Kurdistan Regional Government Ministry of Higher Education and Scientific Research University of Sulaimani College of Medicine Department of Microbiology
THE ROLE OF INFLAMMATORY CELLS IN ALLERGIC RHINITIS A THESIS SUBMETTED TO THE CONCIL COLLEGE OF MEDICINE UNIVERSITY OF SULAIMANI IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF M.Sc. IN MICROBIOLOGY/IMMUNOLOGY
By Sulaf Mousa Issa B.Sc. in Biology H.D. in Microbiology
Supervised by Professor Dr.Kawa Abdulla Mohammad Amin B.Sc., M.Sc., Ph.D. Medical Microbiology
May, 2017
Jozardan, 2717 I
أَﻟَﻢْ ﺗَﺮَ ﻛَﯿْﻒَ ﺿَﺮَبَ اﻟﻠﱠﮫُ ﻣَﺜَﻠًﺎ ﻛَﻠِﻤَﺔً ﻃَﯿﱢﺒَﺔً ﻛَﺸَﺠَﺮَةٍ ﻃَﯿﱢﺒَﺔٍ أَﺻْﻠُﮭَﺎ ﺛَﺎﺑِﺖٌ وَﻓَﺮْﻋُﮭَﺎ ﻓِﻲ اﻟﺴﱠﻤَﺎءِ
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Declaration I am Sulaf Mousa Issa MSc student declare that this thesis is my original work and has never been presented in any other university and that all resources and materials have been duly acknowledge.
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List of Contents Abstract
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List of Figures
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List of Tables
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List of Abbreviations
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Dedication
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Acknowledgements
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Chapter One: Introduction Introduction
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Chapter Two: Literature Review 2.1
Allergic Rhinitis
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2.1.1
Definitions
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2.1.2
Classification
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2.1.3
Prevalence
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Pathophysiology of Allergic Rhinitis
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2.2.1
Sensitization to Allergens
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2.2.2
Early and Late Response
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2.2.3
Minimal Persistent Inflammation
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2.2
VII
2.2.4
Complications of Allergic Rhinitis
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2.3
Genetic of Allergic Rhinitis
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2.4
Diagnosis of Allergic Rhinitis
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2.5
Therapy of Allergic Rhinitis
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2.6
2.6.1
Inflammatory Cells, Proinflammatory Cells, and Mediators in Allergic Rhinitis Eosinophil Cells
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2.6.1.1 Eosinophil Overview
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2.6.1.2 Eosinophil Function
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2.6.1.3 Eosinophil and the Immune System
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2.6.1.4 Eosinophil Cationic Protein
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2.6.2
Mast Cells
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2.6.2.1 Mast Cells Overview
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2.6.2.2 Mast Cells Function
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2.6.2.3 Mast Cells and the Immune System
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2.6.3
Immunoglobulin IgE
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2.6.3.1 Immunoglobulin IgE Overview
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2.6.3.2 IgE in Disease
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2.6.3.3 IgE and the Immune System
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VIII
2.6.4
Cytokines
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T Cells
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2.7.1
T Cells Overview
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2.7.2
T Cells Function
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2.7.3
T Cells and the Immune System
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B Cells and Immunoglobulin Production
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2.8.1
B Cells Overview
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2.8.2
B Cells Function
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2.8.3
B Cells and the Immune System
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2.7
2.8
Chapter Three: Materials and Methods 3.1
Materials
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3.2
Methods
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3.2.1
Patients and Setting of the Study
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3.2.2
Inclusion and Exclusion Criteria
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3.2.3
Biosafety Considerations
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3.2.4
Ethical Considerations
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3.2.5
Specimens
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3.2.6 3.2.7 3.2.8
3.2.9
3.2.10
3.2.11 3.3
Optical Flow Cytometry Kit for Detecting Eosinophil in Human Blood ECLIA Kit for Detecting Immunoglobulin IgE in Human Serum Agglutination Kit for Detecting Rheumatoid Arthritis (RA) Factor/C-Reactive Protein (CRP) in Human Serum ELISA Kit for the Measurement of Human ECP (Eosinophil Cationic Protein) in Serum ELISA Kit for the Measurement of Human Interleukin-17 (IL-17) in Serum ELISA Kit for the Measurement of Human Interleukin-33 (IL-33) in Serum Statistical Analysis
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Chapter Four: Results 4.1 4.1.1
4.1.2
4.2
4.3
4.4
Characteristics of Patients and Healthy Control Comparison of Blood Eosinophil Counts Among Allergic Rhinitis Patients and Healthy Control Comparison of Serum IgE levels Among Allergic Rhinitis Patients and Healthy Control Comparison of Serum ECP Levels Among Allergic Rhinitis Patients and Healthy Control Comparison of Serum IL-17 Levels Among Allergic Rhinitis Patients and Healthy Control Comparison of Serum IL-33 Levels Among Allergic Rhinitis Patients and Healthy Control
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4.5
4.6
4.7
4.8
4.9
Correlation Between ECP Level and IL-17 Level Among the Allergic Rhinitis Patients Correlation Between ECP Level and IL-33 Level Among the Allergic Rhinitis Patients Correlation Between IL-17 Level and IL-33 Level Among the Allergic Rhinitis Patients Correlation Between ECP Level and IgE Level Among the Allergic Rhinitis Patients Correlation Between Symptoms and Different Markers Among the Allergic Rhinitis Patients
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Chapter Five: Discussion 5.1 5.2
5.3
5.4
5.5
5.6
5.7
5.8
Characteristics of Patients and Healthy Control Comparison of Serum ECP Levels Among Allergic Rhinitis Patients and Healthy Control Comparison of Serum IL-17 Levels Among Allergic Rhinitis Patients and Healthy Control Comparison of Serum IL-33 Levels Among Allergic Rhinitis Patients and Healthy Control Correlation Between ECP Level and IL-17 Level Among the Allergic Rhinitis Patients Correlation Between ECP Level and IL-33 Level Among the Allergic Rhinitis Patients Correlation Between IL-17 Level and IL-33 Level Among the Allergic Rhinitis Patients Correlation Between ECP Level and IgE Level Among the Allergic Rhinitis Patients
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5.9
Correlation Between Symptoms and Different Markers Among the Allergic Rhinitis Patients
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Chapter six: Conclusions and Recommendations Conclusions
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Recommendations
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References
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Appendices Questionnaire for Allergic Rhinitis Patients Abstract in Arabic Abstract in Kurdish
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Abstract Background: Allergic rhinitis (AR) is one of the world's most common health problems that affect people of all ages. AR is defined as an inflammation of the lining of the nose. It's characterized by production of allergen-specific IgE, which bind to mast cells and basophils and initiate a cascade of cellular events that affect the respiratory tract. Clinical sign and symptoms of AR are often non-specific and ignored by patients and physicians, and it has a significant impact on patients’ quality of life. Aims of the study: To determine the level of cytokines; Interleukin-17 (IL17) and Interleukin-33 (IL-33) in AR patients. To find the relationship between the level of the cytokines IL-17, IL-33, an anti- Eosinophil cationic protein (ECP) and anti- Immunoglobulin E (IgE) antibody in mediating activation in the eosinophil and mast cell in AR. To find the correlation between the inflammatory cell, its mediators and allergic symptoms. Methods: In this study the blood sample collection were carried out from February 2016 to June 2016. Blood samples were taken from 88 AR patients and 88 healthy controls (HC). Each patient sample was analyzed for eosinophil counts by optical flow cytometry, IgE by electrochemiluminescence immunoassay (ECLIA), ECP, IL-17, and IL-33 by enzyme-linked immunosorbent assay (ELISA). Furthermore, for control sample also analyzed for rheumatoid factors (RF) and C-reactive protein (CRP) by agglutination. Results: There was no significant difference between AR patients and control group in age and gender. Levels of Eosinophils, IgE, ECP, IL-17, IL-33 and the total symptom scores were significantly higher in AR patients than HC. Serum ECP with IL-17, IL-33, and IgE levels were correlated in a patient with AR, respectively. There was no correlation between IL-17 and IL-33. There was a correlation between symptom scores and Eosinophils, and IgE in a patient with AR, respectively. No correlation was observed between symptom scores and ECP, IL-17, and IL-33 in a patient with AR, respectively. Conclusions: Serum levels of ECP, IL-17, and IL-33 are suitable markers to differentiate AR patients from HC subjects. ECP correlates with IL-17, IL-33, and IgE in AR disease. Finally, cytokines (IL-17, IL-33) and ECP plays a key role in regulating immune responses in AR, and their pathway is being used as a novel therapeutic target in diseases associated with eosinophils, mast cells, and basophils.
Key words: Allergic rhinitis, immune system, ECP, IL-17, IL-33, IgE.
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List of Figures
Figure Figure Title No. 2.1 The Biology of Allergic Sensitization and of the Allergic Reaction
Page No. 8
in the Nasal Mucosa Leading to the Generation of Symptoms and to Functional Alterations such as Nasal Hyperresponsiveness 2.2
Effects of the Eosinophil Cationic Protein (ECP)
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2.3
Induction and Effector Mechanisms in Type I Hypersensitivity
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2.4
IL-17 and the IL-17 Receptor Families
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2.5
IL-33 Responses in Necrosis and Apoptosis
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3.1
Typical Standard Curve of Human ECP ELISA Kit
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3.2
Typical Standard Curve of Human IL-17 ELISA Kit
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3.3
Typical Standard Curve of Human IL-33 ELISA Kit
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4.1
Comparison of Blood Eosinophil Counts in AR Patients and HC
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Subjects 4.2
Comparison of Total IgE Level in Blood of AR Patients and HC
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4.3
Histogram Shows the Difference in Serum Level of ECP
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Concentration (ug/L) Between AR Patients and HC 4.4
Comparison Between IL-17 Level in Serum of AR Patients and HC
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4.5
Comparison Between IL-33 Level in Serum of AR Patients and HC
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4.6
Correlation Between Serum ECP and IL-17 Levels in AR Patients
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4.7
Correlation Between Serum ECP and IL-33 Levels in AR Patients
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4.8
Correlation Between Serum IL-17 and IL-33 Levels in AR Patients
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4.9
Correlation Between the Concentration of ECP and IgE Levels in
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AR Patients
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List of Tables
Table No.
Table Title
Page No.
3.1
List of Materials
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3.2
List of Kits
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3.3
List of Equipment
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4.1
4.2
Characteristics of Patients and Healthy Control According to Age, Sex, Symptoms, Serum IgE and Eosinophil Correlation Between Symptoms and Different Markers in AR Patients.
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List of Abbreviations APCs
Antigen-Presenting Cells
AR
Allergic Rhinitis
ARIA
Allergic Rhinitis, and its Impact on Asthma
BAL
Broncho Alveolar Lavage
BCR
B Cell Receptor
C
Constant
CAP
Capsulated Hydrophilic Carrier Polymer
CBC
Complete Blood Count
CCR
Chemokine Receptor
CR
Complement Receptor
CRP
C-Reactive Protein
DCs
Dendritic Cells
DNA
Deoxyribo Nucleic Acid
ECLIA
Electrochemiluminescence immunoassay
ECP
Eosinophil Cationic Protein
EDN
Eosinophil-Derived Neurotoxin
EDTA
Ethylene Diamine Tetra Acetic Acid
ELISA
Enzyme Linked Immunosorbent Assay
ENT
Ear Nose Throat
EPO
Eosinophil Peroxidase
E-selectin
Endothelial Selectin
FcεRI
High-Affinity IgE Receptors I
FcεRII
Low-Affinity IgE Receptors II
GM-CSF
Granulocyte/Macrophage- Colony Stimulating Factor
GWAS
Genome-Wide Association Studies
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H
Heavy
HRP
Horse Radish Peroxidase
HSCs
Hematopoietic Stem Cells
ICAM-1
Intercellular Adhesion Molecule 1
IDO
Indoleamine Dioxygenase
IFN-γ
Interferon Gamma
Ig
Immunoglobulin
IgE
Immunoglobulin E
IL
Interleukin
L
Liter
LT
Leukotriene
MAST
Multiple Allergen Simultaneous Test
MBP
Major Basic Protein
MC (C)
Mast Cell (Chymase)
MC (T)
Mast Cell (Tryptase)
MC (TC)
Mast Cell (Tryptase, Chymase)
MCP
Monocyte Chemoattractant Protein
mg
Milligram
MHC II
Major Histocompatibility Complex Class II
ml
Milliliter
MPI
Minimal Persistent Inflammation
MZ
Marginal Zone
NF-HEV
Nuclear Factor Protein in High Endothelial Venules
NF-kB
Nuclear Factor- kB
NGF
Nerve Growth Factor
NK
Natural Killer Cell
NKT
Natural Killer T
nm
Nanometer XVII
O.D
Optical Density
PAF
Platelet Activating Factor
PAR
Perennial Allergic Rhinitis
pg
Picogram
PGDR
Prostaglandin Receptor
R
Receptor
RA
Rheumatoid Arthritis
RANTES
Regulated Upon Activation Normal T Cell Expressed and Secreted
RAST
Radio Allergo Sorbent Test
RNA
Ribo Nucleic Acid
RNase
Ribo Nuclease
RPM
Round Per Minute
SAR
Seasonal Allergic Rhinitis
SCF
Stem Cell Factor
SCIT
Sub Cutaneous Allergen-Specific Immuno Therapy
sIgE
Specific Immunoglobulin E
SLIT
Sub Lingual Allergen-Specific Immuno Therapy
SPT
Skin Prick Test
TARC
Thymus and Activation Regulated Chemokine
TC cell
Cytotoxic T Cell
TCR
T Cell Receptor
TGF-β
Transforming Growth Factor Beta
TGF-α
Transforming Growth Factor Alpha
TH
T-Helper
TLR
Toll-Like Receptor
TMB
Tetra Methyl Benzidine
TNF-α
Tumor Necrosis Factor Alpha XVIII
Treg cell
Regulatory T Cell
V
Variable
VCAM-1
Vascular Cell Adhesion Molecule 1
VEGF
Vascular Endothelial Cell Growth Factor
WHO
World Health Organization
µg
Microgram
µL
Micro Liter
?m
Micrometer
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Dedicated to the greatest creature….. To the strongest person I know….. To my mother.
