PERIPHERAL BLOOD NEUTROPHIL AND EOSINOPHIL ACTIVITY DURING ALLERGEN-INDUCED LATE-PHASE AIRWAY INFLAMMATION IN ASTHMA Simona Lavinskienė

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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES MEDICAL ACADEMY

Simona Lavinskienė

PERIPHERAL BLOOD NEUTROPHIL

AND EOSINOPHIL ACTIVITY DURING

ALLERGEN-INDUCED LATE-PHASE

AIRWAY INFLAMMATION

IN ASTHMA

Doctoral Dissertation Biomedical Sciences, Biology (01B) Kaunas, 2014

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The doctoral dissertation was prepared during 2010–2014 in the Department of Pulmonology and Immunology, Medical Academy, Lithuanian Univer-sity of Health Sciences.

Scientific Supervisor

Dr. Jolanta Jeroch (Lithuanian University of Health Sciences, Biomedical Sciences, Biology – 01B)

Consultant

Prof. Dr. Kęstutis Malakauskas (Lithuanian University of Health Scien-ces, Biomedical ScienScien-ces, Medicine – 06B)

Dissertation is defended at the Biology Research Council of the Medical Academy of Lithuanian University of Health Sciences:

Chairperson

Prof. Dr. Habil. Vaiva Lesauskaitė (Lithuanian University of Health Sciences, Biomedical Sciences, Biology – 01B)

Members:

Prof. Dr. Dainius Haroldas Pauža (Lithuanian University of Health Sciences, Biomedical Sciences, Biology – 01B)

Prof. Dr. Habil. Virgilijus Ulozas (Lithuanian University of Health Sciences, Biomedical Sciences, Medicine – 06B)

Prof. Dr. Aurelija Žvirblienė (Vilnius University, Biomedical Sciences, Biology – 01B)

Assoc. Prof. Dr. Reinoud Gosens (University of Groningen, Biomedical Sciences, Pharmacy – 08B)

The dissertation will be defended in the open session of the Biology Research Council on December 19, 2014, at noon in the Large Auditorium of the Hospital of Lithuanian University of Health Sciences Kauno Klinikos.

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LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETAS MEDICINOS AKADEMIJA

Simona Lavinskienė

PERIFERINIO KRAUJO NEUTROFILŲ

IR EOZINOFILŲ AKTYVUMAS

ALERGENO SUKELTOS VĖLYVOS

FAZĖS KVĖPAVIMO TAKŲ UŽDEGIMO

METU SERGANT ASTMA

Daktaro disertacija Biomedicinos mokslai,

biologija (01B)

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Disertacija rengta 2010–2014 metais Lietuvos sveikatos mokslų universiteto Medicinos akademijos Pulmonologijos ir imunologijos klinikoje.

Mokslinė vadovė

Dr. Jolanta Jeroch (Lietuvos sveikatos mokslų universitetas, biomedi-cinos mokslai, biologija – 01B)

Konsultantas

Prof. dr. Kęstutis Malakauskas (Lietuvos sveikatos mokslų universi-tetas, biomedicinos mokslai, medicina – 06B)

Disertacija ginama Lietuvos sveikatos mokslų universiteto Medicinos akademijos biologijos mokslo krypties taryboje:

Pirmininkė

Prof. habil. dr. Vaiva Lesauskaitė (Lietuvos sveikatos mokslų univer-sitetas, biomedicinos mokslai, biologija – 01B)

Nariai:

Prof. dr. Dainius Haroldas Pauža (Lietuvos sveikatos mokslų

universi-tetas, biomedicinos mokslai, biologija – 01B)

Prof. habil. dr. Virgilijus Ulozas (Lietuvos sveikatos mokslų universi-tetas, biomedicinos mokslai, medicina – 06B)

Prof. dr. Aurelija Žvirblienė (Vilniaus universitetas, biomedicinos moks-lai, biologija – 01B)

Doc. dr. Reinoud Gosens (Groningeno universitetas, biomedicinos moks-lai, farmacija – 08B)

Disertacija ginama viešame Lietuvos sveikatos mokslų universiteto Me-dicinos akademijos Biologijos mokslo krypties tarybos posėdyje 2014 m. gruodžio 19 d. 12 val. Lietuvos sveikatos mokslų universiteto ligoninės Kauno klinikos Didžiojoje auditorijoje.

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ABBREVIATIONS

AA allergic asthma

APC antigen-presenting cells

AR allergic rhinitis

AHR airway hyperresponsiveness BAL bronchoalveolar lavage CD cluster of differentiation

DTT dithiothreitol

DHR-123 dihydrorhodamine-123

D. pteronyssinus Dermatophagoides pteronyssinus EDN eosinophil derived neurotoxin EDTA ethylene diamine tetra-acetic acid ECP eosinophil cationic protein

ELISA enzyme-linked immunosorbent assay ERS European Respiratory Society

FcεRI high-affinity IgE receptors FEV1 forced expiratory volume in 1 sec FITC fluorescein isothiocyanate

FSC forward light scatter

GINA Global Initiative for Asthma

GM-CSF granulocyte macrophage- colony stimulating factor

HS healthy subjects

Ig immunoglobulin

MBP major basic protein

MFI mean fluorescence intensity MMP matrix metalloproteinase

NADPH nicotinamide adenine dinucleotide phosphate PBS phosphate-buffered saline

PI propidium iodide

RANTES Regulated on Activation, Normal T Cell Expressed

PMN polymorphonuclear

ROS reactive oxygen species S. aureus Staphylococcus aureus

SSC side light scatter

TGF-β transforming growth factor β

Th T helper cells

TNF tumor necrosis factor

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INTRODUCTION

Airway inflammation with eosinophils as well as CD4+ T cells is a cha-racteristic feature of asthma and is considered relevant to the pathogenesis of the disease (1). Allergen challenge in sensitized asthmatics can provoke two phases of temporal airway responses, namely early-phase reaction and late-phase reaction. Late-phase reaction is characterized by the influx of inflammatory cells and associated with an increase in airway hyperrespon-siveness and leads to airway remodeling, which develops as the disease progresses (2). Despite the fact that for the long time eosinophils were relevant in the pathogenesis of asthma, a growing body of evidence shows that neutrophils are also very important cells contributing to the inflammatory process in human asthma (3). The influx of neutrophils to the airways has been reported in severe asthma attacks (4) and after intratracheal allergen challenge in a murine asthma model (1). The increased migration of neutrophils from blood into the airways stimulates the accumulation and abnormal activation of inflammatory cells that are largely responsible for oxidative stress and production of proteases and inflammatory cytokines, contributing to lung injury and chronic inflammation (5, 6). It is suggested that IL-8 plays a key role in the accumulation of neutrophils in the sites of inflammation in severe asthmatic patients (4). However, the mechanisms that control neutrophil accumulation in the airways during allergic airway inflammation have not been completely elucidated yet. Particularly, much attention has been paid to neutrophil functions such as chemotaxis, phagocytic activity, and reactive oxygen species (ROS) production during chronic airway inflammation. Alterations in neutrophil functions lead to an ineffective removal of pathogens and increases inflammation. Despite the data on the importance of neutrophils in chronic airway inflammation, unfortunately, there are only few data about neutrophil activity in allergic diseases. Following airway allergen exposure, the development of airway eosinophilia is associated with increased 5 expression in the sputum, elevated concentrations of IL-5 in luminal fluid and serum, and a heightened capacity of airway cells for ex vivo generation of IL-5 (7–9).Experiments with in vitro allergen as well as endobronchial allergen challenge have shown that blood and bronchoalveolar lavage eosinophils from subjects with asthma have a greater responsiveness to chemoattractants and enhanced chemotaxis (10, 11). During the process of allergic inflammation, eosinophils release not only toxic granule proteins but also ROS, which are known to cause tissue damage (12). It has been demonstrated that allergic patients have

