Uridine, 4-thiouridine and isomaltitol in an asthma-like model

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Linköping University Medical Dissertations No. 1137

Uridine, 4-thiouridine and isomaltitol in an asthma-like model

Anti-inflammatory and modulating effects

Chamilly Evaldsson

Division of Clinical Chemistry

Department of Clinical and Experimental Medicine

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All text, tables and illustrations are © Chamilly Evaldsson, 2009

The original papers included in this thesis have been reprinted with the permission of respective copyright holder:

ISSN: 0345-0082

ISBN: 978-91-7393-596-8

Printed in Sweden by LiU-Tryck, Linköping 2009

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To Martin, Minna and Morris

Aldrig hade jag trott att det skulle vara så fruktansvärt att vara människa – eller så underbart

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Abstract

In chronic inflammatory diseases like asthma or rheumatoid arthritis, erroneous and exaggerated accumulation of leukocytes in a tissue inadvertently causes the body harm. Several efficient anti-inflammatory drugs exist, for example corticosteroids and cyclo-oxygenase inhibitors. However, these drugs have potent and diverse effects and often act by inhibiting events subsequent to initiation of the inflammatory response, leading to more or less severe side effects, especially when used in high doses for long periods of time. For this reason, strategies aimed at early inhibition of recruitment and activation of leukocytes have been suggested as safer and more specific approaches to reduce inflammation.

Leukocyte adhesion to activated endothelium is a prerequisite to the following activation and extravasation, and takes place in the initial phase of inflammation. By using a model that allows leukocytes to adhere to tumour necrosis factor (TNF)-activated endothelial cells, thus mimicking aspects of an inflammatory reaction, we found that uridine, 4-thiouridine and isomaltitol could all reduce adhesion. This suggested that they may have anti- inflammatory potential.

We therefore tried the three substances in a Sephadex-induced lung inflammation model and found that uridine and 4-thiouridine have several anti-inflammatory effects, such as being able to reduce leukocyte accumulation, decrease TNF protein levels and partly inhibit the oedema induced by Sephadex. Isomaltitol turned out to have immunomodulating, rather than anti-inflammatory, effects, which could be of interest in diseases where inadequate inflammatory responses are a problem.

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Sammanfattning på svenska

Vid inflammatoriska sjukdomar som astma och reumatism orsakar vita blodkroppar skada på den egna kroppen. Detta sker på grund av att de felaktigt aktiveras och ansamlas i en vävnad. För att behandla sådana tillstånd används idag flera effektiva anti-inflammatoriska läkemedel, bland andra steroidpreparat (t ex kortison) och prostaglandinsynteshämmare (t ex ipren, voltaren), men de verkar ofta mer brett än önskat eller långt nedströms om de steg som startar inflammationen. Detta leder till biverkningar av olika slag, särskilt när läkemedlen används vid höga doser under längre tid.

Normalt cirkulerar vita blodkroppar i blodet, men vid signaler om skada eller annat ”hot” binder de till ankare som exponerats på blodkärlens insida, aktiveras och vandrar ut i vävnaden där de sedan reglerar och driver inflammationen vidare. Detta händer mycket tidigt vid en inflammation.

Eftersom inbindningen till blodkärlsväggen är ett avgörande steg för de vita blodkropparnas aktivering och vandring ut i vävnaden, har flera strategier för att hindra detta ofta föreslagits som ett sätt att hitta anti-inflammatoriska läkemedel med färre biverkningar.

Genom att använda en modell där vita blodkroppar interagerar med celler från blodkärlsvägg under omständigheter som liknar inflammation fann vi tre substanser – uridin, 4-thiouridin och isomaltitol. De lyckades alla minska antalet blodkroppar som fäste till ankare på cellytan och ansågs därför ha möjligheter att verka anti-inflammatoriskt.

Vi gick vidare med att undersöka substanserna i en astmaliknande lunginflammationsmodell och har visat att två av substanserna – uridin och 4- thiouridin – faktiskt har flera anti-inflammatoriska effekter. Till exempel leder de till minskad invandring av vita blodkroppar, minskar vätskeansamlingen (ödemet) som orsakas av inflammationen och ger lägre nivåer av kroppsegna ämnen som ökar inflammation.

Isomaltitol visade sig ha en modulerande, snarare än anti-inflammatorisk verkan i vår lunginflammationsmodell. Eventuellt skulle isomaltitol kunna vara intressant vid sjukdomar där ansamling av tjockt slem i luftvägarna eller svaga inflammatoriska reaktioner är ett problem.