Sulaf Mousa 2017
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Acknowledgements Praise is to Almighty Allah, the most gracious, the most merciful, for enabling me to accomplish this work. I owe more than thanks to my mother and my sister Kawthar, for their almost unbelievable support and encouragement. Without their support, it is impossible for me to finish my master study. I would like to express my very great appreciation to my research supervisor Professor Dr.Kawa A. M. Amin for his patient guidance, enthusiastic encouragement and useful critiques of this research work. I would like to express my deep gratitude to Assistant Professor Dr.Muaid Ismaiel Aziz (Otolaryngologist) for his valuable and constructive suggestions during the planning and development of this research work. My grateful thanks to Dr.Zana Hama Baqi, Dr.Shukr Hamid Rassam and Dr.Huner Mohamed Hama Ameen in ENT department for their help during period of sample collection. I wish to express my special appreciation and thanks to Dr.Saman Hussein Noori for using his private laboratory (Dr.Saman Laboratory). Special thanks are given to Department of Microbiology/College of Medicine for giving me the opportunity to study the MSc degree. I am deeply grateful to Dr.Raza H. Hussein (University's president) and Ms. Paiman Omar (Registration employe) for their supporting. I am deeply grateful to my friends; Taha Ahmed, Bahadin Jalal, Aram Helmi, Kochar Ibrahim, Khalil Hassan, Hemn Muhedin, Nazhad Hassan, Shno Jalal, Saiwan Maarouf, Dr.Khanda Abdulateef and Dr.Gollshang Ahmed to support me. I would like to thank many of my colleagues in laboratory of Public Health in sulaimani city to support me. Finally I would like to thank my friend Dlovan Jalal Ali for her support, advice and encouragement during my study.
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CHAPTER ONE INTRODUCTION
Chapter One
Introduction
Chapter One Introduction Background Allergic rhinitis (AR) is a symptomatic disorder of the nose affecting 10%-40% of the worldwide population over a lifespan (Liu et al., 2010). Clinical sign and symptoms are often non-specific and ignored by patients and physicians. Most affected individuals do not report their complaints or seek treatments (Mahboub et al., 2014). Accordingly, AR is considered a social problem that negatively affects the patients’ quality of life and performance (Tatar et al., 2013). Recently, AR is divided on the basis of frequency and duration of symptoms into intermittent AR and persistent AR (Konig et al., 2015). The prevalence of AR is increasing all over the world (Min, 2010). The pathophysiology of AR is initiated by binding allergen to mast cellbound Immunoglobulin E (IgE) results in rapid mast cell degranulation, increased levels of inflammatory mediators, accumulation of inflammatory cells in the nasal mucosa, and a recurrence of symptoms several hours after initial allergen exposure. This response can be described as follows: initial allergen sensitization, early and late phase response, and minimal persistent inflammation (Canonica and Compalati, 2009). AR and other allergic diseases result from genetic predisposition in combination with environmental factors (Wang de, 2011). There is a genetic predisposition for the development of allergic symptoms in different societies. In atopic patients, with the strong familial tendency, usually starting in childhood or adolescence, they become sensitized and produce IgE antibodies in response to ordinary allergens (Johansson et al., 2004).
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Chapter One
Introduction
While environmental factors, such as air pollution, temperature, humidity, changed lifestyle, and bacterial/viral infection are frequently quoted as adjuvant factors for allergic sensitization and possible causes of the increased prevalence (Wang, 2005). However, both serum specific IgE (sIgE) antibody test and skin prick test (SPT) results were considered equivalently acceptable for detecting respiratory allergy (Crimi et al., 1999), but there are data indicating that IgE value could be highly specific, but somewhat less sensitive than the SPT (Bousquet et al., 2008). Regarding cells of the immune system, the production of cytokines, chemokines, and other mediators are involved in the inflammatory mechanisms of AR. These mediators are associated with the T-helper cell type 2 (TH2) profile, the release of a variety of preformed and newly generated mediators from mast cells and eosinophils which consider central effector cells in allergic inflammation (Pawankar et al., 2011, Stone et al., 2010). Though, the selective recruitment of mast cells and eosinophils has been shown to be important in the pathogenesis of allergic rhinitis (Amin et al., 2001). Eosinophil cationic protein(ECP) is a mediator of inflammation that is released from the eosinophil upon activation during allergic diseases such as asthma and rhinitis (Woschnagg et al., 2009). ECP possesses cytotoxic ribonuclease (RNase) activity with bactericidal and antiviral properties (Kita, 2013, Munthe-Kaas et al., 2007). The mechanism for killing target cells is by the formation of transmembrane pores and channels (Woschnagg et al., 2009). ECP has been quantified in body fluids of patients with allergic and other inflammatory diseases including serum, bronchoalveolar lavage (BAL) and nasal secretions (Bystrom et al., 2011). Interleukin-17 (IL-17) is a cytokine produced by the T-helper17 (TH17) cells and is involved in host defenses and in the induction of inflammation (Jin and Dong, 2013). Recent progress in knowledge of the TH17 cells suggests a 2
Chapter One
Introduction
pathological role of IL-17 in the allergic responses (Iwakura et al., 2011). However, the involvement of TH17 cells in AR has not been clearly examined (Mo et al., 2013). Interleukin-33 (IL-33), a mediator of inflammation, is a novel cytokine of the IL-1 family that has been involved in the TH2 mediated immune response, host defenses and allergic diseases (Enoksson et al., 2011). IL-33 is produced by different cell types such as macrophages, dendritic cells (DC), mast cells (MC), fibroblasts, smooth muscle cells, endothelial cells and epithelial cells (Saluja et al., 2015). IL-33 is involved in the activation of many cells participating in allergic inflammation such as TH2 cells, MCs, eosinophils, basophils and DCs. Thus this cytokine is considered to play a crucial role in an allergic inflammatory disease such as rhinitis (Rogala and Gluck, 2013).
The Purpose of the Study was to: 1. Evaluate the level of cytokines (IL-17 and IL-33) in allergic rhinitis patients. 2. Find the relationship between the level of the cytokines IL-17, IL-33, an anti-ECP and anti-IgE antibody in mediating activation in the eosinophil and mast cell in allergic rhinitis. 3. Find the correlation between the inflammatory cell, its mediators and allergic symptoms.
Benefits of the Study: 1. Providing evidence for etiological factors of allergic rhinitis diseases. 2. Finding the serum level of IL-33 and inflammatory markers among allergic rhinitis patients in our community.
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CHAPTER TWO LITERATURE REVIEW
Chapter Two
Literature Review
Chapter Two Literature Review 2.1 Allergic Rhinitis 2.1.1 Definitions Allergic rhinitis (AR) is a type of inflammation in the nose, which occurs when the immune system overreacts to allergens in the air. Rhinitis is a global health problem and a symptomatic disorder of the nose induced by allergen exposure of the nasal mucosa via IgE-mediated type I hypersensitivity reactions (Said et al., 2012, Hellgren et al., 2010, Min, 2010). AR is often associated with asthma, sinusitis, otitis media or nasal polyps and has a significant impact on patients’ quality of life (Meymane Jahromi and Shahabi Pour, 2012, Tatar et al., 2013). AR can influence sleep, school attendance and work performance, regardless of gender, age, social and ethnic background (Scadding, 2015, Bousquet et al., 2008).
2.1.2 Classification According to traditional classification, AR has been classified as seasonal allergic rhinitis (SAR) which is known as hay fever, and perennial allergic rhinitis (PAR) based on temporal patterns of symptoms (Sin and Togias, 2011, Rudack, 2004). SAR is caused by outdoor allergens such as pollen and molds occurred during pollen season. The PAR is caused by indoor allergens such as dust mites, molds, cockroaches, animal dander’s and could occur in different seasons. However, this classification was useful but posed several problems. People who are allergic to seasonal pollens may suffer from symptoms over several seasons while people with PAR may experience symptoms for short but recurring periods may suffer from seasonal exacerbations (Todo-Bom et al., 2007). For this reason, the allergic rhinitis and its impact on asthma (ARIA) group in conjunction with the World Health Organization (WHO) have 4
Chapter Two
Literature Review
reclassified AR on the basis of the frequency and duration of symptoms into two categories: Intermittent and persistent. Intermittent AR is defined as experiencing symptoms for <4 days/week or <4 consecutive weeks. Persistent AR is termed as symptoms occurring for more than 4 days/week and more than 4 consecutive weeks (Bauchau and Durham, 2005).
2.1.3 Prevalence Epidemiological studies suggest that the prevalence of AR is increasing globally and currently affecting 40% of the population worldwide (Al-Abri et al., 2014, Zhang and Zhang, 2014). Although AR affecting both adults and children, it remains largely undiagnosed. Up to one-third of patients with allergies never consult a physician, suggesting that AR’s true prevalence may be underestimated (Bauchau and Durham, 2005, Canonica and Compalati, 2009). According to the WHO, prevalence of AR in the United States is 30%, in Europe, 20%, in different parts of the Middle East 9%, in Canada 20% , in Africa 27.3% (Mahboub et al., 2014, Konig et al., 2015, Mbatchou Ngahane et al., 2014).
2.2 Pathophysiology of Allergic Rhinitis 2.2.1 Sensitization to Allergens The sensitization process is initiated in the mucosal surface, when the allergen is processed by antigen-presenting cells (APCs), such as DCs and migrate to lymph nodes, where they presented on the major histocompatibility complex (MHC) class II molecule (Chaplin, 2006). In lymph nodes, this antigen is presented to naive CD4+ T-lymphocytes (T cells) which causes inducing differentiation to the TH2. Activated TH2 cells secrete several cytokines, which promote the differentiation of B cells to plasma cell, then induce immunoglobulin (Ig) synthesis and Ig isotype switching to produce sIgE. Also, 5
Chapter Two
Literature Review
activated TH2 cells induce proliferation of eosinophils, MCs and basophils. Produced antigen-sIgE binds to high-affinity IgE receptors on MCs or basophils (Broide, 2007, Amin, 2012) (Figure 2.1).