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lated oxidative metabolism in blood eosinophils when compared with healthy subjects (13, 14). It leads to the observations that eosinophils isolated from allergic patients might be already activated in peripheral blood streams before they infiltrate the tissue. There are some data about impaired peripheral blood neutrophil and eosinophil apoptosis in allergic patients, and this might contribute to greater airway neutrophilia and eosinophilia. There is no doubt that neutrophils and eosinophils are important cells participating in allergic airway inflammation. Meanwhile, associations between eosino-phil infiltration in the airways and peripheral blood neutroeosino-phil and eosi-nophil activity such as chemotaxis, production of ROS, degranulation, and apoptosis have not been completely elucidated yet. Therefore, the regulation of blood neutrophil and eosinophil activity in asthmatic patients after allergen challenge was investigated in this study.

Aim of study

The aim of this study was to evaluate peripheral blood neutrophil and eosinophil functional activity during allergen-induced late-phase airway inflammation in asthma.

Objectives of the study

1. To evaluate peripheral blood neutrophil activity (chemotaxis, phago-cytosis, spontaneous production of reactive oxygen species, and apop-tosis) during allergen-induced late-phase airway inflammation in patients with asthma.

2. To determine a link between neutrophil activity (chemotaxis and apop-tosis) and airway neutrophilia during allergen-induced late-phase airway inflammation.

3. To determine the changes in peripheral blood eosinophil chemotaxis, production of reactive oxygen species, degranulation, and apoptosis during allergen-induced late-phase airway inflammation in patients with asthma.

4. To evaluate associations of peripheral blood eosinophil chemotaxis and apoptosis with allergen-induced late-phase airway eosinophilia.

Scientific novelty

There is no doubt that eosinophils and neutrophils are important cells participating in asthma pathogenesis. The most prominent feature reflecting asthma pathogenesis is late-phase airway inflammation, which occurs a few hours after allergen inhalation.

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The worldwide published studies on asthma show that most attention is paid to individual, not complex, functions of neutrophils and eosinophils in the airways. Moreover, associations between peripheral blood neutrophil and eosinophil activity and infiltration of these cells in the airways during asthma have not been completely elucidated yet. There are no data about peripheral blood neutrophil and eosinophil activity during allergen-induced late-phase airway inflammation in asthma patients.

Therefore, a successfully applied specific bronchial allergen challenge model allowed us to evaluate dynamic changes in peripheral blood neutro-phil and eosinoneutro-phil activity 7 h and 24 h after bronchial allergen challenge in patients with allergic asthma.

Our findings provide new evidence about neutrophil and eosinophil functional activity during allergen-induced late-phase airway inflammation in asthma patients.

Practical and theoretical significance

Our study provides new information about the pathogenesis of allergic asthma. We found that an inhaled allergen activates peripheral blood neutrophil and eosinophil chemotaxis, phagocytosis, generation of ROS and also reduces apoptosis during late-phase airway inflammation in asthma. Furthermore, altered peripheral blood neutrophil and eosinophil functional activity is related to airway neutrophilia and eosinophilia.

Changes in peripheral blood neutrophil and eosinophil activity observed in this study could be useful for asthma monitoring and predicting disease progression.

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1. REVIEW OF LITERATURE

1.1. Definition and pathogenesis of asthma

Asthma is a chronic inflammatory disorder of the airways in which various cells play a role. Chronic inflammation is associated with airway hyperresponsiveness that leads to recurrent episodes of wheezing, breath-lessness, chest tightness, and coughing, particularly at night or in the early morning. Respiratory symptoms are usually associated with airway smooth muscle contraction, mucus production, and edema of the airway wall, leading to airflow obstruction that is often reversible either spontaneously or following treatment. These episodes are usually associated with widespread but variable airflow obstruction within the lung that is often reversible either spontaneously or with treatment. Asthma is thought to arise from the inter-action of multiple environmental, genetic, and epigenetic factors (15, 16).

Asthma attacks are generally triggered by allergens, viral respiratory infections, and airborne irritants. Uncontrolled asthma can markedly interfere with normal daily activities and seriously impact an individual’s quality of life (17).

There is a growing body of evidence that severe asthma is not a single disease as evidenced by the variety of clinical presentations, physiological characteristics, and outcomes (18). The complexity of the disease together with the lack of a cure for asthma makes this disease a public health burden reflected by increased utilization of health services by asthmatic individuals. The World Health Organization has estimated that 300 million individuals have asthma worldwide, and with current rising trends, this number will reach 400 million by 2025 (19, 20).

Airway inflammation in asthma is a multicellular process involving eosinophils, neutrophils, CD4+ T lymphocytes, and mast cells, with eosinophilic infiltration being the most striking feature (21, 22). The inflammatory process is largely restricted to the conducting airways but as the disease becomes more severe, infiltrate spreads both proximately and distally to include the small airways and adjacent alveoli in some cases (21, 23). The inflammatory response in the small airways appears to occur pre-dominantly outside the airway smooth muscle, where submucosa dominates in the large airways inflammation (21, 24) (Fig. 1.1.1).

Th2 cells involved in chronic allergic inflammatory responses are common at multiple tissue sites and are seen at these sites in patients with asthma, who frequently have comorbidities such as allergic rhinitis (26).

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Fig. 1.1.1. Inflammatory and remodeling processes in asthma (Reproduced from the article by Holgate and Polorosa, 2006)(25) Allergic rhinitis is associated with a complex of symptoms characterized by paroxysms of sneezing, rhinorrhea, nasal obstruction, and itching of the eyes, nose, and palate. It is also frequently related to postnasal drip, cough, irritability, and fatigue (27, 28).

A growing body of data accumulated during the last decades supports a connection between upper and lower airway inflammation, often referred to as “united airways” or “one airway – one disease” (29, 30). Allergic rhinitis occurs in more than 75% of the patients with allergic asthma, and allergic rhinitis is a risk factor for the development of concomitant asthma, and 1 of the 3 patients with allergic rhinitis may develop asthma within 10 years (31). In patients with allergic asthma and without the symptoms of allergic rhinitis, nasal mucosa biopsies showed significantly enhanced eosinophilic inflammation (32). Meanwhile, nasal allergen challenge was associated with increased bronchial hyperreactivity to methacholine (33), upper and lower airway eosinophilia (34), and changes in the cellular composition of inflammatory cells and cytokines in patients with allergic rhinitis (35).

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Moreover, nasal challenge with the house dust mite allergen caused nasal symptoms and late-phase bronchial obstruction in asthma patients. Airway inflammation can be accompanied by a systemic response with the increased maturation of bone marrow stem cells and migration to the site of inflam-mation (36).

