing
strategic basophil gating used for flow cytometry analysis. (A) SSC vs. CD45 Alexa
Fluor 700 (B) SSC vs. HLA-DR APCH7 (C) SSC vs. CD123 (IL-3Rα) PECy7. (D)
Histogram demonstrating isotype vs specific antibody for assessment of receptor
expression on basophils.
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B
100
* P<0.05
R=0.542
80
60
40
20
0
0
10
20
30
% CD203c + 24hr Post-Ag
% CD203c + 7hr Post-Ag
A
40
100
80
60
40
20
0
0
LAR % Fall in FEV 1
10
20
30
LAR % Fall in FEV 1
40
2
4
Doubling Dose PC 20
6
D
% CD203c + 24hr Post-Ag
% CD203c + 7hr Post-Ag
C
* P<0.05
R=0.567
100
80
60
40
20
0
0
2
4
Doubling Dose PC 20
6
100
80
60
40
20
0
0
Figure E3. Correlation between CD203c basophil expression and magnitude of allergeninduced responses. Peripheral blood basophil expression of CD203c at 7 and 24 hours
post-allergen is positively related to maximum % fall in FEV1 during the late asthmatic
response 3-7h post-allergen (A and B) but is not related to the doubling dose shift in
methacholine PC20 from pre- to 24 hours post-allergen (C and D). Data are presented as
meanSEM.
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B
100
%TSLPR+ 24hr Post-Ag
80
60
40
20
0
0
C
%TSLPR+ 7hr Post-Ag
* P<0.05
R=0.426
10
20
30
80
60
40
20
2
4
Doubling Dose PC20
60
40
20
D
100
10
20
30
40
LAR % Fall in FEV 1
100
80
60
40
20
0
0
6
* P<0.05
R=0.516
80
0
0
LAR % Fall in FEV 1
0
0
100
40
%TSLPR+ 24hr Post-Ag
%TSLPR+ 7hr Post-Ag
A
2
4
Doubling Dose PC 20
6
Figure E4. Correlation between TSLPR basophil expression and disease severity.
Peripheral blood basophil expression of TSLPR at 7 and 24 hours post-allergen is
positively related to maximum % fall in FEV1 during the late asthmatic response 3-7h
post-allergen (A and B) but is not related to the doubling dose shift in methacholine PC 20
from pre- to 24 hours post-allergen (C and D). Data are presented as meanSEM.
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A
B
* P<0.05
R=0.412
40
% IL-13 + 24hr Post-Ag
% IL-4 + 24hr Post-Ag
50
30
20
10
0
0
10
20
30
50
40
30
20
10
0
0
40
LAR % Fall in FEV 1
* P<0.05
R=0.629
10
20
30
LAR % Fall in FEV1
40
Figure E5. Correlation between intracellular expression of IL-4 and IL-13 in peripheral
blood basophils and magnitude of allergen-induced late asthmatic response. Peripheral
blood basophil expression of IL-4 (A) and IL-13 (B) is positively related to maximum %
fall in FEV1 during the late asthmatic response 3-7h post-allergen. Data are presented as
meanSEM.
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A
% CD203c +
100
*
80
*
60
40
20
0
% Total Histamine Release
B
100
*
*
80
60
40
20
0
PBS
TSLP (ng/mL)
+
-
+
1
+
10
+
100
Figure E6. The effects of 1 hour stimulation of purified peripheral blood basophils with
PBS (open bars) or increasing concentrations of TSLP (0.1, 1, 10, 100 ng/mL) (black
bars), on the expression of CD203c (A) and histamine release (B). Data expressed as
meanSEM. * P<0.05 compared to PBS control.
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Figure E7. The effects of 18 hours incubation of purified peripheral blood basophils with
PBS (open bars) or increasing concentrations of TSLP (0.1, 1, 10, 100 ng/mL) (black
bars), on the expression of CD203c (A), IL-3Rα (B), CCR3 (C), TSLPR (D), IL-4 (E),
and IL-13 (F). Data expressed as meanSEM. * P<0.05 compared to PBS control.
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Figure E8. The effects of 18 hours incubation of purified peripheral blood basophils with
PBS (open bars) or 10 ng/ml TSLP on the intracellular expression of eosinophilpoietic
cytokines IL-3, IL-5, and GM-CSF. Data expressed as meanSEM. * P<0.05 compared
to PBS control.
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Figure E9. TSLP does not directly induce shape change in basophils measured at 0, 30,
45, 60, and 180 seconds after stimulation with 10 ng/ml TSLP. Data expressed as
meanSEM. * P<0.05 compared to PBS control.
ONLINE REPOSITORY REFERENCES
E1. Cockcroft DW. Measure of airway responsiveness to inhaled histamine or
methacholine; method of continuous aerosol generation and tidal breathing inhalation. In:
Hargreave FE, Woolcock AJ, eds. Airway responsiveness: measurement and
interpretation. Mississauga: Astra Pharmaceuticals Canada Ltd, 1985: 22 - 28.
E2. O'Byrne PM, Dolovich J, Hargreave FE. Late asthmatic responses. Am Rev Respir
Dis 1987; 136 (3):740-51.
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CHAPTER 4: IL-25 and IL-33 promote basophil effector function in allergic asthma
Submitted to Allergy. August 2015.
Emerging reports have shown IL-33 and IL-25 to have potent pro-inflammatory
effects on human basophil activity. For example, IL-25 inhibits basophil apoptosis and
promotes IgE-mediated degranulation [260]. IL-33 can induce IL-4 and IL-13 secretion,
and degranulation independently or in synergy with IL-3 and anti-IgE [83, 284, 314-316].
Our study aimed to expand on these previous findings and examine the effects of IL-25
and IL-33 on basophil activity.
Previous studies have reported that asthmatics express greater levels of IL-25, IL33, and their respective receptors within the peripheral blood and airways, compared to
healthy subjects [215, 257-260, 291-302]. Although previous in vitro studies have
confirmed the surface expression of IL-17RB and ST2 on basophils [83, 284, 314-316,
260], no studies thus far have investigated the change in basophil IL-17RB and ST2
surface expression in the context of allergic asthma. We sought to evaluate the change in
basophil surface expression of IL-17RB, ST2, and intracellular IL-25 following allergen
inhalation in mild allergic asthmatic patients.
We found significant long-term in vitro effects of IL-33 and IL-25 on basophils,
including up-regulation of basophil surface activation marker expression, intracellular
Type 2 cytokine expression, and priming of basophil migration to eotaxin. Following in
vivo allergen inhalation, there was up-regulated IL-17RB, ST2, and intracellular IL-25
expression on basophils. Lastly, due to the difficulty in detecting measurable levels of IL33 and IL-25 within the different biological compartments, we used an alternative assay
to assess the basophil response to the airway microenvironment during the LAR and
determine whether blockade of alarmin cytokines could inhibit this response. Sputum
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supernatant collected 7 hours post-allergen inhalation yielded the most significant
increase in basophil CD203c and Type 2 cytokine intracellular expression, which was
inhibited with blockade of the βc..These findings suggest that following allergen
inhalation, the airway microenvironment has the ability to up-regulate basophil proinflammatory activity, thus promoting the effector role of basophils in the LAR. In
summary, we conclude that the increased pro-inflammatory activity of basophils during
the LAR (reported in Chapters 2 and 3) may be, in part, due to interacting with IL-25 and
IL-33.
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Original Article
Title:
IL-25 and IL-33 Induce Type 2 Inflammation in Basophils from Subjects with Allergic
Asthma
Short Title:
Basophils and alarmin cytokines in allergic asthma
Brittany M. Salter, BSc,1 John Paul Oliveria, BSc,1 Graeme Nusca, BSc,1 Steve G. Smith,
PhD,1 Damian Tworek, MD, PhD,1 Patrick D Mitchell MD,1 Rick M. Watson, BSc,1
Roma Sehmi, PhD,2 Gail M. Gauvreau, PhD1
1
Department of Medicine, McMaster University, Hamilton, ON, Canada
2
Firestone Institute for Respiratory Health, St Josephs, Hamilton, ON, Canada
Corresponding author:
Gail M Gauvreau, McMaster University, HSC 3U26, 1200 Main St West, Hamilton, ON
Canada
Telephone: 905-525-9140 x22791
Fax: 905-528-1807
E-mail: gauvreau@mcmaster.ca
Key Words: Allergic Asthma, Basophils, Alarmin Cytokines, IL-25, IL-33
Abbreviations Used
CCR3, Chemokine Receptor-3
FACS, Fluorescence-activated cell sorting
SSC, Side angle light scatter
FSC, Front angle light scatter
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IL, Interleukin
EAR, Early Asthmatic Response
LAR, Late Asthmatic Response
RT, Room Temperature
H, Hour
Min, Minute
S, Second
ST2, IL-33 Receptor
IL-17RB, IL-25 Receptor
iNKTs, Innate natural killer T cells
βc, Common β-chain
TSLP, Thymic stromal lymphopoietin
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ABSTRACT (250 words)
Background: The alarmin cytokines IL-25 and IL-33 are key promoters of type-2
inflammation. In human studies, basophils respond to alarmin cytokines, however the
relationship of these cytokines with basophil activation and recruitment in allergic
asthmatic responses has not been well characterized. The purpose of this study was to
investigate the effect of IL-25 and IL-33 stimulation on human basophil activity in the
context of allergic asthma.
Methods: 10 mild allergic asthmatics underwent allergen and diluent inhalation
challenges. Bone marrow, peripheral blood, and sputum samples were collected at prechallenge, 7 hours (h), and 24 h post-challenge to measure basophil expression of IL17RB, ST2, and intracellular IL-25. Freshly isolated peripheral blood basophils from
allergic donors were incubated overnight with IL-25 and IL-33, or with sputum
supernatant collected post-allergen to assess pro-inflammatory effects of mediators
released in the airways.
Results: There was elevated expression of IL-17RB, ST2, and intracellular IL-25 in
basophils collected from bone marrow, peripheral blood, and sputum after allergen
inhalation challenge. In vitro stimulation with IL-25 and IL-33 increased intracellular
expression of type 2 cytokines and activation markers, and primed eotaxin-induced
migration of basophils, which was mediated directly through IL-17RB and ST2,
respectively. Stimulation of basophils with sputum supernatants collected post-allergen
challenge up-regulated markers of activation and intracellular type 2 cytokine expression,
which was reversed following blockade of the common β chain (βc).
Conclusion: Our findings indicate that exposure to inhaled allergen leads to activation
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and migration of basophils, which appears to be mediated at least in part through
interaction with IL-33 and IL-25.
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INTRODUCTION
Asthma is a chronic respiratory disease characterized by reversible airway
obstruction, airway inflammation, and airway hyperresponsiveness (AHR). There are
numerous causes of this disorder, and recent evidence indicates that alarmin cytokines
may play a key role in promoting allergic asthma.
IL-25 and IL-33 have been identified as critical immuno-regulatory cytokines that
activate immune cells involved in allergic inflammation. In mouse models, transgenic
over-expression of IL-25 and IL-33 generates airway eosinophilia, up-regulated airway
Type 2 cytokine expression, elevated IgE levels, and mucus hypersecretion.1-4 Moreover,
studies have shown that administration of IL-25 and IL-33 induces airway eosinophilia,
increased airway Type 2 cytokine production, mucus hypersecretion, goblet cell
hyperplasia, and AHR.5, 6 Neutralization of IL-25 and IL-33 in mice leads to reduction of
airway inflammation, IgE levels, Type 2 cytokine expression, goblet cell hyperplasia, and
AHR.7-10
In both human and animal in vitro models, IL-25 and IL-33 have been shown to
exert their effects on progenitor cells, mast cells, granulocytes, lymphocytes, and
dendritic cells.11-20 In human studies of allergic asthma, IL-33 and ST2 expression in
serum, lung tissue, and BALF was found to be higher in asthmatics compared to healthy
controls, and correlated with asthma severity.21-25 Similarly, levels of IL-25 and IL-17RB
mRNA expression are elevated in serum, bronchial mucosa, and the skin, which
correlates with disease severity.21-26
Basophils are involved in the early asthmatic response (EAR) of asthma, acting as
potent sources of histamine and cysteinyl leukotrienes, which contribute to
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bronchoconstriction. Emerging evidence suggests that basophils have an important role in
promoting delayed airway inflammation. Peripheral blood and airway basophils increase
in number and activation in LAR following an allergen inhalation, and they are a
significant source of IL-4 and IL-13 within the periphery.27-30 We have previously
reported that basophil expression of activation markers (CD203c, CCR3, IL-3Rα),
intracellular cytokines (IL-4; IL-13), and TSLPR, significantly increase in peripheral
blood and sputum following allergen inhalation in mild allergic asthmatics for up to 24
h.28 Furthermore, we shown for the first time in vitro that TSLP can promote human
basophil activation via up-regulation of CD203, CCR3, IL-3Rα, and intracellular IL-4
and IL-13 expression, as well as prime migration in response to eotaxin.28 Despite these
findings, it is not well known what factors promote basophil lung homing and effector
activity during the LAR, but various reports suggest that the epithelium may play a role
in regulating basophil activity. Protein and mRNA expression of ST2 and sST2 has been
confirmed on basophils, and becomes up-regulated following stimulation with IL-33 or
IL-3.16,
31-34
In vitro stimulation with IL-33 promotes basophil IgE-dependent and
independent release of histamine, and secretion of cytokines IL-4, IL-8, IL-5, IL-9, IL-6,
IL-13, MCP, MIP.16, 31-34 IL-33 has also been shown to induce human basophil CD11b
expression, adhesion, and prime eotaxin-induced migration, suggesting that IL-33 may
regulate basophil lung-homing.31 Human basophils have been found to produce IL-25
following IgE cross-linking, and constitutively express IL-17RB, which can be upregulated following IL-3 stimulation.35, 36 Lastly, IL-25 can affect human basophils by
inhibiting apoptosis and promoting IgE-mediated degranulation.36
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Despite IL-33 and IL-25 being identified as important promoters of Type 2-type
inflammation in models of allergic asthma, the influence of these cytokines on the
effector role of basophils in allergic asthma needs to be further characterized.