2.2.2 Early and Late Response Allergic reactions develop in two different patterns according to a time sequence. Within minutes of contact with an allergen, IgE-sensitized mast cells degranulate and release a variety of preformed and newly synthesized mediators such as histamine, prostaglandins, leukotrienes and cytokines (Amin, 2012). These mediators cause characteristic symptoms of the classic early-phase response of AR, including sneezing, itching, rhinorrhea, congestion which lasts for about 2-3 hours. However, histamine, which is the major mediator of AR, stimulates the sensory nerve endings of the Vth nerve (trigeminal) and induces sneezing. Histamine also stimulates the mucous glands causing the secretion of mucous (rhinorrhea) and histamine, leukotrienes and prostaglandins act on the blood vessels, causing nasal congestion (Min, 2010, Prussin and Metcalfe, 2006, Pawankar et al., 2011). In addition, AR may be associated with watering, redness of eyes, anosmia, fatigue and dark circles around the eyes (“allergic shiners”) (Blaiss, 1999, Chen et al., 2009, Al-Abri et al., 2014). The late phase of the AR response occurs 4-6 hours after antigen stimulation and is characterized by a recurrence of symptoms but nasal congestion becomes more prominent which lasts for about 18-24 hours (Pawankar et al., 2011). As a result of cytokine or mediator release, the nasal mucosa becomes infiltrated with inflammatory cells, basophils, eosinophils, neutrophils, MCs, TH2 cells and mononuclear cells. A series of mediators are released by these inflammatory cells including leukotrienes, kinins, histamine which cause a
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Chapter Two
Literature Review
continuation of the symptoms and the development of the late phase (Gelfand, 2004, Amin et al., 2001). While cytokines like IL-4, IL-13 from MCs can upregulate the expression of adhesion molecules like vascular cell adhesion molecule 1 (VCAM-1) on the endothelial cells facilitating the infiltration of eosinophils, T cells and basophils into the nasal mucosa. In addition, chemokines like RANTES, eotaxin, MCP-4 and thymus and activation-regulated chemokine (TARC) released from epithelial cells serve as chemoattractants for eosinophils, basophils and T cells (Li et al., 1999, Sekiya et al., 2000). Other cytokines like IL-5 from MCs and granulocyte/macrophage- colony stimulating factor (GM-CSF) which released largely by epithelial cells and T cells enhance the survival of the infiltrated eosinophils (Park and Bochner, 2010). In addition, a variety of proinflammatory mediators released like eosinophil cationic protein (ECP), major basic protein (MBP) and platelet activating factor (PAF) are also implicated in the late phase response (Pawankar et al., 2011) (Figure 2.1).
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Chapter Two
Literature Review
Figure 2.1: The Biology of Allergic Sensitization and of the Allergic Reaction in the Nasal Mucosa Leading to the Generation of Symptoms and to Functional Alterations such as Nasal Hyperresponsiveness (Sin and Togias, 2011).
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Chapter Two
Literature Review
2.2.3 Minimal Persistent Inflammation The term minimal persistent inflammation (MPI) has been used to describe a state when repeated exposure to low levels of allergen produces no allergy symptoms but does elicit a state of heightened sensitivity to subsequent allergen exposure (Ciprandi et al., 1995, Passalacqua et al., 2001). This state which is known as MPI may contribute to common co-morbidities of AR such as asthma. In the patient's nasal mucosa, MPI is characterized by the presence of inflammatory cells (eosinophils, neutrophils) and increased intercellular adhesion molecule 1 (ICAM-1) expression on epithelial cells (Montoro et al., 2007).
2.2.4 Complications of Allergic Rhinitis There are numerous complications of AR include sinusitis, otitis media, hearing impairment, dental problems (overbite) due to excessive mouth breathing, and Eustachian tube dysfunction (Settipane, 1999, Blaiss, 2008). In children, these complications may be associated with reduced appetite, delayed growth and sleep disturbances (apnea), as well as fatigue and mood changes (Ibero et al., 2012).
2.3 Genetic of Allergic Rhinitis According to previous studies, allergic diseases are influenced by genetic predisposition and environmental factors (Wang de, 2011). However, in AR like other atopic diseases, multiple family members often are affected (North and Ellis, 2011), few studies of genes associated with AR have been done in the past decades. Genome-wide association studies (GWAS) which is a rapid way of identifying single nucleotide polymorphisms view there is an association with polymorphisms of the transcription factor GATA-binding protein 3; trans-acting T-cell-specific transcription factor and 9
Chapter Two
Literature Review
interleukin IL-13 with the risk of developing AR was reported in a cohort of children followed up until the age of 10. Furthermore, these studies have not been well replicated, they have been done on small subject numbers and often the subjects had comorbid asthma or atopic dermatitis (Pawankar et al., 2011). Additionally, environmental factors (tobacco smoke, pollution, infection) can have permanent effects on gene regulation and expression through epigenetic mechanisms (North and Ellis, 2011, Hollingsworth et al., 2008, Scadding, 2015). Hence, AR must be evaluated as a complex interplay between genetic and environmental factors (Wang de, 2011).
2.4 Diagnosis of Allergic Rhinitis The diagnosis of AR is based on a typical clinical history of allergic symptoms and diagnostic tests (Bousquet et al., 2008). Allergic nasal symptoms including rhinorrhoea, blockage, sneezing and itching. AR is diagnosed with detailed clinical history by consultanting ear, nose and throat specialist (ENT), including questions about possible asthma and nasal examination, together with the possible inspection of the throat, ears and chest, confirming by either skin prick test (SPT) or serum sIgE. Clinical history includes: symptom type, duration and frequency and exacerbating factors (Scadding, 2015). Skin Prick Test SPT is a standard method of allergy testing. SPT is done by a panel of allergens, including those that are relevant in the patient’s environment (Rondon et al., 2010). However, results are available within 15-20 minutes, SPT is costeffective, slightly uncomfortable (itchy) especially when the subjects are preschool children. Occasionally people will feel dizzy or light-headed and need to lie down (Lee et al., 2014, Sporik et al., 2009). Furthermore, skin tests can be influenced by some drugs, particularly antihistamines, patients’ age and test sites (Bang et al., 1996). 10
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Serum Specific IgE Level Although the first method to detect serum-specific IgE was a radioallergosorbent test (RAST), there is little consensus on which assay method should be used. Commonly used assays include multiple allergen simultaneous test (MAST) and capsulated hydrophilic carrier polymer (CAP) system, each with its own advantages and disadvantages (Finnerty et al., 1989, Min, 2010).
Eosinophil Counts Both eosinophil count and total serum immunoglobulin E (IgE) had been used by many investigators for evaluating allergic disease (Demirjian et al., 2012). However, eosinophilia is associated with several disorders such as allergies, drug reactions, helminth infections, eosinophilic gastrointestinal disorders and hypereosinophilic syndrome (Fulkerson and Rothenberg, 2013), eosinophils are still considered as important effector cells in human allergic diseases due to the release of eosinophil-derived granule proteins, including eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN), eosinophil peroxidase (EPO) and major basic protein (MBP) (Na et al., 2012a, Amin et al., 2016).
2.5 Therapy of Allergic Rhinitis There are several therapeutic approaches including, allergen avoidance (where possible), pharmacotherapy, specific immune therapy and intervention therapy (Rudack, 2004, Ibero et al., 2012). Pharmacological treatment includes: Oral antihistamines, Intranasal antihistamines, Oral corticosteroids, Intranasal corticosteroids, Leukotriene receptor antagonists (LTRAs) and anti-IgE antibody.
Immunotherapy
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immunotherapy (SLIT) and Subcutaneous allergen-specific immunotherapy (SCIT) (Dave et al., 2011, Al-Abri et al., 2014).
2.6 Inflammatory Cells, Proinflammatory Cells, and Mediators in Allergic Rhinitis: Inflammation is the complex biological response of the body to remove harmful stimuli, including damaged cells, irritants, or pathogens and begin the healing process (Chen and Nunez, 2010). Thus the immune system requires not only the humoral and cellular immune response but also mediators known as cytokines that can be synthesized and released from every cell and which develop their biological effects for short distances - frequently only between two cells (Rudack, 2004).
2.6.1 Eosinophil Cells 2.6.1.1 Eosinophil Overview Eosinophils are now considered multifunctional leukocytes that have a role in host protection against parasitic infections, tissue homeostasis and allergy disease by the release of cytotoxic proteins (Jung and Rothenberg, 2014, Amin et al., 2016). Eosinophils derive from the bone marrow and circulate at low levels in healthy individuals, composing 1–5% of peripheral blood leukocytes (Davoine and Lacy, 2014). In pathological conditions, eosinophils are activated and accumulated in inflamed tissues in respiratory mucosa including lungs, in the gastro-intestinal tract, and in lymphocyte-associated organs, the thymus, lymph nodes and the spleen where they release a wide spectrum of inflammatory mediators (Bystrom et al., 2011). Human eosinophils have a bilobed nucleus and distinguished by their cytoplasmic crystalloid granules: secondary (secretory or specific) and primary. Specific granules contain preformed cationic proteins, such as MBP, EPO, ECP 12
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and EDN (Acharya and Ackerman, 2014). In addition, these granules contain a diversity of preformed cytokines, chemokines, enzymes and growth factors, including IL-3, -4, -5, and -13, Interferon gamma (IFN-γ), GM-CSF, RANTES (CCL5), eotaxin-1 (CCL11), etc (Muniz et al., 2012, Amin et al., 2016). Primary granules are formed early in eosinophil development and are enriched in Charcot- Leyden crystal protein. Eosinophils also contain lipid bodies which are cytoplasmic structures and are formed rapidly after activation of eosinophils (Kita, 2013). Eosinophils express number of cell surface receptors, including immunoglobulin receptors for IgG (FcγRII) and IgA (FcαRI); complement receptors (CR and CR3); cytokine receptors (IL-3R, IL-5R,and GM-CSFR) that promote eosinophil development, as well as receptors for IL-1α, IL-2, IL-4, Interferon alpha (IFN-α), and tumor necrosis factor alpha (TNF-α); chemokines (CCR1 and CCR3); leukotriene B4 receptors (LTB4 R); prostaglandin D2 receptors (PGD2 R); platelet-activating factor receptor (PAFR); and toll-like receptors (particularly TLR7/8). Eosinophil expression of FcεRI is minimal that does not activate eosinophils. Eosinophils also express several inhibitory receptors (Munitz and Levi-Schaffer, 2007).
2.6.1.2 Eosinophil Function Traditionally eosinophils have been involved in host protection against parasitic infection (Rosenberg et al., 2013). Recent studies indicate that eosinophils may have a greater role in the protection against viral infections, particularly respiratory viruses and bacterial infections (Percopo et al., 2014, Metcalfe et al., 2016). Moreover, eosinophils have been involved in human allergic inflammation (Kita, 2011).
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2.6.1.3 Eosinophil and the Immune System Eosinophils can perform various immune regulatory functions likely through two main mechanisms: production of a range of cytokines and immunomodulatory molecules; and presentation of antigens (Hogan et al., 2008, Moqbel and Lacy, 2000). For example activated eosinophils can produce IL-3, IL-5, and GM-CSF which are able to act on eosinophils themselves and cause eosinophil proliferation and differentiation (Kita, 2011). While IL-4, IL-6, IL-10 are causing eosinophil activation and recruitment (Moqbel and Lacy, 2000). Other human eosinophil-derived cytokines are eosinophil-derived transforming growth factor beta (TGF-β), which enhances proliferation and collagen synthesis of the lung and dermal fibroblasts (Levi-Schaffer et al., 1999), and transforming growth factor alpha (TGF-α) increases mucin production in airway epithelial cells (Burgel et al., 2001). Other angiogenic factors, such as osteopontin (Puxeddu et al., 2010), vascular endothelial cell growth factor (VEGF) (Puxeddu et al., 2005), nerve growth factor (NGF) (Kobayashi et al., 2002) and tumor necrosis factor (TNF)-α are produced by eosinophil (Moqbel and Lacy, 2000). Furthermore, human eosinophils produce Indoleamine 2,3-dioxygenase (IDO) is an enzyme that catalyzes the oxidative catabolism of tryptophan to kynurenines, which regulates T cell function (Odemuyiwa et al., 2004). While EDN is a chemoattractant and activator of DCs. Consequently, EDN enhances TH2 responses through a Toll-like receptor 2 (TLR2) (Yang et al., 2003, Yang et al., 2004, Yang et al., 2008). In fact, activated mast cells produce and secrete IL-5, PAF and LTB4 known to induce ECP release from eosinophils (Okayama et al., 1994, Bystrom et al., 2011). PGD2 (produced by mast cells) is a chemoattractant for eosinophils and TH2 lymphocytes (Patella et al., 1996, Luster and Tager, 2004). Eosinophils also can act as APCs, present antigen to primed T cells and increase TH2 cytokine production (Padigel et al., 2007, Spencer and Weller, 2010). 14
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2.6.1.4 Eosinophil Cationic Protein ECP Overview The Eosinophil Cationic Protein (ECP), also named as ribonuclease 3 (RNase 3), is a multifunctional protein with both cytotoxic and fibrogenic properties released from the eosinophil upon activation (Jonsson et al., 2010, Bystrom et al., 2012). The molecular mass of ECP is ranging from 16 to 22 kDa largely due to glycosylations of the protein and stored in the granules as a highly glycosylated, non-toxic protein. Upon eosinophil degranulation, ECP is deglycosylated and then acquires cytotoxic capabilities (Metcalfe et al., 2016). However, eosinophils are the major ECP producing cells (they contain 13.5 µg ECP/106 cells), but monocytes produce minute amounts in comparison. Activated neutrophils also produce some ECP at a hundred fold level lesser than the eosinophil, and have the ability to take up further ECP from the surrounding cells transported and stored in their primary granules (azurophil granules) (Bystrom et al., 2011). ECP is released from the eosinophil through interaction with adhesion molecules, stimulation by leukotriene B4 (LTB4), PAF, IL-5, immunoglobulins and complement factors C5a and C3a (Bystrom et al., 2011, Liaskou et al., 2012).