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Original publications

This thesis is based on the following original papers, which are referred to in the text by their Roman numerals:

Paper I Uppugunduri S, Gautam C. Effects of uridine, isomaltitol and 4- thiouridine on in vitro cell adhesion and in vivo effects of 4- thiouridine in a lung inflammation model. Int Immunopharmacol 2004; 4:1241-8.

Paper II Evaldsson C, Rydén I, Uppugunduri S. Anti-inflammatory effects of exogenous uridine in an animal model of lung inflammation.

Int Immunopharmacol 2007; 7:1025-32.

Paper III Evaldsson C, Rydén I, Rosén A, Uppugunduri S. 4-Thiouridine induces dose-dependent reduction of oedema, leucocyte influx and TNF in lung inflammation. Clin Exp Immunol 2009;

155(2):330-8.

Paper IV Evaldsson C, Rydén I, Uppugunduri S. Isomaltitol exacerbates neutrophilia but reduces eosinophilia – new insights into the Sephadex model of lung inflammation. (Manuscript)

Reprints have been made with permission from the publishers.

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Acknowledgements

A thesis does not just happen. The eight years of work it has taken to complete this particular one have been a long and convoluted journey, both in terms of work and personal circumstances. I am indebted to all of those who have helped me even out the great and small bumps along the way.

Some very special thanks (in no special order) go to:

Srinivas Uppugunduri – for the scientific guidance, for all the conversations on literature, Indian mythology and human nature, for your friendliness, and for giving me this opportunity. Namaste.

Anders Rosén – for your encouragement and help as assistant supervisor, and for being an inspiration when it comes to combining scientific skill with personal humbleness.

“...the meek shall inherit the earth; and shall delight themselves in the abundance of peace.”

Margareta Evaldsson – for graciously lending me a desk and metres of shelf space in your home the last year, and for being such a great friend and mother-in-law.

Martin Evaldsson, PhD – “Si hoc legere scis nimium eruditionis habes” – and that is only one of the many reasons to why I love you. Without your early mornings, extraordinary intelligence, unquenchable humour and weekly grocery shopping, we would be lost.

Minna and Morris, the most important, most difficult and most rewarding experiments I have ever undertaken. Constant sunshine makes a desert.

Marianne, Barbro, Rani and the Olaison/Kåreklint family for your support.

All my friends in the Salvation Army.

Friends, colleagues and my boss (former three (soon four) and current) at (what is currently known as) Apoteket Farmaci, especially Salumeh, Malene, Katarina, Britta, Anna H, Karin T, Karin J, Maggan, Kristina and everyone else who have made me feel at home at work.

Christina Ekerfelt at the Department of Clinical and Experimental Medicine (IKE) for being calm and reasonable during turbulent times, Monika Hardmark for the countless times she has guided me through the administrative jungle of IKE, and the kind staff at the Linköping University animal department.

The Medical Research Council of Southeast Sweden (FORSS) and Apoteket AB for funding the work presented in this thesis.

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Abbreviations and synonyms

15-HETE 15-hydroxyeicosatetraenoic acid AHR airway hyperresponsiveness ANOVA analysis of variance

ATP adenosine 5'-triphosphate BALF bronchoalveolar lavage fluid BSA bovine serum albumin

cAMP cyclic adenosine monophosphate CCR CC chemokine receptor

CD cluster of differentiation

cGMP cyclic guanosine monophosphate CNT concentrative nucleoside transporter COPD chronic obstructive pulmonary disease DMEM Dulbecco’s modified Eagle’s medium DNA deoxyribonucleic acid

EAR early asthmatic reaction

EC50 half maximal effective concentration ECGF endothelial cell growth factor ECP eosinophilic cationic protein EDTA ethylene diamine tetraacetic acid EGTA ethylene glycol tetraacetic acid ELISA enzyme-linked immunosorbent assay ENT equilibrative nucleoside transporter

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EpDRF epithelial-derived relaxing factor EPO eosinophil peroxidase

ESL-1 E-selectin ligand 1 FAK focal adhesion kinase FCS foetal calf serum

GINA Global Initiative for Asthma

GM-CSF granulocyte macrophage colony-stimulating factor GPR G protein-coupled receptor

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, a zwitterionic buffer

HEV high endothelial venule

HIV human immunodeficiency virus HSA human serum albumin

HUVEC human umbilical vein endothelial cells i.p. intraperitoneal

i.t. intratracheal i.v. intravenous

ICAM intracellular cell adhesion molecule ICS inhaled corticosteroid

IFN interferon Ig immunoglobulin IL interleukin

IP3 inositol triphosphate

IU international unit, based on measured biological activity or effect

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LAR late asthmatic reaction LT leukotriene, e.g. in LTC4