Furthermore, little is known about the response of basophils to the airway
microenvironment, such as mediators released by the epithelium and other inflammatory
cells, following allergen inhalation. The purpose of this study was to determine the
relationship between basophils and alarmin cytokines in human allergic asthma, and to
examine the basophil response to airway secretions following allergen inhalation in mild
allergic asthmatics.
METHODS
Allergen Challenge Study Design
10 subjects with mild allergic asthma underwent inhalation challenges with
allergen and diluent control (Subject characteristics in Online Supplement, Table E1). All
subjects had a positive skin test to common aeroallergens, methacholine PC20 <16
mg/mL, FEV1 of ≥70% of predicted and, and a dual phase response to allergen (a fall in
FEV1 of ≥20% by 2 h and ≥15% 3-7 h post allergen). Subjects used only short-acting
bronchodilators for control of asthma, and were excluded from the study according to
specific criteria outlined in Online Supplement.
Baseline samples of peripheral blood, bone marrow aspirate, and sputum samples
were collected on day 1 and subjects were randomized to diluent or allergen challenge on
day 2. At 7 h post-challenge blood and sputum was collected, and at 24 h post-challenge
bone marrow, blood and sputum samples were collected. AHR to allergen was measured
by a shift in methacholine PC20 value measured before and 24 h after challenge. Allergen
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and diluent challenges were conducted as previously described (Online Supplement
Methods) and separated by 1-2 weeks.37 Subjects returned after the wash out period to
donate 100 mL blood for in vitro stimulation of basophils with airway samples.
Basophils for IL-25/IL-33 in vitro experiments were purified from 100mL of
blood from 8 donors, confirmed to be allergic through positive skin testing to common
aeroallergens. The study was approved by Hamilton Integrated REB and all subjects
provided signed informed consent.
Bone marrow, Blood, and Sputum Sample Processing
During allergen anddiluent inhalation challenges, 10 mL of blood was collected
into sodium heparin vaccutainers and 10 mL of bone marrow was aspirated from the iliac
crest into heparin (1,000 U/mL). In order to semi-isolate the basophils from bone
marrow, the samples were diluted in McCoys 5A, layered on Lymphoprep and
centrifuged at 2200 rpm for 20 minute (min) at room temperature (RT).
Sputum samples were induced using hypertonic saline and mucous plugs were
selected from samples followed by dispersement with DPBS, as previously described.38
Sputum samples were centrifuged at 790 g 4ºC for 10 min, followed by collection of
supernatant, centrifugation at 1500 g 4ºC for 10 min, and aliquoting of supernatant into
eppendorfs that were frozen at -20ºC until later use for in vitro experiments. Cytospins
were prepared from sputum cells and stained with Diff-Quick for differential cell counts
and with toluidine blue for metachromatic cell counts.
Multiplex Immunoassay kits were used to measure IL-33 and IL-25 in bone
marrow, peripheral blood, and sputum samples collected pre and post-challenges.
Peripheral blood, bone marrow, and sputum cells were immunostained and acquired via
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flow cytometry to measure expression of IL-17RB, ST2, and intracellular IL-25 (Online
Supplement Methods and Fig E1).
Purification of Basophils for Cell Cultures
100 mL of blood was diluted with McCoys 5A media, and layered on
Lymphoprep. Erythrocytes were lysed and basophils were purified via negative selection
with magnetic separation beads. Basophil cell suspensions were >98% pure as
determined by flow cytometry and >90% viable by trypan blue.
18 h Basophil Cultures with IL-25 and IL-33
Purified basophils suspended in RPMI-C collected from 8 allergic subjects were
incubated for 18 h at 37ºC in RPMI-C with monensin, with PBS or IL-3, anti-IgE, or a
pre-determined optimal dose of IL-33 or IL-25 (all 10 ng/mL) (Online Supplement Fig
E2; E3), and with/without neutralizing antibodies to ST2 or IL17RB (both 10 µg/mL).
Basophils were immunostained to measure expression of CD203c, CCR3, ST2, IL-17RB,
TSLPR, as well as intracellular IL-4 and IL-13 via flow cytometry (Online Supplement
Fig E1).
Shape Change Assay with IL-33 and IL-25
To determine if IL-33 and IL-25 had a direct effect on basophil shape change, purified
basophils were incubated with/without IL-33 or IL-25 (0.1, 1, 10, 100 ng/mL) at 37ºC in
RPMI-C for 0, 30, 45, 60 or 180 second (s). To determine if IL-33 or IL-25 primed
basophil shape change response to eotaxin, purified basophils were incubated for 18 h at
37ºC in RPMI-C with PBS, IL-33, or IL-25 (all 10 ng/mL). Following incubation, cells
were stimulated with eotaxin (5 ng/mL) or PBS for 0, 30, 45, 60 or 180 s. Basophils were
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fixed with ice-cold 1% PFA for 10 min to stop the reaction, and cell shape change was
measured by the parameter FSC via flow cytometry.
18 h Basophil Cultures with Airway Samples
Purified basophils resuspended in RPMI-C collected from the mild allergic
asthmatic subjects were incubated for 18 h at 37ºC with monensin and sputum
supernatant previously collected from allergen inhalations at baseline, 7 h, and 24 h postchallenge. To measure the effect of receptor blockade isolated basophils were
additionally treated with 10 µg/mL isotype controls or neutralizing antibodies targeting
the βc, TSLPR, IL-17RB, and ST2. Incubation with PBS served as negative control,
whereas incubation with 10 ng/mL IL-3 was the positive control. Basophils were
immunostained to measure CD203c and intracellular expression of IL-13 and IL-4 by
flow cytometry.
Statistical Analysis
All data are presented as mean ± SEM. Flow cytometry results are expressed as
mean percent positive cells. Statistical analysis for the inhalation challenges was
performed using 2-way ANOVA and post-hoc Bonferroni test. In vitro experiments were
analyzed using 1-way ANOVA with a post-hoc Tukey test. Significance was accepted at
P<0.05.
RESULTS
Allergen inhalation induces airway bronchoconstriction, hyperresponsiveness and
inflammation mild allergic asthmatics
Inhalation of allergen induced a maximum fall in FEV1 of 20.1±5.5% within the
first 2 hours, and a maximum fall in FEV1 of 20.8±5.2% 3-7 h post-challenge, compared
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to no change in FEV1 following diluent inhalation. The methacholine PC20 decreased one
doubling dose post-allergen challenge, compared to no change post-diluent.
Compared to diluent, the allergen inhalation challenge induced a significant
increase in the total cell count and number of granulocytes within the sputum (Online
Supplement Table E2). IL-25 could be detected in the bone marrow of 2 subjects, in the
sputum of 3 subjects, and in the blood of only 7 subjects. IL-33 could be detected in the
bone marrow of 6 subjects, in the sputum of 3 subjects, and in the blood of 4 subjects.
We found no significant change in IL-25 or IL-33 levels in bone marrow, sputum or
blood after allergen challenge (data not shown). The measurable levels of IL-25 or IL-33
showed no correlation with allergen-induced change in AHR to methacholine or
maximum fall in FEV1.
Allergen inhalation up-regulates expression of IL-17RB and ST2 in bone marrow,
peripheral blood, and sputum basophils
In the bone marrow, there was a significant increase in the expression of both IL17RB and ST2 at 24 h post-allergen challenge compared to baseline (Fig 1 A). In the
peripheral blood we observed a significant increase in IL-17RB at 7 h post-allergen and
in ST2 expression at 7 and 24 h post-allergen, compared to the diluent (Fig 1 B).
Peripheral blood ST2 expression at 7 and 24 h post-allergen was significantly greater
compared to the baseline (Fig 1 B). Sputum basophil expression of IL-17RB increased
significantly at 24 h post-allergen, whereas no change was found in ST2 expression,
compared to diluent (Fig 1 C). Basophil intracellular levels of IL-25 increased at 24 h
post-allergen in peripheral blood and remained elevated for up to 24 h post-allergen in the
sputum, compared to diluent (Fig 1). Allergen did not change the expression of
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intracellular IL-25 in bone marrow basophils. We observed a significant positive
correlation between allergen-induced maximum % fall FEV1 3-7 h and basophil
peripheral blood ST2 expression measured 24 h post-allergen (P=0.0116, R=0.7552) (Fig
2 B). No relationship was found between the allergen-induced change in AHR to
methacholine and ST2, IL-17RB or intracellular IL-25 expression.
Basophils express functional receptors for IL-33 and IL-25
Basophils from allergic subjects expressed IL-17RB and ST2, and stimulation
with IL-3 and anti-IgE significantly increased expression of these receptors (Fig 3 A; B).
Stimulation with IL-25 and IL-33 markedly up-regulated the expression of their own
receptors, IL-17RB and ST2, respectively (Fig 3 A; B), whereas IL-25 had no effect on
ST2 expression, nor did IL-33 up-regulate IL-17RB expression (Fig 3 A; B). IL-25 and
IL-33 had no effect on TSLPR expression, however TSLP up-regulated IL-17RB and
ST2 expression (Fig 3 C).
IL-33 and IL-25 exert pro-inflammatory effects on basophils
We examined the effects 18 h incubation of IL-25 and IL-33 on basophil
activation and observed significantly higher intracellular expression of IL-4 and IL-13
(Fig 4), as well as CD203c and IL-3Rα surface expression (Fig 4). Treatment with
neutralizing antibodies to IL-17RB and ST2 significantly inhibited IL-25 and IL-33induced markers of basophil activation compared to the isotype control (Fig 4).
IL-25 and IL-33 prime basophil shape-change in response to eotaxin
Compared to the PBS negative control, incubation with IL-33 and IL-25 for 18 h
induced significant up-regulation of CCR3 expression on basophils (Fig 5 A). IL-33 and
IL-25 did not directly stimulate basophil shape change (data not shown), however
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McMaster University- Medical Sciences
overnight incubation of basophils with IL-25 and IL-33 induced a significantly greater
shape change in response to stimulation with eotaxin (Fig 5 B; C).
The priming effect was observed by 45 s following eotaxin stimulation and
maintained for up to 180 s. Addition of blocking antibodies to IL-17RB and ST2
significantly inhibited the priming effects exerted by IL-33 and IL-25 on eotaxin-induced
basophil shape change compared to the isotype control (Fig 5 D; E).
Sputum supernatant samples collected post-allergen inhalation induce basophil
activation
Commercial multiplex immunoassays were unable to detect IL-33 and IL-25
levels in airway samples, thus we used an alternative assay evaluating the in vitro effect
of airway secretions collected after allergen inhalation on basophil activation. Airway
samples collected 7 h post-allergen had the greatest effect on basophil activity as shown
by increased expression of CD203c, and intracellular IL-4 and IL-13 compared to PBS
control and compared to 0 h samples (Fig 6A). There was no difference between the
effect of IL-3 (positive control) and 7 h post-allergen airway samples on basophil
activation. Incubation with 0 h airway samples significantly induced CD203c and
intracellular IL-4 expression (Fig 6 A; B), whereas 24 h airway samples induced only
intracellular IL-4 expression (Fig 6 B).
To ascertain the mechanisms through which the airway microenvironment
promotes basophil activation post-allergen inhalation, we determined whether airway
sample-induced basophil activation could be inhibited following treatment with
neutralizing antibodies to the βc for IL-3/IL-5/GM-CSF, and the receptors for alarmin
cytokines ST2, and IL-17RB. Blockade of the βc significantly inhibited the effects
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McMaster University- Medical Sciences
induced by 7 h post-allergen airway samples
(Fig 7). Treatment with neutralizing
antibodies to TSLPR (Fig 7 A), ST2 (Fig 7 B), or IL-17RB (Fig 7 B) alone did not inhibit
the stimulatory effects of 7 h post-allergen airway samples on basophil activation.
Although treatment with TSLPR, ST2, or IL-17RB in combination with βc significantly
inhibited the stimulatory effects, it was not significantly more than βc alone (Fig 7).
Discussion
Previous studies have shown that basophil express ST2, which can be upregulated following in vitro stimulation with IL-33 or IL-3.16, 31-34, 36 We expanded on
these findings by demonstrating that IL-25 and IL-33 up-regulated expression of their
own receptor on basophils. Interestingly, IL-3, anti-IgE, and TSLP stimulation upregulated expression of IL-17RB and ST2, however IL-25 and IL-33 had no effect on
TSLPR expression, suggesting that these cytokines act downstream of TSLP. We have
shown that the sensitivity of basophils to IL-33 and IL-25 can be mediated through IgE or
by IgE-independent mechanisms.
Other studies have reported that IL-33 and IL-25 induce pro-inflammatory effects
on human basophil activity.16, 31-34, 36 IL-25 inhibits basophil apoptosis and promote IgEmediated degranulation, but does not induce IL-4 or IL-13 release.36 IL-33 promotes IgEdependent and independent basophil histamine degranulation, and the release of type 2
cytokines, independently or in synergy with IL-3 and anti-IgE.16, 31-34 Although Wang et
al did not find IL-25 to induce basophil Type 2 cytokine release, we observed increased
intracellular IL-4 and IL-13 cytokine expression. Discrepancies between these studies
may be due to differences in time-points (12 h vs. 18h) and the use of intracellular
cytokine measurement via flow cytometry as opposed to ELISA measurement of culture
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Ph.D. Thesis- B.M. Salter
McMaster University- Medical Sciences
supernatants. In addition, is it possible that another stimulus may be required for these
cytokines to be released from basophils. We also confirm previous reports that overnight
incubation of basophils with IL-33 increased intracellular production of IL-4 and IL-13,
but also showed up-regulation of surface activation markers CD203c and IL-3Rα. IL-25
and IL-33-induced basophil activity was inhibited following blockade of IL-17RB and
ST2, demonstrating the effects of these cytokines are, in part, mediated through their own
functional receptors. The up-regulation of intracellular Type 2 cytokine and surface
activation marker expression on human basophils demonstrates a primary role of IL-25
and IL-33 in promoting the pro-inflammatory activity of these cells.
Suzukawa et al. previously demonstrated that stimulation with IL-33 could
enhance basophil migration towards eotaxin,33 however previous studies have not shown
an effect of IL-25 on basophil migration. In this study, using shape change as a surrogate
marker for migration, as previously described,28, 39 we demonstrated that IL-25 and IL-33
prime basophil migration to eotaxin through up-regulation of CCR3 expression.