Role of ECP ECP plays a role against the parasite, bacterial, viral infections (Kita, 2011). Moreover, it has cytotoxic effects against host cells thus it causes severe damage to the host tissues itself (Figure 2.2) (Koczera et al., 2016, Amin et al., 2016). ECP also Induces degranulation of mast cells (Metcalfe et al., 2016).
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Figure 2.2: Effects of the Eosinophil Cationic Protein (ECP) (Koczera et al., 2016).
ECP and the Immune System Early studies are investigating the effects of ECP on the proliferation of T and B lymphocytes, which indicate that the protein could regulate those cells in vivo (Peterson et al., 1986). During allergic inflammation, eosinophils might act as APCs where they interact with T cells, thereby possibly contributing to T cell activation (Bystrom et al., 2011). Both mast cells and eosinophils are effector cells of allergic inflammation. However, ECP cause histamine release from MCs, but the MCs and eosinophils interactions could be attributed to the positive feedback of ECP release (Bystrom et al., 2011). In addition, ECP is associated with damaged epithelium (Amin et al., 2001). Major Basic Protein Overview The major basic protein (MBP) is one of the most highly cationic proteins synthesized by eosinophils, MBP is expressed as two different homologs (MBP1 and MBP2). MBP is a small protein with a molecular mass of 13.8 kDa and has a high isoelectric point, so strongly basic that it cannot be measured 16
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accurately. Its basicity is due to the presence of 17 arginine residues. Both MBP1 and MBP2 stimulate histamine and LTC 4 release from human basophils and MBP1 activates primed mast cells. MBP1is a potent vasodilator and increases permeability (Metcalfe et al., 2016).
2.6.2 Mast Cells 2.6.2.1 Mast Cells Overview Mast cells (MC) are tissue-based inflammatory cells of bone marrow origin that respond to signals of innate and adaptive immunity with the immediate and delayed release of inflammatory mediators (Amin, 2012, Stone et al., 2010). These cells are up to 20 ? m diameters, ovoid or irregularly elongated cells with an oval nucleus and contain abundant metachromatic cytoplasmic granules (Stone et al., 2010). There are three subtypes of human MC known as MC (T) that contain only tryptase, MC (TC) that contain tryptase and chymase, and MC (C) that only contain chymase (Moon et al., 2014, Amin, 2012). MC (T) cells are localized within the mucosa of the respiratory and gastrointestinal tracts and increase with mucosal inflammation. MC (TC) cells are localized within connective tissues such as the dermis, submucosa of the gastrointestinal tract, heart, conjunctivae and perivascular tissues (Metcalfe, 2008). Mast cells express a high-affinity receptor FcεRI for the Fc region of IgE. Other MC receptors are IL-3R, IL-4R, IL-5R, and toll-like receptors (TLRs). MCs may also express C3a and C5a receptors (Stone et al., 2010). Mediators produced by MCs are divided into preformed mediators, newly synthesized lipid mediators and cytokines/chemokines (Park et al., 2014, Amin et al., 2005). Preformed mediators, including histamine, serine proteases (tryptase and chymase), carboxypeptidase A and proteoglycans (heparin) are 17
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stored in cytoplasmic granules. Histamine has effects on smooth muscle (contraction), endothelial cells, nerve endings, and mucous secretion (Moon et al., 2014). Newly synthesized lipid mediators are eicosanoid mediators including thromboxane, PGD2, LTC4, and PAF. These lipid mediators produced rapidly after MCs activation through FcεRI from endogenous membrane arachidonic acid stores, to ensure energy supply and to maintain lipid homeostasis in the body (Dichlberger et al., 2013). TNF-α is a major cytokine stored and released by MCs, increases bronchial responsiveness and has anti-tumor effects. Other cytokines produced by MCs include IL-3, IL-4, IL-5, IL-10, IL-13 and GM-CSF (Metcalfe, 2008, Stone et al., 2010).
2.6.2.2 Mast Cells Function Mast cells are best known for their role in several chronic allergic disorders (including asthma, allergic
rhinitis, and atopic dermatitis),
inflammatory disorders, cancer and autoimmune diseases. In addition, MCs play role in anaphylaxis response (type I hypersensitivity reaction), in wound healing and host defense mechanisms against pathogens (da Silva et al., 2014).
2.6.2.3 Mast Cells and the Immune System Mast cells have a central role in inflammatory and immediate allergic reactions. They are able to produce inflammatory mediators, such as histamine, proteases, chemotactic factors and cytokines that act on the vasculature, smooth muscle, connective tissue, mucous glands and inflammatory cells (Amin, 2012). During an allergic response, IgE release from B-cells will bind to the high-affinity IgE receptors (Fc RI) on MCs. A subsequent exposure to the same allergen cross-links the cell-bound IgE and triggers the release of preformed prostaglandins, histamines and cytokines (Bax et al., 2012). While 18
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MC is activated, newly synthesized mediators are produced such as LTC4, PGD2 and cytokines. Those preformed mediators, newly mediators and cytokines can have profound effects on vascular endothelium, including the alteration of vascular permeability and adhesiveness. This can allow other circulating inflammatory cells to adhere to the endothelium and to migrate into the inflammatory site (He et al., 2013). Histamine cause vasodilation, bronchoconstriction, increased capillary permeability and smooth muscle contraction (Lundequist and Pejler, 2011, Amin, 2012). Tryptase and chymase can damage and activate the bronchial epithelium. Tryptase induces eosinophil and neutrophil accumulation. Chymase induces eosinophil migration (He et al., 2013). MC Cytokines including: IL-4 and IL-13 stimulate the proliferation and differentiation of activated B-cells and induces class switch into IgE antibodies. IL-5 is involved in the maturation, migration and survival of eosinophils. While IL-6, IL-8, and TNF stimulate mast cells migration (Murdoch and Lloyd, 2010). Thus, mast cells play important role in allergic and inflammatory reactions (Lundequist and Pejler, 2011) (Figure 2.3).
Figure 2.3: Induction and Effector Mechanisms in Type I Hypersensitivity (Amin, 2012). AA, allergic asthma; ASM, Airway Smooth Muscle. 19
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2.6.3 Immunoglobulin IgE 2.6.3.1 Immunoglobulin IgE Overview Immunoglobulins are glycoproteins, heterodimeric composed of two heavy (H) and two lights (L) chains produced by plasma cells in response to an antigen. They can be separated functionally into variable (V) domains that bind antigens and constant (C) domains that specify effector functions such as activation of complement or binding to Fc receptors. There are five main classes of heavy chain C domains. Each class defines the IgM, IgG, IgA, IgD and IgE isotypes (Schroeder and Cavacini, 2010). Immunoglobulin E (IgE) is a class of immunoglobulin associated with hypersensitivity and allergic reactions as well as parasitic worm infection (Schroeder and Cavacini, 2010). IgE exists as monomer consists of two identical heavy chains and two identical light chains with variable (V) and constant (C) regions. The heavy chains contain one variable domain and four constant region domains (Pate et al., 2010). It is normally present in human serum in lowest concentration of the five immunoglobulin subtypes. It has the shortest half-life and the expression is tightly regulated in the absence of disease. IgE shows no transplacental transfer (Stone et al., 2010, Chang et al., 2007). Two signals are required for IgE synthesis. The first signal is provided by IL-4/IL-13, which activates the transcription at the IgE isotype specific switch region. The second signal is provided by ligation of CD40 on B cells by CD40L on T cells, which activates deoxyribonucleic acid (DNA) switch recombination. T cells are the principal source of these signals (Prussin and Metcalfe, 2006, Pate et al., 2010). IgE has two types of receptors: the low-affinity IgE receptor (FcεRII also known as CD23) and the high-affinity IgE receptor (FcεRI). FcεRII is expressed on the surface of B cells, T cells, Langerhans cells, macrophage, monocytes, 20
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eosinophils and platelets. FcεRI is expressed on MCs and basophils (MacGlashan, 2008).
2.6.3.2 IgE in Disease IgE plays role in atopic diseases such as atopic dermatitis, followed by atopic asthma and AR. Subsequent antigen-sIgE cross-linking to FcεRI receptors on MCs and basophils are leading to immediate hypersensitivity reactions (Gieras et al., 2015). IgE also plays a role in parasitic infections, viral infections, bacterial infections, inflammatory diseases, hematologic malignancies, cutaneous diseases, cystic fibrosis, nephrotic syndrome and primary immunodeficiency diseases (Stone et al., 2010, Pien and Orange, 2008, Pate et al., 2010).
2.6.3.3 IgE and the Immune System In AR patients, MCs and basophils express an increased number of Fc RI receptors. Inhaled allergens bind to IgE on the surface of MCs and basophils, inducing cross-linkage of IgE, and triggering the release of histamines, leukotrienes, and other inflammatory mediators, leading to the onset of allergic symptoms (Hanf et al., 2004, Pawankar et al., 2011). Following antigen presentation by the APCs to TH2 cells, TH 2 cells will release mediators like IL-4 and IL-13 that will stimulate allergen-specific B cells (which are also stimulated through the B-cell antigen receptor) leading to the differentiation of B cells into IgE-secreting plasma cells (Van Cauwenberge, 1997, Deo et al., 2010). Further contact will result in the production of IgE that will bind to the high-affinity IgE receptors of the MCs and basophils and the low-affinity IgE receptors on several cells (Poole et al., 2005, Galli and Tsai, 2012).
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2.6.4 Cytokines Cytokines are small proteins produced by a wide variety of cells, in response to any immune stimulus. Cytokines are signaling molecules that have a specific effect on the interactions and communications between cells. They regulate immunity, inflammation, cell activation, cell migration, cell proliferation, apoptosis and hematopoiesis (Coondoo, 2011).
Interleukin-17 Overview Interleukin-17: also known as IL-17A, is a cytokine produced by Thelper17 (TH17) subset of CD4 T cells and is associated with the induction of inflammation (Qu et al., 2013, Tsvetkova-Vicheva et al., 2014, Kaiko et al., 2008). Infante-Duarte et al. first demonstrated that IL-17-producing T cells were a distinct TH population of TH1 cells and TH2 cells in both mice and humans (Infante-Duarte et al., 2000). IL-17 has six family members (IL-17A to IL-17F), and five receptors (IL17RA, IL-17RB/IL-25R, IL-17RC, IL-17RD/SEF and IL-17RE) (Gaffen, 2009). In addition to TH17 cells, a wide variety of T cells also produces IL-17A and IL-17F. These cytokines are produced by cytotoxic CD8 T cells (Tc17), γδT (γδ-17) cells, and NKT (NKT-17) cells. Neutrophils, monocytes, natural killer cells (NK), macrophages and MCs have also been shown capable of rapidly producing IL-17A and IL-17F (Cua and Tato, 2010, Pappu et al., 2011, Zhang et al., 2013).
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Figure 2.4: IL-17 and the IL-17 Receptor Families (Iwakura et al., 2011).
Function According to recent studies, IL-17 is involved in the development of allergic, autoimmune diseases, and tumors (Iwakura et al., 2011, Song et al., 2016, Tabarkiewicz et al., 2015). Also play important roles in the host defenses against bacterial, viral and fungal infections as well as inflammation (Onishi and Gaffen, 2010, Golden et al., 2013).
IL-17 and the Immune System In fact, IL-17 is capable of mediating a link between innate and acquired immunity, specifically, macrophage and T-cell functions (Barin et al., 2012, Fang et al., 2011, Liu et al., 2012). IL-17A and IL-17F activate immune cells (T cells, B cells, neutrophils, DCs and macrophages) and nonimmune cells (Epithelial cells, endothelial cells, keratinocytes, smooth muscle cells and fibroblasts), to produce cytokine, chemokines and antimicrobial peptides to recruit immune cells at inflammation sites, promote local tissue destruction and protect from pathogens, resulting in disease development and host protection (Iwakura et al., 2011, Peterson and 23
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Artis, 2014, Oboki et al., 2008). For example, macrophages and many other cells are capable of producing IL-17 in numerous inflammatory, allergy, autoimmune diseases, tumors and in host defense against infection; IL-17 can promote recruitment of macrophage at the site of inflammation and induce their cytokine/chemokine production (Zhang et al., 2013), but a cost of this response can be tissue-damaging inflammation (Cooper, 2009, Salgame et al., 2013).