1.2. Allergen-induced early and late-phase airway inflammation Allergen sensitization is a central element in allergic asthma. In allergic sensitization, the airway is capable of recognizing usual allergens to develop Th2 cytokine responses, which also involves a cascade of inflammation caused by the interaction of respiratory epithelium, innate and adaptive immune system, promoting a chronic inflammatory response (37). Inhaled allergens, such as pollen, house dust mites, and mold are processed by one of the antigen presenting cells (APCs), commonly dendritic cells. Processed allergens are then loaded into to the major histocompatibility complex II (MHC II), a cell surface molecule, which presents the allergens on the surface of cells and enables APC to interact with CD4+ T cells. The MHC II–allergen combination interacts with naive T cells, which initiates sensi-tization and triggers the subsequent immune response to the allergen. Dendritic cells also contribute to the differentiation of the nature of the immune response (Th1/Th2) by producing inflammatory mediators such as IL-12 and type I interferons, which stimulate differentiation of naive T cells into Th1 cells. The production of IL-4 by eosinophils and mast cells inhibits the expression of IL-12 and Th1 development, therefore, directing the immune response to the production of Th2 effector cells. Expression of IL-4, OX40L, and CD86 polarizes T-cell differentiation in favor of Th2 responses, which is the hallmark of allergic response (38). Activated and sensitized T cells then produce a range of cytokines such as IL-3, IL-4, IL-5, IL-6, IL-9, IL-13, and granulocyte-macrophage colony stimulating factor (GM-CSF). IL-4 and IL-13 produced by T cells drive B cells to proliferate and produce allergen-specific antibodies (IgE), which is the main pathway of allergen sensitization. The sensitization results in the formation of IgE memory B-cells and allergen-specific memory T cells, which are respon-sible for future allergic responses (39).

Allergen challenge in sensitized asthmatics can provoke two phases of temporal airway responses, namely early-phase reaction and late-phase reaction.

An early-phase reaction (Fig. 1.2.1) occurs within minutes of allergen exposure and mainly reflects the secretion of mediators by mast cells at the

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receptors (FcεRI) on their surfaces. When crosslinking of adjacent IgE molecules by a bivalent or multivalent allergen occurs, aggregation of FcεRI triggers a complex intracellular signaling process that results in the secretion

of three classes of biologically active products: activated mast cells

subsequently release their granules, which contain inflammatory mediators such as histamine, leukotrienes, ROS, and lipid mediators as well as other mediators (40–42). The outcome of the mediator release includes bronchoconstriction, vasodilatation, increased vascular permeability and mucus production, and enhanced airway responsiveness. The secretion of mediators takes place when the membrane of mast cell cytoplasmic granules fuses with the plasma membrane during degranulation (43).

In allergic patients, an early allergic inflammatory reaction may be followed later by a late-phase reaction (44).

Fig. 1.2.1. Early-phase of allergen-induced airway inflammation (Reproduced from the article by Galli et al., 2008)(2)

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Activated mast cells induce but slower the expression of other cytokines and chemokines (tumor necrosis factor-α [TNF-α], IL-4, and IL-5) up to 72 h after allergen challenge, which contributes to the inflammatory cascade of allergen-induced late-phase reaction (Fig. 1.2.2) (45, 46). Late-phase reaction peaks at 6 to 9 h after allergen provocation. The orchestration of the late-phase reaction is thought to be the result of long-term outcomes of mast cell activation during the early-phase reaction and activation of T cells by an allergen. The release of Th2 cytokines, which is initiated by the activation of T cells after allergen challenge, is an important mechanism of the late-phase response (47). The nature of the late-phase reaction represents an increasing number and enhanced activity of resident cells and leukocytes such as eosinophils, neutrophils, basophils, macrophages, and CD4+ T cells re-cruited to the airways (46, 48–51).

Fig. 1.2.2. Late-phase of allergen-induced airway inflammation (Reproduced from the article by Galli et al., 2008)(2)

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1.3. The role of IL-8 and IL-5 in allergic airway inflammation Interleukin 8 (IL-8) is a member of the CXC subfamily and is a potent chemotactic factor for neutrophils, having a proinflammatory effect (52, 53). IL-8 at high concentrations has been reported to be present at inflammation sites in vivo (54). Many cell types including epithelial and endothelial cells, alveolar macrophages, lymphocytes, fibroblasts, and neutrophils upon proinflammatory stimulation produce and secrete IL-8 (55).

IL-8 participates in the process of neutrophil transmigration, including the shedding of L-selectin, upregulation of β2 integrins, and adhesion to the endothelium. Moreover, various functions such as degranulation and respiratory burst of neutrophils can be regulated by IL-8 (56, 57). Studies have shown that IL-8 is responsible for the secretion of neutrophil elastase and matrix metalloproteinase 9 (MMP-9), which can cause tissue damage (58). Moreover, IL-8 has been suggested as a predictive marker of bacteremia and sepsis (59).

IL-8 has been suggested to play an important role in asthma and especially in its severity (60). Asthma exacerbations following the withdrawal of inhaled corticosteroids have been associated with a signi-ficant influx of neutrophils and an increase in the sputum IL-8 level (61). Increased IL-8 levels have been observed in serum of allergic asthma patients with anti-IgE treatment (62). Other studies demonstrated enhanced IL-8 levels in BAL fluid and induced sputum in patients with asthma when compared with controls (63). It was also reported that nasal allergen challenge resulted in higher secretion of IL-8 in the nasal lavage of rhinitis patients (63). Monteseirin et al. showed that the IgE-dependent mechanism was related to IL-8 release and upregulation of IL-8 mRNA expression by neutrophils from allergic patients (64).

IL-5 has been recognized as the most specific cytokine of the eosinophil origin and has been identified as the key denominator in inflammatory pathways in asthma (65). IL-5 plays a key role in eosinophil proliferation, differentiation, maturation, migration to tissue sites, and survival as well as prevention of eosinophil apoptosis (66, 67). Seys et al. reported that uncontrolled asthma was associated with higher mRNA expression of IL-5 in sputum (68).

Residual eosinophils in the tissues suggest that survival and function may not depend on 5 as eosinophils downregulate the expression of their IL-5-receptor α (IL-5Rα) and that tissue eosinophils may survive in the absence of IL-5 (9). Recently, anti-human IL-5Rα mAb therapy has been introduced due to its potential to eliminate eosinophils localized in the inflammatory tissues by antibody-dependent cell-mediated cytotoxicity (69). Intravenous

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administration of anti-human IL-5Rα mAb resulted in marked reduction of blood eosinophils within 24 h in patients with mild atopic asthma (70).

There are some controversial data regarding anti-IL-5 antibody therapy in animal models with allergic inflammation and asthma patients. Anti-IL-5 treatment has been shown to eliminate airway eosinophilia with no effect on airway hyperresponsiveness (AHR) in an animal model of determined airway inflammation (71). Anti-IL-5 applied to patients with mild asthma decreased blood and sputum eosinophilia without improvement in the symptoms of asthma (72, 73). Other study with anti-IL-5 used in severe asthma demonstrated a reduction in blood eosinophilia, but did not show any influence on clinical parameters (74). Repeated treatment demonstrated only 55% reduction in bronchial mucosa eosinophilia, and a large-scale clinical trial showed no effect on improving symptoms of asthma in patients with moderate persistent asthma (75).

Although it is known that IL-8 and IL-5 are important cytokines in asthma pathogenesis, the importance of these cytokines in allergen-induced late-phase airway inflammation still needs to be clarified. Therefore, this study aimed to investigate the levels of these cytokines during bronchial allergen challenge.