Suzukawa et al. found no increase in basophil CCR3 expression following 1 h
stimulation with IL-33, whereas we found that 18 h of IL-33 stimulation up-regulated
receptor expression. Variation between these findings could be explained by the different
stimulation time-points of the cultures. We have previously shown that following allergen
inhalation, bone marrow basophils had a decrease in CCR3 (and CD203c) expression at
24 h post-allergen, whereas CCR3 (and CD203c) expression was up-regulated on both
peripheral blood and sputum basophils.28 Taken together with increased sputum basophil
numbers post-allergen, this suggests that a subset of activated basophils expressing
CCR3, migrate in response to eotaxin from the bone marrow into the airways following
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McMaster University- Medical Sciences
allergen inhalation. This coincides with the mechanisms of eosinophil migration from the
bone marrow,40 elevated peripheral eotaxin levels by 5 h post-allergen challenge,41 and
with our findings that IL-25 and IL-33 up-regulate CCR3 basophil expression following
incubation for 18 h.
During the LAR, peripheral and luminal basophils increase in number, activation,
and expression of IL-4 and IL-13 post-antigen activation.27-30, 42 We previously reported
that during the LAR from 7 – 24 h post-allergen, circulating and sputum basophils
express up-regulated CD203c, IL-3Rα, as well as intracellular IL-4 and IL-13.28 Given
that our in vitro studies show IL-25 and IL-33 promote basophil intracellular Type 2
cytokine expression, it is possible that the increased expression seen following allergen
inhalation may be due to interaction with alarmin cytokines. Most importantly, we have
shown up-regulation of basophil expression for IL-17RB within the bone marrow,
peripheral blood, and airways, as well as ST2 within the bone marrow and peripheral
blood post-allergen challenge. These findings are similar to our previous reports that
basophil TSLPR expression increases following allergen inhalation for up to 24 h. 28
Moreover, 24 h post-allergen ST2 basophil expression had a significant positive
correlation with allergen-induced bronchoconstriction. This is similar to our previous
reports that peripheral blood TSLPR basophil expression correlates with maximal %
FEV1 fall during the LAR.28 These findings suggest that interaction between basophils
and alarmin cytokines may be a contributing factor to declined lung function during the
LAR. Basophils have been identified as a source of IL-25,35 and we found intracellular
IL-25 expression to be elevated within the periphery and sputum post-allergen, which
may further contribute to Type 2 inflammation. In contrast, we found no change in
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Ph.D. Thesis- B.M. Salter
McMaster University- Medical Sciences
sputum basophil ST2 expression following allergen challenge. It is possible that
increased IL-33 levels within the periphery and airways drive basophil activity rather
than increase receptor expression for IL-33.
The final component of this study was to examine basophil responsiveness during
the LAR to the airway microenvironment, such as mediators released by the epithelium
and other inflammatory cells. Overnight incubation of isolated human basophils with 7 h
post-allergen sputum supernatant yielded the most increase in CD203c, and intracellular
IL-13 and IL-4. The 0 h airway samples also induced significant increases in CD203c and
intracellular IL-4, supporting the notion that allergic asthmatics have underlying type 2
inflammation in the airways compared to healthy individuals. Collectively, these findings
indicate that following allergen inhalation, the airway microenvironment can up-regulate
markers of basophil activation. Blockade of the βc led to inhibition of basophil activation
induced by 7 h airway samples, whereas neutralization of receptors for alarmin cytokines
had no inhibitory effect.
Given that previous murine models have shown that knock out of TSLPR, IL17RB, or ST2 results in decline AHR and eosinophilia, it is interesting that the blockade
of these receptors had no effect on basophil pro-inflammatory activity. We hav been
unable to measure TSLP, IL-33, and IL-25 in sputum samples at baseline or post-allergen
challenge28 and this may be due to alarmin cytokines being unstable and metabolized
quickly, thus requiring more sensitive assays for measurement.43 Secondly, Fux et al. has
demonstrated that IL-33 increases rapidly in BALF after allergen challenge, but declines
to baseline levels at 18 h post-allergen.44 It is possible that although alarmin cytokines are
upstream triggers of type 2 inflammation during the EAR, their levels may not be
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Ph.D. Thesis- B.M. Salter
McMaster University- Medical Sciences
significant during the LAR. Downstream Type 2 cytokines levels such as IL-3, IL-5, and
GM-CSF, may be more prevalent during the LAR within the airways. Therefore,
blockade of βc has a more profound inhibitory effect on basophil activation in vitro, as
opposed to epithelial-derived cytokine receptor neutralization. Lastly, basophils are
known to have an IL-3 autocrine activation loop, and we have previously reported that
TSLP can induce basophil production of IL-3, which could in turn, serve as another
mechanism for TSLP to enhance basophil activation by signaling through IL-3Rα.28 In
this study we have demonstrated that IL-25 and IL-33-induced basophil activation was
blocked following blockade of IL-17RB and ST2, however, we cannot rule out the
possibility, that similar to TSLP, the effects of IL-25 and IL-33 may be mediated in an
IL-3-dependent manner. This could partly explain why of IL-17RB and ST2 had no effect
on airway sample-induced basophil activation, whereas neutralizing the βc had a
dominant inhibitory effect.
Previous studies have shown that airway IL-33 and IL-25 mRNA expression is
increased in asthmatics.23-26 Thus, we expect these cytokines to be elevated in the airways
following allergen inhalation, however future studies are needed to determine the relative
protein levels of these cytokines within the airways. Similarly we could not measure
secreted IL-13 or IL-4 from basophils due to low cell numbers in the in vitro cultures.
Instead we measured intracellular cytokine expression through flow cytometry, and
although this is an established method that demonstrates intracytoplasmic expression of
IL-13 and IL-4, it cannot indicate whether or not these proteins are actually secreted.42
Furthermore, due to limited basophil numbers, migration experiments typically using the
109
Ph.D. Thesis- B.M. Salter
McMaster University- Medical Sciences
trans-well migration technique were replaced with cell shape change as surrogate
measure of migration.39
In summary, we demonstrate that post-allergen inhalation there is an increase in
basophil numbers and sensitivity to IL-25 and IL-33 through up-regulation of IL-17RB
and ST2 surface expression. Furthermore, we show that markers of basophil activity
significantly increase post-allergen in response to the airway microenvironment, and that
this happens in an IL-3-dependent manner. Our previous reports on the increase of
basophil effector function following allergen inhalation may be in part due to stimulation
by IL-25 and IL-33.28 We conclude that the interaction between alarmin cytokines and
basophils may play a central role in stimulating Type 2 inflammation.
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Ph.D. Thesis- B.M. Salter
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25. Azazi E, Elshora A, Tantawy E, et al. Serum levels of Interleukin-33 and its soluble
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27. Gauvreau GM, Lee JM, Watson RM, et al. Increased numbers of both airway
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28. Salter BM, Oliveria J, Nusca G, et al. Thymic Stromal Lymphopoietin Activation of
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30. Ocmant A, Michils A, Schandene L, et al. IL-4 and IL-13 mRNA real time PCR
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33. Suzukawa M, Iikura M, Koketsu R, et al. An IL-1 cytokine member, IL-33, induces
human basophil activation via its ST2 receptor. J Immunol 2008;181:5981-5989.
34. Blom L, Poulsen BC, Jensen BM, Hansen A, Poulsen LK. IL-33 induces IL-9
production in human CD4+ T cells and basophils. PLOS One 2011;6(7):e21695.
35. Wang YH, Angkasekwinai P, Lu N, et al. IL-25 augments type 2 immune responses
by enhancing the expansion and functions of TSLP-DC-activated Th2 memory cells. J
Exp Med 2007;204(8):1837-47.
36. Wang H, Mobini R, Gang Y, et al. Allergen challenge of peripheral blood
mononuclear cells from patients with seasonal allergic rhinitis increase IL-17RB, which
regulates basophil apoptosis and degranulation. Clin Exper Allergy 2010;40:1194-1202.
37. Cockcroft DW. Measure of airway responsiveness to inhaled histamine or
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38. Bafadhel M, McCormick M, Saha S, et al. Profiling of sputum inflammatory
mediators in asthma and chronic obstructive pulmonary disease. Respiration 2010;83:3644.
39. Heineman A, Hartnell A, Stubs EL V, et al. Basophil responses to chemokines
regulated by both sequential and cooperative receptor signaling. J Immunol
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40. Stirling RG, Rensen ELJ, Barnes PJ, Chung KF. Interleukin-5 induces CD34+
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41. Dorman SC, Babirad I, Post J, et al. Progenitor egress from the bone marrow after
allergen challenge: Role of stromal cell-derived factor 1alpha and eotaxin. J Allergy Clin
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42. Devouassoux G, Foster B, Scott LM, et al. Frequency and characterization of antigenspecific IL-4 and IL-13 producing basophils and T cells in peripheral blood of healthy
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43. Nagarkar DR, Poposki JA, Tan BK, et al. Thymic stromal lymphopoietin activity is
increased in nasal polyps of patients with chronic rhinosinusitis. J Allergy Clin Immunol
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44. Fux M, Pecaric-Petkovic T, Odermatt A, et al. IL-33 is a mediator rather than a
trigger of the acute allergic response in humans. Allergy 2014;69(2):216-222.
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FIGURES AND FIGURE LEGENDS
80
*
20
*
*
40
20
0
60
40
20
0
*
20
0
0
Pre
24h Post
Diluent
Allergen
60
40
20
0
100
80
*
60
40
20
0
100
80
60
40
20
80
80
†
*
IL-25+ (%)
IL-25+ (%)
100
100
†
100
80
60
40
0
Sputum
†
IL-17RB+ (%)
100
C
ST2+ (%)
100
80
60
*
60
40
Peripheral Blood
ST2+ (%)
100
80
60
40
20
0
B
Pre
7h
Post
IL-25+ (%)
Bone Marrow
IL-17RB+ (%)
IL-17RB+ (%)
ST2+ (%)
A
24h
100
†
80
†
*
60
*
40
20
0
Pre
7h
Post
24h
Figure 1– Allergen-inhalation induces changes in surface receptor expression for
alarmin cytokines on basophils of mild allergic asthmatics.
Through flow cytometry basophil expression of ST2, IL-17RB, and intracellular
IL-25 in 10 mild allergic asthmatics was measured in (A) bone marrow, (B) peripheral
blood, and (C) sputum at pre-allergen, 7 h, and 24 h post-allergen (black bars), compared
to diluent (open bars). Data are presented as meanSEM. * Significantly different from
pre-challenge for bone marrow and diluent-control for peripheral blood and sputum, †
significantly different from 0 h; P<0.05.
117
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ST2+ (%) 7 Hr Post-Allergen
A
B
100
P=0.32
R=0.125
80
60
40
20
0
0
10
20
30
ST2+ (%) 24 Hr Post-Allergen
Ph.D. Thesis- B.M. Salter
40
LAR Maximum % Fall in FEV 1
100
P<0.05
R=0.7552
80
60
40
20
0
0
10
20
30
40
LAR Maximum % Fall in FEV 1
Figure 2– Correlation ST2 basophil expression and disease severity
Peripheral blood basophil expression in 10 mild allergic asthmatics of (A) ST2 at
7 h post-allergen compared to LAR % fall in FEV1, (B) ST2 at 24 h post-allergen
compared to LAR % fall in FEV1. Data are presented as meanSEM. *Significant
correlation between basophil ST2 expression and disease severity; P<0.05.
118
Ph.D. Thesis- B.M. Salter
A
McMaster University- Medical Sciences
100
*
% IL17RB+
80
60
*
*
*
40
20
0
B
100
% ST2+
80
60
40
*
*
*
*
20
0
C
100
% TSLPR+
80
*
60
40
*
*
20
0
PBS
+
Anti-IgE (ng/mL) IL-3 (ng/mL)
TSLP (ng/mL)
IL-25 (ng/mL)
IL-33 (ng/mL)
-
10
-
10
-
10
-
10
-
10
Figure 3– IL-25 and IL-33 up-regulate their own epithelial-cell derived receptor
expression on basophils.
Isolated human peripheral blood basophils suspended in RPMI-C from a pool of 8
allergic subjects were cultured for 18 h with PBS or optimal concentrations of anti-IgE,
IL-3, TSLP, IL-25, or IL-33 (all 10 ng/mL), then assessed for expression of (A) IL-17RB,
(B) ST2, and (C) TSLPR. Data are expressed as meanSEM. * Significantly different
from PBS negative control; P<0.05.
119
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100
CD203c + (%)
A
80
*
*
*
*
60
†
40
†
20
B
25000
IL-3Ra+ (sMFI)
0
20000
C
100
*
15000
†
10000
*
*
*
†
†
*
5000
0
IL-13+ (%)
80
60
40
*
20
*
*
*
†
†
†
0
D
100
IL-4 + (%)
80
*
60
40
*
*
†
20
†
*
†
0
PBS
Anti-IgE
IL-3
IL-25
Anti-IL-17RB
IL-33
Anti-ST2
Isotype Control
+
-
+
-
+
-
+
+
+
+
-
+
+
+
+
-
Figure 4 – IL-25 and IL-33 effect on long-term function of basophils.
Isolated human peripheral blood basophils from 8 allergic subjects were incubated
for 18 h with or without IL-25 or IL-33 (both 10 ng/mL) and neutralizing antibodies for
IL-17RB and ST2 or isotype controls (both 10 µg/mL). Receptor blockade (light grey
bars) was assessed by comparison to isotype controls (black bars) via measuring: (A)
120
Ph.D. Thesis- B.M. Salter
McMaster University- Medical Sciences
CD203c, (B) IL-3Rα, (C) intracellular IL-13, and (D) intracellular IL-4. Anti-IgE and IL3 (10 ng/mL) served as positive controls (dark grey bars). Data are expressed as
meanSEM. * Significantly different from PBS negative control, † significantly different
from isotype control P<0.05.
Figure 5- IL-25 and IL-33 have priming effects on eotaxin-induced basophil shape
change
Isolated human peripheral blood basophils suspended in RPMI-C from 8 allergic
subjects were incubated with/without IL-25 and IL-33 (10 ng/mL) to assess (A) CCR3
expression and (B; C) shape change was measured via change in FSC post-stimulation
121
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McMaster University- Medical Sciences
with eotaxin (5 ng/mL). The effect of neutralizing antibodies for (D) IL-17RB and (E)
ST2 (light grey bars) on shape change was also assessed. Data are expressed as
meanSEM. ‡ Significantly different from eotaxin, * significantly different from PBS
negative control, † significantly different from isotype control; P<0.05.