LTRA leukotriene receptor antagonist MAPK mitogen-activated protein kinase MBP major basic protein

MIP macrophage inflammatory protein MMP matrix metalloprotease

MPA Medical Products Agency NEAA nonessential amino acids NF- B nuclear factor B

NO nitric oxide

NPP nucleotide pyrophosphatase/phosphodiesterase

NRTI nucleoside analogue viral reverse transcriptase inhibitor OCS orally administered corticosteroids

OPD o-phenylene diamine dihydrochloride OVA ovalbumin

PBS phosphate-buffered saline PDE phosphodiesterase

PKC phosphokinase C PLA phospholipase A PLC phospholipase C

PSGL-1 P-selectin glycoprotein ligand 1

RANTES regulated upon activation normal T cells expressed and secreted RNA ribonucleic acid

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ROS reactive oxygen species S4U 4-thiouridine

SCS systemic corticosteroids TH T helper lymphocyte

TNF tumour necrosis factor, formerly known as TNF-α tRNA transfer RNA

U uridine or the amount of an enzyme that catalyzes the conversion of 1 µmole substrate per minute under specified conditions

UDP uridine 5'-diphosphate UMP uridine 5'-monophosphate UTP uridine 5'-triphosphate UV ultraviolet

VCAM vascular cell adhesion molecule VLA very late antigen

WHO World Health Organisation

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1 Background

1.1 Inflammation

Inflammation is the body’s normal response to harmful stimuli, such as injury or infection. The word itself is derived from the Latin inflammatio, “to set on fire”, and refers to heat (calor) as one of the four cardinal symptoms of an acute inflammation. Heat and the other symptoms, rubor (redness), dolor (pain) and tumor (swelling) were described in the first century of our time by Aurelius Cornelius Celsus¹, but were doubtlessly recognised much earlier. Later, a fifth sign of inflammation, the observation of which is commonly attributed to Galen², was added to the medical books – the functio laesa (loss of function).

The heat and the redness stem from local vasodilation, while swelling is due to increased permeability across the blood vessel wall. Pain is the result of chemical activation of nerve endings. The processes behind this are all triggered by resident cells like dendritic or endothelial cells, and are subsequently maintained by more specialized inflammatory cells, normally dominated by neutrophils, recruited from circulation.

Cytokines make out a large group of secreted proteins with remarkably varied functions in inflammation. Most, if not all, cell types are capable of cytokine production, but types of cytokines expressed and their effects depend on the source cell, target cell and phase of inflammation. Cytokine functions include effects on cell activation, differentiation, growth, trafficking and many more, and may be both pro- and anti-inflammatory. Cytokines that particularly induce migration of leukocytes, so called chemoattraction, have aptly been called chemokines. The effects of cytokines can be autocrine, paracrine or endocrine. Each cytokine is capable of triggering a cascade of cellular events,

1 He gave this famous description in the fourth book of his work De Medicina. As a curiosity, this is also the book where he provides anatomical remarks on the lung, which is depicted as

“spongy” and “divided like the hoof of an ox into two lobes”. (With the modern definition of the word “lobe”, the lung actually consists of five lobes.)

2 The phrase has also been claimed to originate from the English physician Thomas Sydenham (1624-1689) or the German pathologist Rudolf Virchow (1821-1902).

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and as cytokines are often released and act in concert, a wide repertoire of responses can be achieved. [1, 2]

Just like in regular cell trafficking, recruitment of cells during inflammation occurs in a stepwise fashion. What distinguishes inflammation is the extent to which the presence and activation of one inflammatory cell type will lead to the recruitment of another and the usually cascading manner of the process.

The specific mediators that are released at the site of inflammation will activate endothelial cells nearby. Upon activation, the endothelial cells expose adhesion molecules that catch and guide inflammatory cells from circulation through the steps of rolling, adhesion and subsequent extravasation.

These steps involve three families of adhesion molecules, classified by structural features. Selectins, which are glycosylated proteins that bind to other glycosylated proteins and glycolipids, mediate the low-affinity interactions that enable rolling. Integrins are heterodimeric proteins which often function as ligands for immunoglobulin adhesion molecules, and together they mediate firm adhesion, arrest and extravasation [3-5]. Specific sets of adhesion molecules govern which circulatory cells that are recruited (e.g. neutrophils or eosinophils), and this in turn is dictated by the cytokines and chemokines that triggered their exposure. [3, 4, 6]

An acute inflammation normally subsides within the range of some minutes to a few days. In chronic inflammation, on the other hand, the traits of acute inflammation will either be constant over a much longer period of time (weeks to years), or recur intermittently, while reparative processes take place simultaneously. This makes the inflammation much more varied in how it is expressed. The cellular infiltrates will generally include a higher degree of lymphocytes and monocytes, and, as a sign of the continuous tissue repair, angiogenesis and fibroblast proliferation are more notable.