1.4. Activity of neutrophils in asthma

Neutrophils are short-lived cells that first arrive at the site of inflam-mation or infection. Neutrophils account for 50%–75% of the circulating leukocytes in humans, and their numbers are further increased in acute and chronic inflammatory diseases (76). The bulk of their life span is spent proliferating and differentiating in the bone marrow where the cells are stored for a few days and then released into the circulation. The cells circulate in the blood for a short time before they migrate into the tissues where they function as mobile phagocytes. Neutrophils are an important part of the innate immune defense against injury and infection due to their ability to engulf and kill pathogenic microorganisms (77, 78). Any defect in the functionality of phagocytic cells can result in fatal diseases due to the lack of protection from invading pathogens.

The inflammatory response mediated by neutrophils is a multistage process that involves adhesion of circulating neutrophils to the vascular endothelium and transmigration of neutrophils to the inflammatory site, where they become activated and participate in the defense mechanisms (79). Neutrophils can recognize and swallow foreign organisms during phagocytosis, where they kill and destroy microorganisms using ROS and antimicrobial/proteolytic granule proteins. In addition to this, neutrophils

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release cytokines and chemokines. The most common is IL-8, which has a wide spectrum of biological activities and is able to regulate the inflame-matory response of other cells (macrophages, T lymphocytes, and neutro-phils themselves) (80, 81).

A growing body of evidence shows the importance of neutrophils in the pathogenesis of asthma. Neutrophils are found in nocturnal asthmatics, and frequently their number is associated with the severity of nocturnal asthma (82). In addition, neutrophils are found in large amounts in the airways of patients with acute exacerbations of asthma, and this is mostly associated with respiratory tract infections (83). Neutrophil influx in bronchoalveolar lavage (BAL), induced sputum, and the biopsy has been demonstrated in patients with acute asthma exacerbation and with no infection (84, 85).

It has been reported that the number of neutrophils are is greater in patients with more severe asthma and subjects with severe asthma during intubation (86), sudden fatal asthma, and life-threatening asthma (84, 87, 88). Large numbers of neutrophils and no or minimal numbers of eosino-phils have been observed in the airway mucosa of patients with fatal asthma (88–92). In addition, it has been shown that blood neutrophils from allergic asthma patients release increased levels of myeloperoxidase after stimu-lation with N-formyl-methionyl-leucylphenylalanine (fMLP) (93), which can be mediated by activation of IgE receptors (94).

Increased spontaneous production of ROS was observed in neutrophils isolated from asthmatics (95) and also in neutrophils after stimulation (96, 97). Inhalation of ozone (98) or endotoxin (99) and other triggers such smoking (100), organic dust (101), and inflammation or infection (102) increase neutrophilic influx to the airways and are associated with AHR. Moreover, evidence shows that neutrophil consumption is associated with improvement in AHR (98, 103). Inflammatory mediators such as LTB4, IL-17 and IL-8 involved in neutrophil accumulation have also been associated with AHR (104, 105). Neutrophils may play a role in airway remodeling by the release of growth factors such as transforming growth factor β (TGF-β), which leads to the activation of fibroblasts and alterations in the turnover of the extracellular matrix. TGF-β production in the neutrophils of patients with asthma was enhanced when compared with healthy controls (106). Formation of new blood vessels (angiogenesis) is also an important feature of airway remodeling. Neutrophils are an important source of angiogenic factors and release VEGF on stimulation with bacterial products. Then released VEGF can activate endothelial cells to induce angiogenesis (107).

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The influx of neutrophils to the airways has been reported in severe asthma attacks (4) and after intratracheal allergen challenge in a murine asthma model (3). The mechanism is based on the reactivity of neutrophils to chemoattractive signals, a process known as chemotaxis, which is crucial for an efficient control of pathogens. During chemotaxis, cells migrate through barriers (vessel walls or epithelial layers) and tissues toward a site of infection or allergen-induced inflammation (108, 109).

This process step by step can change the functional status of the neutrophil from a passive circulating cell into a highly activated effector cell of innate immunity.

The enhanced migration of neutrophils from blood into the airways activates the accumulation and abnormal activation of inflammatory cells that are largely responsible for oxidative stress and production of ROS, proteases, and cytokines, contributing to lung injury and chronic inflammation (5, 6). This damage occurs when neutrophils accumulate in large numbers, and their activation is inappropriate or uncontrolled.

It is known that IL-8 is a major chemoattractant of neutrophils, produced and released by neutrophils (108, 110), alveolar macrophages (111), and other activated cells. It is suggested that IL-8 plays a key role in the accu-mulation of neutrophils at the sites of inflammation in severe asthmatic patients (4).

However, the mechanisms that control neutrophil accumulation in the airways especially during allergen-induced late-phase inflammation have not been completely elucidated yet.

1.4.2. Phagocytosis

Throughout the phagocytic process, neutrophils release a range of inflammatory mediators that contribute to the local inflammatory process, including lipid mediators (e.g. leukotriene B4), proteolytic enzymes (e.g. neutrophil elastase), ROS (e.g. superoxide), cytokines (e.g. TNF-α) and chemokines (e.g. IL-8) (112, 113). By this mechanism the pathogen can be destroyed and resolution of the inflammatory response.

The mechanism underlying the uptake of pathogens begins with the binding of receptors on the neutrophil membrane with specific molecules on the membrane of the particle to be swallowed. The ligand/receptor complex then activates rearrangements in the cytoskeleton, which leads to inter-nalization of the complex, forming a phagosome. Fusion of the phagosome

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with intracellular granules allows the formation of the phagolysosome. Pathogens in the phagolysosome are killed by exposure to enzymes, ROS, and antimicrobial peptides, and this can be classified as oxygen-dependent or oxygen-independent mechanisms (114).

It is believed that oxidative stress is emerging as a common mechanism altering neutrophil functions that can be reversed by various antioxidant strategies (115).

Bacterial colonization is not a feature of asthma; however, patients with stable asthma tend to develop lower respiratory tract infections caused by bacteria, and there are also reports on defective phagocytosis (116, 117). Macrophages isolated from children with asthma demonstrated decreased phagocytosis to S. aureus, which can be explained by the imbalance in glutathione homeostasis in the airways (118). Other studies, using an inhaled lipopolysaccharide, have shown reduced expression of CD11b and phagocytosis of sputum and blood phagocytes in asthmatic subjects (119). However, phagocytic activity of neutrophils in asthma has not been completely understood yet. Therefore, the regulation of blood neutrophil phagocytic activity in asthmatic patients especially after allergen challenge needs to be clarified.

1.4.3. Oxidative burst and reactive oxygen species

Oxidative stress can be related to inhaled oxidants and elevated amounts of ROS released from inflammatory cells (120). ROS can activate inflammatory responses in the airways through the activation of redox-sensitive transcription factors (121) (122). The process of phagocytosis triggers the production of ROS through the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex and cause cell and tissue damage in asthma (120). The active oxidase at these sites generates superoxide (O2–) as it transfers electrons from cytosolic NADPH to molecular oxygen. Within the phagosome, superoxide anion is dismutated by superoxide dismutase (SOD) to form oxygen and hydrogen peroxide (H2O2). Hydrogen peroxide is converted by enzyme myeloperoxidase (MPO) to other ROS such as hypochlorous acid, chloramines, singlet oxygen, which are effective microbicidal compounds (123, 124).