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McMaster University- Medical Sciences
Figure 6- Effects of 18 h of incubation with post-allergen airway samples
Isolated human peripheral blood basophils suspended in RPMI-C from 8 mild
allergic asthmatic subjects were cultured for 18 h with PBS as negative control (open
bars) or IL-3 (10 ng/mL) as positive control (dark grey bars) or sputum supernatant
collected from dual responders at 0 h, 7 h, and 24 h post-allergen (black bars) then
measured for expression of (A) CD203c, (B) IL-4, and (C) IL-13. Data expressed as
meanSEM. * Significantly different from PBS negative control; p<0.05.
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McMaster University- Medical Sciences
B
A
CD203c + (%)
100
*
*
†
80
100
*
*
*
*
*
60
40
20
*
*
*
*
*
*
60
*
40
20
0
100
80
80
†
*
†
60
*
†
*
*
40
0
†
†
*
*
†
†
*
†
*
*
100
*
IL-13 + (%)
80
*
60
*
40
0
*
*
60
20
*
*
*
20
100
IL-13 + (%)
80
100
IL-4 +(%)
IL-4 + (%)
0
40
80
†
*
60
*
†
*
*
*
40
20
20
Sputum
Isotype Control
Anti-TSLPR
Anti-Beta Chain
IL-3
†
†
†
CD203c + (%)
†
0
-
+
+
-
+
+
-
+
+
-
+
+
-
+ +
+ - +
- +
- -
Sputum
Isotype Control
Anti-IL-17RB
Anti-ST2
Anti-Beta Chain
IL-3
+
+
0
-
+ + +
+ - - + - - +
- - - - -
+
+
-
+
+
-
+ + +
+ - - + - - +
- + +
- - -
+
+
Figure 7- Blocking the βc neutralizes the effects of airway samples on basophils.
Isolated human peripheral blood basophils suspended in RPMI-C from 16 mild
allergic asthmatic subjects were cultured for 18 h with PBS as negative control (open
bars) or 7 h post-allergen dual responder sputum supernatant and neutralizing antibodies
to (A) TSLPR, (B) ST2 or IL-17RB, and (A; B) βc (10 µg/mL) (light grey bars) on
markers of basophil function by expression of CD203c, IL-4, and IL-13. Data expressed
as meanSEM. IL-3 (10 ng/mL) served as positive control (dark greys bars) *
Significantly different from PBS negative control, † significantly different from isotype
control (open bars); P<0.05.
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ONLINE SUPPLEMENT
MATERIALS AND METHODS
Subject Exclusion Criteria
Subject were excluded from the study if they were pregnant or nursing, current
smokers or ex-smokers with more than 10 pack-years, or developed lower respiratory
infections/exacerbations or used inhaled or oral steroids 4 weeks prior to study. Antihistamines, caffeine, and non-steroidal anti-inflammatory agents were prohibited 48 h
before any study visit.
Methacholine Challenge
The methacholine inhalation challenge was carried out via tidal breathing from a
Wright nebulizer.E1 Doubling concentrations of methacholine chloride (Methapharm,
ON) were inhaled orally from a Hans Rudolph valve every 2 min. The FEV1 was
measured following inhalation at 30 s and 90 s or until it stopped falling. The percent fall
was calculated from the post-diluent FEV1 value. The test was terminated when a fall in
FEV1 of at least 20% of the lowest post-saline value occurred. The methacholine PC20
was calculated using linear interpolation.
Allergen and Diluent Challenge
Allergen challenges were conducted by administering inhaled doubling
concentrations of allergen extract, as previously describedE2 and diluent challenges were
conducted using 3 inhalations of 0.9% saline for 2 min each. The EAR was the largest
percent fall in FEV1 between 0 and 2 h, and the LAR was the largest percent fall in FEV1
between 3 and 7 h post-challenge.
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Materials and Manufacturers
1) Cell Isolation: For the layering process we used McCoy's 5A from Gibco and
Lymphoprep from Axis Shield. To further isolate basophils, a negative isolation kit was
used from Stem Cell Tech.
2) Cytospins: Sputum cytospins were stained with Diff-Quick from Sigma Aldrich for
differential cell counts and toluidine blue from Sigma Aldrich for metachromatic cell
counts.
3) Mediator Measurement: To measure IL-33 we used an ELISA kit from R&D systems
and for IL-25 we used Abnova.
4) Basophil Cultures: Golgi block/Monensin was obtained from Biolegend. IL-25, IL-33,
and IL-3 was obtained from R&D Systems, and anti-IgE from Sigma Aldrich. The
neutralizing antibodies for IL-17RB and ST2 were obtained from R&D Systems. Eotaxin
was obtained from R&D Systems.
5) Flow cytometry: Flow Jo Software was obtained from TreeStar and the LSR II flow
cytometer was from BD Biosciences. The antibodies required for flow cytometry
measurement of the basophil markers of interest were obtained from the following: BD
Biosciences for CD45 AF700, HLA-DR APC Cy7, and CCR3 APC; TSLPR PerCP
Cy5.5, IL-3Rα PE Cy7, IL-4 APC, and IL-13 FITC from Ebioscience; CD203c PE from
MACS Miltenyi; IL-25 FITC, ST2 PE, and IL-17RB APC from R&D Systems.
18-h Basophil Cultures
Purified basophils suspended in RPMI-C were incubated for 18 h at 37ºC with the
golgi-blocking agent, monensin, and PBS or increasing concentrations of IL-25 or IL-33
(0.1, 1, 10, 100 ng/mL). Basophils were immunostained to measure extracellular
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McMaster University- Medical Sciences
receptors CD203c, CCR3, IL-17RB, ST2, and TSLPR, and intracellular cytokine
expression of IL-4, IL-13, and IL-25 by flow cytometry.
Flow cytometry staining and analysis
Cells for flow cytometry experiments were immunostained with isotype controls
or specific antibodies to the extracellular receptors CD45, HLA-DR, CCR3, TSLPR, IL3Rα, CD203c for 30 min at 4ºC. To measure intracellular cytokine expression, cells were
washed, fixed and permeabilized, then stained with isotype controls or antibodies to IL-4,
IL-13, and IL-25. Cells were washed and acquired with a LSR II flow cytometer.
Analyses were performed using Flow-Jo software. Basophils were defined as the
CD45+/HLA-DR-/IL-3Rα+ population (Online Repository Fig E1). We first gated around
the CD45+ population in the SSC vs. CD45 AF700 plot (Online Repository Fig E2, A),
followed by gating around the HLA-DR- population in the SSC vs. HLA-DR APCH7
(Online Repository Fig E1, B), and a final gate around the IL-3Rα+ population in the SSC
vs. IL-3Rα PE Cy7 plot (Online Repository Fig E1, B). The isotype control for the
markers of interest was set to 2%, which was compared to the specific markers to detect
the percentage of cells expressing the marker. For staining of blood and bone marrow, at
least 300, 000 CD45+ events were acquired, with collection of the same cell number for
each time-point pre/post-diluent/allergen challenge per subject. In addition, at least 1,
000, 000 events were acquired for sputum samples, with the same cell number collected
for each time-point pre/post-diluent/allergen challenge per subject. Lastly, for the in vitro
culture studies, at least 10, 000 CD45+ events were acquired per culture condition.
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Statistical Analysis
All data in the manuscript are presented as mean ± SEM. In this study the EAR
and LAR were assessed as the maximum allergen-induced fall in FEV1 %, based on
analysis of the minimum FEV1 % remaining over 0-3 h and 3 to 7 h, respectively, postchallenge.
The methacholine PC20 values were log-transformed before analyses, and
expressed as geometric mean and range. The methacholine PC20 between Day 1/Day 3 of
both diluent and allergen inhalation challenges were compared.
Flow cytometry results are expressed as mean percent positive cells. In cases
where basophils constitutively express the marker of interest the results are expressed as
specific Mean Fluorescent Intensity (sMFI). The cytokines measured by ELISA are
shown as concentration (pg/mL).
Statistical analysis for the inhalation challenges was performed using 2-way
ANOVA and post-hoc Bonferroni test. The in vitro experiments were analyzed using 1way ANOVA with a post-hoc Tukey test. Correlation analyses were conducted using a
Pearson correlation test. Significance was accepted at P<0.05.
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ONLINE REPOSITORY TABLES
Table E1: Subject Characteristics
Sex
Age (y)
% Predicted
FEV1
Allergen
Inhaled
M
61
96.00
CAT
Titration
of Inhaled
Allergen
1:32
F
24
80.00
RGW
1:16.15
M
52
95.00
HDM
1:68
F
40
104.00
CAT
1:120
M
25
86.00
RGW
1:83
F
24
93.00
HDM
1:84
M
26
93.00
HDM
1:32
M
20
94.00
GRS
1:4
F
34
70.00
CAT
1:512
M
26
96.00
CAT
1:83
No.
Mean
Mean
M/F 6:4 33.2±13.6 90.7±9.6
F, female; M, male; HDM, house dust mite; RGW, ragweed; GRS, grass. Data presented
as mean ± SEM for Age.
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Table E2: Allergen inhalation induces airway changes in mild allergic asthmatics
Sputum cell count was measured pre and post-challenge to determine effect of
allergen compared to diluent. Data are presented as meanSEM. * Significantly different
than diluent control; P<0.05.
Sputum Total
Cell Count
(x104cells/mL)
Neutrophils
(x104cells/mL)
Eosinophils
(x104cells/mL)
Metachromatic
cells
(x104cells/mL)
Pre-Diluent
Challenge
7h-Post
Diluent
Challenge
24h-Post
Diluent
Challenge
PreAllergen
Challenge
7h-Post
Allergen
Challenge
24h-Post
Allergen
Challenge
1.72±1.2
1.48±0.9
1.46±0.8
1.78±1.2
3.40±2.9
3.14±2.3*
1.02±1.3
1.16±1.3
1.25±1.4
0.88±0.91
2.44±2.3*
1.69±1.3
0.012±0.0
0.022±0.0
0.006±0.0
0.02±0.0
0.34±0.4*
0.33±0.4*
0±0.0
0±0.0
0±0.0
0.01±0.0
0.22±0.3*
0.21±0.2*
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ONLINE REPOSITORY FIGURES
Figure E1- Basophil gating strategy
Representative whole blood dot plots showing strategic basophil gating used for flow
cytometry analysis. (A) Side Scatter (SSC) vs. CD45 Alexa Fluor 700 (B) SSC vs. HLADR APCH7 (C) SSC vs. IL-3Rα PECy7 (D) Histogram demonstrating isotype vs.
specific antibody for assessment of receptor expression on basophils.
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Ph.D. Thesis- B.M. Salter
60
*
*
40
80
20
0
E
*
100
80
*
60
40
20
0
H
100
100
80
80
IL-4+ (%)
10000
5000
60
*
40
*
60
*
40
*
20
20
4000
F
*
0
100
100
I
80
1000
TSLPR+ (%)
2000
60
40
*
*
-
0.1
1
10
100
60
40
20
20
0
IL-33 (ng/mL)
0
80
3000
IL-25 + (%)
CCR3 sMFI
40
0
0
C
60
20
15000
G
100
IL-17RB + (%)
*
80
ST2 + (%)
CD203c + (%)
IL-3Ra+ (sMFI)
B
D
*
100
IL-13+ (%)
A
McMaster University- Medical Sciences
0
0
-
0.1
1
10
100
-
0.1
1
10
100
Figure E2- Basophil activation marker dose response to IL-33
Isolated peripheral blood basophils suspended in RPMI-C from a pool of 8 allergic
subjects were cultured for 18 h with PBS (negative control, open bars) or increasing
concentrations of IL-33 (0.1, 1, 10, 100 ng/mL) (black bars), were assessed for
expression of (A) CD203c, (B) IL-3Rα, (C) CCR3, (D) intracellular IL-13, (E)
intracellular IL-4, (F) intracellular IL-25, (G) IL-17RB, (H) ST2, and (I) TSLPR. Data
expressed as meanSEM. * Significantly different from PBS negative control; P<0.05.
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100
D
*
40
0
0
E
*
10000
5000
60
*
40
*
10000
5000
60
*
40
0.1
1
10
100
100
60
40
20
0
0
-
40
80
20
0
60
0
I
100
80
*
*
20
20
TSLPR+ (%)
*
IL-25 + (%)
*
15000
40
80
0
F
60
100
20
20000
IL-25 (ng/mL)
H
100
ST2 + (%)
15000
80
0
80
IL-4+ (%)
IL-3Ra+ (sMFI)
40
20
0
CCR3+ (sMFI)
*
60
20
100
G
80
*
60
B 20000
C
100
IL-17RB + (%)
80
IL-13+ (%)
CD203c + (%)
A
-
0.1
1
10
100
-
0.1
1
10
100
Figure E3- Basophil activation marker dose response to IL-25
Isolated peripheral blood basophils suspended in RPMI-C from 8 allergic subjects were
cultured for 18 h with PBS (negative control, open bars) or increasing concentrations of
IL-25 (0.1, 1, 10, 100 ng/mL) (black bars), were assessed for expression of (A) CD203c,
(B) IL-3Rα, (C) CCR3, (D) intracellular IL-13, (E) intracellular IL-4, (F) intracellular
IL-25, (G) IL-17RB, (H) ST2, and (I) TSLPR. Data expressed as meanSEM. *
Significantly different from PBS negative control; P<0.05.
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McMaster University- Medical Sciences
ONLINE REPOSITORY REFERENCES
E1. Cockcroft DW. Measure of airway responsiveness to inhaled histamine or
methacholine; method of continuous aerosol generation and tidal breathing inhalation. In:
Hargreave FE, Woolcock AJ, eds. Airway responsiveness: measurement and
interpretation. Mississauga: Astra Pharmaceuticals Canada Ltd, 1985: 22 - 28.
E2. O'Byrne PM, Dolovich J, Hargreave FE. Late asthmatic responses. Am Rev Respir
Dis 1987; 136 (3):740-51.