The composition of cells together with the lesion characteristics are often used to classify chronic inflammation; hence definitions like granulomatous (“forming grains”), fibrinous (“forming threads”), ulcerative (“forming sores”) or purulent (“causing pus”) inflammation.

The subtypes of cells or lesions that appear depend on a variety of factors, such as which organ or compartment of the body that is involved, what the harmful stimuli is (e.g. autoantigen, a foreign body or a pathogen), and

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ultimately the combination inflammatory mediators and adhesion molecules that are exposed to and expressed by involved cells.

Macrophages and lymphocytes are pivotal in the orchestration of all chronic inflammation [7]. Macrophages are named after their ability to “eat”

pathogens, but are also the main producers of many potently pro- inflammatory cytokines, including tumour necrosis factor³ (TNF) is of central importance for recruitment and activation of circulating cells. Lymphocytes are needed to generate antibodies, which allow a highly specific and adaptive host defence, and release a variety of factors that will increase survival and function of other inflammatory cells. Lymphocytes exist in several different subtypes, each with a specialised function and profile of cytokine production.

The proportion, both in numbers and activation, of these subtypes affect the nature of an inflammation. In neutrophilic inflammation, the T helper lymphocyte subtype 1 (TH1) will usually dominate, while a shift towards the TH2 subtype is correlated with a predisposition to allergic disease and asthma.

The cytokines (interleukin (IL)-4, IL-5 and IL-13, to name a few) and other mediators released by TH2 cells increase eosinophil and mast cell function and survival [8, 9]. The latter two cell types are also associated with for example asthma, which is a chronic inflammatory disease.

Inflammation is thus the sum of a huge number of cells and mediators in interaction, all contributing to or being affected by their surrounding chemical milieu. To set the stage, it could therefore be of interest to clarify that this thesis is mainly concerned with a model of acute-type granulomatous lung inflammation with several chronic- and asthma-like features (which will be expounded on below), both in regard to the cells and the mediators involved.

As asthma is one of the western world’s most common chronic inflammatory diseases today and is important both in terms of incidence and cost, this is the disease to which most parallels will be drawn.

3 TNF was formerly known as TNF-α.

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1.2 Asthma

1.2.1 Prevalence, clinical symptoms and definition

For the last thirty years, asthma has been one of the diseases increasing most in the western world, and is today one of the most common chronic inflammatory diseases. According to one estimate, around 300 million people are affected worldwide, a figure that appears to increase in beat with the industrialization of developing countries [10]. In Sweden alone, approximately 8% of the population are reportedly diagnosed with asthma [11].

Although the disease can occur at all ages, the majority of asthmatic patients develop their disease early in life, with a gender preponderance of boys in childhood. [10, 12] Clinically, asthma is often diagnosed based on several types of information, for example patient-reported symptoms, (allergic) disease history, measured respiratory function, subjective effect of medication etc. [10, 11, 13]. Key symptoms include wheezing, breathlessness and coughing, which may be worse at night. These are the effects of three features common to all forms of asthma, namely bronchoconstriction, airway hyper- responsiveness (AHR) and chronic inflammation.

The reliance on clinical symptoms in diagnosing asthma is reflected in attempted definitions of the disease, where little is said of the aetiology. One example is the working definition by the US National Asthma Education and Prevention Program’s Expert Panel Report from 2007, where asthma is described as:

…a chronic inflammatory disorder of the airways in which many cells and cellular elements play a role: in particular, mast cells, eosinophils, neutrophils (especially in sudden onset, fatal exacerbations, occupational asthma, and patients who smoke), T lymphocytes, macrophages, and epithelial cells. In susceptible individuals, this inflammation causes recurrent episodes of coughing (particularly at night or early in the morning), wheezing, breathlessness, and chest tightness. These episodes are usually associated with widespread but variable airflow obstruction that is often reversible either spontaneously or with treatment. The inflammation also causes an associated increase in the existing bronchial hyperresponsiveness to a variety of stimuli. Reversibility of airflow limitation may be incomplete in some patients with asthma. [13]

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Similar definitions have been expressed by other organisations, including the World Health Organisation (WHO), the Global Initiative for Asthma (GINA) and the Swedish Medical Products Agency (MPA) [14, 15].