Interleukin-33 Overview Interleukin-33 is a cytokine that belongs to the IL-1 superfamily and is mainly produced by different types of structural cells, including endothelial cells, epithelial cells, keratinocytes, smooth muscle cells and fibroblasts (Demyanets et al., 2013, Miller, 2011). Moreover, various types of immune cells, such as activated dendritic cells, macrophages and mast cells produced low quantities of human IL-33 (Nakae et al., 2013). Interleukin-33 (IL-33) was identified as a ligand for IL-1RL1 (also called ST2, T1, Der4, and fit-1), which is a member of the Toll/IL-1 receptor superfamily (Borish and Steinke, 2011, Nakae et al., 2013). However, IL-33was originally identified as “DVS27” and as a nuclear factor protein in high endothelial venules; thus, it was called NF-HEV (Rogala and Gluck, 2013), but it is considered an alarm in a molecule due to its release after necrosis or tissue damage. In contrast, apoptosis leads to the inactivation of IL-33 because it is cleaved by caspases. Different stimuli such as bacterial, viral, fungal infections and allergen challenges can trigger the release of IL-33 (Saluja et al., 2015).
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Many cell types express ST2 on their surface and can bind to IL-33. ST2 is expressed on TH2 cells, MCs, macrophages, basophils, eosinophils and involved in the activation of these cells (Schmitz et al., 2005, Oboki et al., 2010, Kurowska-Stolarska et al., 2009). ST2L is also expressed on endothelial and epithelial cells (Aoki et al., 2010, Miller et al., 2008, Mildner et al., 2010). There are two unique characteristics that help IL-33 to consider as a cytokine with dual function, acting both as a traditional cytokine through activation of the ST2L receptor complex and as an intracellular nuclear factor with transcriptional regulatory properties (Miller, 2011) (Figure 2.5).
Figure 2.5: IL-33 Responses in Necrosis and Apoptosis (Lamkanfi and Dixit, 2009).
Function IL-33 has a potent role in various inflammatory diseases associated with TH2 immune responses including allergic diseases such as asthma, anaphylaxis and atopic dermatitis and in host defense against parasite infections (Liew et al.,
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2010). Also, IL-33 has a role in autoimmune diseases, cardiovascular diseases and cancer (pleural malignancy) (Miller, 2011, Kakkar and Lee, 2008).
IL-33 and the Immune System It is well established that IL-33 has a plenipotent and pleiotropic activity. This cytokine is expressed and acts on many cells participating in allergic inflammation such as antigen-experienced TH2 cells, MCs, macrophages, eosinophils, basophils, DCs and induces TH2-type cytokine production by different cells; thus, these facts make a base for the assumption that IL-33 may play a role in the pathogenesis of allergic inflammation such as allergic rhinitis (Smithgall et al., 2008, Oliphant et al., 2011). When IL-33 is binding to the ST2 receptor on MCs and TH2 induces secretion of cytokines IL-4, IL-5 and IL-13 (Lamkanfi and Dixit, 2009). As MCs are activated and signaling pathways are started with subsequent activation of NF-κB and transcription of proinflammatory cytokines such as IL-1β, IL-6, IL-8, IL-13, TNF, chemokines and prostaglandins (Ohno et al., 2012). PGD is a chemoattractant for eosinophils and TH2 lymphocytes. Moreover, IL-33 may induce cytokine production in either the presence or absence of co-stimulation of MCs via IgE/antigen–FcεRI signals (Haenuki et al., 2012, Iikura et al., 2007). In T cells, IL-33 can also induce TH2 cytokine production and chemotaxis of in vitro TH2 cells (Schmitz et al., 2005, Ohno et al., 2012, Komai-Koma et al., 2007). In the classic murine model, antigen-specific TH2 cells stimulated with IL-33 preferentially induce the production of IL-5 and IL13, but not IL-4, thus these T cells are called atypical TH2 cells (KurowskaStolarska et al., 2008). The same results were obtained in BAL fluid after intranasal administration of IL-33 in which IL-5 and IL-13 levels increased and IL-4 26
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did not change, suggesting that IL-33 may be involved in IL-4 independent TH2 cell differentiation (Louten et al., 2011). Notably, IL-33 induces IL-5, IL-13 and IFN-γ production by human TH2 in vitro skewed cells in house dust mite specific T cell culture (Chu et al., 2013). Previous studies demonstrated that IL-33 enhances eosinophils survival, up-regulates cell surface expression of adhesion molecule ICAM-1 on eosinophils, suppresses ICAM-3 and selectin. Also, IL-33 enhances the release of cytokines IL-6, chemokines CXCL8, CCL2 of eosinophils and enhances Siglec-8-mediated apoptosis of eosinophils (Chow et al., 2010, Na et al., 2012b). Thus was suggesting a correlation between blood and pulmonary eosinophilia with elevated IL-33 serum level (Kim et al., 2010). In human endothelial cells, IL-33 induces inflammatory activation through up-regulation of IL-6, IL-8, monocyte chemoattractant protein-1 (MCP1), VCAM-1, ICAM-1, endothelial selectin (E-selectin), increases vascular permeability and promotes angiogenesis (Demyanets et al., 2013).
2.7 T Cells 2.7.1 T Cells Overview The T-cell is a subtype of white blood cell that plays a central role in cellmediated immunity. T cells can be distinguished from other lymphocytes, such as B cells and NK cells, by the presence of a T-cell receptor (TCR) on the cell surface (Grisanti et al., 2011). The precursors of T cells are produced in the bone marrow but leave the bone marrow and mature in the thymus. This process involves the expression of a functional T cell receptor (TCR), the co-receptors CD4 and/or CD8, in addition to other T cell co-receptors (Duan and Morel, 2006, Abul et al., 2007).
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There are several types of mature T cells including T helper cells (TH cells), cytotoxic T cells (TC cells), memory T cells, regulatory T cells (Treg cells), natural killer T cells (NKT cells) and δγ T cells (Grisanti et al., 2011). The TH cells are CD4+ cells that have the CD4 co-receptor and only recognize antigens presented by MHC II molecules. The MHC II molecule is found on all immune cells (Corthay, 2009, Abul et al., 2007). Soon after the exposure by allergen, the naive TH cells (TH0) are activated and differentiated into three main types: TH1, TH2 and TH17 cells depending on the nature of the cytokines present at the site of activation (Sallusto and Lanzavecchia, 2009, MacLeod et al., 2010, Qu et al., 2013).
2.7.2 T Cells Function The two main subtypes are TH and TC cells. The TH cells are important in the activation of B cells, macrophages and TC cells. The TC cells destroy the tumor and viral infected cells. Both subtypes present an important role in the control of intracellular pathogens (Grisanti et al., 2011). Consequently, more studies have demonstrated the role of three main effectors TH cells (TH1, TH2 and TH17), in the immune system. While TH1 cells are involved in cellular immune response and intracellular infections, TH2 cells play an important role in humoral immunity, extracellular parasites, allergic diseases and asthma. Also, TH17 cells play a significant role in adaptive immunity, infectious diseases, autoimmune conditions and allergy (Qu et al., 2013, Wilke et al., 2011). Memory T cells can quickly expand into either TH or TC cells following re-exposure to their cognate antigen(Grisanti et al., 2011). Treg cells functions include prevention of autoimmune diseases by maintaining self-tolerance; suppression of allergy (Corthay, 2009). While δγ T and NKT cells represent a link between the innate and adaptive immune responses (Grisanti et al., 2011). 28
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2.7.3 T Cells and the Immune System Normally, the antigen is processed by the APC into smaller fragments, then presented these fragments in MHC molecules class II, which interact with T cells through the TCR and the CD4 co-receptors (Mesquita Junior et al., 2010). While, a second signal is occurred by the interaction of several other costimulatory receptors present on the surface of the T cell and the APC (Mesquita Junior et al., 2010, Botturi et al., 2011). As a result, several cytokines are produced such as IL-4, IL-5, and IL-10 causing T cell activation and survival (Kopf et al., 2000). However, T cells are the only cells able to recognize antigens after processing by APCs, but they can also regulate and coordinate immune responses in allergic diseases through T-cell subsets (e.g., TH1, TH2, TH17) or induce tolerance through induction of regulatory T cells (Salazar and Ghaemmaghami, 2013). In atopic diseases including allergic rhinitis and asthma, inflammatory conditions are characterized by the increasing of TH2 cell activation and T regulatory cell (Treg) deficiency during allergen exposure (Botturi et al., 2011). Thus, TH2 cells have a central role in the development of mucosal inflammation after allergen exposure (Deo et al., 2010, Sin and Togias, 2011). During an allergic response, TH2 cytokines have a direct effect on B cell, MCs and eosinophils (Maggi, 2010). For example, IL-4 induces B-cell class switching into IgE. Also, IL-4 stimulates mast cell proliferation and degranulation.
IL-5 promotes activation and recruitment of eosinophils
(Mesquita Junior et al., 2010, Kaiko et al., 2008). While IL-10 can inhibit the activity of IFNγ and allowing IL-4 to proceed in the B cell class switching into IgE (Deo et al., 2010). IL-6 mediated differentiation of naive T cells to effector TH2 cells by inducing the production of IL-4. Finally, IL-13 can downregulate TH1 (Rincon et al., 1997, Anthony et al., 2007).
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2.8 B Cells and Immunoglobulin Production 2.8.1 B Cells Overview B cells are types of white blood cells known as lymphocytes, they are also classified as professional antigen-presenting cells (APCs) (Harwood and Batista, 2011). Mature B cells express IgM and IgD on their cell surfaces. They constitute approximately 15% of peripheral blood leukocytes and defined by their production of immunoglobulin (Ig) (Thomas et al., 2009, Vale and Schroeder, 2010). B cells develop from hematopoietic stem cells (HSCs) in the bone marrow. Stages of this process are dependent on contact between the developing B cell precursors and bone marrow stromal cells, but do not require antigen and are regulated by positive and negative selection steps, resulting in the formation of immature B cells (Kurosaki et al., 2010). To complete development, immature B cells migrate from the bone marrow to the peripheral lymphoid tissues and pass through two transitional stages, mainly in the spleen, before differentiation to either follicular or marginal zone (MZ) B-cell depending on the signals received through the B cell receptor (BCR) and other receptors. The majority of immature B cells will become follicular IgM+ IgD+ mature naive cells that recirculate through peripheral lymphoid tissues until they encounter their cognate antigen, after which they can further differentiate into long-lived memory B cells or antibodysecreting plasma cells (Pillai and Cariappa, 2009). While the remaining immature B cells become marginal zone B cells and localize primarily in the marginal sinus of the spleen, in which initiate immune responses against blood borne pathogens, especially encapsulated bacteria (Kato et al., 2013).
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2.8.2 B Cells Function The functional structure of the B cells is responsible for the humoral immunity. In which B cells have the ability to differentiate into antibodysecreting plasma cells which are generally considered to positively regulate immune responses by producing Ag-specific Abs and helping to induce optimal CD4+ T cell activation and also through serving as APCs (Noh and Lee, 2011, Mesquita Junior et al., 2010). Also, B cells seem to negatively regulate allergic diseases, including contact dermatitis, asthma, anaphylaxis, non-IgE-mediated food allergy of atopic dermatitis and negatively regulates autoimmune diseases (Noh and Lee, 2011, Yang et al., 2013).
2.8.3 B Cells and the Immune System Cellular interactions are essential for normally regulated, protective immune responses (Fu and Chaplin, 1999). Generally, B cells immune response to the antigens (Ag) requires T cell-dependent B cell responses and T cellindependent B cell responses (Mesquita Junior et al., 2010). The immune response to an antigen requires antigen recognition by the helper T cell and activates B cell, stimulating its clonal expansion, class switching, the affinity maturation and the differentiation into memory B cell. These antigens are known as T cell-dependent antigens (Abul et al., 2007). Unlike T-cells, B–cells can recognize antigens without requiring that the antigen is processed by an APCs. In which B cells are activated and differentiated
into
short-lived
plasma
cells
without
class
switching.