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CHAPTER 5:
McMaster University- Medical Sciences
DISCUSSION
The alarmin cytokines TSLP, IL-25, and IL-33 have emerged as key mediators of
the allergic immune response. Numerous reports show that these cytokines induce potent
pro-inflammatory effects on a range of cells including DCs, T and B-lymphocytes, HPCs,
granulocytes, and mast cells. Murine models emphasize that alarmin cytokines contribute
to the pathophysiology of allergic disease by enhancing AHR, bronchoconstriction,
airway inflammation, mucus hypersecretion, and airway remodeling.
The objective of this thesis was to explore the interaction between basophils and
alarmin cytokines, and whether this relationship can result in potentiating the effector
role of basophils in the context of allergic asthma. The thesis studies are focused in two
distinct areas, the first was to determine the effector role of basophils in the LAR, and the
second, to examine the effect of alarmin cytokines (TSLP, IL-33, and IL-25) on basophil
pro-inflammatory activity. The overarching hypothesis for the studies found herein is that
alarmin cytokines mediate the pro-inflammatory role of basophils in the LAR of allergic
asthma.
The data presented herein provide convincing evidence for a role of basophils
during airway late phase responses in allergic asthma, and the interaction between
basophils and alarmin cytokines enhancing the activity of these cells. Chapter 2
demonstrates increased peripheral blood basophil surface activation marker expression
during the LAR, coincident with allergen-induced bronchoconstriction. Chapter 3
demonstrates basophil infiltration to the airways and up-regulation of surface activation
marker and Type 2 cytokine intracellular expression during the LAR following allergen
inhalation. Chapters 3 and 4 demonstrate that basophil receptor expression for alarmin
cytokines TSLP, IL-25, and IL-33 become up-regulated following allergen inhalation.
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McMaster University- Medical Sciences
Chapters 3 and 4 also explored whether TSLP, IL-25, and IL-33 can enhance basophil
pro-inflammatory activity in vitro, and demonstrated that these cytokines induce surface
activation marker and Type 2 cytokine intracellular expression, as well as prime
migration to eotaxin. Lastly, we hypothesized that following allergen inhalation the
airway microenvironment contains Type 2 and alarmin cytokines, which promote the proinflammatory activity of basophils. Chapter 4 demonstrated that post-allergen inhalation
the airway microenvironment has the ability to up-regulate basophil surface activation
marker and Type 2 cytokine intracellular expression in vitro via the common βc.
Collectively, our findings not only provide novel insights into the role of basophils in
allergic asthma, but also explain, in part, how alarmin cytokines can promote the proinflammatory activity of these cells.
5.1 Basophils: Important Effector Cells in Allergic Asthma
Basophils have been traditionally known for their ability to produce abundant
quantities of histamine, prostaglandins, proteolytic enzymes, and CysLTs, following IgE
cross-linking with the high affinity FcεR1; these in turn contribute to the hallmark
bronchoconstriction of allergic hypersensitivity reactions. Recent advancements in
basophil research have also allowed us to identify an important association between
basophils and allergic asthma. Peripheral blood and airway basophil numbers are elevated
in asthmatics versus healthy controls [135] and increased basophil numbers correlate with
allergic asthma severity [136-141]. Following allergen exposure basophils migrate from
the peripheral blood to the airways [135-137]. The precise details of the kinetics of
basophil pro-inflammatory activity following allergen inhalation remain unclear.
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McMaster University- Medical Sciences
Basophils are known sources of potent bronchoconstrictors, histamine and
CysLTs, which become released within 1 hour of allergen crosslinking, suggesting that
these cells are primarily involved in the EAR. However, emerging evidence suggests that
basophils have an additional effector role in the LAR. Basophils accumulate at sites of
allergen exposure including the skin, nasal mucosa, and airways during the LAR of
patients with atopic dermatitis, seasonal AR, and asthma, respectively, following an
allergen challenge [120, 142-145]. Furthermore, studies have shown basophil surface
activation marker expression of CD203c to remain up-regulated for up to 24 hours postallergen inhalation [Appendix II], decrease during remission of asthma symptoms, and
correlate with airflow limitation [146]. Lastly, peripheral and airway basophils have been
identified as a source of IL-4 and IL-13 during asthmatic exacerbations [102-105].
Basophils are also potent sources of TSLP and IL-25 [106; 110]. The reported evidence
that basophil number and activation increases during the LAR, in combination with
studies showing basophils are key sources of Type 2 and alarmin cytokines, suggest that
basophils play a role in promoting and maintaining the Type 2 inflammatory response in
allergic asthma. Further research is needed to better understand the exact role of
basophils in the context of allergic asthma and more specifically during the LAR.
5.1.1 Basophil Activation in Allergic Asthma
In recent years, researchers have looked towards basophil biomarker
measurements to identify and monitor allergic disease. CD203c has is a transmembrane
receptor that has the ability to hydrolyze ATP and negatively regulate ATP-induced
basophil and mast cell activation [166]. CD203c becomes up-regulated following IgE
cross-linking and IL-3 stimulation [159; 166] and thus has been used to monitor basophil
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McMaster University- Medical Sciences
activation and assess immediate-type allergic hypersensitivity responses. CD203c
expression on basophils reflects the allergic response to hymenoptera venom, grass
pollens, and cat dander [159-165]. Few studies have looked at the relationship between
CD203c basophil expression and allergic asthma. As such, prior to this thesis, we
proceeded to examine the change of CD203c basophil expression following a whole lung
allergen challenge in mild allergic asthmatics. We demonstrated that peripheral blood
basophil CD203c expression was up-regulated for up to 24 hours post-allergen inhalation
in mild allergic asthmatics [Appendix II]. We further used CD203c expression to
determine the response of basophils following the inhalation of a nicotinic receptor
agonist to treat mild allergic asthma [Appendix II]. From these studies we established that
CD203c basophil expression increases in the circulation following an allergen challenge,
however several unanswered questions remain: (i) how does CD203c expression
compare to other markers of basophil activation; (ii) does basophil activation differ
between those subjects who develop IER compared to DR asthmatics; and, (iii) does the
level of basophil activation reflect the magnitude of allergen-induced LAR?
CD69 and CD63 are surface markers that also reflect basophil activation. CD69
basophil expression is significantly higher in asthmatic individuals compared to healthy
controls, with expression levels being more elevated on basophils in BALF then
peripheral blood of asthmatics [156]. In vitro studies have also shown basophil CD69
expression to up-regulate following exposure to recombinant allergen [157]. Lastly,
CD63 basophil expression is currently used as a biomarker of IgE-mediated allergy [151154]. In Chapter 2, we compared the level of basophil expression of CD203c, CD69, and
CD63 following an allergen challenge in both IER and DR mild allergic asthmatics. We
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Ph.D. Thesis- B.M. Salter
McMaster University- Medical Sciences
initially compared pre-allergen basophil activation marker expression of IER versus DR
and observed a significantly greater pre-allergen level of CD63 basophil expression with
respect to DR. In addition, we found both IER and DR subjects to have a significant
inverse correlation between pre-allergen basophil CD203c expression and percentpredicted FEV1. These findings support previous reports by Ono et al. that asthmatic
patients had a correlation between CD203c basophil expression and % predicted FEV1
values [146]. We additionally measured the change in peripheral blood basophil
expression of CD203c, CD69, and CD63 at 0, 3, 7, and 24 hours post-allergen compared
to diluent challenge. We found that IER had a significant increase in basophil CD203c
expression at 3 hours post-allergen compared to diluent, but no change in CD69 or CD63
expression. CD203c expression returned to baseline levels at 7 and 24 hours postallergen, which is consistent with a lack of the allergen-induced LAR and overall
improvement in lung function. Following allergen inhalation, DR patients experienced a
significant up-regulation of CD203c from 3 to 24 hours post-allergen, and correlated with
the maximum % fall in FEV1. In contrast, CD63 peaked only during the LAR of dual DR
compared to the diluent and no change was detected in CD69 expression. Chapter 2
demonstrates that CD203c is a surface marker that reflects peripheral blood basophil
activation that can be used to differentiate between varying asthmatic responses to an
inhaled allergen, as well as reflect pre-allergen airflow limitation, and allergen-induced
bronchoconstriction during the LAR. Most importantly, these findings further support
that increased basophil activity plays a role in potentiating the LAR. Because of these
findings, we chose to use CD203c as our standard basophil surface activation marker for
subsequent experiments described herein.
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McMaster University- Medical Sciences
The next objective was to extend our findings to determine, how does CD203c
basophil surface expression change across multiple biological compartments following
allergen inhalation? This would explore how allergen exposure changes basophil
activation in different biological compartments, and whether this is consistent with
previous evidence supporting a role for basophils in the LAR. We specifically evaluated
the change in bone marrow, peripheral blood, and airway basophil CD203c expression of
dual responder mild allergic asthmatics following allergen inhalation. In bone marrow
basophils there was a decrease in CD203c expression at 24 hours post-allergen challenge,
whereas CD203c expression was up-regulated on both peripheral blood and sputum
basophils. Given that the number of sputum basophils increased 24 hours post-allergen,
these above findings suggest that a subset of activated basophils migrate from the bone
marrow into the peripheral blood, and finally to the airways following allergen inhalation.
The percentage of circulating basophils expressing CD203c at 7 and 24 hours postallergen had a positive correlation with the maximum % fall in FEV1 during the LAR.
The correlation between CD203c expression and declined lung function is consistent with
the findings of Chapter 2. Our study demonstrated that the level of basophil activation
during the LAR reflects severity of allergen-induced bronchoconstriction, providing
indirect supporting evidence that basophils have an important role in allergic asthma.
Collectively, these studies offer evidence that CD203c expression on peripheral
blood basophils post-allergen challenge can be used to differentiate between IER and DR
asthmatic responses to inhaled allergen, as well as monitor asthma status. Most
importantly, changes in basophil CD203c levels together with a positive correlation
between allergen-induced bronchoconstriction and CD203c expression, suggests that
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Ph.D. Thesis- B.M. Salter
McMaster University- Medical Sciences
activated basophils contribute to the decline in lung function during the LAR. Lastly,
given that CD203c increases on the surface of basophils within the peripheral blood
following an allergen challenge, this is suggesting CD203c is playing a part in negatively
regulating basophil activation. Whether asthmatic subjects have an impaired ability of
CD203c to regulate basophil activity compared to healthy controls needs to be further
investigated.
5.1.2 Basophil Migration in Allergic Asthma
Basophil migration can be induced by several chemo-attractants including IL-8,
eotaxin, RANTES, MCP-3, MCP-1, and MIP-1α [75-79]. The most potent of these is
eotaxin; its effects are mediated through binding to the surface receptor CCR3 on
basophils [75]. The interaction between eotaxin and basophils is thought to be an
important component to the strong influx of these cells to the airways during the EAR
and LAR [70]. To date, the change in CCR3 expression on basophils following allergen
inhalation and how this relates to basophil migration across biological compartments has
not been investigated. The purpose of the study objective was to answer the question:
does allergen inhalation affect basophil CCR3 surface expression and subsequent
infiltration into the airways? The results of this study would determine whether the
change in basophil CCR3 expression coincides with basophil migration and overall
cellular activation following allergen inhalation. In Chapter 3, we specifically measured
the level of CCR3 expression on basophils within the bone marrow, peripheral blood, and
sputum following both an allergen and diluent challenge in mild allergic asthmatics. In
bone marrow basophils there was a decrease in CCR3 expression at 24 hours postallergen, whereas CCR3 expression was up-regulated on both peripheral blood and
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sputum basophils. In-line with the increase of sputum basophil CCR3 expression, we
observed a significant increase in basophil numbers within the sputum at 7 and 24 hours
post-allergen. This is consistent with previous findings of Gauvreau et al. 2000 [120]. In
parallel with a decline in bone marrow basophil CCR3 expression, there was downregulated CD203c expression, which suggests that a subset of activated basophils
expressing CCR3 migrate in response to eotaxin from the bone marrow into the
peripheral blood, followed by influx to the airways post-allergen inhalation. This is
consistent with the mechanisms of eosinophil migration from bone marrow [81], and with
reports of increased peripheral eotaxin levels by 5 hours post-whole lung allergen
challenge and within bronchial biopsies 24 hours after a segmental allergen challenge
[80; 82]. Our findings demonstrate that the transit of basophils to the airways may be
regulated by their expression of CCR3, which becomes up-regulated on peripheral blood
basophils during the LAR, thus allowing these cells to play a role in allergic
inflammation.
5.1.3 Basophil Type 2 Cytokine Expression in Allergic Asthma
Basophils and mast cells are known for their development of the EAR through
IgE-mediated release of histamine and CysLTs, which in turn contributes to immediate
bronchoconstriction. However, recent reports suggest that basophils have an additional
effector role in the EAR and LAR. Deovaussoux and coworkers demonstrated that
following in vitro allergen stimulation with Fel d 1, basophil production of IL-4 and IL13 accounted for a significant frequency of cells producing these cytokines, next to T
cells [103]. Nouri-Aria et al. found basophils to be responsible for producing 72% of IL-4
found in bronchial mucosa 24 hours post-allergen challenge in asthmatics versus healthy
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controls, thus confirming a likely role of this cell in the LAR [105]. A real-time
quantitative polymerase chain reaction-based assay in whole blood also identified
basophils as the most prominent source of IL-4 and IL-13 after in vitro stimulation with
Fel d 1 allergen [104]. IL-4 and IL-13 induce IgE antibody production from B cells,
activate Th2 cells, mast cells, eosinophils, and neutrophils, as well induce goblet cell
hyperplasia, adhesion molecule expression, chemokine release, and AHR [15-21]. In
addition, IL-4 is important in regulating Th0 cell differentiation into Th2 cells [22; 23],
whereas IL-13 alone can drive allergic inflammation and the asthmatic response [24].
Although the aforementioned studies establish that basophils are sources of IL-4
and IL-13 upon allergic antigen stimulation, more in vivo models are required to monitor
the change basophil production of IL-4 and IL-13 following an allergen challenge. The
exact kinetics of basophil Type 2 cytokine expression following an allergen inhalation
challenge across varying biological compartments is unknown. A component of the study
in Chapter 3 was to examine the change in basophil IL-4 and IL-13 intracellular
expression following a whole lung allergen challenge in the bone marrow, periphery,
and airways of mild allergic asthmatics.