1.2.2 Aetiology

Constriction of bronchial smooth muscles, airway hyperresponsiveness and the underlying chronic inflammation are pivotal factors in causing the clinical symptom of airflow limitation. These factors are, as mentioned, common to all forms of asthma and affect each other with inflammation as the main orchestrator. In more progressed stages of the disease, factors such as pronounced airway oedema, potential fibrosis and remodelling of the lung tissue, mucus hypersecretion and reduced mucus clearance further limit airflow [13]. These are also driven by the ongoing inflammation.

What initially causes and perpetuates the airway inflammation is not very well understood, nor how the sometimes unknown triggers of bronchoconstriction act, but most likely these factors differ between patient subgroups. For practical reasons, asthma is often classified by the stimuli that are associated with acute episodes. Another common way of classifying asthma is by whether or not there is an allergic component, since allergy (or

“atopy”), is known to be the single greatest risk factor for developing the disease. The latter type of classification is supported by observations that atopic asthma in a patient usually coincides with the patient’s family history of asthma or other allergic disease, such as rhinitis or eczema, suggesting that there is a considerable heritable component [12]. A large portion of asthmatics do not, however, have any signs of allergy, but suffer from so called intrinsic asthma. These patients are generally older at disease debut and tend to have more severe symptoms, but otherwise share the features of airway inflammation, obstruction and hyperreactivity [16]. In yet a fraction of patients, exacerbations may be triggered by both allergic and non-allergic stimuli like chemical irritants or cold.

Although some polymorphisms coupled to TH2 cell interleukins are associated with atopy and asthma, the exact genetics behind asthma remains ill- understood [9, 10]. So far, more than a hundred genes which individually only slightly increase the risk of asthma have been linked to various traits [17], and it is likely that different phenotypes of asthma may relate to completely different genotypes.

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A frequently suggested explanation, not least to the increasing prevalence of asthma, is the so called “hygiene hypothesis”. This proposes that increased living standards have reduced the number of immunological challenges we encounter early in life. The resulting reduction in numbers of cells and mediators that regulate immune responses during infection (the so called TH1

type response), will tip the balance in favour of “pro-allergic” cells and mediators (a TH2 response), which would thus predispose us to allergy and allergy-related diseases like asthma. Appealing as it may be, the TH2

hypothesis unfortunately fails to shed light on issues like why airway hyperresponsiveness and tissue remodelling are only weakly linked to inflammation or why T-cell repressive treatment is generally clinically ineffective for treating asthma. Other environmental factors in relation to climate zones, air pollution, viral respiratory infections and airborne allergens are also commonly mentioned as possible culprits.

To conclude, a definite cause of asthma has not been established. The many identified risk factors may suggest that asthma is perhaps better defined as a syndrome, rather than a single disease.

1.2.3 Cells, mediators and adhesion molecules in asthma

As has been stated, several types of cells interact to shape and perpetuate inflammation and it is important to stress this complexity. An exhaustive review is not achievable in this context, and therefore only some major cells and mediators involved in asthma will be briefly mentioned. A very schematic overview of the relation between inflammation and clinical symptoms is presented in Figure 1.

Both cells resident to the lungs (such as the macrophages, epithelial cells, endothelial cells, smooth muscle cells and mast cells), and recruited cells (such as eosinophils and TH2 lymphocytes), contribute to the disease-specific symptoms and tissue changes. They maintain inflammation through release of a variety of mediators, including cytokines, chemokines, lipid metabolites and expression of adhesion molecules. Different cell types and mediators will vary

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in importance, depending on which phase or feature of asthma that is studied⁴.

Somewhat counterintuitively, the degree of inflammation (eosinophilia) correlates poorly with asthma severity [17], but prolonged inflammation does lead to long-term morphological changes of the airways as a consequence of the constructive and destructive processes that take place simultaneously. Post mortem studies on airways from patients who have died of asthma reveal submucosal infiltration by eosinophils, collagen deposits under the basement membrane, increased smooth muscle thickness and tissue oedema. The airways may be occluded by mucus plugs filled with inflammatory cells and shed epithelial cells.

1.2.3.1 Central roles for TH2 lymphocytes and macrophages

The importance of TH1/TH2 balance has been extensively debated through the years, and current understanding of T lymphocyte subtypes imply that their roles are far more entwined than previously believed. Despite this, TH2

lymphocytes together with macrophages still retain their position as central players in asthma, although they themselves are not necessarily the main effector cells that induce clinical symptoms.

Macrophages are of general importance in all chronic inflammation, being the producers of potent pro-inflammatory cytokines like IL-1α/β, TNF and interferon (IFN)-γ. TNF, which is of interest in this thesis, has a vast array of effects including inflammatory activation and stimulation of leukocytes, lymphocytes, macrophages, endothelium and many other cell types. Release of TNF protein can be triggered by many stimuli, including lipopolysaccharides, other cytokines, reactive oxygen species, autocrine TNF signalling and, importantly, by IgE-dependent mechanisms [18-20].