Consequently, the immune memory is generally weak (Clem, 2011, Schroeder and Cavacini, 2010, Chaplin, 2010). In fact, B cells play a key role in humoral allergic response through IgE production. IgE synthesis is regulated by IL-4 and IL-13 which are considered 31
Chapter Two
Literature Review
pleiotropic cytokines (Deo et al., 2010, Kato et al., 2013). However, these cytokines are produced by many cells such as MCs, TH2 and human B cells which in turn stimulating the proliferation and differentiation of activated Bcells, and induces class switch into IgE antibodies, but IL-4 alone induces differentiation of naive helper T cells (Th0 cells) to TH2 cells. Then activated TH2 cells inturn cause activation/recruitment of IgE antibody producing B cells, mast cells and eosinophils (Deo et al., 2010). While, MHC class II expressing B cells can function as APCs and drive TH2 cells (ten Broeke et al., 2013). In atopic diseases such as allergic rhinitis and asthma, once synthesized, IgE bind to the FcεRI, on MCs and basophils, or the FcεRII, found on a wide variety of leukocytes. MCs are one of the important effector cells in allergic diseases, though cross-linking of receptor-bound IgE on the surface of these cells leading to degranulation of MCs and release of mediators and to the transcription of cytokines that act on various tissues and inflammatory cells (Murdoch and Lloyd, 2010, Amin, 2012).
32
CHAPTER THREE MATERIALS AND METHODS
Chapter Three
Materials and Methods
Chapter Three Materials and Methods
3.1 Materials: Table 3.1: List of Materials.
Materials
Manufacture
Origin
Disposable sterile syringe-10ml HBM
China
Cotton wool
Xinle
China
Clot activator and EDTA tube
Xinle
China
Eppendorf tube
Jiangsu Province Huaxing
China
Medical Apparatus Industry Plain tube (no anti-coagulant)
Citotest labware
China
Gloves
Vinyl industrial
China
Disposable tips
Yancheng Huida medical
China
instruments Cuvette cup
Roche
USA
Can tube
Barcopharma
China
Filter paper or clean paper
United scientific supplies
USA
IgE Calset
Roche
USA
Precicontrol universal
Roche
USA
Diluent universal
Roche
USA
Procell-system buffer
Roche
USA
Cleancell-cleaning solution
Roche
USA
Syswash-wash water additive
Roche
USA
Assaycup-reaction vessels
Roche
USA
Assaytip- pipette tips
Roche
USA
33
Chapter Three
Materials and Methods
Table 3.2: List of Kits.
Kits Kits Components Optical Flow Cytometry Kit 1. Diluent for Detecting Eosinophil 2. Lytic Reagent 3. Cleaning Solution ECLIA Kit for Detecting 1. M StreptavidinImmunoglobulin IgE coated microparticles 2. R1 Anti-IgEAb~biotin 3. R2 Anti-IgEAb~Ru (bpy) Agglutination Kit for 1. Latex Reagent Detecting Rheumatoid 2. Positive Control Arthritis (RA) Factor 3. Negative Control Agglutination Kit for 1. Latex Reagent Detecting C-Reactive 2. Positive Control Protein (CRP) 3. Negative Control ELISA Kit for the 1. Microwell plate Measurement of Human coated with antiECP (Eosinophil Cationic human ECP antibody Protein) 2. ECP standard 3. Secondary antihuman ECP antibody 4. Conjugate 5. Antibody and conjugate diluent 6. Assay diluent 7. Standard diluent 8. Wash concentrate 9. Substrate 10. Stop solution ELISA Kit for the 1. Microplate coated Measurement of Human with polyclonal Interleukin-17 (IL-17) antibody to IL-17 2. Biotinylated antihuman IL-17 3. Streptavidin-HRP 34
Manufacture Origin Orphee France
Roche
USA
Plasmatec
UK
Plasmatec
UK
Diagnostics Sweden Development, Uppsala,
Abcam
UK
Chapter Three
ELISA Kit for the Measurement of Human Interleukin-33 (IL-33)
Materials and Methods
concentrate 4. Recombinant human IL-17 standard 5. Assay diluent A 6. Assay diluent B 7. Wash buffer concentrate 8. TMB 9. Stop solution 1. Microplate coated with polyclonal antibody to IL-33 2. Biotin-Conjugate anti-human IL-33 polyclonal antibody 3. Streptavidin-HRP 4. IL-33 standard lyophilized 5. Sample diluent 6. Conjugate diluent 7. Calibrator diluent 8. Assay buffer concentrate 9. Wash buffer concentrate 10. TMB substrate solution 11. Stop solution
35
Abcam
UK
Chapter Three
Materials and Methods
Table 3.3: List of Equipment.
Equipment
Manufacture
Origin
Centrifuge
Nuve
Turkey
Micropipette
Slamed
Germany
Multi-channel pipette
Slamed
Germany
Refrigerator
Hitachi
Japan
Incubator shaker
Awareness technology, INC
USA
Water bath
Memmert
Germany
Cobas - e 411
Hitachi
Japan
Colter - Mythic 22
Orphee
France
ELISA Reader
Awareness technology, INC
USA
3.2 Methods 3.2.1 Patients and Setting of the Study: This study was a case-control study, involved 88 patients and 88 subjects control in all age groups and both sexes. The sample collection were performed in the laboratory of Public Health and Ali Kamal Health Center, while the investigations were performed in the Public Health laboratory in sulaimani city for complete blood count (CBC), IgE test, C-reactive protein (CRP) and rheumatoid arthritis (RA) test. Enzyme-linked immunosorbent assay (ELISA) were done in Dr.Saman private Laboratory. The control group consisted of healthy, no allergic rhinitis, asthma and no other respiratory diseases. They were negative to CRP, RA-test, serum IgE level and normal eosinophil count. The sample collection started in February 2016 and the samples were collected in four days in a week and extended until completing the total number of samples in end of the June. Thereafter, samples were subjected to laboratory analysis; some of the investigations were done in the next day like CBC, IgE, CRP and RF test, while others like ELISA technique were performed later. 36
Chapter Three
Materials and Methods
3.2.2 Inclusion and Exclusion Criteria: All the Allergic rhinitis patients were diagnosed by consultant ear, nose and throat specialist (ENT) according to the British Society for Allergy and Clinical Immunology (BSACI) criteria. Allergic Rhinitis based on history, symptoms and clinical examine by using total serum IgE and CBC as markers. Healthy control subjects were normal individuals with no history of any chronic diseases. The control was collected from normal people accompanying patients attending for the allergic rhinitis. Patients who took the treatment of allergies, before the blood was drawn, were excluded.
3.2.3 Biosafety Considerations: All the laboratory waste products including disposable materials such as syringes, gloves, tips and plastic tubes were sealed in a special bag then burned in on incubator according to the safety measures.
3.2.4 Ethical Considerations: Ethical approval for the study was obtained from The Ethic Committee of the Faculty of Medical Science/School of Medicine. Informed consent about the study was obtained from each patient (verbal agreement) agreed to participate in the study.
3.2.5 Specimens: Eight milliliters of venous blood were collected from each patient, three milliliters were kept in ethylenediaminetetraacetic acid (EDTA) tube used for eosinophil test and five milliliters were kept in a clot activator tube and centrifuged for 10 minutes at 4500 round per minute (RPM), then part of the serum used for IgE, RF, CRP tests and the remaining serum put in plain tube and stored at -80oC till using them for further investigations. 37
Chapter Three
Materials and Methods
3.2.6 Optical Flow Cytometry Kit for Detecting Eosinophil in Human Blood: The kit preparation and procedure follow the instruction by manufacture recommendation.
Procedure: 1. Preparations before Analysis: The human blood venous sample must be collected in an EDTA K2 or K3 (Ethylene diamine tetraacetic acid, two or tri potassic) tube in sufficient quantity.
It must be correctly
homogenized before analysis. It is recommended to use a rotary agitator turning between 20 to 30 turns/ minutes during 10 minutes. 2. Sample Analysis: As soon as the temperature is reached, a measurement cycle can be run. Present the tube of the blood sample under the needle and press the start cycle trigger. The cycle LED is located at the top of the needle when becomes red the tube can be removed only when the needle is up. A new cycle can be started again when it turns from green again. Normal range: 1-5 × 10^9/L.
3.2.7 ECLIA Kit for Detecting Immunoglobulin IgE in Human Serum: The kit preparation and procedure follow the instruction by manufacture recommendation.
Procedure: For optimum performance of the assay by the analyzer, resuspension of the microparticles takes place automatically prior to use. The system automatically regulates the temperature of the reagents and the opening/closing of the bottles. The test procedure was done following the steps below: 1. Place the IgE reagent into the reagent tracking. Open the cover of the reagent kit. 38
Chapter Three
Materials and Methods
2. Place the reagent barcode card into the barcode reader and press the scan icon on the screen to read it. Then press the reagent scan icon in order to display the IgE test name in the test list. 3. Place calibration vials (cal 1 and cal 2) and control vials (1 and 2) into positions 1,2,3,4 of the sample tracking respectively. Scan it through sample scan icon. Then press the start icon to complete the process and the IgE reagent will be ready to use. 4. Add the samples serum into cup tubes. Place samples into sample tracking from positions 1 to 30 respectively. Press the start icon on the screen. 5. Run process last for 18 minutes. Results will be read automatically. Normal range:
Age group
IU/mL
ng/mL
Neonates
1.5
3.6
Infants in 1st year of life
15
36
Children aged 1‑5 years
60
144
Children aged 6‑9 years
90
216
Children aged 10‑15 years
200
480
Adults
100
240
39
Chapter Three
Materials and Methods
3.2.8 Agglutination Kit for Detecting Rheumatoid Arthritis (RA) Factor /C-Reactive Protein (CRP) in Human Serum: The kit preparation and procedure follow the instruction by manufacture recommendation.
Procedure: Qualitative Method 1. Allow each component to reach room temperature prior to use. 2. Gently shake the latex reagent to disperse the particles. 3. Add one drop of the latex reagent using the dropper provided (40 µL) to each of the required circles of the agglutination slide. 4. Use the pipette stirrer provided, place a drop of undiluted serum onto a circle of a test slide. 5. Spread the reagent and serum sample over the entire area of the test circle using a separate stirrer for each sample. 6. Gently tilt the test slide backward and forwards approximately once every two seconds to two minutes. Interpret results immediately after two minutes. Extended incubation may lead to false results. Positive and negative controls should be included at regular intervals. Both are ready for use and do not require further dilution. 7. At the end of the test, rinse the test slide with distilled water, dry and store in a sealed bag. Normal laboratory precautions should be maintained while handling potentially infectious patient samples. RA normal level: Adults < 8 IU/ml. CRP normal level: Adults < 6 mg/L.
40
Chapter Three
Materials and Methods
3.2.9 ELISA Kit for the Measurement of Human ECP (Eosinophil Cationic Protein) in Serum: The kit preparation and procedure follow the instruction by manufacture recommendation.
Samples Preparation: The
following
sampling
procedures
are
recommended.
The
standardization of the sampling procedure is very important for the measurements of reproducible levels (Bjork et al., 2000).
Serum 1. Collect blood by venipuncture using Becton Dickinson 4 ml Vacutainer hemogard SST® tubes for serum separation. 2. Leave blood tubes at room temperature until clot. 3. Centrifuge at 4500 RPM for 10 minutes and decant serum into a new tube. 4. Dilute serum samples with Assay diluent. Normally recommend 40 times.
Procedure: Bring all reagents, serum references and controls to room temperature (20-27°C) before use. The test procedure was done following the steps below: 1. Add 100 ? L of samples and standards per well. Securely cover with a plate sealer and incubate for 60 minutes at room temperature. 2. Aspirate and discard the well contents. Wash the wells 4 times with 400 ? L wash buffer and aspirated. After the last wash inverts the plate and blot it against an absorbing tissue. 3. Add 100 ? L of the secondary antibody solution to each well using a multichannel pipette. Securely cover with a plate sealer and incubate for 60 minutes at room temperature.
41
Chapter Three
Materials and Methods
4. Aspirate and discard the well contents. Wash the wells 4 times with 400 ? L wash buffer and aspirated. After the last wash inverts the plate and blot it against an absorbing tissue. 5. Add 100 ? L conjugate solution to each well using a multichannel pipette. Securely cover with a plate sealer and incubate for 60 minutes at room temperature. 6. Aspirate and discard the well contents. Wash the wells 4 times with 400 ? L wash buffer and aspirated. After the last wash inverts the plate and blot it against an absorbing tissue. 7. Add 100 ? L of the substrate solution to each well using a multichannel pipette. Securely cover with a plate sealer and incubate for 10 minutes at room temperature. 8. Add 50 ? L of the stop solution to each well using a multichannel pipette. 9. Read the absorbance of each well at 450 nm within 20 minutes after stopping the reaction by ELISA reader. Detecting the range of the ECP: 2-800 ug/L.
Figure 3.1: Typical Standard Curve of Human ECP ELISA Kit.
42
Chapter Three
Materials and Methods
3.2.10 ELISA Kit for the Measurement of Human Interleukin-17 (IL-17) in Serum: The kit preparation and procedure follow the instruction by manufacture recommendation.