We initially measured the levels of Type 2 and alarmin cytokines within the bone
marrow, peripheral blood, and sputum. Type 2 cytokine levels of IL-4 and IL-13 were
significantly increased within the serum at 7 hours post-allergen, however these cytokine
levels could not be detected within the bone marrow or sputum. In addition, alarmin
cytokines (TSLP, IL-33, IL-25) were not significantly detected in any of the biological
compartments. With respect to basophil intracellular expression of these cytokines,
during the LAR post-allergen, the percentage of basophils expressing intracellular IL-4
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and IL-13 in bone marrow, peripheral blood, and sputum were up-regulated, but lagged
behind elevated serum levels measured at 7 hours post-allergen. These studies
demonstrate intracytoplasmic expression of Type 2 cytokines rather than secretion, but
these findings are still consistent with previous studies showing that basophils are marked
sources of IL-4 and IL-13 during asthmatic exacerbations [98-105, 107]. Lastly,
peripheral IL-4 and IL-13 basophil intracellular expression at 24 hours post-allergen
significantly correlated with maximum % fall in FEV1. As a whole, Chapter 3 showed
that allergen inhalation induced basophil activation, Type 2 cytokine intracellular
expression, infiltration into the airways, and correlated with allergen-induced
bronchoconstriction. Collectively these data strongly suggest that basophils may play a
role in mediating the Type 2 inflammatory response and allergen-induced
bronchoconstriction in allergic asthma.
5.2 Epithelial Cells Secrete a Triad of Alarmin Cytokines that Stimulate Basophils
Regulation of immune responses within the airways has traditionally focused on
leukocytes as the orchestrators of the immune system, however recent studies suggest
that the airway epithelium actively communicates with airway leukocytes. Epithelial cells
express PRRs and TLRs that detect environmental stimuli such as viral and allergic
antigens, and their subsequent activation leads to the release of soluble cytokines,
chemokines, growth factors, and anti-microbial peptides, which alert the immune system
to danger. Among these alarmin cytokines are TSLP, IL-33, and IL-25. It is important to
note that the epithelium has a varying response to different inhaled allergens. For
example, epithelial PRRs and TLRs can recognize and become activated by inhaled
allergens, including HDM, pollen, cockroach, and fungi [329-341]. Hammad et al.
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showed that TLR expression and activation on airway epithelial cells is necessary for the
production of TSLP, IL-25, and IL-33 after exposure to HDM extracts [342]. Given that
HDM is the most prevalent cause of allergic sensitization, with up to 85% of asthmatics
being HDM allergic, it is important to understand the mechanisms through which this
allergen and other common aeroallergens promote asthma following activation of the
airway epithelium [343]. It has become clear that a crucial component to the pathology of
allergic asthma is brought on by multiple allergens activating the airway epithelium to
promote alarmin cytokine release, which in turn mediate Type 2 inflammation. It is also
noteworthy that despite the epithelium being a key source of the alarmin cytokine triad,
there are also multiple immune cell sources of these mediators. For instance, mast cells,
macrophages, granulocytes, and DCs have been found to secrete TSLP [182-190];
granulocytes, mast cells, and Th2 cells release IL-25 [110; 258]; and IL-33 is produced
by DCs, macrophages, and mast cells [291; 299]. Thus, it is important that we account
for these additional sources of alarmin cytokines, when developing therapeutic strategies
to neutralize alarmin cytokines within the airways.
To provide evidence that alarmin cytokines are important biological targets to
treat allergic asthma, we must first understand the relationship between these cytokines
and effector cells. Although there are numerous murine and human studies showing an
influence of alarmin cytokines on basophils, there is still a great deal of unanswered
questions as to how interaction of basophils with these cytokines can potentiate their role
in allergic asthma.
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5.2.1 Basophil Expression of Receptors for Alarmin Cytokines in Allergic Asthma
The receptors for alarmin cytokines have been reported on a number of structural
and immune cells within the airways, including basophils. Protein and mRNA expression
of TSLPR and IL-7Rα have been confirmed in human peripheral basophils [66, 205,
217]. Siracusa et al. further demonstrated that human TSLPR basophil expression is upregulated following overnight incubation with IL-3 [66]. Agrawal and colleagues
reported that basophil TSLPR expression in atopic asthmatics is up-regulated following
IgE receptor ligation with Der p 1 or anti-IgE, and the magnitude of this increase
correlated with total serum IgE levels [217]. Human peripheral basophils also express IL17RB and ST2, which become up-regulated following stimulation with IL-3 [260, 284,
312, 314-316]. Although the above findings demonstrate that basophil expression of
receptors for alarmin cytokines can be promoted via IgE-dependent and IgE-independent
mechanisms in vitro, it remains unclear as to how in vivo exposure to allergen can
modulate the expression of these receptors. In Chapters 3 and 4, we aimed to answer the
question of does basophil expression of receptors for alarmin cytokines change in
response to inhaled allergen across varying biological compartments? We specifically
measured the change in TSLPR, IL-17RB, and ST2 basophil expression in the bone
marrow, peripheral blood, and airways following allergen inhalation in mild allergic
asthmatics.
Basophil TSLPR and IL-17RB expression were up-regulated within the bone
marrow, peripheral blood, and airways for up to 24 hours post-allergen. ST2 basophil
expression increased significantly within the bone marrow and peripheral blood for up to
24 hours post-allergen. Conversely, no change was found in the sputum, suggesting that
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1) basophil sensitivity to IL-33 increases primarily within the peripheral blood following
allergen exposure rather than within the airways or 2) increased IL-33 levels within the
airways contribute to basophil activity rather than increase sensitivity to IL-33. Although
we were not able to measure IL-33 protein levels within the airways, Fux et al. has
reported that following a segmental allergen challenge, IL-33 protein levels within the
BALF, measured via an ELISA, increase significantly within the EAR (10 minutes postallergen challenge), but decline to baseline during the LAR (18 hours post-allergen
challenge) [344]. These findings suggest that IL-33 plays a role in potentiating basophil
activity following allergen inhalation predominately during the EAR.
Given that basophils are a source of IL-25 [110], the change in basophil IL-25
expression following allergen exposure was measured. Interestingly, intracellular
expression of IL-25 in basophils increased significantly following allergen inhalation in
the peripheral blood and sputum. The mechanism driving basophil IL-25 intracellular
expression can be, in part, explained by the findings of Wang et al. who determined that
human basophils cultured alone with IL-3 did not produce IL-25, however IgE crosslinking triggered strong IL-25 production by basophils [110]. Furthermore, activated
basophils from allergic subjects produced two-fold more IL-25 than non-allergic subjects
following IgE cross-linking [110]. This was additionally supported by our findings that
overnight stimulation with IL-3, IL-25, IL-33, or TSLP had no effect on IL-25
intracellular expression, whereas anti-IgE had a significant effect (data not shown). As
such, these data suggest that production of IL-25 from basophils is promoted via an IgEdependent mechanism that further aids the epithelium in promoting and mediating Type 2
inflammation. Lastly, we found that peripheral blood basophil expression of TSLPR at 7
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and 24 hours, as well as ST2 at 24 hours post-allergen had a significant positive
correlation with the maximum % fall in FEV1 during the LAR.
The above findings demonstrate that in vivo exposure to allergen increases
sensitivity of basophils to alarmin cytokines by up-regulating receptor expression for
TSLP, IL-25, and IL-33 on basophils. This in turn may further contribute to increased
allergen-induced bronchoconstriction, as well as allergen-induced activity of basophils
during the LAR that was reported in Chapters 2 and 3. Collectively, we hypothesize that
increased basophil sensitivity for alarmin cytokines further potentiates basophil
activation, type 2 cytokine intracellular expression, and migration following allergen
inhalation, thereby contributing to the pathology of allergic asthma.
5.2.2 The Influence of Alarmin Cytokines on Basophil Pro-Inflammatory Activity
TSLP, IL-25, and IL-33 can regulate basophil pro-inflammatory activity. TSLP,
in the presence or absence of IL-3, can modulate various basophil activation markers,
such as increased CD69, L-selectin, CD11b, IL-3Rα, and ST2 surface expression in the
murine model [66]. Wang et al. reported that following stimulation with IL-25, human
basophils show significant inhibition of apoptosis and increased IgE-mediated
degranulation [260]. There are also reports that murine and human basophils produce
TSLP and IL-25, suggesting that basophils have an autocrine feedback loop [106; 110].
Lastly, various studies have reported that in vitro stimulation with IL-33 can induce
human basophil production of type 2 cytokines (IL-5, IL-6, IL-13, IL-8, IL-9, and IL-4),
degranulation of histamine, up-regulated expression of CD11b, and prime eotaxininduced migration [83, 284, 312, 314-316].
The aim of Chapters 3 and 4 was to specifically examine the in vitro effect of
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TSLP, IL-25, and IL-33 on human basophils, including surface activation marker
expression (CD203c, IL-3Rα, CCR3), Type 2 cytokine intracellular expression (IL-4 and
IL-13), and migration. The findings of these experiments would help determine what
factors may have been contributing to the up-regulation of basophil activity and
infiltration to the airways following allergen inhalation observed in Chapters 2 and 3.
Firstly, stimulation with TSLP, IL-25, and IL-33 promoted up-regulation of their
own receptors on basophils. IL-3 and anti-IgE, as well as TSLP could also promote upregulation of IL-17RB and ST2 expression. IL-25 and IL-33 had no effect on TSLPR
expression, suggesting these cytokines act downstream of TSLP. These data are
consistent with previous reports that TSLPR, ST2, and IL-17RB expression on basophils
become up-regulated in response to IL-3 and IgE crosslinking [66, 83, 217, 260, 284,
312, 314-316,]. In addition these data support the findings of Siracusa et al., showing that
TSLP-stimulated murine basophils have increased responsiveness to IL-3 and IL-33 [66].
Most importantly, these results demonstrate that both IgE-dependent and IgE–
independent mechanisms could have contributed to the up-regulation of receptors for
alarmin cytokines on basophils post-allergen challenge observed in Chapters 3 and 4.
Secondly, we demonstrated that overnight incubation of basophils with TSLP, IL25, and IL-33 increased intracellular expression of type 2 cytokines (IL-4 and IL-13) and
up-regulated activation marker expression CD203c and IL-3Rα. These findings help
support previous studies demonstrating that IL-33 can induce basophil type 2 cytokine
secretion [83, 284, 312, 314-316], but also further expand on the relationship between
TSLP and IL-25 with basophils. The aforementioned effects of TSLP, IL-25, and IL-33
on basophils were shown to not be significantly different in potency and shared similar
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levels to that of the traditional mediators of basophil activation IL-3 and anti-IgE. Lastly,
TSLP, IL-25, and IL-33 induced up-regulated basophil CCR3 expression, which was
associated with increased priming to eotaxin-induced migration, as shown through shape
change. TSLP, IL-25, and IL-33 were similar in potency with respect to up-regulating
basophil CCR3 expression, however TSLP was found to have a significantly higher
effect on priming basophil shape change to eotaxin compared to IL-25 and IL-33.
It is interesting that we found, for the most part, the level of potency between the
alarmin cytokines to be the same with respect to inducing basophil activity. There are
several studies with animals, which have attempted to answer the question of which
cytokine is the most critical to initiating and mediating allergic asthma. Barlow et al.
compared the importance of IL-25 and IL-33 by observing the effect on
bronchoconstriction following the knockout of IL-17RB, ST2 or both [346]. In the
absence of ST2 there was a more marked drop in bronchoconstriction as opposed to IL17RB knockout. They further showed in an ex vivo model that administration of IL-33
induced greater airway contraction then IL-25. Lastly, IL-33 was found to appear earlier
within the airways versus IL-25, and was responsible for the expansion of IL-13producing ILC2s. These findings suggest that IL-33 may play a more critical role in the
rapid induction of contraction by, in part, inducing expansion of IL-13-producing ILC2s,
whereas IL-25-induced responses are slower and less potent. Chu and colleagues
measured the change in TSLP, IL-25, and IL-33 levels within the lungs following
exposure to HDM and the effect in a murine model of allergic asthma following knockout
of their respective receptors [347]. TSLP levels increased within the airways 3 days
following exposure to HDM, however knockout of TSLPR or neutralization of TSLPR
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showed similar levels of serum IgE, airway inflammation and T cell activity to the
wildtype HDM-exposed model. Similar findings were shown for IL-17RB knockout.
Conversely, ST2 knockout mice had a marked decline in serum IgE, mucus production,
airway inflammation, CD4+ T cell activation, and eosinophilia. Their data suggests that
IL-25 and TSLP are redundant and although exposure to an allergen increases levels of
these cytokines within the airways, which may in part contribute to the pathogenesis,
unlike IL-33, they are not central mediators of allergic inflammation. Lastly, Toki and
colleagues demonstrated that TSLPR-deficient mice had decreased airway levels of IL-33
protein, ILC2s, eosinophils, IL-5 and IL-13 [348]. They concluded that TSLP is
important in regulating IL-33 levels but that IL-33 is a key inducer of Type 2
inflammation and controls ILC2s numbers and activity. Thus, TSLP may be acting more
upstream of IL-33 and can control the release of IL-33. Collectively, the above findings
indicate that IL-25 and TSLP are redundant cytokines that may contribute to the
pathogenesis of asthma but are not central mediators like IL-33. Our own data indicates
that alarmin cytokines play redundant roles in promoting basophil activity. However,
whether or not IL-33 is more critical to basophil activity as opposed to IL-25 and TSLP
remains to be clear. More studies will need to be done in the future to determine what
cytokine is most important to basophil activity in an in vivo model of allergic asthma.
Novel findings from Chapters 3 and 4 demonstrate that the interaction between
basophils and alarmin cytokines leads to up-regulation of basophil activity with potency
similar to anti-IgE and IL-3. One may reasonably conclude that increased basophil
activity during both the EAR and LAR, following exposure to allergen, are in part due to
interaction between basophils and the alarmin cytokines TSLP, IL-25, and IL-33. Thus,
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the relationship between these cytokines and basophils may play a role in mediating Type
2 immune responses in allergic asthma.
5.2.3 The Influence of the Airway Microenvironment on Basophil Effector Activity
Despite the many reports that basophils are a source of IL-4 and IL-13 within the
airways following exposure to allergen [98-105], it is unknown whether the interaction
between basophils and the airway microenvironment induces Type 2 cytokine release.