The enhanced generation of ROS is related to oxidation of proteins, DNA, and lipids, which may cause organ/tissue injury or may activate a number of cellular responses through the formation of secondary metabolic ROS. ROS is likely to alter remodeling of extracellular matrix, apoptosis and mitochondrial respiration, cell proliferation, maintenance of surfactant

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and the antiprotease screen, effective repair responses, and immunomo-dulation (78, 125).

It has been demonstrated that neutrophils purified from the BAL fluid and peripheral blood of patients with allergic asthma release significantly higher quantities of ROS (97). Fukunaga et al. reported that neutrophils from house dust mite-sensitized asthmatic patients produced higher amounts of ROS (126). Therefore, the regulation of blood neutrophil generation of ROS in asthmatic patients after bronchial allergen challenge needs to be investigated.

1.4.4. Apoptosis

A neutrophil lifespan is generally thought to be in the range of 8–20 h, though recent data with in vivo labeling suggest a lifespan of 5.4 days under physiological conditions in humans (127). Aged neutrophils die by apoptosis. This mechanism is substantive to maintain the balance of cellular homeostasis under physiological conditions (128). Apoptosis makes neut-rophils unresponsive to extracellular stimuli and leads to expression of “eat-me” signals, so that neutrophils can be recognized and removed by macrophages in the spleen, bone marrow, and Kupffer cells in the liver (129, 130). Although the neutrophil-mediated inflammatory response is crucial for resolution of infection, timely removal of these phagocytes is required as they are armed with potent cytotoxic components like ROS and granule products that can cause tissue injury.

Phosphatidylserine (membrane phospholipid) is expressed on the surface of apoptotic neutrophils, which interacts with macrophage receptor CD36. This leads to phagocytic uptake by macrophages and timely removal of neutrophils, which helps in resolution of inflammation (131). Morphological changes during apoptosis include chromatin condensation, nucleolar disruption, cytoplasmic contraction, and membrane blebbing. If apoptotic neutrophils are not removed by macrophages, these cells can undergo secondary necrosis resulting in cell disruption and leakage of toxic cellular contents causing tissue injury (132). Delayed apoptosis can be the main reason for increased neutrophil viability in tissues, which causes resolution of airway inflammation.

Many factors can have an impact on neutrophil apoptosis. During bacterial infections, cytokines such as IL-6, IL-8, G-CSF, and GM-CSF or bacterial products like lipopolysaccharides reduce neutrophil apoptosis via activation of proinflammatory signaling pathways. These include nuclear factor κB (NF-κB), signal transducer and activator of transcription (STAT), phosphoinositide 3-kinase (PI 3K), and mitogen-activated protein kinases

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(MAPK). This results in upregulation of antiapoptotic proteins of the intrinsic pathway Mcl-1, Bcl-XL, and A1 enhancing neutrophil survival and suggests that enhanced neutrophil survival is desirable at early stages of inflammation to promote clearance of a pathogen. The intrinsic pathway of apoptosis can be activated by genotoxic stress, withdrawal of growth factors, and ultraviolet radiation. Glucocorticoids also enhance neutrophil survival or delay apoptosis by upregulating Mcl-1 in neutrophils (123, 133). TNF-α possesses both pro- and antiapoptotic activities. At low doses, it induces survival while it is proapoptotic at higher doses. Also, neutrophil survival is influenced by the process of phagocytosis. Phagocytosis along with the production of ROS mediates neutrophil apoptosis. Neutrophils are exposed to both pro- and antiapoptotic factors, and the net result on neutrophil survival depends on the balance between these two factors (134).

1.5. Activity of eosinophils in asthma

Eosinophils are multifunctional leukocytes known as one of the most important cells involved in asthma pathogenesis (135). Activated eosino-phils release granule-derived proteins such as major basic protein (MBP) and eosinophil peroxidase (EPO) during degranulation, lipid-derived me-diators such as LTC4 and platelet-activating factor (PAF), and ROS (136). Eosinophils can regulate the immune function by the synthesis of cytokines such as IL-4, IL-5, and GM-CSF, chemokines such as eotaxin, IL-8, and RANTES (Regulated on Activation, Normal T Cell Expressed) and growth factors such as TGF-α and -β (136). In the airways of asthmatic subjects, eosinophils have the ability to prolong inflammatory responses by the release of inflammatory mediators. MBP can cause epithelial damage, airway constriction, and AHR. MBP instillation into mouse airways in vivo can induce AHR causing damage to the respiratory epithelium (137).

The main processes in the development and recruitment of eosinophils includes hematopoietic development, release from the bone marrow, endo-thelial adhesion, chemotaxis, and survival. IL-5 is responsible for eosinophil growth, differentiation, and mobilization from the bone marrow and also can prolong their survival (138).

In a mouse model, it was demonstrated that the deficiency of IL-5 can result in decrease in the number eosinophils after allergen sensitization and challenge with ovalbumin. Moreover, mice also did not develop AHR to methacholine (138). The study with inhalation of IL-5 in patients with allergic asthma showed increased blood eosinophilia and serum ECP (139).

IL-3 and GM-CSF have also been found to be important in the priming and activation of eosinophils (140). Cytokine stimulation of eosinophils

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results in their rapid migration and further production of inflammatory mediators (141).

An increased eosinophil count in sputum, bronchial biopsies, and blood has been found to be related to asthma severity, symptoms, and risk of the exacerbations (142). There is no doubt that eosinophils are multifunctional cells playing a dual role as both effectors and immunoregulatory cells. Effector eosinophils cause damage to the airway mucosa and the associated nerves by releasing cytotoxic granules and lipid mediators, which may cause bronchoconstriction. In addition, eosinophils demonstrate immune regu-latory functions, such as production of cytokines and chemokines that can lead to the mucus hypersecretion, exacerbation of inflammation, and airway remodeling (143–147).

Moreover, eosinophils can release IL-4, which recruit T cells to the airways in the development of asthma (148, 149). The development of eosinophils is mediated by transcription factors including GATA-1, PU.1 and C/CBP as well as cytokines, particularly GM-CSF, IL-3, IL-9, and IL-5 (143, 150, 151). It is thought that IL-5 is the most specific cytokine having an impact on eosinophil activity (152).

1.5.1. Chemotaxis

Eosinophil chemotaxis to the lungs during allergic airway inflammation represents a major part of the inflammation process (153). Transmigration of the eosinophil through the vascular endothelium is a multistep process; rolling, tethering, firm adhesion, and transendothelial migration are regu-lated by the coordinated interaction between networks involving chemokine, cytokine, and adhesion molecules (154, 155). Adhesion is the initial step of eosinophil recruitment, which is mediated by interaction between a P-selectin-glycoprotein ligand on eosinophils with P-selectin and E-selectin on endothelial cells. The next step involves rolling where eosinophils may adhere to the endothelium and start to roll (143).

The firm attachment of eosinophils to the endothelium is mediated by very late activation antigen 4 (VLA-4) (integrin α4β1) and CD11b/CD18 binding to vascular adhesion molecule 1 and intercellular adhesion mole-cule 1 (156). The expression of VLA-4 on eosinophils can be upregulated by eotaxin (67). This chemokine is one of the most important chemokines involved in eosinophil migration into inflammatory tissues. The principal receptor involved in eosinophil attraction to eotaxin is CCR3 (157). Two additional eosinophil selective CC chemokines have also been described:

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eotaxin-2 and eotaxin-3. Both these eotaxins also activate eosinophil lipid body formation and increases the production of LTC4 (158).