4 Generally, clinical studies have been performed with patients or animal models mimicking classic allergic TH2 type asthma with eosinophilia, which is what most of the discussion here will refer to, but it should be mentioned that other forms of asthma exist even if the understanding of them is far from extensive. It has recently been suggested that asthma should be endotyped based on inflammatory cell profiles found in sputum, rather than as atopic or intrinsic. The suggested classifications are eosinophilic, neutrophilic, mixed (both neutrophils and eosinophils) and paucigranulocytic (few or no granulocytes) asthma [17].

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Figure 1. Schematic view of the relations between inflammation and clinical symptoms in classic, TH2-driven, allergic asthma.

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1.2.3.2 The importance of an intact epithelium

The normal function of the airway epithelium is to act as a physical barrier to pathogens and to keep the airways open through mucus clearance by its ciliated cells. In asthma, massive shedding of epithelial cells is observed, which may account for some of the hypersensitivity to inhaled bronchoconstrictors. The partial or extensive denudation of the epithelium also exposes nerve endings, which has led to speculations that some of the potent effects by β2-agonist (see section 1.2.5) come from triggering the release of epithelial-derived relaxing factor (EpDRF). In inflammation, the epithelial cells become an important source of bronchoconstrictive mediators like endothelin-1, the cysteinyl leukotrienes and 15-HETE. [21]

1.2.3.3 Neutrophils and the early asthmatic reaction

Neutrophil influx correlates with acute episodes of aggravated airway inflammation, the so called early asthmatic reaction (EAR) [22], and have been implicated to have a role especially in severe non-allergic asthma where their release of e.g. reactive oxygen species and matrix metalloproteases (MMPs) may contribute to tissue damage [15, 23].

1.2.3.4 Selective accumulation of eosinophils

Although neutrophils are the dominating cell type in many kinds of chronic inflammation they correlate primarily with TH1 responses, and in general, the chronic asthmatic inflammation is instead highly associated with eosinophilia and a TH2 response [24]. Eosinophil accumulation parallels the late reaction (LAR), which is the aggravation in symptoms that often occur hours after an acute bronchoconstrictive attack. Several types of findings, including the post mortem histological analyses mentioned above, support the hypothesis that eosinophils are central to the pathogenesis and pathophysiology of asthma, even if their role is likely to be more complex than previously believed⁵. While TH2 lymphocytes are generally thought to be the main recruiters of

5 It was previously supposed that anti-IL-5 antibodies could be beneficial to asthmatic patients since IL-5 have profound effects on eosinophil terminal differentiation, control their release from bone marrow and prolong their survival. Trials with monoclonal antibodies did indeed suppress the asthma-induced eosinophilia, but surprisingly did not suppress the allergen LAR or airway hyperresponsiveness, which contradicts to the eosinophil hypothesis and suggests that eosinophils are not required for these symptoms [22].

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eosinophils, eosinophilia occurs even in intrinsic asthma, which is not associated with a TH2 response. [25]

The specific accumulation of eosinophils in the airway mucosa, which may outnumber neutrophils by a factor of tens to hundreds, is potentiated at several levels. Mechanisms include increased levels of circulating eosinophils, selective adhesion through e.g. very late antigen (VLA)-4/vascular cell adhesion molecule (VCAM)-1 interaction, intracellular cell adhesion molecule (ICAM)-3 and P-selectin [26], selective migration and prolonged survival [27].

Both the number of cells in circulation and prolonged survival in tissues involve the actions of cytokines such as IL-3, IL-5 and granulocyte macrophage colony-stimulating factor (GM-CSF). IL-4, IL-5 and IL-13 are essential for eosinophil growth, activation and survival. These cytokines are produced in large amounts by another cell type abundantly present in asthmatic mucosa, namely the TH2 lymphocyte, and it is believed that the TH2

cells direct the recruitment and production of eosinophils.

Eosinophils are capable of phagocytosis, but their main role in host defence relates to release of toxic proteins from secretory vesicles and production of reactive oxygen species (ROS) [28]. Important secreted proteins include eosinophilic cationic protein (ECP), major basic protein (MBP), and eosinophil peroxidase (EPO), of which ECP and EPO are unique to eosinophils. These proteins are deleterious to pathogens and parasites, but also to cells and tissues of the host. For example, MBP is known to damage the airway epithelium and to impair ciliary motility [22], but interestingly, eosinophils could also be involved in tissue repair by promoting collagen synthesis and deposition, something which has been suggested as (one of) the mechanism behind the subepithelial fibrosis sometimes found in asthma patients [27-29].