Within the airway microenvironment both epithelial cells and immune cells produce proinflammatory cytokines that have the potential to promote the effector role of basophils.
When this happens and what specific cytokines are most crucial for the up-regulation of
basophil activity following allergen inhalation remains unclear. An additional
justification to look at the interaction between the airway microenvironment and
basophils in vitro was due to the difficulty in measuring levels of alarmin cytokines
within the bone marrow, peripheral blood, and bone marrow. We employed an alternative
assay in Chapter 4 to assess 1) basophil pro-inflammatory responses to airway secretions
collected post-allergen inhalation and 2) whether the mechanism of this effect was
mediated through alarmin cytokines within the airway secretions. Chapter 4 showed that
isolated human basophils incubated overnight with 7 hour post-allergen sputum
supernatant showed the most significant increase in CD203c expression, and intracellular
expression of IL-13 and IL-4. The 0 hour airway samples also induced significant
increases in CD203c and intracellular IL-4, supporting the notion that allergic asthmatics
have underlying Type 2 inflammation in the airways compared to healthy individuals.
To further ascertain the mechanisms through which the airway microenvironment
promotes basophil function following allergen inhalation, basophils treated with blocking
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antibodies to the βc, TSLPR, ST2, and IL-17RB were examined. Blockade of the βc
significantly inhibited the effects induced by 7 hour post-allergen sputum supernatant on
basophil activation, whereas blockade of TSLPR, ST2, and IL-17RB had no sufficient
effect. Collectively these findings suggest that the interaction between the airway
microenvironment and basophils further promotes the effector role of basophils through a
pathway mediated by IL-3/IL-5/GM-CSF. Given that our in vitro studies have shown a
marked pro-inflammatory effect of alarmin cytokines on basophils, it is interesting that
blockade of their receptors had no effect on inhibiting airway sample-induced basophil
activity. However, we feel this does not refute or lessen the importance of alarmin
cytokines in allergic asthma or the impact of these cytokines on basophils.
There are several possibilities as to why blockade of alarmin cytokines had no
effect on airway sample-induced basophil activity. Firstly, our lab and other researchers
have found it difficult to measure alarmin cytokines within sputum at baseline and postallergen challenge (Chapters 3 and 4). The difficulty in measuring these cytokines can be
supported by the ineffectiveness of blocking alarmin cytokines to inhibit 7 hour airway
sample induced basophil activation. This may be due to alarmin cytokines being unstable
and metabolized quickly [345], thereby reducing the amount of available cytokines
present within the airway samples to activate basophils in vitro. Secondly, Fux et al. has
demonstrated through an ELISA that in response to allergen, IL-33 protein levels
increased rapidly within the BALF but declined to baseline levels at 18 hours postallergen [344]. As such, it is possible that although alarmin cytokines are upstream
triggers of Type inflammation during the EAR, downstream Type 2 cytokines such as IL3, IL-5, and GM-CSF play a more prevalent role in elongating the allergic response
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during the LAR within the airways. It is plausible that downstream Type 2 cytokines are
at higher levels within the sputum at 7 hours post-allergen as opposed to upstream
alarmin cytokines, thus blockade of βc has a more profound inhibitory effect on basophil
activity in vitro, as opposed to epithelial-derived cytokine receptor blockade. Lastly,
basophils are known to have an IL-3 autocrine activation loop. We have shown that
TSLP can induce basophil production of IL-3, which could in turn serve as another
mechanism for TSLP to enhance basophil activation by signaling through the IL-3R (α
and β) (Chapter 3). In Chapter 4, IL-25 and IL-33-induced basophil activation was
inhibited following blockade of IL-17RB and ST2. The possibility, that similar to TSLP,
the effects of IL-25 and IL-33 may be additionally mediated through an IL-3-dependent
mechanism cannot be excluded. This could partly explain why anti-IL-17RB and antiST2 had no effect on airway sample-induced basophil activation, whereas blocking the βc
had a dominant inhibitory effect. The proposed theories warrant further investigation,
which has been added to the future directions section of this thesis.
As a whole, we have shown that airway samples up-regulate basophil activity following
allergen inhalation, suggesting that within the airway microenvironment, potent Type 2
cytokines including IL-3, IL-5, and GM-CSF promote basophil activity, which may in
turn further contribute to the pathology of allergic asthma.
5.3
Therapeutic Implications: Basophil/Epithelial-Derived Cytokine Axis as a
Target in Allergic Asthma
TSLP, IL-33, and IL-25 have been implicated as key drivers of allergic
inflammation, with elevated levels correlating to disease severity. Data from murine
models provide compelling evidence for a critical role for these alarmin cytokines in the
pathogenesis of asthma, as shown by their ability to induce AHR, bronchoconstriction,
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airway inflammation, and tissue remodeling [219, 236-241, 245-268, 317-328].
Unfortunately, there have been few human studies to support the claims made in murine
models. The only human clinical trial to date has been carried out by Gauvreau et al.,
which involved determining the efficacy of the mAb AMG 157 in blocking the
interaction between TSLP with its specific receptor in mild allergic asthmatics [241].
Gauvreau and colleagues demonstrated that treatment with AMG 157 in subjects with
mild allergic asthma significantly attenuated allergen-induced EAR and LAR in parallel
with a reduction in blood and sputum eosinophils. They postulated that the effects of
AMG 157 were partially due to the indirect effects of anti-TSLP treatment on mast cell
and basophil activation, and subsequent release of potent mediators that in turn recruit
activated eosinophils to the airways. This hypothesis seems plausible given that they
observed a significant decrease in blood and sputum eosinophils and exhaled nitric oxide
at baseline following treatment with AMG 157. We further conclude that this hypothesis
is likely, given the following: 1) basophils are key sources of histamine and CysLTs
during the EAR, and 2) in Chapters 2 and 3 we demonstrated that basophil activation,
Type 2 cytokine intracellular expression, and infiltration within the airways are distinct
features of the LAR. Unfortunately, Gauvreau et al. did not specifically measure the
effect of in vivo AMG 157 treatment on basophil pro-inflammatory activity and airway
infiltration, so it is unclear whether blocking the TSLP/TSLPR pathway had a direct
effect on basophils, which could have been partially responsible for the subsequent
changes in FEV1 and eosinophilia.
Given the promising findings of the AMG 157, we decided to include a study
component in Chapters 3 and 4 to determine whether treating human peripheral basophils
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with blocking antibodies to TSLPR, ST2, and IL-17RB could inhibit basophil activation
induced by TSLP, IL-25, and IL-33, respectively, in vitro. In Chapter 3 TSLP-induced
basophil activation was down-regulated by blocking antibodies to TSLPR, IL-3Rα, and
βc, but not through blockade of IL-5Rα or GM-CSFRα. Basophils have been known to
have an IL-3 autocrine activation loop [92], thus the ability of TSLP to also induce
basophil production of IL-3, could in turn, serve as another mechanism for TSLP to
enhance basophil activity by signaling dependently through IL-3R (α and β). We
conclude that initiation and promotion of basophil pro-inflammatory activity following
allergen inhalation in human allergic asthma is at least, in part, due to activation by
TSLP, which can inhibited by blockade of TSLPR or IL-3R (α and β).
It is important to note the differences with respect to murine and human models
pertaining to the effect of TSLP on basophils. Siracusa and colleagues demonstrated that
TSLP induces basophil differentiation from murine progenitor cells independently of IL3 [66]. They further showed that TSLP-elicited basophils had a distinct phenotype
compared to IL-3-elicited basophils, where they had greater expression of IL-3Rα and
ST2, and produced more IL-4 or IL-13 in response to subsequent stimulation with IL-3 or
IL-33. Collectively, these findings indicate that in a murine model, TSLP can act
independently of IL-3 to create a specific subset of basophils with increased sensitivity to
IL-3 and IL-33. On the other hand, Hui et al. has found that in the presence of IL-3,
TSLP promotes basophil differentiation from human CD34+ cells [68]. TSLP was not
able to promote differentiation independently of IL-3. Collectively, these studies suggest
that there are key differences between the effects of TSLP exerted on basophils across
species. Our own thesis results, where TSLP-induced activation can be inhibited
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following blockade of TSLPR or IL-3R (α and β), have supported the notion of Hui et al.
that the effect of TSLP on basophils may be, in part, IL-3-dependent. It will be important
in the future to determine if there are other marked differences between human and
animal models with respect to the effects of alarmin cytokines on basophils and other
immune cells. Furthermore, it will be important to determine if both targeting of IL-3 and
TSLP is needed to neutralize basophil activity and if this is the same for IL-33 and IL-25.
In Chapter 4 IL-25 and IL-33-induced basophil activity was inhibited by
treatment with blocking antibodies to IL-17RB and ST2. This demonstrates that the
effects of these cytokines are mediated through a pathway dependent on their own
functional receptors. However, the possibility that similar to TSLP, the effects of IL-25
and IL-33 may also be mediated in an IL-3-dependent manner cannot be ruled out. This is
supported, in part, by the findings in Chapter 4 that showed blockade of IL-17RB and
ST2 had no effect on airway sample-induced basophil activity, whereas blocking the βc
had a dominant inhibitory effect. Thus, we suggest that a future direction of this thesis is
to identify any additional pathways that IL-25 and IL-33 may act through to regulate
basophil activity.
In this thesis, we provide a mechanism through which AMG 157 may operate to
decrease basophil numbers and activity at sites of inflammation. Furthermore, we
demonstrate a mechanism by which murine models involving the blockade of IL-25 and
IL-33 have shown decreased airway inflammation. Additional studies are warranted to
determine whether the inhibition of allergen-induced responses through blockade of
alarmin cytokines is due to the decreased activity and infiltration of basophils within the
airways.
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Therapeutic avenues for asthma have traditionally focused on preventing either
inflammation or bronchoconstriction. Inhaled glucocorticoid treatment is one of the gold
standard anti-inflammatory and bronchodilativeo treatments for asthma. Unfortunately,
corticosteroids, at high doses, are associated with side effects including osteoporosis,
growth suppression, adrenal suppression, and oral candidiasis [349-351]. In some cases
patients do not respond to corticosteroids, highlighting a need to develop a treatment that
encompasses both corticosteroid-resistant and non-resistant asthmatics that can inhibit
bronchoconstriction and airway inflammation. Based on the capacity of AMG 157 to
attenuate baseline indices of inflammation, it is possible that anti-TSLP therapy could
provide a new promising therapeutic avenue to treat asthma. However, the full clinical
value of AMG 157 remains to be determined. In addition, although there are promising
results from murine models involving the blockade of IL-25 and IL-33, no studies to date
have been conducted in humans. The important roles that have been identified for alarmin
cytokines underscores the need to further investigate the targeting of these cytokines to
attenuate allergic asthma.
5.4
Limitations
Limitations in the studies presented herein are acknowledged. In Chapters 3 and
4, given the invasiveness of bone marrow collection we could only collect samples at two
time-points. No bone marrow samples were drawn during the diluent challenge for
statistical comparison. Conclusions would be stronger if diluent-controlled bone marrow
samples were collected. However, the baseline and 24 hour post-allergen bone marrow
samples were both collected in the morning to control for diurnal variation. Secondly, in
Chapters 3 and 4, we were unable to detect secreted TSLP, IL-25, and IL-33 levels within
the bone marrow, periphery, or sputum. It would have been beneficial to our study if we
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could have examined whether there were associations between these cytokines and the
level of basophil activity, and if this correlated with lung function. We postulate that the
inability to measure these cytokines was either due to instability or local degradation of
the protein, which is consistent with the inability to detect these cytokines in biological
fluids reported by others [345]. With respect to TSLP, the frequency of subjects with
detectable levels of TSLP in blood was 10%. Although this is lower then another study
reporting measurement of 33% of TSLP in blood, this may be due to differences in the
population studies or performance of the assays utilized [352]. In the introduction, we
described previous studies that have shown airway Type 2 cytokine, as well as TSLP, IL25, and IL-33 mRNA expression is increased in asthmatics and correlates with disease
severity, and that blockade of these cytokines can attenuate allergen-induced airway
responses. Thus, we expect these cytokines to be elevated in the airways following
allergen inhalation. Future studies are needed to determine the kinetics of protein levels
of TSLP, IL-25, and IL-33 within the airways following an allergen challenge using more
sensitive detection assays. Similarly we could not detect secreted IL-13 or IL-4 from
basophil cultures possibly due to the low cell numbers available for the assay. Instead we
measured intracellular cytokine expression via flow cytometry. This does not directly
reflect cellular secretion of these cytokines, however intracytoplasmic expression of IL13 and IL-4 is a viable alternative and has been described previously in basophils [353;
354].
Thirdly, due to limited basophil cell numbers, it was difficult to conduct migration
experiments using a Boyden chamber in Chapters 3 and 4, thus we assessed cell shape
change as surrogate measure of responsiveness to eotaxin. Given that previous studies
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have used shape change to examine granulocyte migration, we feel that this technique is
an acceptable alternative for basophil migration.
Fourthly, in Chapter 4, we found that 7 hour post-allergen sputum supernatant
significantly induced basophil activity. We did not specifically measure what mediators
were contained within these airway samples and we acknowledge that measuring a wide
panel of cytokines and chemokines would have strengthened our conclusion. In the past,
our lab has found it quite difficult to measure adequate levels of mediators in sputum
supernatant. This is demonstrated in Chapters 3 and 4, where we were unable to measure
IL-13, IL-5, IL-4, TSLP, IL-25, and IL-33 within sputum supernatant. Instead, we
conducted in vitro studies that involved the blockade of Type 2 cytokines, known to have
a profound effect on basophil activity, as well as the more novel cytokines of interest
TSLP, IL-25, and IL-33. The inability to inhibit the effect of the sputum supernatant
samples of basophil activity by blocking receptors for alarmin cytokines further supports
the above notion that these cytokines are unstable and transient within the sputum.
Lastly, it is increasingly clear that asthma is complex and made up of disease
variants with a number of different pathophysiologies. Research over the last two decades
has sought to better understand the heterogeneous clinical nature of asthma. Older
attempts to phenotype asthma have emphasized differences between allergic vs. nonallergic asthma. However, more recently asthma phenotypes have been further redefined
by including information regarding the underlying pathophysiological mechanisms
present across different endotypes. Given the large number of idenitified asthma
endotypes, there is a growing need to develop targeted treatments. In order to generate
targeted treatments it is important to understand the underlying mechanisms and
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identification of cellular or molecular biomarkers that are characteristic of each endotype.