Experiments with in vitro allergen as well as endobronchial allergen challenge have shown that blood and BAL eosinophils from subjects with asthma have a greater responsiveness to chemoattractants and enhanced chemotaxis (10, 11). However, more investigations in order to understand eosinophil chemotaxis in allergen-induced late-phase airway inflammation are needed to be carried out.

1.5.2. Oxidative burst and reactive oxygen species

The oxidative burst in eosinophils can produce 10 times more superoxide anions than neutrophils (159). During the process of allergic inflammation, eosinophils release not only toxic granule proteins but also ROS, which are known to cause tissue damage (12). ROS generation in eosinophils is dependent on activation of the NADPH oxidase complex, which is located in the plasma membrane and has close similarities to the NADPH oxidase complex in neutrophils (160, 161). Upon activation, NADPH oxidase transports electrons across the membrane to reduce molecular oxygen, O2, into superoxide, O2–, which undergoes nonenzymatic or superoxide dismutase (SOD)-catalyzed dismutation to hydrogen peroxide, H2O2.

It has been demonstrated that allergic patients have upregulated oxidative metabolism in blood eosinophils when compared with healthy subjects (13, 14). ROS production is elicited by several stimuli such as immunoglobulins and cytokines (162). Moreover, the signal from adhesion molecules plays a critical role in ROS production by eosinophils (163). It is known that besides chemotaxis, CC chemokines, especially eotaxin and RANTES, can prime ROS production by eosinophils (13).

Consequently, evaluation of ROS production in peripheral blood eosinophils during allergen-induced late-phase airway inflammation is important for understanding the pathogenesis of eosinophilic inflammation in the airways.

1.5.3. Degranulation

In response to various stimuli, including cross-linking of different subclasses of immunoglobulin receptors, interferon γ (INF-γ), and the chemokines, eotaxin and RANTES, eosinophils are recruited from the circulation to inflammatory foci, where they modulate immune responses through the extracellular release of granule-derived products (164, 165).

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Eosinophil granule proteins such as eosinophil-derived neurotoxin (EDN), ECP, MBP, and EPO are the proteins mostly implicated in asthma pathophysiology (166).

Therefore, the increased numbers of eosinophils and their secreted products in the asthmatic lung often correlates with disease severity and exacerbation of disease (167). The study by Taniuchi et al., which involved atopic dermatitis patients, demonstrated that serum EDN levels in mild, moderate, and severe groups were significantly greater than in the control group (168). Moreover, serum EDN levels correlated with skin scores. In terms of clinical utility, EDN levels are a more accurate biomarker of the underlying pathophysiology of asthma (i.e., eosinophilic inflammation); consequently, they provide an objective measure of eosinophil secretory activity.

Serum EDN has been shown to reflect eosinophil degranulation in vivo and in vitro, and therefore, it is thought be a good tool for monitoring the disease (169, 170). Particularly, it is of importance to evaluate changes in EDN levels during allergen-induced late-phase airway inflammation in asthma patients.

1.5.4. Apoptosis

In healthy individuals, eosinophils are short-living cells. Under in vitro conditions, blood eosinophils undergo spontaneous apoptosis in a few days but under physiological conditions, they tend to migrate and accumulate into the liver and the spleen, where they are likely to live longer than a few days (171-173). Apoptosis represents an ideal means to deplete eosinophils from the airways. Apoptosis is a noninflammatory way of cell death characterized by cell shrinkage, chromatin condensation, DNA fragmentation, and maintenance of membrane integrity (174).

Eosinophil longevity may be enhanced up to 1–2 weeks by proin-flammatory cytokines such as IL-5, IL-3, and GM-CSF present in inflamed airways (175). Indeed, blood and tissue eosinophils from patients with asthma have been shown to live longer when eosinophils from healthy individuals (176, 177).

Eosinophil removal from the airways is useful to reduce eosinophilic inflammation and alleviate the symptoms of asthma (178). Eosinophil apoptosis can be facilitated by Fas activation (179). Fas ligands are sig-nificant proapoptotic agents for eosinophils in vivo, and it has been demonstrated that Fas ligand neutralization is related to enhanced airway eosinophilia in a mouse model of allergic asthma (180). Nitric oxide (NO) is produced in high amounts in the lungs of asthmatic patients and is able to

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regulate eosinophil apoptosis in a complex manner. It has been demons-trated that NO can have both anti- and pro-apoptotic effects on eosinophils (181, 182) and both enhancing and reducing properties regarding lung eosinophilia (183, 184).

Many mediators such as glucocorticoids as well as theophylline and cysteinyl leukotriene receptor antagonists can prolong eosinophil apoptosis in the absence and presence of eosinophil survival-prolonging cytokines (174, 185) and the proapoptotic effects of these drugs may contribute to their clinical efficacy (186–188). However, there is a lack of information about peripheral blood eosinophil apoptosis in allergen-induced late-phase airway inflammation.

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2. MATERIALS AND METHODS

2.1. General design of experiments

The study included 155 nonsmoking adults: 60 patients with intermittent or mild-to-moderate persistent allergic asthma, defined according the GINA criteria (189), 55 patients with mild-to-moderate persistent allergic rhinitis, defined according to the Allergic Rhinitis and its Impact on Asthma criteria (190), who comprised the comparative group, and 40 healthy subjects who comprised the control group. The patients were recruited from the Depart-ment of Pulmonology and Immunology, Hospital of the Lithuanian Uni-versity of Health Sciences, Kaunas. The study consisted of two parts:

 Neutrophil activity investigation based on the project funded by a grant from the Research Council of Lithuania: 2010–2012 “The role of phenotype of inflammatory cells and their functions for the predic-tion of allergic airway diseases” (LIG-18/2010). The study protocol was approved by the Regional Biomedical Research Ethics Committee of the Lithuanian University of Health Sciences (P1-48/2004), and each participant gave his/her informed written consent.

 Eosinophil activity investigation based on the project funded by a grant from the Research Council of Lithuania: 2012–2014 “The role

of Th9 cells expression and eosinophils activity in the course of allergic airway diseases” (LIG-08/2012). The study protocol was approved by the Regional Biomedical Research Ethics Committee of the Lithuanian University of Health Sciences (BE-2-23), and each participant also gave his/her informed written consent.

2.1.1. Characterization of study population

Patients with allergic asthma and allergic rhinitis had a clinical history of the disease for ≥1 year, current symptoms, and positive results of skin prick test (≥3 mm) with Dermatophagoides pteronyssinus (D. pteronyssinus) for the first part of the study and with D. pteronyssinus, birch pollen allergens, or 5 grass mixture allergens for the second part of the study. All patients were not using inhaled, nasal, or oral steroids at least 1 month before visits, short-acting β2 agonists at least 12 h and long-acting β2 agonists at least 48 h prior the lung function test, and antihistamines and antileukotrienes, 7 days before the skin prick test and prior the lung function test. None of the patients had a history of smoking. Baseline forced expiratory volume in one second (FEV1) was more than 70% of the predicted value in all patients. All

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healthy subjects were nonsmokers, without symptoms of rhinitis or asthma, with normal findings of spirometry, and all showed negative results of skin prick test.

All patients were screened for allergy by the skin prick test using stan-dardized allergen extracts (Stallergenes S.A., France) for the following aller-gens: D. pteronyssinus, D. farinae, cat and dog dander, 5 mixed grass pol-len, birch polpol-len, mugwort, Alternaria, Aspergillus, and Cladosporium. Histamine hydrochloride (10 mg/mL) was used for a positive control. Skin testing was read 15 minutes after application. The results of the skin prick test were considered positive if the mean wheal diameter was ≥3 mm (191).