In addition to the secreted proteins, inflammatory mediators produced by this cell type include cysteinyl leukotrienes, which are potent bronchoconstrictors and are associated with oedema, several interleukins (1α, 2, 3, 4, 5, 6, 8, 10, 13), RANTES, macrophage inflammatory protein (MIP)-1α and TNF. Many of these mediators have autocrine functions, but they also affect surrounding cells and tissues. E.g. IL-4 and IL-13, though produced mainly by TH2 cells, induce transcription of the eotaxin gene in the airway epithelium. This CC chemokine has chemotactic activity for eosinophils and stimulates their degranulation. In addition, serum eotaxin levels actually correlate with severity of asthma [22].

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1.2.3.5 Mast cells

Mast cells are another cell type associated with asthma, and allergic asthma in particular. Unlike many circulating inflammatory cells they mature in the tissue, where they can reside for long periods of time. Mast cells are found in close proximity to surfaces that are exposed to the exterior, such as the gut and respiratory epithelium or the skin. In asthmatic airways they are considerably more numerous than in healthy subepithelial tissue [13]. Mast cells are also found close to blood vessels and nerves, where their release of mediators can directly affect local circulatory and nervous signals.

Tissue mast cells have high affinity receptors for the IgE antibody Fc RI region and on activation through these they will release a variety of preformed granule-contained mediators such as histamine, TNF, IL-4, IL-5, GM-CSF and lipid mediators like LTC4. Apart from antibody-mediated immunity, they also participate in innate immunity responses against certain bacterial infections and parasitic infestations⁶. [30]

1.2.3.6 Leukotrienes generated by cells present in asthma

All leukotrienes are implicated in asthma. They are derived from arachidonic acid, which is normally found esterified to the mammalian cell membrane phospholipids. Once cleaved from the membrane, arachidonic acid is metabolized to one of several types of eicosanoids⁷, depending on cell type and situation. The different members of the leukotriene family are usually metabolized rapidly in a stepwise manner. LTA4 is converted to LTB4 or LTC4, the latter which is then the precursor of the other two cysteinyl leukotrienes, D4 and E4.

6 As remarked, eosinophils are also important in fighting parasites, but the mechanisms involved are overlapping rather than identical to those of mast cells. Interestingly, it appears as if some types of parasite infestations may protect against the development of asthma. In my personal view this implies that the “hygiene hypothesis”, which mainly relates to lymphocytes, should perhaps be explored and expanded in regard of mast cell and eosinophil function too. (If it hasn’t already.)

7 The word stems from the Greek word eikosi, which means “twenty”, and refers to the twenty carbon atoms that the precursor molecules contain.

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The half-life of the three cysteinyl leukotrienes LTC4, LTD4 and LTE4, is short, which makes their impact dependent on when and where they are formed.

Their importance in asthma is bolstered by the fact that eosinophils and mast cells, which are present in high numbers, also have a great capacity to generate leukotrienes. As mentioned previously, the cysteinyl leukotrienes are significantly linked to bronchoconstriction and tissue oedema.

LTB4 is an important chemoattractant for neutrophils, but also attracts activated eosinophils and increases cellular rolling and adhesion.

A number of the mediators involved in asthma are coded by regions on chromosome 5q (11q in mice), a chromosome with several known genotypes associated with the disease. These encompass several cytokines, e.g. IL-5 [9], but also LTC4 synthase [31], which is massively over-expressed in aspirin- intolerant asthmatic patients [32].

1.2.3.7 TNF amplifies inflammation on many levels

Other genetic regions of interest include areas on chromosome 6p (coding for human leukocyte antigen and TNF genes), 12q (coding for IFN-γ and mast cell growth factor genes), and 14 (T cell receptor area) [33]. TNF expression was originally considered a TH1 type response, but the cytokine is now recognized to play a central role in amplifying most kinds of inflammation, including asthma where it activates other cells, trigger physiological responses and induce the expression of several mediators and adhesion molecules. The main producers of TNF in asthma are probably macrophages and mast cells [18], but several other cell types are capable of TNF synthesis as well, including the above mentioned epithelial cells and eosinophils. Like many of the mast cell mediators, TNF is directly and indirectly associated with bronchospasm and airway hyperreactivity. This cytokine also up-regulates the endothelial surface expression of adhesion molecules like VCAM-1 and ICAM-1, which are important for eosinophil accumulation, and stimulates the production of other consequential mediators, e.g. endothelin-1 and eotaxin. A link has been suggested between TNF and subepithelial fibrosis, too, and its expression has been shown to be elevated in the sputum of asthmatic patients during antigen challenge. [18, 20]

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1.2.4 Asthma vs. other inflammatory airway diseases

The combination of a chronic lower airway inflammation, recurrent and often acute episodes of airway obstruction with wheezing, and an apparently⁸ non- bacterial/non-viral cause of the acute symptoms are the main features distinguishing asthma from other inflammatory lung diseases. The diagnosis is often apparent from the symptoms but in a small, yet significant, number of cases the symptoms may resemble or be confused with any of the differential diagnoses listed in Table 1. An example is prolonged or even chronic cough in children; another is chronic obstructive pulmonary disease (COPD) due to tobacco smoking, which is the most important differential diagnosis for adults.