This may in turn aid in predicting clinical responses to various asthma therapies and
create a more individualized approach to diagnosis and treatment. An inherent limitation
to this thesis is that our findings pertain to the context of the eosinophilic-driven allergic
asthma endotype. We acknowledge that our study is limited in the sense where we have
only addressed one distinct endotype and thus cannot relate our findings to the entire
population of allergic asthmatics. Similarly, we are limited by studying a specific range
of asthma severity: mild to moderate. It is clear that there are different underlying
mechanisms across asthma severities. Thus, it will be important to determine if basophils
play a role in other endotypes or severities of asthma, or if they perhaps drive their own
distinct form of asthma. Furthermore, whether alarmin cytokines are critical mediators in
other endotypes or severities of asthma remains to be clear and if the interaction with
basophils is an important component to the pathophysiological mechanism of these
endotypes.
5.5
Summary
The overall objective of this thesis was to elucidate whether the interaction
between basophils and alarmin cytokines could potentiate the effector role of these cells
in the LAR of allergic asthma. Our initial objective was to determine the role of basophils
in the LAR following allergen inhalation in mild allergic asthmatics. The results suggest
that a subset of activated basophils migrate from the bone marrow into the peripheral
blood and finally into the airways following exposure to allergen inhalation. This study
was the first to analyze basophil activity within sputum samples collected from asthmatic
subjects via flow cytometry. Our results showed that following allergen inhalation, for up
to 24 hours, airway basophils expressed up-regulation of surface activation markers and
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intracellular Type 2 cytokines, supporting the notion that basophils have the ability to
play an effector role during late phase asthmatic responses. Furthermore, in Chapter 2, we
demonstrated that the surface marker CD203c is a primary measurement for detecting
basophil activation following allergen inhalation. We were able to demonstrate
distinctive differences in the kinetics of basophil activation between IER and DR. We
showed that basophil CD203c expression is associated with allergen-induced
bronchoconstriction, suggesting a role for basophils in the LAR. In addition, we found
Type 2 cytokine intracellular expression in Chapters 2 and 3 to correlate significantly
with the maximum % fall in FEV1 during the LAR, further suggesting a role for a
basophils in the potentiation of the allergen-induced LAR.
We next examined the change in basophil receptor expression for TSLP, IL-25,
and IL-33 following allergen inhalation. This study was the first to demonstrate a marked
upsurge in TSLPR, IL-17RB, and ST2 basophil expression in varying biological
compartments including the bone marrow, peripheral blood, and airways following an
allergen challenge. A key finding to this study was the correlation between peripheral
TSLPR and ST2 expression with maximum % fall in FEV1 during the LAR, suggesting
that increased sensitivity of basophils to alarmin cytokines, may in part contribute to
allergen-induced decline in lung function.
To expand on the above findings, we investigated whether TSLP, IL-25, and IL33 can enhance the activity of basophils in vitro, and demonstrated that these cytokines
up-regulate basophil activation marker and Type 2 cytokine intracellular expression, and
eotaxin-induced migration. We conclude that the up-regulated basophil activity during
the LAR following allergen inhalation could be partially due to interaction with alarmin
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cytokines. This is further supported by our in vitro studies demonstrating that blockade of
signaling between TSLP, IL-25, and IL-33 and their respective receptors attenuated
basophil activity.
Finally, given that we showed basophil activity within the sputum to increase
following allergen inhalation, we hypothesized that following allergen inhalation the
airway microenvironment contains Type 2 and alarmin cytokines, which can promote the
effector role of basophils. We found that post-allergen inhalation, the airway
microenvironment, has the ability to up-regulate basophil activation and Type 2 cytokine
intracellular expression in vitro through a IL-3/IL-5/GM-CSF-dependent mechanism.
Collectively, these studies presented within this thesis provide evidence that basophils
have an effector role in allergic asthma, and that interaction with the epithelium-derived
cytokines, can further promote this role following allergen inhalation. Our findings are
summarized in Figure 1.
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Figure 1. The Basophil/Epithelial Alarmin Axis
Allergen
TLRs
PRRs
Epithelial Cells
IL-33
TSLPR
+
Eotaxin
TSLP
ST2
IL-17RB
+
IL-25
+
+
Basophil
FcεR1
Cross-linking
+
Migra on
IgE
CCR3
Th2 Cell
+
IL-3,
IL-4,
IL-13,
IL-5,
IL-9,
GM-CSF
Smooth muscle cells
+
IL-3Rα
B Cell
IL-13
IL-4
IL-3
IL-5
Airway Hyperresponsiveness
Bronchoconstric on
Airway Inflamma on
Adapted from Chapter 3, Figure 7: TSLP-basophil axis in the airways during
allergic stimulation. Inhaled allergen binds to TLR and PRR receptors on the surface of
epithelial cells, which in turn produce chemokines (eotaxin) and alarmin cytokines
(TSLP, IL-25, IL-33) that can affect basophil activity within the airways. Alarmin
cytokines can bind to their respective receptors on basophils and induce up-regulation of
these receptors, as well as stimulate long-term Type 2 intracellular cytokine and
activation marker expression of basophils. In addition, alarmin cytokines can increase
CCR3 expression and enhance the chemotactic response of basophils to eotaxin, thus
promoting further infiltration into the airways. B cell-derived IgE can cross-link with
high-affinity FcεR1 and further up-regulate immediate basophil activity through the
release of histamine and CysLTs. Furthermore, the release of potent Type 2 cytokines
from activated Th2 cells can promote long-term basophil effector activity. Continued
release of pro-inflammatory mediators via both IgE-dependent and IgE–independent
mechanisms from basophils can lead to initiation and prolongation of the hallmark AHR,
bronchoconstriction, and airway inflammation of allergic asthma.
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5.6
McMaster University- Medical Sciences
Future Directions: Blockade of Basophil/Epithelial-Cell Derived Cytokine
Axis in Allergic Asthma
Given the more readily available isolation techniques and specific markers to
identify basophils, there has been an upward shift in basophil research in allergic disease.
Despite our findings that basophil number and activity increases within the airways
following allergen inhalation, the question still remains: How integral are basophils to
the development and propagation of allergic asthma?
In the past there has been a lack of true basophil knockout mouse models
developed due to the inability to identify a basophil-specific growth factor. However,
recently there have been some promising results from basophil depletion studies, which
have demonstrated the importance of basophils in driving late allergen-induced
responses. For example, Matsuoka et al. developed a basophil-deficient mouse model that
specifically targeted CD203c expression, which led to complete abolishment of ear
swelling in IgE-mediated chronic allergic inflammation, which normally involves
massive eosinophil infiltration [134]. These findings suggest that basophils act as an
important initiator and effector of chronic inflammation. Mukai and colleagues further
reported that the transfer of FcεR1-expressing basophils into FcεR1-deficient mice
restored the development of the delayed-onset allergic inflammation, as demonstrated by
ear swelling [100]. Conversely, transfer of FcεR1-expressing mast cells into FcεR1deficient mice restored acute or early phase allergic inflammation. They concluded that
basophils are indispensible for chronic allergic inflammation, whereas mast cells are
indispensible for the acute phase of allergic inflammation. This study highlights the
nonredundant roles of basophils that are distinct from mast cells. Obata et al., generated a
mAb specific to basophils and found that treatment with this mAb did not effect passive
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cutaneous anaphylaxis or contact hypersensitivity but did completely abolish the
development of IgE-mediated chronic allergic dermatitis [126]. Wada et al. used the
Mcpt8DTR mouse model to test the role of basophils in acquired resistance to tick
infection. They demonstrated that following treatment with DT there was a greater then
90% depletion in basophil numbers for 5 days and subsuequent abolishment of the
secondary infection, however transfer of basophil-enriched CD49b+ splenocytes isolated
from sensitized mice was sufficient to confer resistance in naive mice. Reber et al.
demonstrated that Mcpt8DTR mice given DT or treatment of wild-type mice with basophildepleting antibody Ba103 had significant reduction in peanut-induced hypothermia [133].
Lastly, Noti and colleagues demonstrated that using CD200R3 antibody-mediated or
Mcpt8DTR depletion of basophils resulted in the reduction of esophagitis eosinophilia and
symptoms of eosinophilic esophagitis in a murine model [130]. These findings suggest
that basophils play an important role in promoting eosinophilic infiltration to sites of
inflammation. Although the aforementioned studies suggest that basophils play a pivotal
role in the development of IgE-mediated chronic allergic inflammation, no animal studies
thus far have studied the effect of basophil depletion in the context of allergic asthma.
Thus a future direction could be to develop true basophil knockout or depletion mouse
models to determine the effect of basophil deficiency on AHR, bronchoconstriction,
eosinophilia, and Type 2 inflamamtion. The findings of these studies would provide an in
vivo model to study the distinct role of basophils in the context of allergic asthma.
Furthermore, despite there being multiple murine models that knockout receptors for
alarmin cytokines, to our knowledge, none thus far have investigated the subsequent
effect on basophil activity. It is important to also determine how critical the basophil-
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epithelial-derived cytokine axis is to allergic asthma, and whether there are differences
between mouse and human models.
As mentioned in Chapter 1, Tsai and et al. demonstrated CD203c to negatively
regulate ATP-driven activation of basophils and mast cells. IgE cross-linking resulted in
the production of ATP from mast cells and basophils, which could then act back on these
cells in an autocrine manner to activate themselves. CD203c-deficient mice expressed
greater sensitivity to allergen and increased cellular infiltration to the airways. They
concluded that CD203c is an important regulator of basophil and mast cell activity in the
context of asthma. Given that we have shown alarmin cytokines to promote basophil
activity, it would be beneficial to determine if these cytokines can induce ATP
production. This could then be taken further to assess whether blockade of CD203c could
inhibit the effect of alarmin cytokines on basophils. This may prove to be a beneficial
therapeutic avenue to inhibit both IgE-dependent and independent activation of basophils.
In this thesis we have established that alarmin cytokines can promote basophil
activity, but some questions remain: Are the cytokines TSLP, IL-25, and IL-33 equally
important influencers of basophil activity or is one more important than the other? Is
one of these cytokines more upstream then the others? Additional mechanistic studies
will help elucidate the relationship between alarmin cytokines and basophils.
Furthermore, determining the most important and/or upstream alarmin cytokine inducer
of basophil activity will allow us develop the most efficient targets for treating allergic
asthma.
As mentioned earlier, another avenue of investigation is to determine whether IL33 and IL-25 signaling is in part mediated via an IL-3-dependent mechanism in
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basophils. The findings of these experiments may help explain what we observed in
Chapter 4, where blockade of the βc inhibited airway sample-induced basophil activation
but neutralizing ST2 and IL-17RB had no effect. It is possible that although TSLP, IL-33,
and IL-25 may have been present in the sputum, they could have subsequently indued
basophil IL-3 expression, which could have in turn further stimulated basophil activity
through autocrine feedback. Therefore, neutralization of βc was more effective at
inhibiting the downstream effects on basophil activity that were induced by alarmin
cytokines within the sputum samples. As we have shown in Chapter 3, TSLP can induce
basophil activation marker expression and intracellular expression of Type 2 cytokines
IL-4 and IL-13, which was inhibited following blockade of IL-3Rα and the βc.
Furthermore, Smithgall et al. reported that IL-33 stimulates IL-5 and GM-CSF release
from basophils, suggesting that these cytokines may act back on basophils in an autocrine
manner to magnify basophil activity [284]. Thus additional experiments are required to
establish if these cytokines induce IL-3 release in basophils and whether blockade of IL3Rα has an effect on IL-33 or IL-25-induced basophil activity.
Although we have established that TSLP can affect human basophil activity, it is
important to determine the specific intracellular signaling pathways that are mediated by
the TSLP/TSLPR axis on basophils. Thus, future experiments could include determining
whether the TSLPR and IL-7Rα are both required for TSLP signaling and what specific
intracellular signaling pathways are involved. This will in turn identify additional targets
for impairing basophil function in allergic asthma.
Given that murine models have shown blockade of alarmin cytokines to attenuate
AHR and inflammation, it is important to determine if these same results can be
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translated to human studies. Future experiments are required to thoroughly investigate the
efficacy of blocking TSLP, IL-25, and IL-33 to treat allergic asthma. Ultimately, a better
understanding between the interaction of the epithelium and immune cells will help lead
the way to the development of novel therapies that target the source of inflammation
underlying the pathology of allergic asthma.
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CHAPTER 6:
McMaster University- Medical Sciences
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APPENDIX I
Copyright Permissions to Reprint Published Materials
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March 11, 2015
Dear Brittany
Thank you for your email. Please ignore my previous email as there was a copy-paste-mistake in
it:
As to your request, I am pleased to inform you that permission is granted herewith to use your
article
Inhibition of Allergen-induced Basophil activation by ASM-024, a nicotinic receptor ligand
Int Arch Allergy Immunol 2014;165:255-264 (DOI:10.1159/000370068)
to be reproduced in your upcoming PhD thesis, provided that proper credit will be given to the
original source and that S. Karger AG, Basel will be mentioned.
Please note that this is a non-exclusive permission, hence any further use or distribution, either
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McMaster University- Medical Sciences
May 11, 2015
Hi There,
I have recently had the following manuscript published in JACI:
Manuscript Number: JACI-D-14-01529R1
Title: Thymic Stromal Lymphopoietin Activation of Basophils in Allergic Asthma is IL-3
Dependent
I would like to re-print this paper in my thesis since this manuscript was a component of
my PhD thesis. How do I go about obtaining permission to re-print this manuscript?
Thank you
May 11, 2015
Dear Brittany:
As an Elsevier journal author, you retain various rights including Inclusion in a thesis or
dissertation (provided that this is not to be published commercially); see
https://www.elsevier.com/about/policies/lightbox_scholarly-purposes for more
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Best of luck with your thesis and best regards,
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JACI Publication Re-Print Policy:
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APPENDIX II
Reprinting of:
Inhibition of allergen-induced basophil activation by ASM-024, a nicotinic
receptor ligand
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209
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