Procedure: Bring all reagents, serum references and controls to room temperature (20-27°C) before use. The test procedure was done following the steps below: 1. Add 100 ? L of each standard and sample into appropriate wells. Cover well and incubate for 2.5 hours at room temperature with gentle shaking. 2. Discard the solution and wash 4 times with a 1X wash solution. Wash by filling each well with 1X wash solution (400 ? L) using a multi-channel pipette and aspirated. After the last wash, remove the wash buffer, invert the plate and blot it against clean paper towels. 3. Add 100 ? L of 1X biotinylated IL-17 detection antibody to each well. Incubate for 1 hour at room temperature with gentle shaking. 4. Discard the solution. Repeat the wash as in step 2. 5. Add 100 ? L of 1X HRP-streptavidin solution to each well. Incubate for 45 minutes at room temperature with gentle shaking. 6. Discard the solution. Repeat the wash as in step 2. 7. Add 100 ? L of TMB one-step substrate reagent to each well. Incubate for 30 minutes at room temperature in the dark with gentle shaking. 8. Add 50 ? L of stop solution to each well. Read at 450 nm immediately by ELISA reader. Detecting the range of the IL-17: 15.63-1000 pg/ml.
43
Chapter Three
Materials and Methods
Figure 3.2: Typical Standard Curve of Human IL-17 ELISA Kit.
3.2.11 ELISA Kit for the Measurement of Human Interleukin-33 (IL-33) in Serum: The kit preparation and procedure follow the instruction by manufacture recommendation.
Procedure: Bring all reagents, serum references and controls to room temperature (20-27°C) before use. The test procedure was done following the steps below: 1. Wash the microplate twice with approximately 400 ? L 1X wash buffer per well with the thorough aspiration of microplate contents between washes. 2. After the last wash step, empty wells and tap microplate on an absorbent pad or paper towel to remove excess 1X wash buffer. Use the microplate strips immediately after washing. 3. Add 50 ? L of sample diluent to all wells. 4. Add 50 ? L of each standard dilution to appropriate wells, including the no protein control. 44
Chapter Three
Materials and Methods
5. Add 50 ? L of each sample to the appropriate wells. 6. Cover with adhesive film and incubate at room temperature for 2 hours (microplate can be incubated on a shaker set at 400 rpm). 7. Remove adhesive film and empty wells. Wash microplate strips 6 times, according to step 1. 8. Add 100 ? L of 1X biotin-conjugated antibody to all wells. 9. Cover with an adhesive film and incubate at room temperature for 1 hour (microplate can be incubated on a shaker set at 400 rpm). 10. Remove adhesive film and empty wells. Wash microplate strips 6 times according to step 1. 11. Add 100 ? L of 1X streptavidin-HRP to all wells, including the blank wells. 12. Cover with an adhesive film and incubate at room temperature for 1 hour (microplate can be incubated on a shaker set at 400 rpm). 13. Remove adhesive film and empty wells. Wash microplate strips 6 times according to step 1. 14. Pipette 100 ? L of TMB substrate solution to all wells. 15. Incubate the microplate strips at room temperature for 30 minutes. Avoid direct exposure to intense light. 16. Stop the enzyme reaction by adding 100 ? L of stop solution to each well. Results must be read immediately at 450 nm by ELISA reader. Detecting the range of the IL-33: 7.8-500 pg/ml.
45
Chapter Three
Materials and Methods
Figure 3.3: Typical Standard Curve of Human IL-33 ELISA Kit.
3.3 Statistical Analysis Statistical analyses were performed using the GraphPad version 5. Results were expressed as mean ± standard error (mean ± SE) and analyzed using Student’s t-test or Mann-Whitney-U test for two groups or one-way analysis of variance (ANOVA). The Pearson product-moment correlation coefficient (r) was used to assess the relationship between cytokines and eosinophil levels or IgE levels. A P-value equal or less than (0.05) was considered statistically significant (S), less than (0.01) highly significant (HS), else it was regarded as non-significant (NS).
46
CHAPTER FOUR RESULTS
Chapter Four
Results
Chapter Four Results
4.1 Characteristics of Patients and Healthy Control In this study, we have taken 176 subjects of different ages, including female and male, 88 of them were allergic rhinitis patients and 88 of subjects representing apparently healthy control. Table 4.1 illustrate the basic characteristics of patients and healthy controls. Of the 88 patients with AR (75% female and 25% male) whose mean age was (33.16 + 11.89) years. The mean IgE level in AR was (160.98), eosinophil (3.5), respectively. The mean allergic symptom score of the total population was (1.80 + 0.40).
Table 4.1: Characteristics of Patients and Healthy Control according to Age, Sex, Symptoms, Serum IgE and Eosinophil.
Allergic Rhinitis
Healthy subjects
(n =88)
(n =88)
P value
Age (year), (M+SD) 33.16 + 11.89
34 + 11.62
Sex (Female/Male)
66 / 22
62 / 26
160.98 + 220.81
34.88 + 25.52
0.0001
3.5 + 2.9
2.29 + 1.05
0.0004
1.80 + 0.40
0.01
0.0001
Serum total IgE (IU/ml), (M+SD) Eosinophil (10^9/L) (M+SD) Symptom
47
0.363
Chapter Four
Result
4.1.1 Comparison of Blood Eosinophil Counts Among Allergic Rhinitis Patients and Healthy Control Regarding the standard normal eosinophil value in the blood (15×10^9/L), all 88 allergic rhinitis patients (AR) values were more than the normal rate (75% female and 25% male) (3.5 + 2.9). All the 88 healthy control (HC) subjects were within the normal rate to eosinophil count (2.29 + 1.05). The eosinophil values were significantly higher in AR patients compared to HC
No of eosinphil in blood (10^9/L)
subjects (P-value = 0.0004) as shown in (Figure 4.1). 10
Eosinophil level (P = 0.0004)
8 6 4 2 0
A
R
H
C
Figure 4.1: Comparison of Blood Eosinophil Counts in AR Patients and HC Subjects.
4.1.2 Comparison of Serum IgE Levels Among Allergic Rhinitis Patients and Healthy Control The IgE level was considered positive over 100 IU/ml and detected in all the AR patients (160.98 + 220.81). While all the HC subjects were within the normal range to IgE test (34.88 + 25.52) considering the standard normal IgE level in serum. A significant difference was found in mean IgE levels between AR and HC groups (P-value = 0.0001) as shown in (Figure 4.2).
48
Chapter Four
Result
800 600 400 200
HC
0
AR
IgE level in blood (IU/ml)
IgE level (P = 0.0001) 1000
Figure 4.2: Comparison of Total IgE Level in Blood of AR Patients and HC.
4.2 Comparison of Serum ECP Levels Among Allergic Rhinitis Patients and Healthy Control Levels of ECP were detected in all the AR patients. Statistical analysis showed serum ECP levels (detecting range: 2-800ug/L) in the AR patients (3.80 + 6.83) were significantly higher than the HC subjects (1.70 + 2.53) with (Pvalue = 0.0001) as shown in (Figure 4.3).
49
serum ECP concentration (ug/L)
Chapter Four
Result
ECP (P = 0.0001)
25 20 15 10 5 0 A
R
H
C
Figure 4.3: Histogram Shows the Difference in Serum Level of ECP Concentration (ug/L) Between AR Patients and HC.
4.3 Comparison of Serum IL-17 Levels Among Allergic Rhinitis Patients and Healthy Control Levels of IL-17 were detected in 46 of the 88 AR patients. Figure 4.4 Shows a significant difference in serum levels of IL-17 (detecting range: 15.631000pg/ml) between AR group (169.82 + 240.12) and HC group (26.97 +
Serum IL-17 concetration (pg/ml)
30.183) with (P-value = 0.0001). IL-17 (P = 0.0001) 1000 800 600 400 200 0
R A
C H
Figure 4.4: Comparison Between IL-17 Level in Serum of AR Patients and HC.
50
Chapter Four
Result
4.4 Comparison of Serum IL-33 Levels Among Allergic Rhinitis Patients and Healthy Control Levels of IL-33 were detected in 67 of the 88 AR patients. Figure 4.5 Shows serum levels of IL-33 (detecting range: 7.8-500pg/ml) which is significantly different in the AR group (1.18 + 1.17) than the HC group (0.64 +
Serum IL-33 concentration (pg/ml)
1.10) as shown below (P-value = 0.0001). IL-33 (P = 0.0001) 5 4 3 2 1 0 A
R
C H
Figure 4.5: Comparison Between IL-33 Level in Serum of AR Patients and HC.
4.5 Correlation Between ECP Level and IL-17 Level Among the Allergic Rhinitis Patients Figure 4.6 shows positive significant correlation between serum ECP levels and IL-17 levels of the 88 AR patients (P-value = 0.041, R= 0.42).
51
Serum IL-17 concentration (pg/ml)
Chapter Four
Result
Allergic Rhinits (P = 0.041, R = 0.42) 1000 800 600 400 200 0 0
10
20
30
40
50
Serum ECP concentration (ug/L)
Figure 4.6: Correlation Between Serum ECP and IL-17 Levels in AR Patients.
4.6 Correlation Between ECP Level and IL-33 Level Among the Allergic Rhinitis Patients In Figure 4.7 shows that there was a positive significant correlation between serum ECP levels and IL-33 levels of the 88 AR patients (P-value =
Serum IL-33 concentration (pg/ml)
0.0001, R= 0.80). Allergic Rhinits (P = 0.0001, R = 0.80) 5 4 3 2 1 0 0
10
20
30
40
50
Serum ECP concentration (ug/L)
Figure 4.7: Correlation Between Serum ECP and IL-33 Levels in AR Patients.
52
Chapter Four
Result
4.7 Correlation Between IL-17 Level and IL-33 Level Among the Allergic Rhinitis Patients Statistical analysis showed no correlation between serum IL-17 and IL-33 levels of the 88 patients with AR (P-value = 0.091, R= 0.182) as shown in (Figure 4.8).
Figure 4.8: Correlation Between Serum IL-17 and IL-33 Levels in AR Patients.
4.8 Correlation Between ECP Level and IgE Level Among the Allergic Rhinitis Patients Figure 4.9 shows positive significant correlation between serum ECP levels and IgE levels of the 88 AR patients (P-value = 0.017, R= 0.45).
53
Chapter Four
Result
IgE level in blood (IU/ml)
Allergic Rhinits (P = 0.017, R = 0.45) 800 600 400 200 0 0
5
10
15
20
Serum ECP concentration (ug/L)
Figure 4.9: Correlation Between the Concentration of ECP and IgE Levels in AR Patients.
4.9 Correlation Between Symptoms and Different Markers Among the Allergic Rhinitis Patients Table 4.2 shows that there was a positive significant correlation between symptoms and eosinophil levels, and also between symptoms and IgE levels in AR patients, but no correlation between symptoms and each of ECP, IL-17 and IL-33 levels.
Table 4.2: Correlation Between Symptoms and Different Markers in AR Patients. * = significant, ns = non significant, P-value <0.05 (significant)
Eosinophil IgE level ECP IL-17 IL-33 level in blood in blood positive positive positive
Symptoms
Ratio
0.51
0.60
0.64
-0.14
0.42
P value
0.0259
0.0054
0.277
0.098
0.35
P value
*
**
ns
ns
ns
54
CHAPTER FIVE DISCUSSION
Chapter Five
Discussion
Chapter Five Discussion
5.1 Characteristics of Patients and Healthy Control Atopic diseases, including asthma and AR are common diseases with the great global health problem, thus we investigated the characteristics of AR patients through different markers. In this study, the AR group included 88 patients; their age range was 17-62 years. The female to male ratio was 3:1. There was no significant difference in age between AR patients compared to HC subjects. Our result showed that age do not play role in the development of AR disease. The mean eosinophil values in this study as shown in (Table 4.1) were significantly different in AR patients compared to HC subjects (P-value = 0.0004) regarding the standard normal eosinophil value in blood. A similar finding was also observed by Beppu, who showed that the mean of the blood eosinophil count was significantly higher in patients with AR compared to HC subjects (Beppu et al., 1994). While, Anand found that 94% of AR patients had normal blood eosinophil count according to the standard criteria, thus he suggested there was a need to revise the present standard value (Anand and Tapan, 2014). However, the eosinophils also are produced in healthy people, but in allergic diseases, where the secretion of various mediators such as IL-5, IL-3, and GM-CSF, promotes the production and activation of eosinophils which in turn secrete mediators increase the allergic inflammation (Deo et al., 2010). In the current study, the mean IgE levels were significantly higher in AR patients as compared to HC group (P-value = 0.0001) as shown in (Table 4.1) and (Figure 4.2). The same result was also obtained by other researchers, that showed the difference in mean IgE levels between AR patients and HC subjects 55
Chapter Five
Discussion
was significant (Kramer et al., 2000, Agha et al., 1997, Sin et al., 1998). Dehlink had another opinion in pediatric patients stating that serum IgE did not necessarily reflect the total body IgE. Thus, even in the absence of elevated levels of serum IgE, cell-bound IgE could be detected on peripheral blood cells in a subgroup of patients, thus these immune effector cells could travel to site of allergen contact and cause allergic symptoms despite normal or low serum IgE level (Dehlink et al., 2010). Allergic disease is caused when the immune system overreacts to the allergen by producing IgE. In which, these IgE can bind to immune cells that release inflammatory chemical mediators, causing allergic symptoms (Galli and Tsai, 2012). The total nasal symptom scores mean (sneezy-runny nose or blocked nose) in this study were significantly higher in AR patients than HC subjects (Pvalue <0.0001). This result is near to the study done by Amizadeh, who found symptom in patients with AR were higher compared to control group (Amizadeh et al., 2013). Amizadeh also cited the percentage of blocked nose much lower compared to sneezy and runny nose and that is the same result obtained in the current study (81% sneezy-runny nose and 19% blocked nose) (Amizadeh et al., 2013, Said et al., 2012). The nasal symptoms of AR start after the exposure to an allergen. These symptoms occasionally improve with time, but this can take many years naturally in infants and children, and probably will not disappear completely mostly in adults (Bousquet et al., 2008).