Pulmonary function was tested using a pneumotachometric spirometer CustovitM (Custo Med, Germany). FEV1, forced vital capacity (FVC), and FEV1/FVC ratio were recorded as the highest of three reproducible measu-rements. The results were compared with the predicted values matched for age, body height, and sex according to the standard methodology (192).

Airway responsiveness was assessed as changes in airway function after challenge with inhaled methacholine using a reservoir method (193). Methacholine was nebulized into a 10-L reservoir with a pressure nebulizer (Pari Provocation I; Pari, Stanberg, Germany). Aerolized methacholine was inhaled through a one-way valve at 5-minute intervals starting with 15-μg methacholine dose and doubling it until a 20% decrease in FEV1 from the baseline or the total cumulative dose of 3.87 mg was achieved. The bronchoconstricting effect of each dose of methacholine was expressed as a percentage of decrease in FEV1 from the baseline value. The provocative dose of methacholine causing a ≥20% fall in FEV1 (PD20) was calculated from the log dose-response curve by linear interpolation of two adjacent data points. Characteristics of study population are presented in Tables 2.1.1.1 and 2.1.1.2.

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Table 2.1.1.1. Demographic and clinical characteristics of the studied subjects in the first part (n=80)

Characteristic Patients with allergic asthma (n=30) Patients with allergic rhinitis (n=30) Healthy subjects (n=20) Age, median (range), years 34 (21–50) 30 (18–49) 30 (22–45)

Sex (male/female), n 14/16 19/11 12/8

Wheal diameter induced by D. pteronyssinus, median (range), mm

6.2 ± 0.9 (4–12)

7.9 ± 0.7 (4–15)

0 FEV1, mean ± SEM (range), % of

predicted 98.0 ± 7.9† (82–116) 112.0 ± 10.2 (97–141) 104.9 ± 13.6 (79–114) PD20, mean ± SEM (range), mg 0.4 (0.1–0.7) 2.8* –

FEV1, forced expiratory volume in the first second; PD20, provocative dose of methacholine

causing a 20% fall in FEV1.

*PD20 was estimated only for 4 patients with allergic rhinitis.

†P < 0.05, vs. patients with allergic rhinitis.

Table 2.1.1.2. Demographic and clinical characteristics of the studied subjects in the second part (n = 75)

Characteristic Patients with allergic asthma (n = 30) Patients with allergic rhinitis (n = 25) Healthy subjects (n = 20) Age, median (range), years 31 (18–51) 28 (22–53) 27 (21–50)

Sex (male/female), n 20/10 8/17 6/14

Wheal diameter induced by D. pteronyssinus, median (range), mm Sensitization to D. pteronyssinus/ birch/5 grass mixture allergen, n

6.7 (4–12) 21/5/4 6.1 (4–14) 15/6/4 0 0

FEV1, mean ± SEM (range), % of

predicted 96 ± 12† (73–118) 102 ± 11 (84–127) 102 ± 8 (89–110) PD20, mean ± SEM (range), mg 0.31

(0.03–0.38)

0.52* –

FEV1, forced expiratory volume in the first second; PD20, provocative dose of methacholine

causing a 20% fall in FEV1.

*PD20 was estimated only for 4 patients with allergic rhinitis.

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30 2.1.2. Study design

On a screening visit, all subjects were informed about participation in the study, informed written consent was obtained, inclusion/exclusion criteria were verified, and also physical examination, spirometry, bronchial responsiveness to methacholine, and the skin prick test were performed. At 24 hours before bronchial challenge with specific allergen, spirometry was performed, and peripheral blood and induced sputum were collected; these data were used as baseline values. Bronchial challenge was performed with D. pteronyssinus, birch pollen allergens, or five grass mixture allergens (Stallergenes SA, France) at different concentrations (0.01 index of reacti-vity (IR)/mL, 0.1 IR/mL, 1.0 IR/mL, 10 IR/mL, 33.3 IR/mL) using a KoKo DigiDoser nebulizer (Sunrise Medical, Somerset, PA) (194).

The methods of spirometry, bronchial responsiveness to methacholine, and the skin prick test are described in one of our previous publication “Sputum Neutrophil Count After Bronchial Allergen Challenge is Related to Peripheral Blood Neutrophil Chemotaxis in Asthma Patients” (Annex 3).

Bronchial challenge with a specific allergen was performed at 8:00 AM, and spirometry was reassessed every 10 min within the first hour and later on every hour for subsequent 6 hours. Peripheral blood and induced sputum were repeatedly collected 7 h and 24 h after bronchial challenge. The study design is presented in Fig. 2.1.2.1.

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2.2.1. Peripheral blood

Peripheral blood samples were collected into sterile vacutainers without anticoagulants (for serum collection) or with ethylene diamine tetra-acetic acid (EDTA) (for neutrophil and eosinophil isolation).

2.2.2. Sputum induction and processing

Of the 155 subjects, 54% (n=78) were enrolled in sputum analysis. In the first part of the study, 36 adults were examined: 15 patients with allergic asthma, 13 patients with allergic rhinitis, and 8 healthy subjects. In the second part of the study, 42 adults were enrolled: 18 patients with allergic asthma, 14 with allergic rhinitis, and 10 healthy subjects. A total of 34 subjects from the first part of the study and 33 subjects from the second part of the study were excluded because of their inability to expectorate sputum of sufficient quality and quantity necessary for further processing.

Subjects inhaled 10 mL of sterile hypertonic saline solution (3%, 4%, or 5% NaCl, Ivex Pharmaceuticals, USA) at room temperature from an ultrasonic nebulizer (DeVilbiss Health Care, USA). The duration of each inhalation was 7 min, and it was stopped after expectoration an adequate amount of sputum. In order to detect a possible decrease in FEV1, spiro-metry was performed after each inhalation. Sputum was poured into a Petri dish and separated from saliva. A 4-fold volume of freshly prepared 0.1% dithiothreitol (DTT; Sigma-Aldrich) was added. The mixture was vortexed and placed on a bench rocker for 15 min at room temperature. Next, an equal volume of phosphate-buffered saline solution (PBS; Sigma-Aldrich) was added to DTT. The cell pellet was separated using a 40-μm cell stainer (Becton Dickinson, USA). The mixture was centrifuged for 10 min at 4°C; the supernatant was aspirated and stored at –70°C for later assay. Total cell counts, percentage of epithelial cells, and cell viability were investigated using a Neubauer hemocytometer (Heinz-Herenz; Germany) under a micro-scope (B5 Professional, Motic, China) by employing the trypan blue exclusion method. The cytospin samples of induced sputum were prepared using a cytofuge instrument (Shandon Southern Instruments, USA).

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32 2.2.3. Neutrophil purification

Peripheral blood samples (10 mL) were dilute by adding the same volume of PBS. The suspension was carefully layered over Ficoll-Paque (ρ=1.077 g/mL) in conical tubes and centrifuged at 1000g for 30 min at 20°C in a swinging bucket rotor without brake. Granulocytes were separated by hypotonic lysis of erythrocytes. The cells were centrifuged at 300g for 10 min. The pellet was resuspended in 1 mL of RPMI 1640. Later, 10 µL of the cell solution was added to 10 µL of trypan blue dye (Mediatech Inc., Herndon, VA), and neutrophils were counted using a Neubauer hemocyto-meter (Heinz-Herenz; Germany).