In this COPD variant, the airway obstruction is however chronic and non- reversible, not episodic as in asthma. In addition, COPD patients seldom respond very well to bronchodilators and show distinct lung tissue alterations in x-ray.

Table 1. Differential diagnoses for asthma

Modified from “Expert Panel Report 3: Guidelines for the Diagnosis and Management of

Asthma-Summary Report 2007” J Allergy Clin Immunol 2007; 120:S94-138.

Infants and children Adults

Upper airway disease (e.g. allergic rhinitis and sinusitis)

Obstruction involving large airways (e.g. foreign body or tumour in trachea or bronchus) Obstruction involving small airways (e.g. viral bronchiolitis, cystic fibrosis)

Other causes (e.g. recurrent cough not due to asthma)

COPD (e.g. chronic bronchitis or emphysema)

Congestive heart failure Pulmonary embolism Airway obstruction due to tumours

Cough secondary to drugs (e.g.

angiotensin-converting enzyme inhibitors)

8 Bacterial or viral upper airway infections temporarily cause airway inflammation, which may increase the propensity of acute asthma episodes, but the infections do not appear to trigger bronchoconstriction themselves. Although common colds are correlated with asthma exacerbations, it is not known if they cause the disease [10], [34].

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1.2.5 Treatment

There is no available cure for asthma, but to see fatal asthma attacks is fortunately an unusual event today⁹. From being regarded as a principally psychosomatic disease in the early 1900s [35], the last decades of research and increased understanding of the underlying mechanisms have led to vast achievements in controlling and managing asthma on a long-term basis.

Pharmacologically, treatment regimens aim at two goals: bronchodilation in acute episodes and reduction or control of the chronic airway inflammation.

Bronchodilators include β2 adrenergic receptor agonists (e.g. salbutamol), anticholinergics and theophylline, where β2-agonists are by far the most used, due to their effectiveness.

Neither of the bronchodilators, however, reduce airway inflammation, and other drugs are therefore needed for prophylactic long-term control. The most successful drugs used for this purpose are inhaled corticosteroids (ICS) [34].

Compared to orally administered steroids (OCS), they have the benefit of achieving a more localized effect at a lower dose, thus reducing the systemic side effects [36]. The use of ICS in children has been much disputed, but is now considered safe at normal doses [37]. See Table 2 for a more complete list of bronchodilator and controller therapies.

Pharmacological treatment of asthma usually follows an individualized plan designed after the severity of the disease. The Swedish MPA suggests a stepwise approach (see Table 3) where β2 agonists together with corticosteroids at different doses form the core regimens [38]. If needed, these are complemented with for example antileukotrienes.

In addition to the purely pharmacological aspect of asthma management, proper monitoring and assessment of asthma severity in combination with control of environmental factors and patient education are pivotal in

9 How asthma attacks could express themselves before the era of efficient bronchodilators and inhalation steroids is well illustrated in the movie Diarios de motocicleta from 2004, where Ernesto “Che” Guevara (played by Gael García Bernal) nearly dies of asthma after contracting a cold. The film is worth seeing for other reasons too, of course.

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ble 2. Bronchodilator and controller therapies for asthma treatment d mainly on the therapies described in Harrison’s principles of internal medicine, 17th ed., Goodman & Gilman’s Pharmacology, 11th ed. and akologisk behandling vid astma – Behandlingsrekommendation (2007), from the Swedish Medical Products Agency. Short-acting β2-agonists (e.g. salbutamol, terbutalin) Functional antagonist of smooth muscle constriction Inhaled Fast onset of effect Duration of 3-6 h Used only for symptom relief Long-acting β2-agonists (e.g. salmeterol, formoterol) Functional antagonist of smooth muscle constriction Administered orally Duration of approximately 12 h Anticholinergics (ipratropium) Inhibit the cholinergic reflex component of smooth muscle constriction Inhaled Always used in combination with fast-acting β2-agonist Only used in patients where asthma is less well controlled

Uridine, 4-thiouridine and isomaltitol in an asthma-like model