56
Chapter Five
Discussion
5.2 Comparison of Serum ECP Levels Among Allergic Rhinitis Patients and Healthy Control In the current study, serum ECP detected in the AR patients regarding the ECP detecting range level. A significant difference in the ECP level was found between AR group and the control group. Similar results were reported, when the serum ECP concentration in patients with AR group was compared to HC group (Beppu et al., 1994, Sin et al., 1998, Do et al., 1998). In a review data performed by Bystrom, which confirmed that the elevated ECP concentration in serum of AR patients is associated with human epithelial cell damage in the respiratory tract inflammatory disease; thus, the immunotherapy is the only treatment that modified fundamental allergic mechanisms reduce the inflammation, but it is effect becomes apparent after years (Bystrom et al., 2011).
5.3 Comparison of Serum IL-17 Levels Among Allergic Rhinitis Patients and Healthy Control In this study, the IL-17 protein detected in the serum of AR patients and it was significantly higher in AR patients compared to HC group. The same result was obtained by Tsvetkova-Vicheva and Lu, they found that serum IL-17 level was higher in AR patients compared to the HC (Tsvetkova-Vicheva et al., 2014, Lu et al., 2011). Vocca, detected IL-17 plasma levels in patients with AR. He found that IL-17 was significantly higher than the HC group (Vocca et al., 2015). While Milovanovic, showed that the IL-17/ TH17 cells have been implicated in allergy through experimental study present evidence that IL-17 enhances IgE production by human B cells in allergic patients suffered from asthma and atopic dermatitis compared with normal controls, they found that after the removal of the TH17 cells in vitro from peripheral blood mononuclear 57
Chapter Five
Discussion
cells (PBMCs) of allergic patients, IgE levels decrease, while addition of recombinant IL-17 to PBMCs restore it (Milovanovic et al., 2010). Serum IL-17 high levels may be associated with nasal inflammation, so targeting IL-17 could be useful in the treatment of AR disease (Makihara et al., 2014).
5.4 Comparison of Serum IL-33 Levels Among Allergic Rhinitis Patients and Healthy Control The results of the present study showed that serum levels of IL-33 are significantly different in the AR patients than in the HC group (P=0.0001). Chai, agreed with the result of the present study (Chai et al., 2017). Gluck, found serum levels of IL-33 were significantly higher in patients with intermittent allergic rhinitis (IAR) group compared to HC group (Gluck et al., 2012). In another study done by Vocca, who found that plasma concentration of IL-33 level was significantly higher in AR patients compared to HC subjects (Vocca et al., 2015). Ketelaar, showed that IL-33 is present at very low levels in serum of asthma patients using ELISA technique, and this is in agreement with our finding; we also detect low levels of the IL-33 in serum of patients with AR using the same technique (Ketelaar et al., 2016). While Rogala and Gluck reviewed data and reported that IL-33 is an interesting cytokine because it has either proinflammatory or anti-inflammatory properties. However, IL-33 is involved in TH2 mediated inflammatory responses in AR disease, but it has also protective effect such as in cardiovascular disease (Rogala and Gluck, 2013). The elevated IL-33 cytokine enhances the AR inflammation through activated various cells including TH2, MC and eosinophils through the ST2 receptor. Thus, the IL-33/ST2 pathway also might be an important as a therapeutic target to decrease allergic inflammation (Saluja et al., 2015).
58
Chapter Five
Discussion
5.5 Correlation Between ECP Level and IL-17 Level Among the Allergic Rhinitis Patients In the current study, a significant correlation has been found between serum ECP and IL-17 levels in the AR patients. However, the ECP and IL-17 have been found in AR patients (Bystrom et al., 2012), but there were no previous studies proving a correlation between ECP and IL-17 in the AR disease. While IL-17 known to promote eosinophils survival and degranulation via GM-CSF (Bystrom et al., 2012). Our findings demonstrate that IL-17 cytokine and ECP protein may augment nasal inflammation of AR patients.
5.6 Correlation Between ECP Level and IL-33 Level Among the Allergic Rhinitis Patients In the present study, we also found a significant correlation between serum ECP and IL-33 levels in the AR patients. However, IL-33 can directly enhance eosinophils survival and degranulation (Bystrom et al., 2012), but there were no previous studies proving a correlation between ECP and IL-33 in the AR disease. These findings indicated that IL-33 and ECP plays a key role in nasal inflammation of AR patients and may be useful in assessing the severity.
5.7 Correlation Between IL-17 Level and IL-33 Level Among the Allergic Rhinitis Patients Our result showed that the concentrations of the IL-17 and IL-33 cytokines were detected in patients with AR, but IL-33 found at low levels in serum of these patients. Among AR patients, the correlation was not observed between serum IL-17 and IL-33 levels. Our finding was in agreement with a study done by Vocca. However, he found that these cytokines significantly increased in patients with AR, but he could not find the correlation between them (Vocca et al., 2015). Despite the involvement of IL-33 and IL-17 59
Chapter Five
Discussion
cytokines in the pathogenesis of AR disease, together, maybe not have influence on the severity of the disease.
5.8 Correlation Between ECP Level and IgE Level Among the Allergic Rhinitis Patients In this study, we found a significant correlation between serum ECP and IgE levels in the AR patients. There were no previous studies proving a correlation between ECP and IgE in the AR disease. The explanation of this finding; the eosinophil may degranulates and release mediators such as ECP after contact with IgE through surface receptors in allergic diseases.
5.9 Correlation Between Symptoms and Different Markers Among the Allergic Rhinitis Patients We found a significant correlation between symptom scores and eosinophil count. Eosinophils play a role in the development of symptoms in the late phase response, as described in several previous studies. Eosinophils with other inflammatory cells as soon as attracted by chemical mediators to the target site, damage nasal tissue and this results in a nasal obstruction which is one of the main symptoms of the AR disease (Davoine and Lacy, 2014, Min, 2010, Kay, 2001). Apar showed a significant correlation between eosinophil count and symptom severity score for AR (Apar et al., 2012). Chen also observed a correlation of eosinophil count with symptom severity score of perennial allergic rhinitis (PAR) (Chen et al., 2006). We also found a significant correlation between IgE levels and symptom scores. The reason could be that AR symptoms either sneezing-rhinorrhea or nasal obstruction are resulting due to the release of the allergic mediators such 60
Chapter Five
Discussion
as histamine upon cross-linking of the allergen-specific IgE bound to the surface of mast cells in AR patients (Pawankar et al., 2011). Moreover, the study done by Chen, shows that levels of IgE have been correlated with symptom severity score in PAR patients (Chen et al., 2006). Statistically, there is no correlation between ECP, IL-17, IL-33 levels and symptom scores in patients with AR, respectively. This result is in agreement with the study by (Do et al., 1998), who found the symptom scores which was not correlated with serum ECP concentrations. While Chen found the symptom severity score was correlated with serum ECP levels in PAR patients (Chen et al., 2006). Ciprandi and Gluck found significant correlation between symptom severity score and IL-17 and also IL-33, respectively (Ciprandi et al., 2009, Lu et al., 2011, Gluck et al., 2012). However, serum levels of ECP, IL-17 and IL33 have been increased in pateints with AR, but they cannot be used as an indicator to judge the severity of the symptoms.
61
CHAPTER SIX CONCLUSIONS AND RECOMMENDATIONS
Chapter Six
Conclusions and Recommendations
Chapter Six Conclusions
The Following Points are Noted from the Present Study: 1. This study shows that the prevalence of AR does not differ between genders from all ages. 2. Serum levels of ECP, IL-17 and IL-33 are suitable markers to differentiate AR patients from HC subjects. 3. There is a positive correlation between elevated serum ECP and cytokines (IL-17, IL-33) and IgE antibody in AR disease, respectively. 4. There is no correlation between IL-17 and IL-33 in AR disease. 5. The nasal symptoms correlate with levels of eosinophils and IgE, but not with levels of ECP, IL-17 and IL-33 in AR disease, respectively. 6. Finally, high levels of cytokines (IL-17, IL-33) and ECP in AR support the idea of the multifactorial nature of the allergic disease and the role of these markers in the pathogenesis of AR.
62
Chapter Six
Conclusions and Recommendations
Recommendations 1. Further researches are recommended to improve our understanding of the role of cytokines such as IL-17, IL-33 clinically and to know more about inflammatory mechanism involved in the AR disease for an adequate prevention and treatment in the future.
2. Measurement ECP, IL-17 and IL-33 concentration in nasal secretions of AR patients.
3. Study on genetic predisposition of AR disease.
4. These observations may provide basis for future studies targeting the correlation of ECP with cytokines (IL-17, IL-33), for understanding their role in diseases associated with eosinophils, mast cells, basophils and for future therapeutic approaches in the management of AR disease.
Limitation of the Study: There were no ELISA instruments in a research center in the school of medicine so we had to do our practical procedure in another laboratory.
63
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85
APPENDICES
Questionnaire for Allergic Rhinitis (AR) Patients Name: ( ) Season
Date: (
) Out of season
Patient code number: Age:
Gender: (
) Male
(
) Female
Occupation: Address: Phone: Date of diagnosis with AR: Type of AR: Residence: ( (
(
) Intermittent
(
) Persistent
) Urban ( ) Rural ) Exposure to tobacco smoke
Family history of AR: (
) Siblings
(
(
) Parents
) Exposure to animals (
) Grandparents
(
) others
Co-morbidities: ( ) Bronchial asthma ( ) Sinusitis ( ) Atopic dermatitis ( ) Allergic conjunctivitis ( ) aspirin sensitivity ( ) Chronic rhino sinusitis ± polyposis Predominant symptoms: (
) Sneezers-runners
(
) Blockers
Exaggerated factors (type of allergen): ( ) Polyvalent house dust ( ) House dust mites ( ) Fungi ( ) Insects ( ) Food ( ) irritant material "perfume, detergent, chlorine" Medication: ( ( Imaging: (
) Antihistamine ) other
(
) Montlukast
(
( (
) Pollens ) pets
) Steroid "local, oral"
) CT scan
Past surgical history: ( ) Sinusitis surgery ( ) Tonsillectomy and Adenoidectomy ( ) Nasal deviation surgery ( ) Rhinoplasty ( ) Ear surgery "myringotomy ± grommets"
E . 33 3317 17; 33 17 E .2016 2016 3317 E . 3317 E . E3317 33 17 . E .3317 33 17 33 17 33 17 E
2017
1438
3317 33 17 2016 8888 33 17 17 33 33 17 33 17 33 17
33 17 33 17 33 17
ﻟﺔﻻﻳﺔﻥ ﺳﺆﻻﻅ ﻣﻮﺳﻰ ﻋﻴﺴﻰ ﺩﺑﻠﺆﻣﻲ ﺑﺎﻵ ﻟﺔ ﻣﺎﻳﻜﺮﻭَﺑﺎﻳﻮَﻟﻮَﺟﻲ
ﺑﺔﺳﺔﺭﺛﺔﺭﺷﺘﻲ ﺛﺮﻭَﻓﻴﺴﻮَﺭ ﺩﻛﺘﻮَﺭ ﻛﺎﻭﺓ ﻋﺒﺪ ﺍﷲ ﻣﺤﻤﺪ ﺍﻣﻴﻦ
ﺯﺍﻳﻨﻲ2017
ﻛﻮﺭﺩﻯ2717