The neutrophil count was derived as follows: PMN/mL = (number of neutrophils counted × dilution × 104) / number of large squares counted. Blood neutrophil viability was assessed by trypan blue exclusion and it was found to be 98%.

2.2.4. Eosinophil purification

Peripheral blood samples (20 mL) were diluted by adding the same volume of PBS. The suspension was carefully layered over Ficoll-Paque (ρ=1.077 g/mL) in conical tubes and centrifuged at 1000g for 30 min at 20°C in a swinging bucket rotor without brake. Granulocytes were separated by hypotonic lysis of erythrocytes. Later, the granulocyte pellet was resuspended in cold MACS buffer (containing: PBS pH 7.2, 0.5% bovine serum albumin (BSA) and 2 mM EDTA by diluting MACS BSA Stock Solution 1:20 in autoMACSR Rinsing Solution) (40 μL per 107 total cells) and incubated with Biotin-Antibody Cocktail (biotin-conjugated mono-clonal antibodies against CD2, CD14, CD16, CD19, CD56, CD123, and CD235a (Glycophorin A) (10 μL per 107 total cells) for 10 min. After incubation, 20 μL of Anti-Biotin MicroBeads (conjugated to monoclonal anti-biotin antibodies (isotype: mouse IgG1) per 107 total cells was added, mixed, and incubated for additional 15 min at 4°C. A LS column (Miltenyi Biotec, USA) was prepared during this time by placing LS column in the magnetic field of MACS Separator and washing it with 2 mL of MACS buffer. A preseparation filter (30 μm Miltenyi Biotec, USA) was rinsed with MACS buffer and placed on the top of the column. The cells were then applied to the preseparation filter/LS column, and the magnetically nonla-beled eosinophils depleted by retaining them on a column in the magnetic field of a Separator, while the unlabeled eosinophils pass through the column. Cell fraction was eluted with 5 mL of MACS buffer.

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Cell fractions were centrifuged (400g, 10 min, 4°C), and the pellet resuspended in 1 mL of RPMI 1640. Later, 10 µL of the cell solution was added to 10 µL of trypan blue dye (Mediatech, Inc, Herndon, VA), and eosinophils were counted using a hemocytometer (Heinz-Herenz; Germa-ny). The eosinophil count was derived as follows: PMN/mL = (number of eosinophils counted × dilution × 104) / number of large squares counted. Blood eosinophil viability was assessed by trypan blue exclusion and was found to be 98%.

2.3. Neutrophil and eosinophil activity assay methods 2.3.1. Chemotaxis of peripheral blood neutrophil and eosinophil

Neutrophil and eosinophil chemotaxis in vitro was performed in a 10-well cell transmigration chamber (Neuro Probe, USA). The lower and upper wells of chamber were isolated by a polyvinylpyrrolidone-treated polycar-bonate track-etch membrane, containing 2×106 3-μm/mm2 pores (Neuro Probe). The lower wells were prefilled with isotonic Percoll (GE Health-care) and chemotactic factors. For neutrophil chemotaxis, IL-8 at different concentrations (10, 30, or 100 ng/ml) was used; for eosinophil chemotaxis, eotaxin at concentrations of 10, 100, and 1000 ng/mL was used. RPMI 1640 was used as a negative control. The upper wells were filled with neutrophil (2×106

/mL) or eosinophil (1×104/mL) culture suspension and incubated for 2 h (37°C, 5% CO2). After incubation, the suspensions of upper and lower

wells were resuspended in tubes for flow cytometry.

Nonmigrated cells remained in the upper wells. The migration rate was calculated from the total number of neutrophils or eosinophils harvested from the lower well and expressed as percentage of the total input of neutrophils or eosinophils into the upper compartment of the well. The number of migrated cells was calculated by flow cytometry using liquid counting beads (BD Biosciences, USA), according to the manufacturer’s recommendations. The kit contains thiazole orange (TO) solution to stain all cells and propidium iodide (PI) to stain dead cells. Neutrophils and eosinophils were gated using their density and size in side angle light scatter and forward angle light scatter respectively and FL1 and FL3 channels were used. The total cell count was determined and the amount of migrated neutrophils or eosinophils was expressed in percentages.

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2.3.2. Peripheral blood neutrophil phagocytosis

Phagocytic activity of peripheral blood neutrophils in vitro was deter-mined in sterile 96-well microplates (Falcon, BD, USA). To evaluate phagocytic capacity, neutrophils were stimulated with fluorescein isothio-cyanate (FITC)-labeled Staphylococcus aureus bacteria. Neutrophils were stimulated with ratios of 2, 6, 18, 56, and 167 FITC-labeled S. aureus bacteria per neutrophil. The plates were filled with phagocytosis-stimulating factors; neutrophil cultures were added at a concentration of 2×106

/mL and incubated for 2 h (37°C, 5% CO2).

After incubation, the cell suspension was added to flow cytometer tubes.

For each determination, 104 events were acquired. Neutrophils were gated using their density and size in side angle light scatter and forward angle light scatter, respectively. Neutrophil phagocytic activity was evaluated by a flow cytometer determining the mean fluorescence intensity using FL1 channel (excitation wavelength, 488 nm).

2.3.3. Analysis of reactive oxygen species in peripheral blood neutrophils and eosinophils

Spontaneous ROS production in peripheral blood neutrophils and eosinophils was performed in sterile 96-well microplates (Falcon, BD, USA). For the detection of generated ROS, dihydrorhodamine-123 (DHR-123, 750 ng/mL final, Invitrogen, USA), a nonfluorescent dye, was added. DHR-123, interacting with intracellular ROS, is oxidized to the green-fluorescent rhodamine-123. The plates were filled with cell cultures and incubated for 45 min (37°C, 5% CO2).

The relative amount of generated ROS was measured flow cytometrically by determination of mean green fluorescence intensity in the cell population in the FL1 channel.

2.3.4. Peripheral blood eosinophil degranulation

Eosinophil degranulation was analyzed by the levels of an eosinophil- derived neurotoxin (EDN) in the serum. EDN levels in serum samples were determined by the enzyme-linked immunosorbent assay (ELISA) using a commercial EDN ELISA Kit (Immunodiagnostik, Germany) following the manufacturer’s instructions. Assay standards, controls and prediluted patient samples containing human EDN were added to wells of a microplate that was coated with a high-affinity monoclonal anti-human EDN antibody. After the first incubation period, antibody immobilized on the wall of

PERIPHERAL BLOOD NEUTROPHIL AND EOSINOPHIL ACTIVITY DURING ALLERGEN-INDUCED LATE-PHASE AIRWAY INFLAMMATION IN ASTHMA Simona Lavinskienė

Simona Lavinskienė

PERIPHERAL BLOOD NEUTROPHIL

AND EOSINOPHIL ACTIVITY DURING

ALLERGEN-INDUCED LATE-PHASE

AIRWAY INFLAMMATION

IN ASTHMA

Simona Lavinskienė

PERIFERINIO KRAUJO NEUTROFILŲ

IR EOZINOFILŲ AKTYVUMAS

ALERGENO SUKELTOS VĖLYVOS

FAZĖS KVĖPAVIMO TAKŲ UŽDEGIMO

METU SERGANT ASTMA

CONTENTS

ABBREVIATIONS

INTRODUCTION

1. REVIEW OF LITERATURE

2. MATERIALS AND METHODS